CN114072508A

CN114072508A - Novel lactic acid bacterium

Info

Publication number: CN114072508A
Application number: CN202080021187.7A
Authority: CN
Inventors: A·叶德烈约夫斯基; C·弗雷莫; S·范迪伦; T·德斯福格雷; M·C·若多; D·卢根
Original assignee: DuPont Nutrition Biosciences ApS
Current assignee: DuPont Nutrition Biosciences ApS
Priority date: 2019-03-14
Filing date: 2020-03-13
Publication date: 2022-02-18
Also published as: AU2020236909A1

Abstract

The present invention relates to compositions comprising a polypeptide encoding beta-galactosidaselacZGene (A)lacZ ^FS) The polynucleotide of (a), said beta-galactosidase being characterized by a specific profile with respect to its lactose hydrolysis efficiency. The invention also relates to a composition comprisinglacZ ^FSStrains of streptococcus thermophilus and bacterial compositions thereof of the alleles, and their use for obtaining fermented milks that do not undergo post-acidification.

Description

Novel lactic acid bacterium

Technical Field

Background

The food industry uses bacteria to improve the taste and texture of food or feed products. In the dairy industryIn this case, lactic acid bacteria are generally used, for example, to achieve acidification of milk (by fermentation of lactose) and to texture the product into which it is incorporated. For example, Streptococcus thermophilus (S. thermophius) Species of lactic acid bacteria are widely used alone or in combination with other bacteria for the manufacture of fresh fermented dairy products, such as cheese or yoghurt.

One of the limitations of using lactic acid bacteria in dairy technology is post-acidification, i.e. the production of lactic acid by lactic acid bacteria after the target pH (the pH required for the technology) has been obtained. Therefore, to avoid this post acidification phenomenon, it is necessary for the dairy manufacturer to rapidly cool the fermented product immediately after the target pH is obtained. Therefore, it would be advantageous for dairy manufacturers to lack flexibility during the manufacturing process, while having the possibility to keep the fermented product at the fermentation temperature for a period of time. Furthermore, the cooling step is energy consuming, and therefore bypassing the cooling step is both economical and environmentally friendly.

WO90/05459 describes mutant strains of Lactobacillus bulgaricus which are selected on the basis of their colour phenotype on a medium containing X-gal. This application reports the identification of temperature-conditioned Lactobacillus bulgaricus mutants (blue at 37 ℃ C., but white at 4 ℃ C.) and pH-sensitive Lactobacillus bulgaricus mutants (blue at pH 7, but white at pH4.5 or 5). However, WO90/05459 does not mention any mutation in the lacZ gene. Furthermore, WO90/05459 describes mutants characterized by an enzyme having an activity which is at least 90% of the activity of the wild-type enzyme under production conditions (processing temperature or processing pH) while having an activity which is at least 20% lower than the activity of the wild-type enzyme under storage conditions. However, the teaching of WO90/05459 is insufficient with respect to any enzymatic activity, and in particular with respect to β -galactosidase activity; indeed, as shown in examples 4 and 5 of the present application, at pH4.5 or pH6, there is no recognized reference β -galactosidase activity in the strain. Thus, the characterization of the mutants described in WO90/015459 is not possible without any reference values or reference strains.

WO2010/139765 describes a method for manufacturing fermented dairy products using a weakly post-acidified culture based on a specific lactobacillus bulgaricus strain. Since the culture is characterized by weak lactic acid production at fermentation temperatures, the pH is substantially stable and the cooling step can be avoided. However, WO2010/139765 does not characterize exemplary lactobacillus bulgaricus strains.

WO2015/193459 proposes other solutions to overcome the post-acidification problem: for example, by controlling the concentration of lactose in the milk before fermentation by adding lactase, lactic acid bacteria that cannot hydrolyze lactose (lactose deficient lactic acid bacteria) are provided. However, these solutions are not satisfactory for dairy manufacturers, as they require the addition of exogenous enzymes (such as lactase) in the milk before fermentation, making the manufacturing process more complex and expensive, or the addition of carbohydrates (such as sucrose) to the milk, which is not in line with the increasing demand for healthier products without additives.

There is therefore a need to provide dairy manufacturers with means for producing lactic acid bacteria based fermented products with satisfactory results and a high degree of flexibility in the manufacturing process.

Drawings

FIG. 1 is a graph representing the acidification curves (pH versus time) in the milk of (A) DGCC7984 strain and its two subclones DGCC12455 and DGCC12456, and (B) the evolution of the acidification rate of strain DGCC12456 over time (mUpH/min over time).

FIG. 2 is a graph representing the evolution of the acidification curve (pH versus time) and (B) acidification speed over time (mUpH/min versus time) in (A) milk of strain DGCC 715.

FIG. 3 is a representative strain 715^R354C(iii) acidification profile (pH versus time) and (B) graph of acidification rate versus time (mUpH/min versus time) in milk of (A).

FIG. 4 is a graph representing the evolution of the acidification curve (pH curve over time) and (B) acidification speed over time (mUpH/min over time) in (A) milk of the strain DGCC 11231.

FIG. 5 is a representative strain 11231^R354C(iii) acidification profile (pH versus time) and (B) graph of acidification rate versus time (mUpH/min versus time) in milk of (A).

FIG. 6 is a graph representing the beta-galactosidase activity of four strains of Streptococcus thermophilus at pH6 and pH 4.5.

FIG. 7 shows representative strains DGCC715 and 715^R354CStrain DGCC11231, strain 11231^R354CAnd β -galactosidase activity of strain DGCC12456 at pH6 and pH 4.5.

FIG. 8 shows representative strains DGCC715 and strain 715^R354CStrain DGCC11231, strain 11231^R354CAnd LacS at pH6 and pH4.5 of strain DGCC 12456: graph of LacZ ratio.

FIG. 9 shows representative strains DGCC715 and strain 715^R354CStrain DGCC11231, strain 11231^R354CAnd the difference in lactose hydrolysis efficiency (Δ EH) between pH6 and pH4.5 for strain DGCC 12456.

FIG. 10 is a graph representing (A) the viscosity measured at day 14 and (B) the evolution over time of the pH of stirred yoghurts (stored at 10 ℃) made with the strain DGCC12456 and packaged at a temperature of 20 ℃ or 35 ℃.

FIG. 11 is a graph representing the evolution of pH over time of yoghurts (stored at 10 ℃) made with the strain DGCC12456 (flat line) and with the reference culture (dashed line).

Disclosure of Invention

In one aspect, the invention relates to a polypeptide encoding a beta-galactosidase^FSThe polynucleotide of (a), which, when inserted in place of the allele of the lacZ gene of strain DGCC715 (deposited at DSMZ at 12.2.2019 with access number DSM33036), produces a polypeptide characterized by LacS_pH4.5：LacZ_pH4.5DGCC 715-derived strain in a ratio greater than 8, in which LacS_pH4.5Represents the lactose import activity of LacS permease calculated by assay A at pH4.5, and LacZ_pH4.5Represents the lactose hydrolysis activity of beta-galactosidase calculated by assay B at pH 4.5. Thus, the present invention relates to a method for encoding beta-galactosidase^FSDefined as the lacZ allele that compares the activity of the lactose import of LacS permease in DGCC715 derivatives, calculated by assay a at pH4.5, to the activity of lactose hydrolysis of beta-galactosidase, calculated by assay B at pH 4.5: (LacS_pH4.5：LacZ_pH4.5Ratio) to more than 8, said DGCC715 derivative being the strain DGCC715 (deposited at DSMZ with access number DSM33036 on 12/2 of 2019) in which its lacZ gene is encoded by said encoded beta-galactosidase^FSThe polynucleotide substitution of (3).

In one aspect, the invention relates to compositions comprising a polypeptide encoding a beta-galactosidase^FSA polynucleotide of a portion of at least 100 nucleotides of said polynucleotide, wherein said nucleotide portion encompasses a polynucleotide corresponding to said beta-galactosidase^FSCodon for residue 354.

In one aspect, the invention relates to a vector comprising a polynucleotide of the invention.

In one aspect, the invention relates to a streptococcus thermophilus strain comprising an allele of the lacZ gene, which is a gene according to the invention encoding a beta-galactosidase^FSlacZ of^FSAn allele.

In one aspect, the present invention relates to a bacterial composition comprising the streptococcus thermophilus strain of the present invention.

In one aspect, the present invention relates to a food or feed product comprising a streptococcus thermophilus strain according to the invention or a bacterial composition according to the invention.

In one aspect, the present invention relates to a method of making a fermentation product comprising: a) inoculating a substrate with the streptococcus thermophilus strain of the invention or the bacterial composition of the invention; and b) fermenting the inoculated substrate obtained from step a) to obtain a fermented product, preferably a fermented dairy product.

In one aspect, the present invention relates to the use of a streptococcus thermophilus strain according to the invention or a bacterial composition according to the invention for the manufacture of a food or feed product, preferably a fermented food product, more preferably a fermented dairy product.

In one aspect, the present invention relates to the use of a polynucleotide or vector of the present invention to obtain a streptococcus thermophilus strain that has a complete STOP phenotype when used in fermented milk by assay C.

In one aspect, the invention relates to the preparation of a compound having a complete STOPA method of producing a phenotypic streptococcus thermophilus strain comprising: a) streptococcus thermophilus strains are provided having a LacS of less than 5_pH4.5：LacZ_pH4.5Ratio of wherein LacS_pH4.5Represents the lactose import activity of LacS permease calculated by assay A at pH4.5, and LacZ_pH4.5Represents the lactose hydrolysis activity of beta-galactosidase calculated by assay B at pH 4.5; b) replacement of said Streptococcus thermophilus strain of step a) with a polynucleotide of the inventionlacZAlleles of genes or replacement of said Streptococcus thermophilus strain of step a) with the corresponding polynucleotide according to the inventionlacZA part of an allele of a gene, or modifying the S.thermophilus strain of step a)lacZThe sequence of the gene may have the same sequence as the polynucleotide of the present inventionlacZ ^FSAn allele; and C) recovering the Streptococcus thermophilus strain having the complete STOP phenotype when used in fermented milk by assay C. Accordingly, the present invention relates to a method for producing a streptococcus thermophilus strain with a complete STOP phenotype, comprising: a) streptococcus thermophilus strains are provided having a ratio of the activity of lactose import of LacS permease calculated by assay A at pH4.5 to the activity of lactose hydrolysis of beta-galactosidase calculated by assay B at pH4.5 (LacS) of less than 5_pH4.5：LacZ_pH4.5Ratio); b) replacement of said Streptococcus thermophilus strain of step a) with a polynucleotide of the inventionlacZAlleles of genes or replacement of said Streptococcus thermophilus strain of step a) with the corresponding polynucleotide according to the inventionlacZA part of an allele of a gene, or modifying the S.thermophilus strain of step a)lacZThe sequence of the gene may have the same sequence as the polynucleotide of the present inventionlacZ ^FSAn allele; and C) recovering the Streptococcus thermophilus strain having the complete STOP phenotype when used in fermented milk by assay C.

In one aspect, the invention relates to the identification of a nucleic acid encoding a beta-galactosidase^FSIs/are as followslacZ ^FSA method of alleles comprising: a) inserted into a test objectlacZAllele instead of strain DGCC 715: (Deposited at DSMZ with access number DSM33036 on 12.2.2019)lacZ(ii) alleles of genes to obtain DGCC 715-derived strains; and b) determining the lactose import Activity of LacS permease by assay A at pH4.5 (LacS)_pH4.5) And the lactose hydrolysis activity of beta-galactosidase was determined by assay B at pH4.5 (LacZ)_pH4.5) (ii) a Wherein LacS_pH4.5：LacZ_pH4.5A ratio greater than 8 indicateslacZThe allele is a gene encoding beta-galactosidase^FSIs/are as followslacZ ^FSAn allele.

Detailed Description

General definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present disclosure.

The headings provided herein are not limitations of the various aspects or embodiments of the disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below may be more fully defined by reference to the entire specification.

As used herein, the term "polynucleotide" is synonymous with the term "nucleotide sequence" and/or the term "nucleic acid sequence". Unless otherwise indicated, any nucleic acid sequence is written left to right in the 5 'to 3' direction.

As used herein, the term "protein" includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "protein". In the present disclosure and claims, the name of the amino acid, the conventional three letter code or the conventional one letter code of the amino acid residue is used. It is also understood that a protein may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code. Unless otherwise indicated, any amino acid sequence is written left to right in the amino to carboxyl direction.

In the present invention, specific numbering of amino acid residue positions in β -galactosidase can be employed. By aligning the amino acid sequence of the sample β -galactosidase with the β -galactosidase of SEQ ID No. 2, a number can be assigned to an amino acid residue position in said sample β -galactosidase which corresponds to the amino acid residue position or number of the amino acid sequence shown in SEQ ID No. 2 of the present invention.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the terms "comprising," "comprises," and "comprising" are synonymous with "including," "includes," or "containing," "contains," and are inclusive or open-ended and do not exclude additional, non-stated members, elements, or method steps. The terms "comprising," including, "and" comprising "also include the term" consisting of.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

It was surprisingly found in the present invention that mutations that alter lactose flux can be used to design strains of streptococcus thermophilus that can be used to produce fermented milk that does not undergo post-acidification when stored at fermentation temperatures.

In one aspect, the invention provides methods for identifying a nucleic acid encoding a beta-galactosidase^FSIs/are as followslacZ ^FSA method of alleles comprising:

a) inserted into a test objectlacZAllelic replacement of strain DGCC715lacZ(ii) alleles of genes to obtain DGCC 715-derived strains; and

b) determination of the lactose import Activity of LacS permease by assay A at pH4.5 in the DGCC 715-derivative of step a) (LacS_pH4.5) And the lactose hydrolysis activity of beta-galactosidase was determined by assay B at pH4.5 (LacZ)_pH4.5)；

Wherein LacS_pH4.5：LacZ_pH4.5A ratio greater than 8 indicateslacZThe allele is a gene encoding beta-galactosidase^FSIs/are as followslacZ ^FSAn allele.

In one embodiment, the method further comprises determining the lactose hydrolysis activity of beta-galactosidase by assay B at pH6 in DGCC 715-derivative (LacZ)_pH6) And wherein LacS_pH4.5：LacZ_pH4.5Ratio greater than 8 and LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min.) indicateslacZThe allele is a gene encoding beta-galactosidase^FSIs/are as followslacZ ^FSAn allele.

As used herein, the expression "Alleles of the lacZ gene"means that found in a particular Streptococcus thermophilus strainlacZThe form of the gene. For the majority of the bacterial genes, the nucleotide sequence of the gene may vary, and the alleles represent different sequences of the same gene.

Of strains of Streptococcus thermophiluslacZA gene is understood herein to meanlacLocated within the operon coding for the lactose permease lacSlacSNucleotide sequence encoding beta-galactosidase downstream of the gene [ Schroeder CJ et al, J Gen Microbiol. 1991 Feb;137(2):369-80]. Word (word) "beta-galactosidase enzyme"in this context can be associated with words"Beta-half Lactosidase"used interchangeably.

Method for producing streptococcus thermophiluslacZAn example of an allele of a gene is DGCC715 strain (DSM33036)Is/are as followslacZAlleles of the gene as shown in SEQ ID NO 1. The allele as defined in SEQ ID NO. 1 encodes the beta-galactosidase as shown in SEQ ID NO. 2.

Method for producing streptococcus thermophiluslacSAn example of an allele of a gene is of DGCC715 strainlacSAlleles of the gene as shown in SEQ ID NO 30. The allele as defined in SEQ ID NO. 30 encodes the lactose permease LacS as shown in SEQ ID NO. 31.

Encoding beta-galactosidase ^FS lacZ of ^FS Alleles

The inventors have shown thatlacZSome of the alleles code for beta-galactosidase when inserted in place of DGCC715 strainlacZThe activity at pH4.5 of the allele (SEQ ID NO:1) is greatly reduced but not zero (as determined by assay B). By "beta-galactosidase activity is not zero at pH 4.5" is meant beta-galactosidase activity at pH4.5 (LacZ) when determined by assay B as described herein_pH4.5) Is detectable.

As shown in examples 4 and 5 below, the β -galactosidase activity in the streptococcus thermophilus strains is highly variable from one strain to another, so that it is technically irrelevant to mention β -galactosidase activity without any reference value or without any reference strain. Furthermore, and as shown in example 6, lacZ was carried compared to DGCC715 strain^FSThe decrease in beta-galactosidase activity at pH4.5 in the allelic DGCC715 derived strain occurred together with an increase in LacS activity (as determined by assay a). Taken together, these results have led the inventors to characterize the decrease of β -galactosidase at pH4.5 by a robust and reproducible parameter, which is the activity of lactose import of LacS permease calculated by assay a at pH4.5 and the activity of lactose hydrolysis of β -galactosidase calculated by assay B at pH4.5Ratio (LacS)_pH4.5：LacZ_pH4.5Ratio). Thus, the inventors have shown that when inserted in place of DGCC715 strainlacZAlleles of the genes (SEQ ID NO:1), theselacZOne of the alleles results in LacS_pH4.5：LacZ_pH4.5The ratio is greater than 8. TheselacZAlleles are defined herein as "lacZ ^FSAn allele ". From theselacZ ^FSThe allele-encoded protein is referred to herein as "beta-galactosidase^FS". In other words,lacZ ^FSallele ratio of the activity of lactose import of LacS permease calculated by assay a at pH4.5 to the activity of lactose hydrolysis of beta-galactosidase calculated by assay B at pH4.5 in DGCC715 derivatives (LacS)_pH4.5：LacZ_pH4.5Ratio) to greater than 8, said DGCC715 derivative strain being the strain DGCC715 (DSM33036) in which its lacZ gene is encoded by said encoding β -galactosidase^FSA polynucleotide substitution of (a); LacS in DGCC715 derivatives as defined in the application_pH4.5：LacZ_pH4.5The ratio was "increased" by comparison with LacS of strain DGCC715 (DSM33036)_pH4.5：LacZ_pH4.5The ratio is compared to determine.

Thus, it resulted in LacS in DGCC 715-derived strains_pH4.5：LacZ_pH4.5Any ratio greater than 8 (as defined herein)lacZ ^FSAlleles (encoding beta-galactosidase)^FS) Are part of the present invention. Thus, LacS in DGCC 715-derived strains_pH4.5：LacZ_pH4.5Any increase in the ratio to greater than 8 (as defined herein)lacZ ^FSAlleles (encoding beta-galactosidase)^FS) Are part of the present invention. In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio is greater than 9 (as defined herein). In one embodiment, the inventionlacZ ^FSAlleles (encoding. beta. -halfLactosidase^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio is greater than 10 (as defined herein). In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio is greater than 11 (as defined herein). In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio is greater than 12 (as defined herein). In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio is selected from greater than 9, greater than 10, greater than 11, and greater than 12. Accordingly, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio (as defined herein) is increased to greater than a value selected from greater than 9, greater than 10, greater than 11, and greater than 12.

As mentioned elsewhere, beta-galactosidase activity at pH4.5 (LacZ)_pH4.5) Is not zero, i.e. detectable when determined by assay B; in one embodiment, and with respect to LacS as defined herein_pH4.5：LacZ_pH4.5Any combination of minimum values of the ratios, of the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5Ratio (as defined herein) of less than 100 (or LacS in DGCC 715-derivative_pH4.5：LacZ_pH4.5The ratio is increased to less than 100).

In one embodiment, as defined hereinlacZ ^FSCharacteristics of the alleles (except LacS)_pH4.5：LacZ_pH4.5Ratio) also lies in the fact that it encodes a beta-galactosidase^FSWhen is coming into contact withIs inserted intolacZ ^FSAllelic replacement of DGCC715 StrainlacZAllele of the gene, the beta-galactosidase^FSActivity at pH6 (as determined by assay B) (LacZ)_pH6) Is at least 7.10^-8mol/(mg total protein extract. min). Accordingly, as defined hereinlacZ ^FSCharacteristics of the alleles (except LacS)_pH4.5：LacZ_pH4.5Ratio) also lies in the fact that it encodes a beta-galactosidase^FSSaid beta-galactosidase^FSActivity at pH6 in DGCC 715-derived Strain (as determined by assay B) (LacZ)_pH6) Is at least 7.10^-8mol/(mg total protein extract. min.) said DGCC715 derivative strain is strain DGCC715 in which its lacZ gene is encoded by saidlacZ ^FSAnd (4) allele replacement. In one embodiment of the process of the present invention,lacZ ^FSallele encoding beta-galactosidase^FSSaid beta-galactosidase^FSActivity at pH6 (LacZ)_pH6) Is at least 8.10^-8mol/(mg total protein extract. min). In one embodiment of the process of the present invention,lacZ ^FSallele encoding beta-galactosidase^FSSaid beta-galactosidase^FSActivity at pH6 (LacZ)_pH6) Is at least 9.10^-8mol/(mg total protein extract. min). In one embodiment of the process of the present invention,lacZ ^FSallele encoding beta-galactosidase^FSSaid beta-galactosidase^FSActivity at pH6 (LacZ)_pH6) Is at least 1.10^-7mol/(mg total protein extract. min). In one embodiment, as defined hereinlacZ ^FSCharacteristics of the alleles (except LacS)_pH4.5：LacZ_pH4.5Ratio) also lies in the fact that it encodes a beta-galactosidase^FSWhen inserting saidlacZ ^FSAllelic replacement of DGCC715 StrainlacZAllele of the gene, the beta-galactosidase^FSActivity at pH6 (as measured by assay B)Stator) (LacZ)_pH6) Is selected from at least 7.10^-8At least 8.10^-8At least 9.10^-8And at least 1.10^-7mol/(mg total protein extract. min) (i.e., in DGCC715 derivative, the DGCC715 derivative is strain DGCC715, in which its lacZ gene is replaced by the lacZ genelacZ ^FSAllelic replacement).

Thus, in one embodiment, LacS is caused in DGCC 715-derived strains_pH4.5：LacZ_pH4.5A ratio greater than 8 (as defined herein) and results in LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min) (as defined herein) of anylacZ ^FSAlleles (encoding beta-galactosidase)^FS) Are part of the present invention. In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5A ratio selected from greater than 9, greater than 10, greater than 11, and greater than 12 (as defined herein), and results in LacZ in the DGCC 715-derivative strain_pH6Is selected from at least 7.10^-8At least 8.10^-8At least 9.10^-8And at least 1.10^-7mol/(mg total protein extract. min) (as determined by assay B). In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) Resulting in LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio (as defined herein) is less than 100. Thus, LacS was isolated in DGCC 715-derived strains_pH4.5：LacZ_pH4.5The ratio increased to greater than 8 (LacS with strain DGCC 715)_pH4.5：LacZ_pH4.5Ratio comparison) and results in LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min) (as defined herein) of anylacZ ^FSAlleles (encoding beta-galactosidase)^FS) Are part of the present invention. In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio is increased to greater than a value selected from greater than 9, greater than 10, greater than 11, and greater than 12 (as defined herein) and results in LacZ in the DGCC 715-derivative strain_pH6Is selected from at least 7.10^-8At least 8.10^-8At least 9.10^-8And at least 1.10^-7mol/(mg total protein extract. min) (as determined by assay B). In one embodiment, the inventionlacZ ^FSAlleles (encoding beta-galactosidase)^FS) LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5The ratio (as defined herein) increases to less than 100.

The beta-galactosidase is disclosed below^FSAre described herein.

Notably, in the present invention, LacS and LacZ activity (at pH4.5 and pH 6) were calculated by assay a and assay B, respectively, as described herein.

When inserted in place of DGCC715 strainlacZAllelic forms of the genes do not result in LacS_pH4.5：LacZ_pH4.5A ratio (as defined herein) greater than 8lacZAlleles are not to be considered according to the inventionlacZ ^FSAn allele. In other words, LacS in DGCC715 derivative was not used_pH4.5：LacZ_pH4.5Ratio (as defined herein) increased to greater than 8lacZAlleles are not to be considered according to the inventionlacZ ^FSAllele, said DGCC715 derivative being strain DGCC715, in which its lacZ gene is replaced by said lacZ allele.

LacS activity, LacZ activity and ratio

The present invention relies on the determination of the activity of the lacS permease for lactose import and/or the determination of the lactose hydrolysis activity of beta-galactosidase at a specific pH (pH 4.5 and/or pH 6). These activities are determined in a particular strain, such as, for example, in DGCC715 strain or in a DGCC 715-derivative strain, as defined herein.

Lactose import Activity of LacS permease at specific pH (pH X) in the textIs called "LacS _pHx". In one embodiment, the activity is determined at pH4.5 (LacS)_pH4.5). In one embodiment, the activity is determined at pH6 (LacS)_pH6). In a specific embodiment, the lactose import activity of the LacS permease is determined by assay a at a specific pH (such as pH4.5 or pH 6).

The lactose hydrolysis activity of beta-galactosidase at a specific pH (pH X) is referred to herein as "LacZ _pHx". In one embodiment, the activity is determined at pH4.5 (LacZ)_pH4.5). In one embodiment, the activity is determined at pH6 (LacZ)_pH6). In a specific embodiment, the lactose hydrolysis activity of β -galactosidase is determined by assay B at a specific pH (such as pH4.5 or pH 6).

Determination of LacS_pH4.5：LacZ_pH4.5Ratios used to qualify the inventionlacZ ^FSOne way of determining the allele is to determine the allele in which it is presentlacZAlleles of genes have been testedlacZThe activity of lactose import of LacS permease at pH4.5 in the allele-substituted DGCC715 strain (referred to herein as "DGCC 715-derivative"), and the activity of lactose hydrolysis of β -galactosidase at pH4.5 in the same DGCC 715-derivative was determined, and the ratio of the two activities was calculated.

Assay A (LacS Activity)

Streptococcus thermophilus strains were grown overnight at 37 ℃ on M17 medium containing 30g/L sucrose as sole carbon source. When the cells reached stationary phase, they were transferred (at 0.05 uDO/mL) to 1 volume of M17 medium containing 30g/L lactose as the sole carbon source and incubated at 42 ℃ for 2 hours. The strain cultures were centrifuged at room temperature (3500 g), the supernatant removed, and the cells resuspended in 0.5 volume of 4% (w/v) glycerophosphate. This washing step was applied twice. 1.8 mL of the cell suspension in 4% glycerophosphate was incubated at 42 ℃ for 2 minutes. Then, 0.2 mL of lactose solution (70 g/L lactose + 0.1M potassium phosphate buffer) was added [ according to the desired measurement, lactose was previously dissolvedThe solution pH is adjusted at pH4.5 or pH 6). The mixture was incubated at 42 ℃ for a further 3 minutes. The reaction was blocked by removing cells by filtration on a 0.22 μm filter. The lactose in the filtered solution was then determined on HPLC using the following protocol. The solution was diluted 10-fold in water and 10 μ Ι _ was injected on Agilent 1200 HPLC (high performance liquid chromatography). The elution was carried out in isocratic mode with pure water at 0.6 mL/min. The molecule was separated to Pb within 40 min²⁺Ion exchange columns (SP-0810 Shodex 300 mm x 8 mm x 7μm) columns. The sugars were detected with a refractometer. Quantification was done by external calibration.

The lactose import activity of LacS permease was calculated as follows:

LacS activity = ([ lactose ])]_Initial- [ lactose]_3min) V (DO x time), expressed in μmol/(uDO. min), where:

- [ lactose]_InitialInitial concentration in μmol/mL

- [ lactose]_3minIs the concentration in. mu. mol/mL after 3 minutes at 42 ℃

DO is the density of bacteria in uDO/mL

Time is the duration of the experiment in minutes (in this case, 3 minutes).

Assay B (LacZ Activity)

A fresh overnight culture of the Streptococcus thermophilus strain to be determined in M17 containing 30g/L lactose was obtained and used to inoculate 10 ml of fresh M17 containing 30g/L lactose at 1% (v/v). After 3 hours of growth on M17 containing 30g/L lactose at 42 ℃, the cells were harvested by centrifugation (6000 g, 10 min, 4 ℃), washed in 1.5 ml cold lysis buffer (KPO40.1M) and resuspended in 300. mu.l cold lysis buffer. EDTA-free protease inhibitor "cOmplete" was prepared as described by the supplier^TM"(Roche, supplier reference 04693132001) was added to the lysis buffer. Cells were disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 250 μ l of resuspended cells and shaking for 6 minutes in a MM200 shaker grinder (Retsch, Haan, Germany) at a frequency of 30 cycles/s. Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4 ℃), and the supernatant was discardedThe solution was transferred to a clean 1.5 mL centrifuge tube kept on ice. Total protein content was determined by using the FLUKA protein quantification kit-Rapid (ref 51254). Beta-galactosidase activity in cell extracts was determined spectrophotometrically by monitoring the hydrolysis of O-nitro-phenol-beta-galactoside (ONPG) to galactose and O-nitro-phenol (ONP). mu.L of the cell extract was mixed with 135. mu.L of reaction buffer (NaPO)₄ 100 mM; KCl 10 mM; MgSO ₄1 mM ONPG 3 mM + beta mercaptoethanol 60 mM, pH = 6). The production of ONP results in yellow color entering the tube. When yellow color appeared, stop buffer (Na) was added by 250. mu.L₂CO₃1M) to block the reaction. The optical density at 420 nm was recorded using a Synergy HT multi-detection microplate reader (BIO-TEK). One unit of beta-galactosidase corresponds to the amount of enzyme that catalyzes the production of 1. mu. mol ONP per minute under the assay conditions. Beta-galactosidase activity was calculated as follows:

LacZ activity = dOD x V/[ dt x l x ɛ x Qprot ], expressed in mol/(mg total protein extract. min), where:

dOD is the change in Optical Density (OD) at 420 nm between blank and test sample

V is the volume of the reaction in which the optical density is measured (250. mu.L herein)

-dt = represents the duration in minutes between the addition of 20 μ Ι _ of bacterial extract and the addition of 250 μ Ι _ of stop buffer

-l = optical path length (0.73 cm in this text)

- ɛ = molar attenuation coefficient of ONP (4500 cm herein)² / µmol)

-Qprot = amount of protein in cuvette (in mg).

Ratio calculation

Once LacS and LacZ activities have been calculated as defined herein, the activity LacS_pHX：LacZ_pHXThe ratio is calculated as follows: [ LacS as defined herein_pHXLacZ as defined herein_pHX] x10^-6。

It is noted that when reference is made to LacS_pHX：LacZ_pHXIn the ratios, both LacS and LacZ activities are in the same strain, in particularCalculated in the same DGCC 715-derivative strain.

lacZ variant alleles encoding beta-galactosidase variants

lacZAlleles which 1) encode a beta-galactosidase whose sequence has at least 95% identity with SEQ ID NO 2, and 2) when inserted in place of DGCC715 strainlacZAllelic Gene leads to LacS_pH4.5：LacZ_pH4.5A ratio (as defined herein) of less than 5, referred to herein aslacZVariant alleles (encoding β -galactosidase variants). In other words,lacZalleles which 1) encode a beta-galactosidase the sequence of which has at least 95% identity with SEQ ID NO 2, and 2) do not convert LacS in DGCC715 derived strains_pH4.5：LacZ_pH4.5The ratio (as defined herein) is increased to 5 or greater than 5, referred to herein aslacZA variant allele (encoding a β -galactosidase variant), said DGCC715 derivative being strain DGCC715, in which its lacZ gene is replaced by said lacZ variant allele; LacS in DGCC715 derivative as described previously_pH4.5：LacZ_pH4.5The ratio was "increased" by comparison with LacS of strain DGCC715 (DSM33036)_pH4.5：LacZ_pH4.5The ratio is compared to determine. Expression'Beta-galactosidase variants"and expression"Beta-galactosidase variants having at least 95% identity to SEQ ID NO 2"may be used interchangeably.

In one embodiment, when inserted in place of DGCC715 strainlacZWhen the allele of the gene is present,lacZvariant alleles result in LacS_pH4.5：LacZ_pH4.5Ratio (as defined herein) of less than 4 (or not to LacS in DGCC715 derivative as defined herein)_pH4.5：LacZ_pH4.5The ratio is increased to 4 or greater than 4). In one embodiment, when inserted in place of DGCC715 strainlacZWhen the allele of the gene is present,lacZvariant alleles result in LacS_pH4.5：LacZ_pH4.5Ratio (as defined herein) of less than 3 (or not to DGCC715 derivative as defined herein)LacS in (1)_pH4.5：LacZ_pH4.5The ratio is increased to 3 or more than 3).

And to the above LacS_pH4.5：LacZ_pH4.5Any combination of the embodiments of the ratios is,lacZvariant alleles are also defined which encode a β -galactosidase variant having a sequence at least 95% identical to SEQ ID No. 2. By "at least 95% identity to SEQ ID No. 2" is meant at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In one embodiment, a variant of beta-galactosidase enzyme (consisting oflacZVariant allele codes) has a sequence with at least 96% identity to SEQ ID No. 2. In one embodiment, a variant of beta-galactosidase enzyme (consisting oflacZVariant allele codes) has a sequence with at least 97% identity to SEQ ID No. 2. In one embodiment, a variant of beta-galactosidase enzyme (consisting oflacZVariant allele codes) has a sequence with at least 98% identity to SEQ ID No. 2. In one embodiment, a variant of beta-galactosidase enzyme (consisting oflacZVariant allele codes) has a sequence with at least 99% identity to SEQ ID No. 2.

In one embodiment, the β -galactosidase variant is the same size as the β -galactosidase protein as defined in SEQ ID NO:2 (1026 amino acid residues), in combination with the percentage identity; thus, in one embodiment,lacZvariant alleles are additionally defined as β -galactosidase variants which encode the 1026-amino acid.

In one embodiment of the process of the present invention,lacZvariant alleles are defined herein as:

1) encodes a beta-galactosidase variant having a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID No. 2; and

2) when inserted to replaceDGCC715Of bacterial strainslacZAllele of the gene, resulting in LacS_pH4.5：LacZ_pH4.5The ratio (as defined herein) is less than 5, less than 4, or less than 3.

Therefore, the temperature of the molten metal is controlled,lacZvariant alleles are defined herein as:

2) LacS in DGCC715 derivatives as defined herein is not to be included_pH4.5：LacZ_pH4.5The ratio is increased to 5 or more than 5, to 4 or more than 4 or to 3 or more than 3.

Non-limiting examples of β -galactosidase variants are disclosed in table 2 and their sequences are as defined in SEQ ID nos 6, 9, 12, 15, 18, 21, 24 and 27.

Replacement of the allele of the lacZ gene of strains of Streptococcus thermophilus, in particular of the DGCC715 strain

By means of a test object to be testedlacZReplacement of particular strains of Streptococcus thermophilus by alleleslacZAlleles of a gene are carried out using conventional techniques in molecular biology and are within the ability of one of ordinary skill in the art. In general, suitable conventional methods include substitution via homologous recombination.

Expression'Insertion of lacZ allele instead of allele of lacZ gene"and expression"By lacZ to be tested Allele substitution of lacZ Gene allele"is synonymous. Expression'lacZ ^FS Allele insertion in place of lacZ Gene, etc Site gene"and expression"By lacZ ^FS Allele substitution of lacZ Gene allele"is synonymous.

By replacement (or insertion instead) is meant replacement bylacZAlleles (to be tested)lacZAllele) encoding a beta-galactosidase different from that of the streptococcus thermophilus strainlacZThe sequence of beta-galactosidase encoded by the alleles of the gene. Thus, substitution (or insertion in place) means of a strain of S.thermophiluslacZThe coding sequence of the gene (from the 1 st nucleotide of the start codon to the last nucleotide of the stop codon) is testedlacZA corresponding coding sequence substitution for an allele.

In DGCC715 strainBy replacing (or inserting instead) is meant in the case oflacZAlleles (to be tested)lacZAllele) encoding a beta-galactosidase protein different from that of DGCC715 strainlacZThe gene encodes a beta-galactosidase sequence. Thus, substitution (or insertion in place) means of DGCC715 strainlacZThe coding sequence of the gene (from nucleotide 1 of the start codon to the last nucleotide of the stop codon, i.e.nucleotides 1 to 3081 of SEQ ID NO:1) is testedlacZA corresponding coding sequence substitution for an allele. It is composed oflacZGenes have been testedlacZAlleles (such aslacZ ^FSAlleles orlacZVariant allele) substituted DGCC715 strain is defined herein as "DGCC 715-derived strain”。

DGCC715 strain

The Streptococcus thermophilus DGCC715 strain has been deposited under Budapest treaty at Leibniz-institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Inhoffentr. 7B, D-38124 Braunschweig) by DuPont Nutrition Biosciences ApS on 12.2.2019 and has received the deposit number DSM 33036. Conditions for culturing the strain are provided in the examples section. The applicant claims that samples of deposited microorganisms described herein are only available to experts until the date of patent grant.

Expression'DGCC715 strain"and"DGCC 715-derived strain"respectively with expression"DSM33036 strain"and"DSM 33036-derived strain"may be used interchangeably.

Generation of lacZ alleles to be tested (including lacZ ^FS Alleles)

To be testedlacZAlleles (in particularlacZ ^FS Alleles) Whether random or directed mutagenesis is used or notlacZ ^FSOf alleleslacZAlleles, in particular from the coding sequence of beta as defined in SEQ ID NO 2Of galactosidaselacZAlleles (e.g., SEQ ID NO:1) or fromlacZVariant alleles are generated. In one embodiment, to be testedlacZAlleles (in particularlacZ ^FSAlleles) were generated by random mutagenesis. In another embodiment, to be testedlacZAlleles (in particularlacZ ^FSAlleles) can be generated by directed mutagenesis. Suitable mutagenesis protocols for random or directed mutagenesis are well known and described in the literature.

Identification may be used as defined hereinlacZ ^FSMethod of screening alleles for assays generated therebylacZAn allele.

Beta-galactosidase enzyme ^FS Protein sequence

According to the inventionlacZ ^FSAlleles-as part of the polynucleotides of the invention or comprised in the lactic acid bacteria of the invention-in addition to leading to LacS_pH4.5：LacZ_pH4.5A ratio greater than 8 (as defined herein) (or LacS_pH4.5：LacZ_pH4.5Ratio increase to greater than 8) and optionally results in LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min) (as defined herein), can be defined by its nucleotide sequence or by the amino acid sequence of its encoded beta-galactosidase.

In one embodiment, as defined hereinlacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSIs different from SEQ ID NO 2. In one embodiment, as defined hereinlacZ ^FSAlleles-as part of the polynucleotide of the invention or comprised in the lactic acid bacterium of the invention-are defined by the fact that: it leads to LacS in DGCC 715-derived strain_pH4.5：LacZ_pH4.5A ratio greater than 8 (as defined herein) (or lacS_pH4.5：LacZ_pH4.5Ratio increased to greater than 8), and optionally results in LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min) (as defined herein), and which encodes a beta-galactosidase^FSSaid beta-galactosidase^FSIs different from SEQ ID NO 2. Described elsewhere in this application with regard to LacS_pH4.5：LacZ_pH4.5Ratio and LacZ_pH6Are similarly applicable in the present context.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSComprising amino acid inhibition (i.e., inhibition of one or more amino acids), amino acid addition (i.e., addition of one or more amino acids), amino acid substitution (i.e., substitution of one or more amino acids), or amino acid inhibition and addition (i.e., inhibition and addition of one or more amino acids) relative to a beta-galactosidase selected from the group consisting of:

a) beta-galactosidase having an amino acid sequence as defined in SEQ ID NO 2; and

b) a variant beta-galactosidase protein as defined herein having at least 95% identity with SEQ ID No. 2. A variant beta-galactosidase protein as defined herein fromlacZVariant allele codes, when inserted in place of DGCC715 strainlacZAlleles of a gene, thelacZVariant alleles result in LacS_pH4.5：LacZ_pH4.5Ratio of less than 5 (as defined herein) (or not to LacS in DGCC715 strain as defined herein_pH4.5：LacZ_pH4.5The ratio is increased to 5 or more than 5). In thatlacZIn the context of variant alleles described elsewhere in this application with regard to LacS_pH4.5：LacZ_pH4.5Specific embodiments of ratios, percent identity, and sizes are similarly applicable in the present context.

In one embodiment of the process of the present invention,lacZ ^FSallele encoding beta-galactosidase^FSSaid beta-galactosidase^FSInclusion of amino acid inhibitors relative to beta-galactosidase selected from the group consisting ofPreparing: a) a beta-galactosidase having an amino acid sequence as defined in SEQ ID No. 2, and b) a beta-galactosidase variant as defined herein having at least 95% identity with SEQ ID No. 2; in a specific embodiment, the beta-galactosidase^FSIs characterized by inhibition of at least one amino acid, in particular 1, 2, 3, 4 or 5 amino acids. In a specific embodiment, the beta-galactosidase^FSIs characterized by the inhibition of one amino acid. In a specific embodiment, the beta-galactosidase^FSIs characterized by inhibition of 2, 3, 4 or 5 amino acids. In a specific embodiment, the beta-galactosidase^FSIs characterized by inhibition of 2, 3, 4 or 5 consecutive amino acids.

In one embodiment of the process of the present invention,lacZ ^FSallele encoding beta-galactosidase^FSSaid beta-galactosidase^FSComprising amino acid additions relative to a beta-galactosidase selected from the group consisting of: a) a beta-galactosidase having an amino acid sequence as defined in SEQ ID No. 2, and b) a beta-galactosidase variant as defined herein having at least 95% identity with SEQ ID No. 2; in a specific embodiment, the beta-galactosidase^FSIs characterized by the addition of at least one amino acid, in particular 1, 2, 3, 4 or 5 amino acids. In a specific embodiment, the beta-galactosidase^FSIs characterized by the addition of one amino acid. In a specific embodiment, the beta-galactosidase^FSIs characterized by the addition of 2, 3, 4 or 5 amino acids. In a specific embodiment, the beta-galactosidase^FSIs characterized by the addition of 2, 3, 4 or 5 consecutive amino acids.

In one embodiment of the process of the present invention,lacZ ^FSallele encoding beta-galactosidase^FSSaid beta-galactosidase^FSComprising amino acid substitutions relative to a beta-galactosidase selected from the group consisting of: a) beta-half having the amino acid sequence as defined in SEQ ID NO 2Lactosidase, and b) a β -galactosidase variant as defined herein having at least 95% identity with SEQ ID No. 2; in a specific embodiment, the beta-galactosidase^FSIs characterized by the substitution of at least one amino acid, in particular 1, 2, 3, 4 or 5 amino acids. In a specific embodiment, the beta-galactosidase^FSIs characterized by the substitution of one amino acid. In a specific embodiment, the beta-galactosidase^FSCharacterized by 2, 3, 4 or 5 amino acid substitutions. In a specific embodiment, the beta-galactosidase^FSThe length is 1026 amino acids.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSWherein said beta-galactosidase^FSDoes not comprise arginine at position 354, wherein the amino acid sequence shown in SEQ ID NO 2 is used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSWherein said beta-galactosidase^FSDoes not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine and lysine, wherein the amino acid sequence shown in SEQ ID NO. 2 is used for numbering. In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSWherein said beta-galactosidase^FSDoes not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine, wherein the amino acid sequence shown in SEQ ID NO. 2 is used for numbering.

In one embodiment, thelacZ ^FSThe allele encodes a beta-galactosidase comprising a cysteine or equivalent amino acid at position 354^FSWherein the amino acid sequence shown in SEQ ID NO 2 is used for numbering. "The equivalent amino group thereof Acid(s)"means polarity, charge, solubility at the residueAny amino acid with similarity in degree, hydrophobicity, hydrophilicity and/or amphipathic nature, as long as it encodes the beta-galactosidase enzyme^FSIs/are as followslacZ ^FSAlleles, when inserted in place of DGCC715 strainlacZAllele of the gene, resulting in LacS_pH4.5：LacZ_pH4.5A ratio greater than 8 (as defined herein) and optionally resulting in LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min) (as defined herein). In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSAn amino acid residue selected from cysteine, alanine and serine is included at position 354, wherein the amino acid sequence shown in SEQ ID NO. 2 is used for numbering.

In one embodiment, thelacZ ^FSThe allele encodes a beta-galactosidase comprising a cysteine at position 354^FSWherein the amino acid sequence shown in SEQ ID NO 2 is used for numbering.

In a specific embodiment of any of these embodiments, the beta-galactosidase enzyme^FSThe length is 1026 amino acids.

In one embodiment, the inventionlacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity to SEQ ID NO. 2, but differs from SEQ ID NO. 2.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity to SEQ ID No. 2, but differs from SEQ ID No. 2 and does not comprise arginine at position 354, wherein the amino acid sequence shown in SEQ ID No. 2 is used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2 and does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine and lysine, wherein the amino acid sequence shown in SEQ ID No. 2 is used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2 and does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine, wherein the amino acid sequence shown in SEQ ID No. 2 is used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2 and comprises a cysteine or equivalent amino acid at position 354, wherein the amino acid sequence shown in SEQ ID No. 2 is used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2 and comprises an amino acid residue at position 354 selected from the group consisting of cysteine, alanine and serine, wherein the amino acid sequences shown in SEQ ID No. 2 are used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2 and comprises a cysteine at position 354, wherein the amino acid sequence shown in SEQ ID No. 2 is used for numbering.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSComprises the following steps:

a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an arginine at position 354 (SEQ ID NO:5, wherein position 354 is not an arginine); or

b) An amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID No. 2 (beta-galactosidase variant as defined herein), but which does not comprise an arginine at position 354. Beta-galactosidase enzyme^FSAs defined in

SEQ ID nos

7, 10, 13, 16, 19, 22, 25 and 28, wherein position 354 is not arginine.

a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine and lysine (SEQ ID NO:5, wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine and lysine); or

b) An amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID No. 2 (beta-galactosidase variant as defined herein), but which does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine and lysine. Beta-galactosidase enzyme^FSAs defined in

SEQ ID nos

7, 10, 13, 16, 19, 22, 25 and 28, wherein position 354 is not an amino acid residue selected from arginine, histidine, glutamine and lysine.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSThe beta-Galactosidase enzyme^FSComprises the following steps:

a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine (SEQ ID NO:5 wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine); or

b) An amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID No. 2 (beta-galactosidase variant as defined herein), but which does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine. Beta-galactosidase enzyme^FSAs defined in

SEQ ID nos

7, 10, 13, 16, 19, 22, 25 and 28, wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine.

a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises a cysteine or equivalent amino acid thereof at position 354 (SEQ ID NO:5, wherein position 354 is a cysteine or equivalent amino acid); or

b) An amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO:2 (beta-galactosidase variant as defined herein), but which comprises a cysteine or equivalent amino acid thereof at position 354. Beta-galactosidase enzyme^FSAs defined in

SEQ ID nos

7, 10, 13, 16, 19, 22, 25 and 28, wherein position 354 is cysteine or its equivalent amino acid.

a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises an amino acid residue at position 354 selected from cysteine, alanine and serine (SEQ ID NO:5, wherein position 354 is selected from cysteine, alanine and serine); or

b) An amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID No. 2 (beta-galactosidase variant as defined herein), but which comprises an amino acid residue at position 354 which is selected from cysteine, alanine and serine. Beta-galactosidase enzyme^FSAs defined in

SEQ ID nos

7, 10, 13, 16, 19, 22, 25 and 28, wherein position 354 is an amino acid residue selected from cysteine, alanine and serine.

a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises a cysteine at position 354 (SEQ ID NO: 4); in one embodiment, thelacZ ^FSAlleles are shown in SEQ ID NO. 3; or

b) An amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO:2 (beta-galactosidase variant as defined herein), but which comprises a cysteine at position 354. Beta-galactosidase enzyme^FSAs defined in SEQ ID NOs 8, 11, 14, 17, 20, 23, 26 and 29.

In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSBeta-galactosidase with the sequence shown in SEQ ID NO:2 was obtained by substituting arginine (R354C) with cysteine at position 354.

In one embodiment, thelacZ ^FSAllelic stateGene encoding beta-galactosidase^FSSaid beta-galactosidase^FSA β -galactosidase variant having at least 95% identity to SEQ ID NO:2 (a β -galactosidase variant as defined herein) was obtained by substituting arginine (R354C) with cysteine at position 354. In one embodiment, thelacZ ^FSAllele encoding beta-galactosidase^FSSaid beta-galactosidase^FSA variant β -galactosidase as shown in SEQ ID NO 6, 9, 12, 15, 18, 21, 24 or 27 was obtained by substituting arginine (R354C) with cysteine at position 354.

Amino acid numbering

In the present application, the specific numbering of amino acid residue positions is used to characterize β -galactosidase. By reacting beta-galactosidase with^FSAlignment of the amino acid sequence of the protein or beta-galactosidase variant with the beta-galactosidase protein defined in SEQ ID NO 2, numbers can be assigned to the beta-galactosidase protein in question, respectively^FSOr amino acid residue positions in the beta-galactosidase variant which correspond to the amino acid residue positions or numbering of the amino acid sequence shown in SEQ ID NO 2.

An alternative way of describing the numbering of amino acids as used in this application is to say that the amino acid position is identified by an amino acid position 'corresponding' to a specific position in the amino acid sequence shown in SEQ ID NO. 2. This should not be construed to mean that the sequences of the present invention must include the amino acid sequence shown in SEQ ID NO. 2. The skilled person will readily understand that the β -galactosidase sequence varies between different bacterial strains. Reference to the amino acid sequence shown in SEQ ID NO 2 is only used to enable the identification of a particular amino acid position within any particular β -galactosidase. Such amino acid positions can be identified routinely using sequence alignment programs, the use of which is well known in the art.

Multiple cores of the inventionAcid of lactic acid

In one aspect, the present invention provides a composition comprisinglacZ ^FSAlleles [ encoding the beta-galactosidase of the invention^FS]Or a polynucleotide consisting thereof. In one embodiment, the polynucleotide is of the inventionlacZ ^FSAlleles [ coding for beta-galactosidase^FS]. In one embodiment, the polynucleotide of the invention encodes a beta-galactosidase as defined herein^FS. In one embodiment, a polynucleotide of the invention is at least 3063 nucleotides, at least 3066 nucleotides, at least 3069 nucleotides, at least 3072 nucleotides, at least 3075 nucleotides, at least 3078 nucleotides or at least 3081 nucleotides in size. In one embodiment, the polynucleotide of the invention is less than 5 kb or less than 4 kb in size. In one embodiment, the polynucleotide ranges in size from a minimum size selected from at least 3063 nucleotides, at least 3066 nucleotides, at least 3069 nucleotides, at least 3072 nucleotides, at least 3075 nucleotides, at least 3078 nucleotides or at least 3081 nucleotides to a maximum size selected from 4 kb and 5 kb. In one embodiment, the polynucleotide is 3078 or 3081 nucleotides in size.

In one embodiment, the polynucleotide of the invention consists oflacZ ^FSAllelic composition, oflacZ ^FSThe alleles are independently flanked on one side (at 5 'and at 3') or on both sides by nucleotide regions ranging from 500bp to 1 kb.

In one aspect, the invention provides a composition comprising a polypeptide encoding a beta-galactosidase as defined herein^FSA portion of at least 100 nucleotides of a polynucleotide of (a), or a polynucleotide consisting of said portion of nucleotides, wherein said portion of nucleotides encompasses a polynucleotide corresponding to said beta-galactosidase^FSCodon for residue 354. Expression'Corresponding to the beta-galactosidase ^FS Codon of residue 354 Seed of Japanese apricot"means as defined hereinlacZ ^FSCodon 354 of an allele, wherein the codon isThe child corresponds to beta-galactosidase^FSResidue 354 of (1), wherein the amino acid sequence shown in SEQ ID NO:2 is used for numbering.lacZ ^FSPosition of codon 354 of the allele and beta-galactosidase^FSThe position of residue 354 can be readily determined by one skilled in the art by aligning a portion of at least 100 nucleotides or a beta-galactosidase peptide encoded by such a portion of at least 100 nucleotides with SEQ ID NO:1 or SEQ ID NO:2, respectively. In one embodiment, the polynucleotide comprises a polynucleotide consisting oflacZ ^FSA portion of a polynucleotide of allelic composition, wherein the nucleotide portion encompasses a nucleotide sequence corresponding to an encoded beta-galactosidase^FSCodon for residue 354.

In one embodiment, the nucleotide moiety comprises or consists of at least 100 consecutive nucleotides of a polynucleotide comprising a polynucleotide as defined hereinlacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 200 consecutive nucleotides of a polynucleotide comprisinglacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 300 consecutive nucleotides of a polynucleotide comprisinglacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 400 consecutive nucleotides of a polynucleotide comprisinglacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 500 consecutive nucleotides of a polynucleotide comprisinglacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 1000 consecutive nucleotides of a polynucleotide comprisinglacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 1500 consecutive nucleotides of a polynucleotide, saidThe polynucleotide compriseslacZ ^FSAn allele or consists thereof. In one embodiment, the nucleotide moiety comprises or consists of at least 2000 consecutive nucleotides of a polynucleotide comprisinglacZ ^FSAn allele or consists thereof.

In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), wherein the residue corresponding to residue 354 is not arginine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), wherein the residue corresponding to residue 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine and lysine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), wherein the residue corresponding to residue 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid, and asparagine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of a portion of at least 100 nucleotides of the polynucleotide of (a), wherein the residue corresponding to residue 354 is cysteine or its equivalent amino acid. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of a portion of at least 100 nucleotides of the polynucleotide of (a), wherein the residue corresponding to residue 354 is cysteine, alanine, and serine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprisesCode beta-galactosidase^FSOr consists of a portion of at least 100 nucleotides of the polynucleotide of (a), wherein the residue corresponding to residue 354 is cysteine.

In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSHas at least 95% identity to SEQ ID NO. 2, but differs from SEQ ID NO. 2. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSHas at least 95% identity to SEQ ID No. 2, but differs from SEQ ID No. 2, and wherein the residue corresponding to residue 354 is not arginine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2, and wherein the residue corresponding to residue 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine and lysine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2, and wherein the residue corresponding to residue 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid, and asparagine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSPolynucleotide of (4)A part of at least 100 nucleotides of an acid, or consisting thereof, said beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2, and wherein the residue corresponding to residue 354 is cysteine or its equivalent amino acid. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2, and wherein the residue corresponding to residue 354 is cysteine, alanine and serine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSHas at least 95% identity with SEQ ID No. 2, but differs from SEQ ID No. 2, and wherein the residue corresponding to residue 354 is cysteine.

In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSThe amino acid sequence of (a) is an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an arginine at position 354 (SEQ ID NO:5, wherein position 354 is not an arginine); or b) an amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO. 2 (beta-galactosidase variant as defined herein) but which does not comprise an arginine at position 354; in one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprises a nucleotide sequence encoding a beta-galactosidase as defined in

SEQ ID Nos

7, 10, 13, 16, 19, 22, 25 or 28^FSOf at least 100 nucleotides of the polynucleotide of (a), or consists ofPosition 354 is not arginine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSThe amino acid sequence of (a) is an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine and lysine (SEQ ID NO:5, wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine and lysine); or b) an amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO 2 (beta-galactosidase variant as defined herein) but which does not comprise an amino acid residue at position 354 selected from arginine, histidine, glutamine and lysine; in one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprises a nucleotide sequence encoding a beta-galactosidase as defined in SEQ ID Nos 7, 10, 13, 16, 19, 22, 25 or 28^FSOr a portion of at least 100 nucleotides of (a), wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, and lysine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSThe amino acid sequence of (a) is an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine (SEQ ID NO:5, wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid and asparagine); or b) an amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO 2 (beta-galactosidase variant as defined herein), but which is(ii) does not comprise an amino acid residue at position 354 selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid, and asparagine; in one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprises a nucleotide sequence encoding a beta-galactosidase as defined in SEQ ID Nos 7, 10, 13, 16, 19, 22, 25 or 28^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), wherein position 354 is not an amino acid residue selected from the group consisting of arginine, histidine, glutamine, lysine, glutamic acid, and asparagine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSThe amino acid sequence of (a) is an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises a cysteine or equivalent amino acid thereof at position 354 (SEQ ID NO:5, wherein position 354 is a cysteine or equivalent amino acid thereof); or b) an amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO. 2 (beta-galactosidase variant as defined herein) but which comprises a cysteine or equivalent amino acid at position 354; in one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprises a nucleotide sequence encoding a beta-galactosidase as defined in

SEQ ID Nos

7, 10, 13, 16, 19, 22, 25 or 28^FSOr consists of a portion of at least 100 nucleotides of the polynucleotide of (a), wherein position 354 is cysteine or its equivalent amino acid. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSIs a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises an amino acid residue at position 354 selected from the group consisting of cysteine, alanine and serine (SEQ ID NO: 2)ID NO 5, where position 354 is selected from cysteine, alanine, and serine); or b) an amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO 2 (beta-galactosidase variant as defined herein) but which comprises an amino acid residue at position 354 selected from cysteine, alanine and serine; in one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprises a nucleotide sequence encoding a beta-galactosidase as defined in SEQ ID Nos 7, 10, 13, 16, 19, 22, 25 or 28^FSOr consists of a portion of at least 100 nucleotides of the polynucleotide of (a), wherein position 354 is an amino acid residue selected from the group consisting of cysteine, alanine, and serine. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSThe amino acid sequence of (a) is an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises a cysteine at position 354 (SEQ ID NO: 4); or b) an amino acid sequence which is otherwise one of the beta-galactosidase variants having at least 95% identity to SEQ ID NO. 2 (beta-galactosidase variant as defined herein), but which comprises a cysteine at position 354; in one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide portion of the codon for residue 354 comprises a nucleotide sequence encoding a beta-galactosidase as defined in SEQ ID NOS: 8, 11, 14, 17, 20, 23, 26 and 29^FSOr consists of a portion of at least 100 nucleotides of the polynucleotide of (a).

In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSBeta-galactosidase obtained by substituting arginine (R354C) with cysteine at position 354 having the sequence shown in SEQ ID NO:2. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSA β -galactosidase variant (β -galactosidase variant as defined herein) having at least 95% identity to SEQ ID NO:2, obtained by substituting arginine (R354C) with cysteine at position 354. In one embodiment, the coverage corresponds to the beta-galactosidase^FSThe nucleotide moiety of codon 354 of (1) comprises a nucleotide sequence encoding a beta-galactosidase^FSOr consists of at least a 100 nucleotide portion of the polynucleotide of (a), said beta-galactosidase^FSA variant β -galactosidase as shown in SEQ ID NO 6, 9, 12, 15, 18, 21, 24 or 27 was obtained by substituting arginine (R354C) with cysteine at position 354.

Generally, polynucleotides encompassed by the scope of the present invention are prepared using recombinant DNA techniques (i.e., recombinant DNA), as described herein. However, in an alternative embodiment of the invention, the polynucleotides may be synthesized in whole or in part using chemical methods well known in the art (see Caruthers MH et al, (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al, (1980) Nuc Acids Res Symp Ser 225-232).

Encoding is as defined hereinlacZ ^FSThe polynucleotide of the protein may be identified and/or isolated and/or purified from any lactic acid bacterium. Various methods for identifying and/or isolating and/or purifying polynucleotides are well known in the art.

By way of example, once a suitable polynucleotide has been identified and/or isolated and/or purified, PCR amplification techniques may be used to prepare further copies of the polynucleotide.

By way of further example, a method from producing beta-galactosidase can be used^FSThe genomic DNA library is constructed from the chromosomal DNA of the lactic acid bacterium of (1). Based on beta-galactosidase^FSCan be synthesized and used to identify the sequences fromProtein-encoding clones of genomic libraries made by lactic acid bacteria.

Alternatively, the polynucleotides of the invention may be prepared synthetically by established standard methods, for example, the phosphoramidite method described by Beucage S.L. et al, 1981, Tetrahedron Letters 22: 1859-. In the phosphoramidite method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, annealed, ligated and cloned in an appropriate vector.

The polynucleotides may be prepared by Polymerase Chain Reaction (PCR) using specific primers, for example as described in US 4,683,202 or in Saiki R K et al, 1988, Science 239: 487-491.

Polynucleotides and nucleic acids encompassed by the present invention can be isolated or substantially purified. By "isolated" or "substantially purified" is meant that the polynucleotide is substantially or essentially free of components normally found in association with the polynucleotide in its native state. Such components include other cellular material, culture media from recombinant production, and various chemicals used to chemically synthesize nucleic acids.

An "isolated" polynucleotide or nucleic acid is typically free of nucleic acid sequences (such as coding sequences present at the 5 'or 3' end) that flank the nucleic acid of interest in the genomic DND of the organism from which the nucleic acid is derived. However, the molecule may include some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

Carrier

The present invention also relates to vectors comprising the polynucleotides of the invention. In one embodiment, the vector is a plasmid.

In one embodiment, the vector contains one or more selectable marker genes, such as genes that confer antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol, or tetracycline resistance). In one embodiment, the vector comprises a nucleotide sequence that enables the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUbl 10, pE194, pAMBl and pIJ 702.

The vectors of the invention may be used to engineer the lactic acid bacteria of the invention.

Streptococcus thermophilus strains comprising a polynucleotide of the invention

The present invention relates to Streptococcus thermophilus strains comprising a polynucleotide of the inventionlacZ ^FSAlleles [ coding for beta-galactosidase^FS]Or consist thereof. In one embodiment, the Streptococcus thermophilus strain comprises the Streptococcus thermophilus of the inventionlacZ ^FSAlleles [ coding for beta-galactosidase^FS]。

For the avoidance of doubt, the species Streptococcus thermophilus is understood to be Streptococcus salivarius subsp thermophilus (S.salivarius)Streptococcus salivarius subsp. thermophilus) And (3) strain.

In one embodiment, the Streptococcus thermophilus strain of the invention is a galactose negative Streptococcus thermophilus strain. Expression'Galactose negative"means a streptococcus thermophilus strain that is not able to grow on galactose as the sole source of carbohydrates, in particular on M17 medium supplemented with 2% galactose. In a specific embodiment,' A "Galactose anion Property of (2)"phenotype is determined by: an overnight culture of the streptococcus thermophilus strain to be tested was inoculated at 1% to M17 liquid medium containing 2% galactose and incubated at 37 ℃ for 20 hours, and wherein a pH of 6 or higher at the end of the incubation indicates a galactose negative phenotype.

As used herein, "comprisesComprising lacZ ^FS Alleles or polynucleotides composed thereof'or'Comprising lacZ ^FS Etc. of Site gene"means contained in the genome of the Streptococcus thermophilus strainlacZThe only allele of the gene islacZ ^FSAn allele. In one embodiment, the Streptococcus thermophilus strain of the invention compriseslacZ ^FSAlleles or polynucleotides composed thereof as suchlacZA unique allele of a gene. Thermophilic chain not contemplated for the inventionThe coccus strain compriseslacZSeveral alleles of a gene.

Such a Streptococcus thermophilus strain can be engineered as follows:

a) by incorporating the inventionlacZ ^FSReplacement of alleles or polynucleotides composed thereoflacZAn allele of a gene; or

b) Replacement thereof with the corresponding polynucleotidelacZPart of an allele of a gene, said polynucleotide comprising a polynucleotide encoding a beta-galactosidase as defined herein^FSWherein said nucleotide moiety encompasses or consists of a portion of at least 100 nucleotides corresponding to said beta-galactosidase^FSCodon for residue 354. "corresponding polynucleotide" is meant to encompass a polynucleotide corresponding to said beta-galactosidase^FSCodon for residue 354lacZThe same part of the allele.

The substitution may be made using conventional techniques as defined herein.

In one embodiment, the Streptococcus thermophilus of the invention (comprisinglacZ ^FSAllele) is also characterized by its ability to result in an acidification slope between pH6 and 5.3 of at least-0.005 UpH/min when tested by assay C. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.006 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.007 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.008 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.009 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.01 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.02 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.03 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.04 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.05 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is selected from at least-0.005, -0.006, -0.007. -0.008, -0.009, -0.01, -0.02, -0.03, -0.04, and-0.05 UpH/min.

Assay C (acidification kinetics in milk)

UHT semi-skimmed milk "Le Pet Vend meen" containing 3% (w/v) powdered milk (BBA, Lactalis) previously pasteurized at 90 ℃ for 10 minutes at 1% (v/v, about 10%⁷CFU/ml) was inoculated with a culture of the streptococcus thermophilus strain to be assayed (M17-carbohydrate-free resuspension cells from an overnight culture grown in M17 supplemented with 3% sucrose). The inoculated milk bottle was incubated statically in a water bath at 43 ℃ for 24 hours (start of fermentation experiment) to obtain fermented milk. The acidification properties of the streptococcus thermophilus strains were assessed by recording the change in pH over time during milk fermentation. The pH was monitored for 24 hours using a CINAC system (Alliance Instruments, France; pH electrode Mettler 405 DPAS SC, Toledo, Spain), as described previously. The pH was measured and recorded every 5 minutes. Using the CINAC v2.07 software, the following descriptors were calculated:

-slope between pH6.0 and pH 5.3 (UpH/min) [ slope pH6-5.3 ];

time corresponding to Vmax (where V_maxIs the maximum rate obtained during the fermentation experiment; t is_Vmax) Time (in minutes) from the start of the fermentation experiment;

pH corresponding to the pH at V0_STOPWherein V0 corresponds to a rate that becomes unambiguously undetectable, i.e. below 0.1 mupH/min (0.0001 UpH/min); "Is definitely changed into"means that the rate remains less than 0.1 mUpH/min for the remainder of assay C (i.e., up to 24h at fermentation temperature); and

corresponds to pH_STOPTime of (TpH)_STOP) [ therefore, the time corresponding to V0 was calculated from the beginning of the fermentation experiment]。

In one embodiment, Streptococcus thermophilus of the invention (comprisinglacZ ^FSAlleles) are also characterized by their textural properties. Thus, the Streptococcus thermophilus of the invention may be characterized in that it is produced when it is used to obtain fermented milkE.g. by assay D (i.e. at 350 s)^-1At a shear rate of).

In one embodiment, the shear stress value generated in the fermented milk obtained with streptococcus thermophilus of the invention is at least 60, at least 120, at least 180 or at least 240 Pa, as determined by assay D. In one embodiment, the shear stress value generated in the fermented milk obtained with streptococcus thermophilus of the invention is less than 60, less than 120, less than 180 or less than 240 Pa, as determined by assay D. In one embodiment, the shear stress value generated in the fermented milk obtained with streptococcus thermophilus of the invention is at least 60 or at least 120 and less than 180 or less than 240 Pa, as determined by assay D.

In one embodiment, the shear stress value produced in the fermented milk obtained with streptococcus thermophilus of the invention is in the range selected from 0 to 59 Pa, 60 to 119 Pa, 120 to 179 Pa, 180 to 239 Pa and 240 to 300 Pa, as determined by assay D.

For reference, the shear stress values generated in the fermented milk obtained with the strain DGCC715 (DSM33036) were determined by assay D and showed a range of 0-59 Pa. As a further reference, the shear stress values generated in the fermented milk obtained with the strain DGCC7710 (deposited under DSM28255) were determined by assay D and were shown to be in the range of 120-179 Pa, more particularly about 150. + -.15 Pa. The Streptococcus thermophilus DGCC7710 strain has been deposited by Danisco Deutschland GmbH on 14.1.2014 under the Budapest treaty at Leibniz-institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Inhoffentr. tr.7B, D-38124 Braunschweig) and has received the access number DSM 28255. We have demonstrated here that the depositor Danisco Deutschland GmbH (Busch-Johannsen-Strasse 1, D-25899 Niebull, Germany) has granted the applicant (DuPont Nutrition Biosciences ApS) reference to the deposited biological material in the present application. The applicant claims that samples of deposited microorganisms described herein are only available to experts until the date of patent grant.

Assay method D

Preparation of inoculum of the strain: 1.8 ml of stock culture stored at-80 ℃ was inoculated into 100 ml of bulk starter medium (bulk starter medium) in a 250 ml flask and incubated at 37 ℃ for 18 hours. Bulk starter culture medium was obtained by adding 10% high heat skim milk powder (BBA Lactalis) to water and stirring for 30 minutes at room temperature; then, the medium was heat-treated at 120 ℃ for 20 minutes.

Milk preparation: 93% (w/w) of fresh commercial milk [ Candia, Lait frais de montane grade Lait enter: 3.6% fat, 3.2% protein ] and 7% (w/w) sucrose; the mixture was heat treated in a water bath at 90 ℃ for 10 minutes. Just before inoculation of the strain, 1 g/100L (w/v) of sodium formate was added.

Fermentation: the inoculum of the strain was added to milk at 1% (v/v), the inoculated milk was poured into a 125ml yoghurt pot and incubated at 43 ℃ to reach a pH of 4.6 (pH was followed by CINAC system; Alliance Instruments, France; pH electrode Mettler 405 DPAS SC, Toledo, Spain). The fermented milk was then slowly cooled to 6 ℃ in a well ventilated low temperature incubator. The samples were stored at 6 ℃ for 7 days.

Before the shear stress measurement, the sample was brought to 8 ℃ and stirred 5 times/5 s with a spoon (1 revolution = 1 s). A rest time of 5 minutes (equilibration time) was applied just before the measurement. The shear stress of the samples was evaluated using a Rheometer (MCR Modular Compact Rheometer type 302, Anton Paar GmbH, Germany) equipped with a CC27 coaxial measurement system (standards DIN 53019 and ISO 3219) and a Peltier system C-PTD200-SN 81154777. The viscosity method is adopted for testing from 0.1s at 31 points^-1To 350s^-1And from 350s at 31 points^-1To 0.1s^-1A varying shear rate ramp is performed. The shear stress was continuously recorded. And (3) setting the duration of a logarithmic variable measuring point, setting an Up-curve initial value to be 10s, setting a final value to be 3s, setting a Down-curve initial value to be 3s, and setting the final value to be 10 s. Select 350s on the rising curve^-1The shear stress value at (a) characterizes the texture properties of the S.thermophilus strain of the invention.

The inventors have shown that the invention is embodiedlacZ ^FSAllelic strains of Streptococcus thermophilus may be used not only in acceptable mannerFermented milk, but also fermented milk that does not undergo post-acidification at the fermentation temperature. The inventors have well shown that these strains of Streptococcus thermophilus (comprising the strains of the invention)lacZ ^FSAlleles) can be detected by LacS as defined herein in this strain_pH4.5：LacZ_pH4.5Ratio and LacS as defined herein_pH6：LacZ_pH6A ratio. In fact, LacS_pH6：LacZ_pH6The ratio represents the ability of the strain of the invention to utilize lactose and thus acidify (lactate production) the lactate to the target pH at the start of the manufacturing process, whereas LacS_pH4.5：LacZ_pH4.5The ratio represents the ability of this same strain to utilize lactose less efficiently when the target pH is reached and thus not produce lactic acid. Thus, the inventors have shown that formula (I) as described herein can be used to characterize strains that exhibit acidification kinetics in milk without post-acidification. In one embodiment, the Streptococcus thermophilus of the invention (comprisinglacZ ^FSAlleles) are also characterized by the difference in hydrolysis Efficiency (EH) of the input lactose calculated by the following formula (I)_pH6 - EH_pH4.5) Less than-0.5:

in the formula (I), LacS_pH6And LacS_pH4.5Represents the lactose import activity of LacS permease calculated by assay A at pH6 and pH4.5, respectively, and LacZ_pH6And LacZ_pH4.5Represents the lactose hydrolysis activity of beta-galactosidase calculated by assay B at pH6 and pH4.5, respectively.

Thus, a Δ EH of less than-0.5 as defined herein means the efficiency of hydrolysis of the input lactose at pH4.5 (EH)_pH4.5) [ (i.e., lactose is delivered into the bacteria by LacS permease and subsequently hydrolyzed by beta-galactosidase)]Efficiency with hydrolysis at pH6 (EH)_pH6) Compared with the prior art, the method is greatly reduced. In one embodiment, the Streptococcus thermophilus of the invention (comprisinglacZ ^FSAllele) is characterized by a Δ EH [ as calculated by formula (I) ]selected from]: less than-0.6, less than-0.7, less than-0.8, less than-0.9, less than-1, less than-1.1, less than-1.2, less than-1.3, less than-1.4, and less than-1.5.

Conversely, Δ EH being slightly positive, about 0, or slightly negative means that the efficiency of hydrolysis of the input lactose is as effective at pH4.5 as at pH6. This Δ EH is characteristic of streptococcus thermophilus strains which, when used in fermented milk, result in fermented milk that undergoes post-acidification.

The invention also resides, in part, in a Streptococcus thermophilus strain as defined herein (comprising a strain according to the invention)lacZ ^FSAlleles) are also characterized by their ability to ferment milk at acceptable industrial times, followed by fermentation of the fermented milk without post-acidification at fermentation temperatures. This ability is defined herein as the "complete STOP" phenotype and can be determined by assay C as defined herein.

Thus, the complete STOP phenotype is characterized by the fact that when the strain of the invention is inoculated to a milk substrate and fermented according to assay C, the milk is fermented such that the pH of the fermented milk STOPs between 4 and 4.8 (pH)_STOP) And T is_VmaxAnd TpH_STOPThe time between is less than 600 minutes. In one embodiment, T_VmaxAnd TpH_STOPThe time between is less than 550 minutes. In one embodiment, T_VmaxAnd TpH_STOPThe time between is less than 500 minutes.

In one embodiment, the pH obtained by assay C using the strain of the invention, alone or in combination with the time between Vmax and V0_STOPIs comprised between 4 and 4.6. In one embodiment, the pH obtained by assay C using the strain of the invention_STOPIs comprised between 4 and 4.5. In one embodiment, the pH obtained by assay C using the strain of the invention_STOPIs comprised between 4 and 4.4.

In one embodiment, the complete STOP phenotype is characterized by the fact that when the strain of the invention is inoculated to a milk substrate and tested according to assay CUpon fermentation, the milk is fermented such that the pH of the fermented milk stops between a range selected from between 4 and 4.8, between 4 and 4.6, between 4 and 4.5 and between 4 and 4.4, and T_VmaxAnd TpH_STOPThe time between is selected from less than 600 minutes, less than 550 minutes and less than 500 minutes.

Thus, once the pH stops significantly quickly, the fermented dairy product can be kept at the fermentation temperature for at least 24 hours without the pH of the fermented product decreasing (which gives a high degree of flexibility during the manufacturing process).

In a particular embodiment, the Streptococcus thermophilus strain of the invention as defined herein carries a gene encoding a beta-galactosidase as defined in SEQ ID NO 4^FSIs/are as followslacZ ^FSAlleles, in particular as defined in SEQ ID NO 3lacZ ^FSThe allele was used as its lacZ gene.

In a particular embodiment, the Streptococcus thermophilus strain of the invention as defined herein carries a gene encoding a beta-galactosidase^FSIs/are as followslacZ ^FSAllele as its lacZ gene, the beta-galactosidase^FSAt least 95% identical to SEQ ID NO. 2, but comprising a cysteine at position 354.

In a particular embodiment, the Streptococcus thermophilus strain of the invention as defined herein carries a gene encoding a beta-galactosidase^FSIs/are as followslacZ ^FSAllele as its lacZ gene, the beta-galactosidase^FSIs one of the beta-galactosidase variants having at least 95% identity with SEQ ID NO:2 (beta-galactosidase variant as defined herein), but which comprises a cysteine at position 354. In a particular embodiment, the Streptococcus thermophilus strain according to the invention carries a gene encoding a beta-galactosidase as defined in SEQ ID NO 8, 11, 14, 17, 20, 23, 26 or 29^FSIs/are as followslacZ ^FSThe allele was used as its lacZ gene.

In a particular embodiment, the invention relates to a Streptococcus thermophilus strain corresponding to the Streptococcus thermophilus strain DGCC7984, wherein said Streptococcus thermophilus strainMethod for producing Streptococcus thermophilus strain DGCC7984lacZThe gene has been encoded for beta-galactosidase as defined in SEQ ID NO 4^FSIs/are as followslacZ ^FSAlleles, in particular as defined in SEQ ID NO 3lacZ ^FSAnd (4) allele replacement. The Streptococcus thermophilus DGCC7984 strain has been deposited under the Budapest treaty at Leibnizz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Inhoffentr. 7B, D-38124 Braunschweig) by Danisco Deutschland GmbH on 14.1.2014 and has received the access number DSM 28257. We have demonstrated here that the depositor Danisco Deutschland GmbH (Busch-Johannsen-Strasse 1, D-25899 Niebull, Germany) has granted the applicant (DuPont Nutrition Biosciences ApS) reference to the deposited biological material in the present application. The applicant claims that samples of deposited microorganisms described herein are only available to experts until the date of patent grant. Expression'DGCC7984 strain"can and expression"DSM28257 strain"used interchangeably.

Uses and methods based on the polynucleotides or vectors of the invention

In one embodiment, the present invention relates to the use of a polynucleotide or vector of the present invention to obtain a streptococcus thermophilus strain with a complete STOP phenotype when used in fermented milk by assay C.

Thus, the polynucleotide or vector is used such that the resulting Streptococcus thermophilus strain compriseslacZ ^FSAlleles as unique in their genomelacZA gene. In one embodiment, the polynucleotide or vector is used to make a strain of Streptococcus thermophiluslacZReplacement of an allele of a gene or a portion thereof with a polynucleotide of the invention; the substitution may be made using conventional techniques as defined herein.

In one aspect, the present invention relates to a method of making a streptococcus thermophilus strain with a complete STOP phenotype comprising:

a) streptococcus thermophilus strains are provided having an activity of less than 5 of the lactose import of the LacS permease calculated by assay A at pH4.5 and an activity of less than 5 of the lactose import calculated by assay B at pH4.5Calculated ratio of lactose hydrolysis Activity of beta-galactosidase (LacS)_pH4.5：LacZ_pH4.5)；

b) With the polynucleotides of the invention (comprisinglacZ ^FSAlleles or their components) replacing the Streptococcus thermophilus strainlacZA gene; and

c) the streptococcus thermophilus strain with the complete STOP phenotype when used in fermented milk by assay C was recovered.

In one embodiment, step b) comprises the use of a composition of the inventionlacZ ^FSReplacement of said strain of Streptococcus thermophilus with a polynucleotide consisting of an allelelacZA gene.

a) streptococcus thermophilus strains are provided having a ratio of the activity of lactose import of LacS permease calculated by assay A at pH4.5 to the activity of lactose hydrolysis of beta-galactosidase calculated by assay B at pH4.5 (LacS) of less than 5_pH4.5：LacZ_pH4.5)；

b) Replacement of said Streptococcus thermophilus strain with the corresponding polynucleotidelacZA portion of a gene, said polynucleotide comprising a polynucleotide encoding a beta-galactosidase as defined herein^FSWherein said nucleotide moiety encompasses or consists of a portion of at least 100 nucleotides corresponding to said beta-galactosidase^FSCodon for residue 354. "Corresponding multiple cores Acid of lactic acid"is meant to encompass the beta-galactosidase enzyme corresponding to^FSCodon for residue 354lacZThe same portion of the allele; and

a) streptococcus thermophilus strains are provided having an activity of less than 5 of lactose import of LacS permease calculated by assay A at pH4.5 andthe ratio of the lactose hydrolysis activity of beta-galactosidase (LacS) calculated by assay B at pH4.5_pH4.5：LacZ_pH4.5)；

b) Modification of said strains of Streptococcus thermophiluslacZGene to have a gene of the present inventionlacZ ^FS(ii) sequences that are allelic identical; and

c) the lactic acid streptococcus thermophilus strain with the complete STOP phenotype when used in fermented milk by assay C was recovered.

In one embodiment, any of the methods described herein for preparing a streptococcus thermophilus strain with a complete STOP phenotype are performed on a medium containing lactose as the sole source of carbohydrates.

In the use or method of the invention, LacS_pH4.5：LacZ_pH4.5Ratios were determined as described herein. In one embodiment, the Streptococcus thermophilus strain of step a) has a LacS of less than 5_pH4.5：LacZ_pH4.5A ratio. In one embodiment, the Streptococcus thermophilus strain of step a) has a LacS of less than 4_pH4.5：LacZ_pH4.5A ratio. In one embodiment, the Streptococcus thermophilus strain of step a) has a LacS of less than 3_pH4.5：LacZ_pH4.5A ratio.

In one embodiment, the Streptococcus thermophilus strain of step a) is further characterized by its ability to result in an acidification slope between pH6 and 5.3 of at least-0.005 UpH/min when tested by assay C. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.006 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.007 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.008 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.009 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.01 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.02 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.03 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.04 UpH/min. In one embodiment, the acidification slope between pH6 and 5.3 is at least-0.05 UpH/min. In one embodiment, the Streptococcus thermophilus strain of step a) is further characterized by its ability to cause an acidification slope between pH6 and pH4.5 when tested by assay C selected from at least-0.005, -0.006, -0.007, -0.008, -0.009, -0.01, -0.02, -0.03, -0.04 and-0.05 UpH/min.

In a further aspect, the present invention relates to a streptococcus thermophilus strain obtained by the use or method of the present invention.

In a still further aspect, the present invention provides a strain of streptococcus thermophilus according to the invention, produced by the method of the invention.

Bacterial compositions

The present invention also relates to a bacterial composition comprising or consisting of at least one, preferably one, streptococcus thermophilus strain according to the invention. In one embodiment, the bacterial composition is a pure culture, i.e. comprises or consists of a single strain of streptococcus thermophilus of the invention. In another embodiment, the bacterial composition is a mixed culture, i.e. comprising or consisting of the streptococcus thermophilus strain of the invention and at least one further microorganism, in particular at least one further bacterial strain. In one embodiment, the bacterial composition is a pure culture, i.e. comprises or consists of a single strain of streptococcus thermophilus of the invention. In another embodiment, the bacterial composition is a mixed culture, i.e. comprising or consisting of the streptococcus thermophilus strain of the invention and at least one further bacterial strain. By "at least" one other bacterial strain is meant 1 or more, in particular 1, 2, 3, 4 or 5 strains.

In one embodiment of any of the bacterial compositions defined herein, the bacterial composition further comprises a food acceptable component, such as a sugar (sucrose, trehalose), maltodextrin or a mineral, as a pure culture or a mixed culture. In a particular embodiment, the bacterial composition as defined herein does not comprise lactose.

In one embodiment, the bacterial composition of the invention comprises or consists of a streptococcus thermophilus strain of the invention and one or more further lactic acid bacteria of a species selected from the group consisting of lactococcus species, streptococcus species, lactobacillus species (including lactobacillus acidophilus), enterococcus species, pediococcus species, leuconostoc species, bifidobacterium species and oenococcus species or any combination thereof. The lactococcus species includes lactococcus lactis, including lactococcus lactis subspLactococcus lactis subsp. lactis) Lactococcus lactis subspecies cremoris (A)Lactococcus lactis subsp. cremoris) And lactococcus lactis lactosyllactis diacetyllactic acid biological variants (Lactococcus lactis subsp. lactis biovar diacetylactis). The Bifidobacterium species includes Bifidobacterium animalis (Bifidobacterium animalis) In particular Bifidobacterium animalis subspBifidobacterium animalis subsp lactis). Other species of lactic acid bacteria include Leuconostoc species, Streptococcus thermophilus, Lactobacillus delbrueckii subspLactobacillus delbrueckii subsp. bulgaricus) And Lactobacillus helveticus (Lactobacillus helveticus)。

In one embodiment, the bacterial composition comprises or consists of the streptococcus thermophilus strain of the invention and at least one streptococcus thermophilus strain different from the streptococcus thermophilus strain of the invention and/or at least one strain of lactobacillus species and/or any combination thereof. In a particular embodiment, the bacterial composition comprises or consists of a streptococcus thermophilus strain of the invention, one or several strains of the species lactobacillus delbrueckii subsp. In a particular embodiment, the bacterial composition comprises or consists of the streptococcus thermophilus strain of the invention, at least one strain of the species streptococcus thermophilus different from the streptococcus thermophilus strain of the invention and a strain of the species lactobacillus delbrueckii subsp. In another specific embodiment, the bacterial composition comprises or consists of a streptococcus thermophilus strain of the invention and a strain of the species lactobacillus delbrueckii subsp.

In one embodiment, the bacterial composition comprises or consists of a streptococcus thermophilus strain, a lactococcus lactis subspecies lactis and/or a lactococcus lactis subspecies cremoris of the invention.

In a particular embodiment of any of the bacterial compositions defined herein, said bacterial composition further comprises at least one probiotic bacterial strain, such as bifidobacterium animalis subsp lactis, lactobacillus acidophilus, lactobacillus paracasei or lactobacillus casei, as a pure culture or as a mixed culture.

In a particular embodiment, the bacterial composition as a pure culture or as a mixed culture as defined above is in frozen, dried, freeze-dried, liquid or solid form, in the form of granules or frozen particles, or in the form of a powder or a dried powder. In a particular embodiment, the bacterial composition of the invention is in the form of a frozen form or granules or frozen granules, in particular contained in one or more boxes or sachets. In another embodiment, the bacterial composition as defined herein is in the form of a powder, such as a dried or freeze-dried powder, in particular contained in one or more boxes or sachets.

In a particular embodiment, the bacterial composition of the invention, as a pure culture or mixed culture as defined above, and whatever the form (frozen, dried, freeze-dried, liquid or solid form, in the form of granules or frozen granules or in the form of a powder or dried powder), comprises the Streptococcus thermophilus strain of the invention, in a concentration comprised between 10⁵To 10¹²cfu (colony forming units)/gram of bacterial composition (cfu/g). In a particular embodiment, the concentration of the Streptococcus thermophilus strain in the bacterial composition according to the invention is between 10⁷To 10¹²cfu/g bacterial composition, and in particular at least 10⁷At least 10⁸At least 10⁹At least 10¹⁰Or at least 10¹¹cfu/g bacterial composition. In a specific embodimentIn the case of a frozen or dried concentrate, the Streptococcus thermophilus strain according to the invention is present in the bacterial composition in a concentration of 10 (as a pure culture or as a mixed culture)⁸To 10¹²cfu/g frozen concentrate or dried concentrate, and more preferably at least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹Or at least 10¹²cfu/g frozen concentrate or dried concentrate.

Production of products Using the Streptococcus thermophilus strains according to the invention

In a further aspect, there is provided a method of making a fermentation product comprising: a) inoculating a substrate with a streptococcus thermophilus strain or a bacterial composition according to the invention, and b) fermenting the inoculated substrate to obtain a fermentation product. In a particular embodiment, the streptococcus thermophilus strain of the invention is inoculated as a bacterial composition as defined herein, such as a pure culture or a mixed culture. Preferably, the matrix is a milk matrix, more preferably milk. By "milk base" is meant milk of animal and/or vegetable origin. In a particular embodiment, the milk base is of animal origin, in particular of any mammal, such as a cow, a goat, a sheep, a buffalo, a zebra, a horse, a donkey or a camel, etc. The milk may be in a natural state, reconstituted milk, skim milk or milk supplemented with compounds necessary for the growth of bacteria or for the subsequent processing of fermented milk. Preferably, the milk base comprises a solid article. Preferably, the solid article comprises or consists of fruit, chocolate products or cereals. Preferably, the fermented product is a fermented dairy product.

In a further aspect, the present invention also provides the use of a streptococcus thermophilus strain or a bacterial composition according to the invention for the manufacture of a food or feed product, preferably a fermented dairy product.

The invention also relates to fermented dairy products obtained using the lactic acid bacterial strains or bacterial compositions of the invention, in particular obtained or obtainable by the methods of the invention. Accordingly, the present invention relates to a fermented dairy product comprising the streptococcus thermophilus strain of the present invention. In a particular embodiment, the fermented dairy food product of the invention is fresh fermented milk.

The streptococcus thermophilus strain or the bacterial composition according to the invention has an advantageous use in various dairy applications as a specific embodiment of the method for manufacturing the fermentation product described herein.

In one aspect, the Streptococcus thermophilus strain or bacterial composition according to the invention may be used for the manufacture of stirred yoghurt. The manufacture of stirred yoghurt comprises fermenting a milk base previously inoculated with a streptococcus thermophilus strain or a bacterial composition according to the invention, optionally storing the stirred yoghurt in a storage tank, and finally packaging the stirred yoghurt in a package. The method comprises cooling the stirred yoghurt between the end of fermentation (i.e. once the target pH has been reached) and the packaging step to stop further acidification of the stirred yoghurt so that the stirred yoghurt is packaged at a temperature between 15 and 22 ℃. Because this cooling step is time and resource (energy) consuming, yogurt manufacturers desire to package stirred yogurt at higher temperatures; packaging at higher temperatures also has the advantage of improving the texture of the stirred yoghurt in the package (see example 8); however, packaging at higher temperatures is not acceptable for yoghurt manufacturers using bacterial compositions currently on the market, as stirred yoghurt has been shown to be too acidic. The streptococcus thermophilus strain or bacterial composition according to the invention solves this problem, enabling yoghurt manufacturers to package stirred yoghurt at higher temperatures while obtaining a product with an acceptable pH. This can be achieved by cooling the stirred yoghurt at a temperature above 22 ℃ or by-passing the cooling step. The invention therefore also relates to the use of a streptococcus thermophilus strain or a bacterial composition according to the invention for the manufacture of a stirred yoghurt. In a particular embodiment, the invention also relates to the use of the streptococcus thermophilus strain or the bacterial composition according to the invention for the manufacture of a stirred yoghurt, wherein the packaging step of the stirred yoghurt is carried out at a temperature of at least 23 ℃. The invention also relates to a method of manufacturing stirred yoghurt comprising (a) fermenting a milk substrate, in particular milk, inoculated with a streptococcus thermophilus strain or a bacterial composition according to the invention, to obtain a stirred yoghurt (pH of 4.2 to 4.7, more preferably 4.45 to 4.6), (b) cooling the stirred yoghurt and (c) packaging the stirred yoghurt, wherein the cooling and packaging temperature is at least 23 ℃ (the cooling and packaging temperature is one temperature). "at least 23 ℃ in the context of cooling and packaging temperatures" means at least 24 ℃, at least 25 ℃, at least 26 ℃, at least 27 ℃, at least 28 ℃, at least 29 ℃, at least 30 ℃, at least 31 ℃, at least 32 ℃, at least 33 ℃, at least 34 ℃, at least 35 ℃, at least 36 ℃, at least 37 ℃, at least 38 ℃, at least 39 ℃ and at least 40 ℃. In one embodiment, the temperature of cooling and packaging is equal to or lower than the fermentation temperature (i.e., typically lower than 43 ℃). In a particular embodiment, the cooling and packaging temperature is at least 23 ℃ and equal to or lower than 43 ℃. As shown in example 8, packaging at a temperature of 35 ℃ provides a similar change in pH over time as a stirred yogurt packaged at 20 ℃ while improving the texture of the stirred yogurt. The invention also relates to a method of manufacturing a stirred yoghurt comprising (a) fermenting a milk substrate, in particular milk, with a streptococcus thermophilus strain or bacterial composition according to the invention to obtain a stirred yoghurt (pH between 4.2 and 4.7, more preferably between 4.45 and 4.6), and (b) packaging the stirred yoghurt, wherein the method does not comprise any cooling step between the end of fermentation and the packaging. In this embodiment, the temperature of cooling and packaging is equal to the fermentation temperature (i.e., typically 42-43 ℃). In one embodiment, the method of making a stirred style yogurt as described herein further comprises transferring the package into a refrigerated compartment (i.e., below 8 ℃).

In another aspect, the streptococcus thermophilus strain or the bacterial composition according to the invention may be used for the manufacture of set yoghurt. The manufacture of set yoghurt comprises cooling the package containing the set yoghurt to stop further acidification of the product once the desired pH is obtained (pH 4.2 to 4.7, more preferably 4.45 to 4.6; deemed to be the end of fermentation). This cooling step is performed in a cooling chamber (also called cooling box or cooling tunnel) and the packages are then transferred into a cold storage chamber (i.e. below 8 ℃). For conventional starter cultures, it is important to stop further growth rapidly after fermentation, which means that a temperature of about 35 ℃ should be reached within 30 minutes after the end of fermentation and 18-20 ℃ after a further 30-40 minutes. Typically, the total cooling time for small packages is about 65-70 minutes and the total cooling time for large packages is about 80-90 minutes. Since this cooling step is time and resource (energy) consuming, yogurt manufacturers wish to reduce the time spent in the cooling chamber; however, reducing this time is not acceptable for yoghurt manufacturers using bacterial compositions currently on the market, as yoghurt products have been shown to be too acidic. The streptococcus thermophilus strain or bacterial composition according to the invention solves this problem by enabling yoghurt manufacturers to be less concerned about the period of time to reach a temperature of 18-20 ℃ while obtaining a product with an acceptable pH. In a particular embodiment, the present invention relates to the use of a streptococcus thermophilus strain or bacterial composition according to the invention in the manufacture of set yoghurt, wherein the time required for the set yoghurt contained in the package to reach a temperature of 18-20 ℃ (from the end of fermentation) is increased compared to the time of 65-70 minutes for a small package (defined herein as a size of 0.1 to 0.2 kg) and the time of 80-90 minutes for a large package (defined herein as a size of 0.4 to 0.6 kg). In a particular embodiment, the time required for the set yogurt contained in the package to reach a temperature of 18-20 ℃ is at least 100 minutes, at least 120 minutes, at least 180 minutes or at least 240 minutes. This can be achieved in several ways that provide the dairy manufacturer with a high degree of flexibility, e.g. by bypassing the cooling step (i.e. bypassing a step in the cooling chamber) or by delaying the time between the end of fermentation and the time of entering the cooling chamber. The present invention relates to a method of manufacturing set yoghurt comprising a) packaging a milk substrate, in particular milk, inoculated with a streptococcus thermophilus strain or a bacterial composition according to the invention into a package, (b) fermenting the inoculated milk substrate (contained within the package) to obtain set yoghurt (pH of 4.2 to 4.7, more preferably 4.45 to 4.6), and c) treating the package such that the time required for the set yoghurt in the package to reach a temperature of 18-20 ℃ is at least 100 minutes, at least 120 minutes, at least 180 minutes or at least 240 minutes. In a specific embodiment, the method of making set yogurt as described herein further comprises d) transferring the package into a refrigerated compartment (i.e., below 8 ℃). In one embodiment, the present invention relates to a method of manufacturing set yoghurt comprising a) packaging a milk substrate, in particular milk, inoculated with a streptococcus thermophilus strain or a bacterial composition according to the invention in a package, and b) fermenting the inoculated milk substrate to obtain set yoghurt (pH between 4.2 and 4.7, more preferably between 4.45 and 4.6), wherein the method does not comprise a cooling step in a cooling chamber. In a specific embodiment, the method of making set yogurt as described herein further comprises c) transferring the package into a refrigerated compartment (i.e., below 8 ℃). In one embodiment, the present invention relates to a method of manufacturing set yoghurt comprising a) packaging a milk substrate, in particular milk, inoculated with a streptococcus thermophilus strain or a bacterial composition according to the invention in a package, b) fermenting the inoculated milk substrate to obtain set yoghurt (pH of 4.2 to 4.7, more preferably 4.45 to 4.6), c) after the end of the fermentation, keeping the set yoghurt in the package at room temperature (i.e. above 20 ℃) for at least 30 minutes, at least 45 minutes or at least 60 minutes; and d) incubating the package in a cooling chamber to bring the set style yogurt contained in the package to a temperature of 18-20 ℃.

In another aspect, the streptococcus thermophilus strain or the bacterial composition according to the invention may be used for storing fermented milks, such as stirred and set yoghurts. At the end of the manufacturing process (including packaging and cooling), the fermented milk is stored in a cold room, at a temperature generally lower than 8 ℃, until distribution. As shown in example 9, yoghurts manufactured with the strains of the invention stored at 10 ℃ maintain a stable pH up to 45 days (stable means a change in pH of less than 0.1 unit). The present invention therefore also relates to a method for the manufacture and storage of fermented milk comprising a) fermenting a milk substrate, in particular milk, with a streptococcus thermophilus strain or a bacterial composition according to the invention to obtain a fermented milk (pH 4.2 to 4.7, more preferably 4.45 to 4.6), b) optionally cooling the fermented milk to a temperature of 18-20 ℃, and c) storing the fermented milk-containing package, the packaging step taking place before or after the fermentation step, but before the optional cooling step, wherein the storage is carried out at a temperature above 8 ℃; in one embodiment, the storage is carried out at a temperature equal to or higher than 10 ℃ and optionally lower than 20 ℃, preferably lower than 15 ℃. In a particular embodiment, the storage time at a temperature higher than 8 ℃ (preferably at a temperature equal to or higher than 10 ℃, and optionally lower than 20 ℃, preferably lower than 15 ℃) is less than 24 hours.

Product(s)

Any product prepared from, containing or comprising the streptococcus thermophilus strain or bacterial composition of the invention is contemplated according to the invention.

Suitable products include, but are not limited to, food or feed products.

These include, but are not limited to, fruits, legumes, forage crops and vegetables, including derived products, grains and grain-derived products, dairy foods and dairy-derived products, meat, poultry, and seafood. Preferably, the food or feed product is a dairy product, a meat or a cereal product.

The term "food" is used in a broad sense and includes feed, food ingredients, food supplements and functional foods. The term "food" is used herein in a broad sense-and covers food for humans as well as food for animals (i.e. feed). In a preferred aspect, the food is for human consumption.

As used herein, the term "food ingredient" includes preparations which are or can be added to food, and includes preparations which can be used at low levels in a wide variety of products where acidification or emulsification is desired, for example.

As used herein, the term "functional food" means a food that is capable of not only providing a nutritional effect and/or taste satisfaction, but also delivering additional benefits to the consumer. Although there is no legal definition of functional foods, most aspects of interest in this field agree that there are foods sold with specific health effects.

The streptococcus thermophilus strain of the invention may be-or may be added to-a food ingredient, a food supplement or a functional food.

The food may be in the form of a solution or as a solid-depending on the use and/or mode of application and/or mode of administration.

The streptococcus thermophilus strains of the invention can be used for preparing food products, such as confectionery products, dairy products, meat products, poultry products, fish products or bakery products.

By way of example, the streptococcus thermophilus strain may be used as an ingredient to prepare soft drinks, fruit juices or drinks containing whey protein, tea, cocoa, milk and lactic acid bacteria drinks, yoghurt, drinking yoghurt and wine.

Preferably, the food as described herein is a dairy product. More preferably, the dairy product as described herein is one or more of: yoghurt, cheese (such as acid curd cheese, hard cheese, semi-hard cheese, farmer cheese), buttermilk, quark milk (quark), sour cream, kefir (kefir), fermented whey based beverages, kefir (koumiss), milk drinks, yoghurt drinks, fermented milk, matured cream, cheese, fromage frais, milk, dairy retentate, processed cheese, cream dessert or baby milk.

Preferably, the food as described herein is a fermented food product. More preferably, the food as described herein is a fermented dairy product-such as fermented milk, yoghurt, cream, matured cream, cheese, fromage frais, milk drink, processed cheese, cream dessert, farmer cheese, yoghurt drink, dairy retentate or baby milk.

Preferably, the dairy product according to the invention comprises milk of animal and/or vegetable origin.

Milk is understood to mean milk of animal origin, in particular milk of any mammal, such as cow, goat, sheep, buffalo, zebra, horse, donkey or camel, etc. The term milk also applies to so-called vegetable milks, that is to say extracts of plant materials which have been treated or otherwise processed, such as leguminous plants (soybeans, chickpeas, lentils, etc.) or oilseeds (rape, soybeans, sesame, cotton, etc.), which extracts contain proteins in solution or colloidal suspension which can be coagulated by chemical action, by acid fermentation and/or by heat. Finally, the word milk also denotes a mixture of animal milk and vegetable milk.

In one embodiment, the term "milk" means a commercial UHT milk supplemented with 3% (w/w) semi-skimmed milk powder, which is pasteurized by heating at 90 ℃ +/-0.2 ℃ for 10 min +/-1 min.

In the field of dairy applications, it is advantageous to use fermented milks, such as yoghurts, manufactured with the streptococcus thermophilus strain or the bacterial composition according to the invention when mixed with a warm flavour, such as coffee or chocolate flavour; in fact, not only the high pH of the yoghurt obtained with the strain of the invention but also the stability of this pH (without post-acidification) inhibits the acidic sensation in the final product and improves its mildness; these advantages make the use of a warming flavour, such as coffee or chocolate flavour, compatible with the manufacture of flavoured yoghurt. In another embodiment, the streptococcus thermophilus strain or the bacterial composition according to the invention is advantageous when used for the manufacture of Ryazhenka type products (eastern europe), also known as "Brown yoghurt" (asian countries) (overcooked milk fermentation, producing caramel aroma); in fact, traditional starter cultures that produce sour sourness are not compatible with such fermented milk products.

Percent identity of beta-galactosidase

A percent identity of at least 95% to SEQ ID No. 2 means a percent identity selected from at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

In one embodiment, the β -galactosidase variant is the same size as the β -galactosidase as defined in SEQ ID NO:2 (1026 amino acid residues), although the sequence of the β -galactosidase differs from SEQ ID NO: 2.

Comparison of sequences can be performed by eye or, more commonly, by means of readily available sequence comparison programs. These commercially available or freely available computer programs can calculate similarity or identity values between two or more sequences.

Percent identity can be calculated over aligned contiguous sequences, i.e., one sequence is aligned relative to the other sequence and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called a "no gap" alignment. Typically, such gap-free alignments are performed only on relatively short residues.

Although this is a very simple and consistent approach, it is not considered, for example, that in an otherwise identical pair of sequences, an insertion or deletion will cause misalignment of downstream amino acid residues, and thus may result in a substantial reduction in identity when globally aligned. Thus, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalising overall identity scores. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity. These more sophisticated methods assign a "gap penalty" to each gap that occurs in the alignment, such that for the same number of identical amino acids, a sequence alignment with as few gaps as possible-reflecting a higher correlation between the two compared sequences-will achieve a higher score than one with many gaps. An "affine gap cost" is typically used that charges a relatively high cost for the presence of a gap and a small penalty (gap extension penalty) for each subsequent residual in the gap. This is the most commonly used vacancy scoring system. High gap penalties will of course result in an optimized alignment with fewer gaps. Most alignment programs allow for modification of gap penalties. However, when using such software for sequence comparisons, default values may be used, as in most cases, these default values have been adjusted to provide a correlation result. Therefore, the calculation of the maximum percent identity first requires that an optimal alignment be produced with gap penalties in mind. A suitable computer program for carrying out this alignment is Vector NTI (Invitrogen Corp.). Examples of software that can be used to perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al, 1999, Short Protocols in Molecular Biology, 4 th edition, Chapter 18).

Although the quality of the alignment can be measured in terms of identity, the alignment process itself is typically not based on an all or no pair-wise comparison. Instead, a scaled similarity score matrix is typically used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix-the default matrix of the BLAST suite of programs. The Vector NTI program typically uses public default values or custom comparison tables (if provided) (see user manual for further details). Alternatively, the percent similarity can be calculated based on an algorithm similar to CLUSTAL using the multiple alignment feature in Vector NTI (Invitrogen Corp.) (Higgins DG & Sharp PM (1988), Gene 73(1), 237-.

Once the software has produced an optimal alignment, it is possible to calculate the percentage of sequence similarity, preferably the percentage of sequence identity. Software typically does this as part of the sequence comparison and generates numerical results.

In one embodiment, the degree of identity with respect to a protein (amino acid) sequence is determined over at least 50 contiguous amino acids, at least 100 contiguous amino acids, at least 150 contiguous amino acids, at least 200 contiguous amino acids, or at least 250 contiguous amino acids.

In one embodiment, the degree of identity with respect to the amino acid or protein sequence can be determined over the complete sequence of SEQ ID NO. 2.

In one embodiment, the sequences [ the sequence of the beta-galactosidase to be compared and SEQ ID NO:2] are aligned by a global alignment program and sequence identity is calculated by identifying the number of exact matches identified by the program divided by the length of the sequence of the beta-galactosidase to be compared.

In one embodiment, the degree of sequence identity between the sequence of the beta-galactosidase to be compared and SEQ ID NO:2 is determined by: 1) aligning the two sequences by any suitable alignment program using a default scoring matrix and a default gap penalty, 2) identifying the number of exact matches, wherein an exact match is the position in the two aligned sequences at a given position in the alignment at which the alignment program has identified the same amino acid, and 3) dividing the number of exact matches by the length of the sequence of β -galactosidase to be compared.

In one embodiment, the global alignment program is selected from CLUSTAL and BLAST, particularly CLUSTAL, which uses default parameters and calculates sequence identity by identifying the number of exact matches identified by the program divided by the length of the subject sequence.

In one embodiment, the global alignment program is CLUSTAL using default parameters, and Sequence identity is determined with BioEdit software (http:// www.mbio.ncsu.edu/BioEdit/bioidit. html) [ select "Sequence" pull-down menu, then select "Pairwise alignment" sub-menu, then select "calcium identity/similarity for two sequences" menu item ].

General recombinant DNA methodology techniques

The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology, and recombinant DNA, which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J.Sambrook, E.F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Books 1-3, Cold Spring Harbor Laboratory Press, Ausubel, F.M. et al (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13 and 16, John Wiley & Sons, New York, N.Y.); B.roe, J.Crabtree, and A.Kahn, 1996, DNA Isolation and Sequencing: analytical technologies, John Wiley & Sons; M.J. Gate (eds.), 1984, Oligoruclein Synthesis: A.P.Auricular, PCR, Molecular Biology, and method of DNA, Molecular Biology, 2 and DNA of Molecular Biology, P.E.J. gap, 1984, Molecular Biology, Analysis, P.S.. Each of these general texts is incorporated herein by reference.

The invention will now be further described by way of examples, which are intended to assist those of ordinary skill in the art in carrying out the invention, and are not intended to limit the scope of the invention in any way.

Materials and methods

Strains and growth conditions

The Streptococcus thermophilus Strains (ST) disclosed in the present application were grown at 37 ℃ in M17 liquid medium (Oxomasum, supplier reference CM0817) supplemented with 30g/L lactose and, if necessary, 15 g/L Agar bacteriological Type A (Biokar, supplier reference # A1010HA), or in milk (UHT semi-skimmed milk "Le Pet tend" + 3% powdered milk BBA Lactalis) at 43 ℃. The autoclaved M17 liquid medium was supplemented with 0.2 μ M filtered lactose, sucrose, galactose or glucose. Frozen stocks of ST strains were obtained by half-diluting overnight cultures grown in M17 liquid medium supplemented with 30g/L sucrose in M17 containing 50% glycerol and stored at-20 ℃.

The DGCC12456 strainlacZTransfer of the allele into the genomes of 2 other strains of S.thermophilus

DGCC 12456-carrying strains were obtained using primers lacZ _ F5 (5'-GTAACTTCGTAGGATACAGTG-3') and lacZ _ R6 (5'-CAGAGTTACCCATTGTGTGC-3')lacZ1198-bp PCR product of the gene. The PCR products were then purified using the QIAquick PCR purification kit (Qiagen) and eluted in dnase-free water. The concentration of the PCR product was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, Mass.). The size and purity of the PCR products were verified by gel-based capillary electrophoresis QIAxcel ® system (Qiagen, Hilden, Germany). Strains DGCC715 and DGCC11231 were transformed with 1198-bp PCR products by natural competence according to Dandoy et al (2011). Select itlacZGene encoded by DGCC12456 strainlacZMutant of allele replacement (checking of DGCC12456 Strain by sequencing)lacZThe presence of an allele).

Validation of DGCC12456 by sequencinglacZPresence of alleles

PCR-amplification of the β -galactosidase gene was performed using primers lacS _ F1 (5 'GTAACTTCGTAGGATACAGTG-3') and lacZ _ R7 (5'-CAGAGTTACCCATTGTGTGC-3') [ incubation step at 98 ℃, 5 min, followed by 33 cycles of 98 ℃, 45 s; at 58 ℃ for 30 s; 68 ℃ for 3 min, and a final extension step at 72 ℃ for 7 min. The 1198-bp PCR product was then treated with Illustra ExoProStar according to the manufacturer's instructions (GE Healthcare). Sequencing reactions were performed using BigDye Terminator v3.1 cycle sequencing kit (Life Technologies) according to the manufacturer's instructions using AB3500 (Applied biosystems) and the primers listed in Table 1.

TABLE 1: for amplification and sequencing for transformationlacZPrimer list of fragments of (a).

LacS Activity [ assay A ]

Streptococcus thermophilus strains were grown overnight at 37 ℃ on M17 medium containing 30g/L sucrose as sole carbon source. When the cells reached stationary phase, they were transferred (at 0.05 uDO/mL) to 1 volume of M17 medium containing 30g/L lactose as the sole carbon source and incubated at 42 ℃ for 2 hours. The strain cultures were centrifuged at room temperature (3500 g), the supernatant removed, and the cells resuspended in 0.5 volume of 4% (w/v) glycerophosphate. This washing step was applied twice. 1.8 mL of the cell suspension in 4% glycerophosphate was incubated at 42 ℃ for 2 minutes. Then, 0.2 mL of lactose solution (70 g/L lactose + 0.1M potassium phosphate buffer) was added [ the lactose solution pH was previously adjusted at pH4.5 or pH6 according to the desired measurement). The mixture was incubated at 42 ℃ for a further 3 minutes. The reaction was blocked by removing cells by filtration on a 0.22 μm filter. The lactose in the filtered solution was then determined on HPLC using the following protocol. The solution was diluted 10-fold in water and 10 μ Ι _ was injected on Agilent 1200 HPLC (high performance liquid chromatography). The elution was carried out in isocratic mode with pure water at 0.6 mL/min. The molecule was separated to Pb within 40 min²⁺Ion exchange columns (SP-0810 Shodex 300 mm x 8 mm x 7μm) columns. The sugars were detected with a refractometer. Quantification was done by external calibration.

The lactose import activity of LacS permease was calculated as follows:

- [ lactose]_InitialInitial concentration in μmol/mL

- [ lactose]_3minIs at 42 DEG CConcentration in μmol/mL after the next 3 minutes

DO is the density of bacteria in uDO/mL

Time is the duration of the experiment in minutes (in this case, 3 minutes).

LacZ Activity [ assay B ]

A fresh overnight culture of the Streptococcus thermophilus strain to be determined in M17 containing 30g/L lactose was obtained and used to inoculate 10 ml of fresh M17 containing 30g/L lactose at 1% (v/v). After 3 hours of growth on M17 containing 30g/L lactose at 42 ℃, the cells were harvested by centrifugation (6000 g, 10 min, 4 ℃), washed in 1.5 ml cold lysis buffer (KPO40.1M) and resuspended in 300. mu.l cold lysis buffer. EDTA-free protease inhibitor "cOmplete" was prepared as described by the supplier^TM"(Roche, supplier reference 04693132001) was added to the lysis buffer. Cells were disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 250 μ l of resuspended cells and shaking for 6 minutes in a MM200 shaker grinder (Retsch, Haan, Germany) at a frequency of 30 cycles/s. Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4 ℃) and the supernatant was transferred to a clean 1.5 mL centrifuge tube kept on ice. Total protein content was determined by using the FLUKA protein quantification kit-Rapid (ref 51254). Beta-galactosidase activity in cell extracts was determined spectrophotometrically by monitoring the hydrolysis of O-nitro-phenol-beta-galactoside (ONPG) to galactose and O-nitro-phenol (ONP). mu.L of the cell extract was mixed with 135. mu.L of reaction buffer (NaPO)₄ 100 mM; KCl 10 mM; MgSO ₄1 mM ONPG 3 mM + beta mercaptoethanol 60 mM, pH = 6). The production of ONP results in yellow color entering the tube. When yellow color appeared, stop buffer (Na) was added by 250. mu.L₂CO₃1M) to block the reaction. The optical density at 420 nm was recorded using a Synergy HT multi-detection microplate reader (BIO-TEK). One unit of beta-galactosidase corresponds to the amount of enzyme that catalyzes the production of 1. mu. mol ONP per minute under the assay conditions. Beta-galactosidase activity was calculated as follows:

-l = optical path length (0.73 cm in this text)

- ɛ = molar attenuation coefficient of ONP (4500 cm herein)² / µmol)

-Qprot = amount of protein in cuvette (in mg).

Lactobacillization Properties [ determination C ]

The acidification properties of the streptococcus thermophilus strains were assessed by recording the change in pH over time during milk fermentation as follows: UHT semi-skimmed milk "Le Pet Vend meen" containing 3% (w/v) powdered milk (BBA, Lactalis) previously pasteurized at 90 ℃ for 10 minutes at 1% (v/v, about 10%⁷CFU/ml) was inoculated with a culture of the streptococcus thermophilus strain to be assayed (M17-carbohydrate-free resuspension cells from an overnight culture grown in M17 supplemented with 3% sucrose). The inoculated flasks were incubated at rest in a water bath at 43 ℃ for 24 hours. pH was monitored during incubation using a CINAC system (Alliance Instruments, France; pH electrode Mettler 405 DPAS SC, Toledo, Spain), as previously described. The pH was measured and recorded every 5 minutes.

Results

Example 1: streptococcus thermophilus exhibiting the complete-stop phenotype was isolated.

Dilutions of cultures of DGCC7984 strain were plated on the surface of M17 supplemented with 5g/L sucrose agar plates. After 48 hours of incubation at 37 ℃,2 colonies of the isolated DGCC7984 strain were picked and propagated in M17 liquid medium supplemented with 20g/L sucrose for 24 hours at 37 ℃. These two subclones of DGCC7984 strain were named DGCC12455 and DGCC 12456. The acidification properties of strains DGCC12455 and DGCC12456 were investigated as follows: 2 strains were inoculated into M17 liquid medium supplemented with 30g/L lactose and then incubated overnight at 37 ℃. Cultures were incubated in tryptone salt solution (pancreatic eggs) as followsPeptone 1g/L, NaCl 8.5g/L) (v/v): the culture was centrifuged at 4000 rpm for 5 minutes; the pellet was resuspended in 10 mL tryptone salt solution. The washed culture was inoculated at 1% (v/v) into 100 mL of UHT skim milk containing 3% (w/v) milk powder and pasteurized at 90 ℃ for 10 minutes. The flasks were incubated in a water bath at 43 ℃ and the pH was measured and recorded online using a CINAC system (fig. 1A). The slope (-UpH/min) between pH6.0 and pH 5.3 was calculated, representing the rate between pH6 and pH 5.3 (the slope of the linear model (Δ pH/Δ time) derived as the evolution of pH over time for pH values between 6 and 5.3). In addition, the assay corresponded to a rate of V0 (which corresponded to becoming unambiguously undetectable, i.e., less than 0.1 mupH/min (0.0001 UpH/min))]pH of pH value of_STOP。

It was found that DGCC12455 and DGCC7984 were all similar in acidification of milk along the kinetics. In contrast, DGCC12456 exhibited a unique acidification profile (fig. 1A). Indeed, after about 600 min of fermentation with DGCC12456, the pH tends to stabilize around 4.37 and there is no change until the end of the fermentation time (pH)_STOP=4.37), whereas for the DGCC12455 and DGCC7984 strains the pH remains reduced after 600 min of fermentation and reaches values around 4.1 and 4.2 at the end of the fermentation time. This particular acidification profile with pH stabilization is called the full-STOP phenotype. However, despite this particular kinetics at the end of fermentation, the acidification slope between 6 and 5.3 is 106 mUpH/min, which is the acidification rate expected in industrial dairy fermentations.

Example 2: of DGCC12456lacZIdentification of genetic differences of genes

The genomes of strains DGCC7984 and DGCC12456 were sequenced and compared. Wherein, inlacZDifferences between the two strains were identified in the genes.lacZThe gene is described (van den Bogaard et al, 2000; Vaughan et al, 2001) as encoding a beta-galactosidase, an enzyme responsible for the hydrolysis of lactose to glucose and galactose. In the context of the DGCC12456 genome,lacZthe C base at position 1060 of the gene was replaced with a T base, resulting in a non-conservative amino acid change, and the arginine at position 354 of β -galactosidase was replaced with cysteine (R354C substitution). Thus, DGCC7984 having coding beta-galactosidaselacZAlleles, the sequence of the beta-galactosidase being as defined in SEQ ID NO 2, and the DGCC12456 strain having the coding beta-galactosidaselacZAlleles, the sequence of the beta-galactosidase being as defined in SEQ ID NO 4. In contrast, of strain DGCC12455lacZSequencing of the gene revealed that its lacZ sequence was identical to that of DGCC7984 (i.e., encoding β -galactosidase, the sequence of which is defined as SEQ ID NO: 2). Taken together, these results show thatlacZMutations in the gene may be responsible for the acidification profile specific to DGCC 12456.

To further investigate this hypothesis, comparisons were made between strains of other S.thermophiluslacZThe gene encodes beta-galactosidase. The R354C substitution found in DGCC12456 was not found in any of the β -galactosidase sequences of other streptococcus thermophilus strains, confirming that the substitution is unique to DGCC 12456.

Most of the tested strains of Streptococcus thermophilus carry a gene encoding beta-galactosidaselacZAlleles, the sequence of the beta-galactosidase is as defined in SEQ ID NO 2. In some strains of S.thermophilus, amino acid differences compared to SEQ ID NO 2 have been identified. These identified amino acid differences are conservative substitutions and have led to the identification of 8 different β -galactosidase variant types (as defined herein) having the sequences as set forth in SEQ ID NOs 6, 9, 12, 15, 18, 21, 24 and 27 [ variants 1 to 8-table 2]As defined in (1).

TABLE 2: comparative amino acid sequence analysis of beta-galactosidase encoded by strains of Streptococcus thermophilus. Numbering of amino acid positions is according to SEQ ID NO 2. Indicates a different position 354 in SEQ ID NO 4.

Example 3: comparison of the acidification profiles of Streptococcus thermophilus strains DGCC715 and DGCC11231 and their derivatives encoding beta-galactosidase with the sequence SEQ ID NO:4 instead of SEQ ID NO:2 (substitution R354C).

The constructs were named 715, respectively^R354CAnd 11231^R354CDerivatives of strains DGCC715 and DGCC 11231. Inserted into DGCC12456lacZGene encoding beta-galactosidase with cysteine (C) at position 354 in place of that of strains DGCC715 and DGCC11231lacZA gene. In fact, PCR amplification from DGCC12456 DNAlacZA gene. Competent cells of DGCC715 or DGCC11231 were prepared and transformed with the amplified DNA. Transformants were verified by sequencing.

Evaluation of Streptococcus thermophilus strains DGCC715, DGCC11231, 715^R354CAnd 11231^R354CAbility to ferment milk, e.g. materials and methods section [ assay C ]]The method as described in (1). The pH was recorded over time using a CINAC apparatus and the results are presented in fig. 2A, 3A, 4A and 5A. The following descriptors (table 3) were calculated:

-slope between pH6.0 and pH 5.3 (UpH/min) [ slope pH6-5.3 ]; and

the rate corresponding to V0 (which corresponds to becoming unambiguously undetectable, i.e. below 0.1 mupH/min (0.0001 UpH/min))]pH of pH value of_STOP。

TABLE 3: descriptors of the acidification kinetics of milk of DGCC715, DGCC11231 and their constructed derivatives calculated from the acidification curves.

The results show that the derivative 715^R354CAnd 11231^R354CThe difference between the acidification curves of (see fig. 3A and 5A) and their respective parental strains (fig. 2A and 4A, respectively) is the stabilization of pH after 10 to 12 hours of incubation. Stabilization of the pH (pH)_STOP) For 11231^R354COccurs around pH 4.27, and for 715^R354COccurs around pH 4.38, while the parent strain continues to acidify the milk after 12 hours of incubation, reaching pH 4.19 and 4.10, respectively, at the end of the incubation time. The results also show that despite the substitution of cysteine for arginine at position 354 of β -galactosidase, the acidification slope between pH6.0 and 5.3 is not negatively affected. Thus, the constructed derivative strain remainsIs suitable for fermenting dairy products in industrial settings.

A second set of descriptors is also considered to characterize the full-STOP phenotype. This second set of descriptors was also determined against DGCC12456 strain. For this purpose, the evolution of the rate (acidification speed) over time was calculated and the results are presented in fig. 1B, 2B, 3B, 4B and 5B. From these curves the following descriptors (table 4) were determined:

time (T) to maximum rate obtained during fermentation experiment_Vmax) Time (in minutes) from the start of the fermentation experiment;

to pH_STOPTime of (TpH)_STOP) [ time to reach V0 as defined above]Time (in minutes) from the start of the fermentation experiment;

- TpH_STOPand T_VmaxTime difference between (in minutes).

TABLE 4: descriptors of rate kinetics of fermentation for DGCC715, DGCC11231 and its constructed derivatives and DGCC12456 calculated from rate curves.

The results show that for derivative 715^R354CAnd 11231^R354C，TpH_STOPAnd T_VmaxThe time difference between these was 410 and 480 minutes, compared to 695 and 840 minutes for their respective parent strains (table 4). The results also show that DGCC12456 strain has the same strain as the derivative 715^R354CAnd 11231^R354CThe same curve. These results indicate that derivative 715 is^R354CAnd 11231^R354CTpH of (1)_STOPAnd T_VmaxThe time difference between them was significantly reduced compared to the time difference of their respective parent strains (285 and 360 min difference, respectively). These data reflect derivative 715^R354CAnd 11231^R354CIn a shorter time (from T) when used in fermented milks_Vmax) A higher stable pH (pH) is obtained_STOP) The ability of the cell to perform. These results confirm that the R354C substitution in the beta-galactosidase of DGCC12456 is responsible for the full-STOP phenotype.

Thus, the strain carrying the lacZ allele encoding a beta-galactosidase with a cysteine at position 354 opens up the possibility of producing fermented milk, which not only reaches its target pH (pH) within an acceptable industrial time (about 600 minutes)_STOP) But also to stabilize their pH at fermentation temperatures for up to 24 hours. In contrast, the parent strain continues to acidify the milk until 700 to 800 minutes and at a lower pH, thus requiring the fermentation process to be stopped by a cooling step before the pH drops too low.

Example 4: beta-galactosidase Activity of various strains of Streptococcus thermophilus at pH6 and pH4.5

Determination of beta-galactosidase carrying the nucleic acid encoding the amino acid sequence as defined in SEQ ID NO:2 by assay B (as defined in materials and methods)lacZAllelic beta-galactosidase activity of various strains of Streptococcus thermophilus at pH4.5 and pH6. The results are presented in fig. 6.

First, these data show that for a particular strain, its β -galactosidase activity at pH4.5 is always lower than its β -galactosidase activity at pH6.0, indicating that β -galactosidase activity decreases with decreasing pH.

Furthermore, these data show that there is an important variability in β -galactosidase activity between strains carrying the same lacZ allele, not only at pH6.0 [ from 9.93x10 ]^-8To 1.74x10^-7mol/(mg Total protein extract. min)]And at pH4.5 [ from 6.7X10^-8To 1.15x10^-7mol/(mg Total protein extract. min)]. This variability can be explained by the genetic background specific to each strain. These data are questionable for the following facts: the beta-galactosidase activity alone (at pH4.5 and/or pH 6) can be used as a reliable descriptor for the characterization of the strains of the invention (with a full-STOP phenotype).

Example 5: streptococcus thermophilus strains 715 and ST11231, derivatives 715 thereof^R354CAnd 11231^R354CAnd comparison of beta-galactose Activity at pH6 and pH4.5 of Strain DGCC12456

After identifying the R354C substitution in β -galactosidase and its role in the specific kinetics of acidification of milk by DGCC12456 (full-STOP phenotype), the β -galactosidase activity at pH6 and pH4.5 of strains DGCC715, DGCC11231, their respective constructed derivatives and DGCC12456 was determined by assay B (as defined in materials and methods). The results are presented in fig. 7.

These data confirm that the vector carries a polypeptide encoding a beta-galactosidase as defined in SEQ ID NO:4 (cysteine at position 354)lacZThe strain of the allele has a beta-galactosidase activity at pH4.5 which is less than the beta-galactosidase activity at pH 6.0.

Notably, the difference in beta-galactosidase activity between pH6 and pH4.5 for the protein carrying the polypeptide encoding beta-galactosidase as defined in SEQ ID NO 4lacZAllelic ratios for strains carrying a beta-galactosidase as defined in SEQ ID NO:2lacZStrains of alleles are more important. Thus, carrying a polypeptide encoding a beta-galactosidase as defined in SEQ ID NO 4lacZThe strain of the allele has a lower beta-galactosidase activity at pH4.5 than the strain carrying the gene encoding beta-galactosidase as defined in SEQ ID NO. 2lacZBeta-galactosidase activity at pH4.5 of the strain of the allele).

However, the variability of beta-galactosidase activity at pH4.5 that existed between strains carrying the same lacZ allele [ from 1.65X10 for strains carrying the lacZ allele encoding beta-galactosidase as defined in SEQ ID NO:4^-8To 3.94x10^-8mol/(mg Total protein extract. min)]It was demonstrated that beta-galactosidase activity, even at ph4.5, could not be used as the only parameter for optimal characterization of the strains of the invention with a complete STOP phenotype.

Example 6: study of lactose permease Activity (LacS)

In the case of the streptococcus thermophilus, the bacteria,lacZthe gene islacPart of an operon (encoding a lactose permease)lacSTogether) and both lactose permease and beta-galactosidase are involved in the catabolism of lactose (by importing lactose (LacS) and then hydrolyzing it to glucose and galactose (lacZ).

LacS activity of strains DGCC715, DGCC11231, their respective derivatives and DGCC7984 and DGCC12456 at pH6.0 and pH4.5 was determined by assay a (as defined in materials and methods). The results are presented in table 5 (together with the β -galactosidase activity determined in example 4).

TABLE 5: the LacS activity, LacZ activity and ratios at pH4.5 and pH6 of DGCC715, DGCC11231, constructed derivatives thereof, and DGCC7984 and DGCC12456 strains.

Although the lactose permease (LacS) activity at pH4.5 is reduced compared to pH6.0 for the strain encoding beta-galactosidase as defined in SEQ ID NO:2, these activities are increased for the strain encoding beta-galactosidase as defined in SEQ ID NO:4 (715)^R354CAnd 11231^R354C) Or unchanged (DGCC 12456). It is hypothesized that to compensate for beta-galactosidase^FSThe lactose hydrolysis is reduced and the lactose permease imports more lactose.

Thus, LacS at pH4.5 and pH6 was calculated: the LacS ratio (LacS/LacZ, which represents the efficacy of the strain to hydrolyze imported lactose = EH) (as defined herein) and is given in table 5 and fig. 8. Carrying a beta-galactosidase encoding the enzyme as defined in SEQ ID NO 2lacZThe allelic strain exhibits a LacS/LacZ ratio at pH4.5 with a similar or slightly reduced value compared to pH 6.0. In contrast, for a vector carrying a beta-galactosidase encoding the enzyme as defined in SEQ ID NO 4lacZAllelic strains, these ratios are significantly increased at pH4.5 compared to pH 6.0. These results reflect the expression of the gene encoding beta-galactosidase as defined in SEQ ID NO 2lacZThe strains of the invention have a reduced efficacy in lactose (i.e. hydrolysis of the lactose input) at pH4.5 using the medium compared to strains of the allele.

Carrying a beta-galactosidase encoding the enzyme as defined in SEQ ID NO 2lacZThe LacS/LacZ ratio at pH4.5 of the strains of the alleles combined with the strain carrying the coding sequence beta-galactose as defined in SEQ ID NO 4Of glycosidaseslacZThe difference between the ratios of strains of alleles is highly significant, so that this parameter can be reliably used to characterize the strains of the invention.

It has been shown that the LacS/LacZ ratio at pH4.5 of strain DGCC715 and its derivatives is sufficiently resolved to use the DGCC715 strain to identify the additional lacZ allele encoding a beta-galactosidase according to the invention ((ii) ((II) ((III))lacZ ^FSAn allele).

Example 7: streptococcus thermophilus strains 715 and ST11231, derivatives 715 thereof^R354CAnd 11231^R354CAnd the lactose-importing hydrolysis Efficiency (EH) of strain DGCC12456

Finally, the inventors have determined additional descriptors that represent the overall behaviour of the streptococcus thermophilus strain of the invention with respect to lactose metabolism during the entire course of milk fermentation. Thus, the following formula (I) was developed, which represents the difference in the hydrolysis efficiency of the input lactose between pH6.0 and pH4.5 (EH)_pH6 - EH_pH4.5)：

In this equation, a Δ EH value of about 0 or slightly positive or slightly negative means that the hydrolysis efficiency of the input lactose is similar at pH6.0 and pH4.5 (i.e., the hydrolysis efficiency is independent of pH). Conversely, a significantly negative Δ EH value means that the hydrolysis efficiency of the input lactose is lower at pH4.5 than at pH6.0 (i.e., as pH decreases, the hydrolysis efficiency decreases significantly).

Based on the β -galactosidase activity and lactose permease activity reported in table 5, Δ EH was calculated for strains DGCC715, DGCC11231, their respective derivatives and DGCC12456 using this formula. The results are presented in fig. 9.

As shown in FIG. 9, and as expected, 2 carry a gene encoding beta-galactosidase as defined in SEQ ID NO:2lacZAllelic strains of S.thermophilus have slightly positive Δ EH values (0.44 and 0.56). In contrast, 3 carry a gene encoding beta-galactosidase as defined in SEQ ID NO 4lacZOf allelesThe strains of S.thermophilus have significantly negative values for Δ EH (from-1.23 to-1.97).

In addition to the LacS at pH4.5 defined above: in addition to the LacZ ratio, the Δ EH values as defined by formula (I) are reliable parameters enabling the characterization of the strains of the invention with a full-STOP phenotype.

Example 8: influence of the packaging temperature during the manufacture of stirred yoghurt

By using the previously described DGCC12456 strain (at least 10)⁷cfu/ml) and Lactobacillus bulgaricus (about 10)³cfu/ml) inoculated milk matrix (protein 3.9%, fat 1.5% and sucrose 6%) and the inoculated milk was incubated at 43 ℃ until pH =4.60 was reached, preparing stirred yoghurt. Immediately thereafter, the yogurt was stirred. The stirred yoghurt was then cooled and packaged at 20 ℃ or 35 ℃ and then stored at 10 ℃ for the entire shelf life (45 days).

The pH during shelf life was measured using a single probe portable pH meter.

With the aid of Brookfield DV-I^TMThe viscosity on day 14 (after the end of fermentation) was measured with a Prime viscometer (AMETEK Brookfield) using spindle S-05 and speed 10 rpm; after 30 seconds, the viscosity number (centipoise; cP) was determined.

As shown in fig. 10A and as expected, packaging at 35 ℃ gave stirred yoghurts a higher texture at day 14 (fig. 10A) than packaging at 20 ℃. Interestingly, the pH of the stirred yogurt remained at a high level for at least 45 days regardless of the packaging temperature (fig. 10B).

These results demonstrate that the streptococcus thermophilus strains of the invention having the complete STOP phenotype are highly attractive to stirred yoghurt manufacturers, as the texture of stirred yoghurt can be improved by increasing the packaging temperature, while at the same time the pH during storage is not compromised.

Example 9: post-acidification of yogurt at 10 deg.C

By using (A) the previously described DGCC12456 strain (at least 10)⁷cfu/ml) and Lactobacillus bulgaricus (about 10)³cfu/ml) or (B) having a high content of Streptococcus thermophilus and Lactobacillus bulgaricus strain (the same Lactobacillus bulgaricus strain as composition A)Post-acidification control performance reference starter cultures were inoculated with milk matrix (3.9% protein and 1.5% fat; no sugar added) and yogurt was prepared by incubating the inoculated milk at 43 ℃ until pH =4.60 was reached. Immediately thereafter the yoghurt was cooled at 22 ℃ and then stored at 10 ℃ for the entire shelf life (45 days). The pH during shelf life was measured using a single probe portable pH meter.

As shown in fig. 11, both cultures showed a relatively high pH during the shelf life. The reference starter culture showed a rapid pH drop to 4.34 until day 14, followed by a pH plateau from day 14 to day 45 (dashed line); in contrast, cultures containing the DGCC12456 strain showed a stable pH (pH between 4.48 and 4.5) throughout the shelf life from day 1 to day 45 (flat line).

These results demonstrate that the Streptococcus thermophilus strains of the invention with a complete STOP phenotype are highly attractive to the fermented milk manufacturer, since the fermented milk product can be stored at temperatures above the conventional refrigerator temperature (typically below 8 ℃) without affecting the pH.

In summary, the streptococcus thermophilus strains according to the invention offer the manufacturers of fermented milks and yoghurts new possibilities to improve their process and to reduce their costs, for example by exploiting the pH stability at fermentation temperature up to 24 hours in the manufacture of set-style yoghurts, by exploiting texture improvement and pH stability at high temperature packaging in the manufacture of stirred yoghurts, or by exploiting the pH stability at 10 ℃ for at least 45 days in the storage of their fermented milks.

SEQUENCE LISTING

<110> DuPont Nutrition Biosciences ApS

<120> novel lactic acid bacterium

<130> NB41527-WO-PCT

<150> EP19162856

<151> 2019-03-14

<150> CN201910406044

<151> 2019-05-16

<150> EP19214119

<151> 2019-12-06

<160> 38

<170> PatentIn version 3.5

<210> 1

<211> 3081

<212> DNA

<213> Streptococcus thermophilus

<400> 1

atgaacatga ctgaaaaaat tcaaacttat ttaaacgatc caaagattgt tagcgttaat 60

actgttgatg ctcactcaga tcataagtat tttgaatctc ttgaagaatt ttctgaaggg 120

gagatgaagt taagacaatc tcttaatgga aaatggaaaa ttcactatgc tcagaataca 180

aatcaggttt taaaagactt ttataaaaca gaatttgatg aaactgattt gaatttcatc 240

aatgtaccag gtcatttaga gcttcaaggt tttggttctc cacaatatgt gaatacccaa 300

tatccttggg atggtaaaga attccttcgt ccacctcaag ttcctcaaga atcaaatgct 360

gttgcatcat acgttaaaca ttttaccttg aatgatgcat taaaagataa aaaagtattt 420

atctcattcc aaggggttgc tacttccatc tttgtatggg tcaatggtaa cttcgtagga 480

tacagtgaag attcatttac acctagtgaa tttgaaatta gtgattacct tgttgaaggt 540

gataacaagt tggcggtagc tgtttatcgt tactctacag caagctggtt ggaagaccaa 600

gacttctgga gactttacgg tatttttaga gatgtttact tgtatgctat tccaaaagtt 660

cacgttcaag atctctttgt taagggagat tatgattacc aaacaaaagc aggtcaattg 720

gatattgatt tgaagactgt tggtgattat gaagacaaga agattaaata tgttctttca 780

gattatgaag gcatcgttac agaaggtgat gcatctgtta atggtgacgg tgaactatct 840

gtaagtcttg aaaatcttaa aatcaaacct tggagtgctg aaagtcctaa actttacgat 900

ttgatccttc atgttttgga tgatgaccaa gttgttgaag tcgttccagt taaagttgga 960

ttcagacgct ttgaaattaa agataaactt atgcttttga atggtaagag aattgtcttt 1020

aaaggggtta acagacacga atttaacgct agaacaggac gttgtatcac tgaagaagat 1080

atgctttggg atatcaaagt gatgaagcaa cataacatca atgctgttcg tacttcacac 1140

tatcctaacc aaacacgttg gtatgaattg tgtgatgaat atggacttta tgttatcgat 1200

gaagccaacc ttgaaacaca cggtacatgg caaaaacttg gtctatgcga accttcatgg 1260

aatatcccag ctagtgaacc agaatggttg cctgcttgtt tggatcgtgc caataacatg 1320

ttccaacgcg ataagaacca cgctagtgtt atcatttggt cttgtggtaa tgaatcatat 1380

gctggtaaag atattgctga catggctgat tacttccgta gtgttgacaa tactcgtcca 1440

gttcactatg aaggtgttgc atggtgtcgt gagtttgatt acattacaga catcgaaagt 1500

cgtatgtatg cgaaaccagc tgatatcgaa gaatacctca caactggtaa actagttgat 1560

ctttcaagcg ttagtgataa acactttgct tcaggtaacc taactaacaa acctcaaaaa 1620

ccttatattt catgtgaata catgcacaca atgggtaact ctggtggtgg attgcaactc 1680

tacactgact tagagaaata tccagaatac caaggtggat ttatttggga cttcattgac 1740

caagctattt acaaaacact tccaaatggt agcgaattcc tatcatatgg tggtgactgg 1800

catgatagac cttctgacta cgaattttgt ggaaatggta tcgtctttgc agatcgtacc 1860

ctaactccaa aacttcaaac agttaaacat ctttactcta atattaagat tgctgttgat 1920

gaaaaatcag taactatcaa gaatgataat ctcttcgaag atctttctgc ttatactttc 1980

ctagctagag tttacgaaga tggtagaaaa gttagtgaaa gtgaatatca ctttgatgtt 2040

aaaccaggcg aagaagcaac attcccagtt aactttgtag tcgaggcttc aaattctgaa 2100

caaatttacg aagttgcttg tgttctgagg gaagcaactg aatgggctcc taaaggtcat 2160

gaaattgttc gtggtcaata tgttgttgaa aagattagca ctgaaacacc agttaaagca 2220

cctttgaatg ttgttgaagg cgacttcaac atcggtattc aaggacaaaa cttctcaatc 2280

ttgctttcac gtgcacaaaa tactttagta tctgctaagt ataatggtgt tgaattcatt 2340

gagaaaggtc ctaaacttag cttcactcgt gcttacactg acaacgatcg tggtgctgga 2400

tatccattcg aaatggcagg ctggaaggtt gctggaaact atagtaaagt tacagatact 2460

caaattcaaa tcgaagacga ctctgttaaa gtgacttatg ttcatgaatt gccaggcttg 2520

tctgatgtcg aagttaaggt aacttatcaa gttgattaca agggtcgaat ctttgttact 2580

gcaaactatg atggtaaagc aggtttgcca aacttccctg aatttggtct agaatttgct 2640

atcggttcac aatttacaaa ccttagctat tatggatacg gtgcagaaga aagctaccgt 2700

gataaacttc ctggtgccta tcttggtcga tatgaaacat ctgttgaaaa gacatttgct 2760

ccatatctaa tgccacaaga atctggtaat cactatggta ctcgtgaatt cacagtatct 2820

gatgataacc ataatggtct taaattcacc gcacttaata aagcattcga attcagtgct 2880

ttgcgtaaca gtactgaaca aattgaaaat gctcgtcacc aatatgagtt gcaagaatct 2940

gatgctacat ggattaaagt tcttgctgct caaatgggtg taggtggtga cgacacatgg 3000

ggtgctccag ttcatgacga attcttgctt agctcagcag atagctatca attaagcttc 3060

atgattgaac cactaaatta g 3081

<210> 2

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 2

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 3

<211> 3081

<212> DNA

<213> Streptococcus thermophilus

<400> 3

atgaacatga ctgaaaaaat tcaaacttat ttaaacgatc caaagattgt tagcgttaat 60

actgttgatg ctcactcaga tcataagtat tttgaatctc ttgaagaatt ttctgaaggg 120

gagatgaagt taagacaatc tcttaatgga aaatggaaaa ttcactatgc tcagaataca 180

aatcaggttt taaaagactt ttataaaaca gaatttgatg aaactgattt gaatttcatc 240

aatgtaccag gtcatttaga gcttcaaggt tttggttctc cacaatatgt gaatacccaa 300

tatccttggg atggtaaaga attccttcgt ccacctcaag ttcctcaaga atcaaatgct 360

gttgcatcat acgttaaaca ttttaccttg aatgatgcat taaaagataa aaaagtattt 420

atctcattcc aaggggttgc tacttccatc tttgtatggg tcaatggtaa cttcgtagga 480

tacagtgaag attcatttac acctagtgaa tttgaaatta gtgattacct tgttgaaggt 540

gataacaagt tggcggtagc tgtttatcgt tattctacag caagctggtt ggaagaccaa 600

gacttctgga gactttacgg tatttttaga gatgtttact tgtatgctat tccaaaagtt 660

cacgttcaag atctctttgt taagggagat tatgattacc aaacaaaagc aggtcaattg 720

gatattgatt tgaagactgt tggtgattat gaagacaaga agattaaata tgttctttca 780

gattatgaag gcatcgttac agaaggtgat gcatctgtta atggtgacgg tgaactatct 840

gtaagtcttg aaaatcttaa aatcaaacct tggagtgctg aaagtcctaa actttacgat 900

ttgatccttc atgttttgga tgatgaccaa gttgttgaag tcgttccagt taaagttgga 960

tttagacgct ttgaaattaa agataaactt atgcttttga atggtaagag aattgtcttt 1020

aaaggggtta acagacacga atttaacgct agaacaggat gttgtatcac tgaagaagat 1080

atgctttggg atatcaaagt gatgaaacaa cataacatca atgctgttcg tacttcacac 1140

tatcctaacc aaacacgttg gtatgaattg tgtgatgaat atggacttta tgttatcgat 1200

gaagccaacc ttgaaacaca cggtacatgg caaaaacttg gtctatgcga accttcatgg 1260

aatatcccag ctagtgaacc agaatggttg cctgcttgtt tggatcgtgc caataacatg 1320

ttccaacgcg ataagaacca cgctagtgtt atcatttggt cttgtggtaa tgaatcatat 1380

gctggtaaag atattgctga catggctgat tacttccgta gtgttgacaa tactcgtcca 1440

gttcactatg aaggtgttgc atggtgtcgt gagtttgatt acattacaga catcgaaagt 1500

cgtatgtatg cgaaaccagc tgatatcgaa gaatacctca caactggtaa actagttgat 1560

ctttcaagcg ttagtgataa acactttgct tcaggtaacc taactaacaa acctcaaaaa 1620

ccttatattt catgtgaata catgcacaca atgggtaact ctggtggtgg attgcaactc 1680

tacactgact tagagaaata tccagaatac caaggtggat ttatttggga cttcattgac 1740

caagctattt acaaaacact tccaaatggt agcgaattcc tatcatatgg tggtgactgg 1800

catgatagac cttctgacta cgaattttgt ggaaatggta tcgtctttgc agatcgtacc 1860

ctaactccaa aacttcaaac agttaaacat ctttactcta atattaagat tgctgttgat 1920

gaaaaatcag taactatcaa gaatgataat ctcttcgaag atctttctgc ttatactttc 1980

ctagctagag tttacgaaga tggtagaaaa gttagtgaaa gtgaatatca ctttgatgtt 2040

aaaccaggcg aagaagcaac attcccagtt aactttgtag tcgaggcttc aaattctgaa 2100

caaatttacg aagttgcttg tgttctgagg gaagcaactg aatgggctcc taaaggtcat 2160

gaaattgttc gtggtcaata tgttgttgaa aagattagca ctgaaacacc agttaaagca 2220

cctttgaatg ttgttgaagg cgacttcaac atcggtattc aaggacaaaa cttctcaatc 2280

ttgctttcac gtgcacaaaa tactttagta tctgctaagt ataatggtgt tgaattcatt 2340

gagaaaggtc ctaaacttag cttcactcgt gcttacactg acaacgatcg tggtgctgga 2400

tatccattcg aaatggcagg ctggaaggtt gctggaaact atagtaaagt tacagatact 2460

caaattcaaa tcgaagacga ctctgttaaa gtgacttatg ttcatgaatt gccaggcttg 2520

tctgatgtcg aagttaaggt aacttatcaa gttgattaca agggtcgaat ctttgttact 2580

gcaaactatg atggtaaagc aggtttgcca aacttccctg aatttggtct agaatttgct 2640

atcggttcac aatttacaaa ccttagctat tatggatacg gtgcagaaga aagctaccgt 2700

gataaacttc ctggtgccta tcttggtcga tatgaaacat ctgttgaaaa gacatttgct 2760

ccatatctaa tgccacaaga atctggtaat cactatggta ctcgtgaatt cacagtatct 2820

gatgataacc ataatggtct taaattcacc gcacttaata aagcattcga attcagtgct 2880

ttgcgtaaca gtactgaaca aattgaaaat gctcgtcacc aatatgagtt gcaagaatct 2940

gatgctacat ggattaaagt tcttgctgct caaatgggtg taggtggtga cgacacatgg 3000

ggtgctccag ttcatgacga attcttgctt agctcagcag atagctatca attaagcttc 3060

atgattgaac cactaaatta g 3081

<210> 4

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 4

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 5

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 5

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 6

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 6

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Ala Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Thr Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Ile Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 7

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 7

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Ala Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Thr Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Ile Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 8

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 8

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Ala Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Thr Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Ile Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 9

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 9

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Ile Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Ser Trp Gly Ser Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 10

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 10

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Ile Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Ser Trp Gly Ser Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 11

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 11

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Ile Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Ser Trp Gly Ser Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 12

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 12

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Ser Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 13

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 13

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Ser Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 14

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 14

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Ser Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 15

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 15

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Lys Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 16

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 16

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Lys Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 17

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 17

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Lys Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 18

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 18

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Phe Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 19

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 19

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Phe Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 20

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 20

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Phe Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 21

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 21

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ser Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 22

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 22

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ser Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 23

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 23

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ser Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 24

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 24

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Thr Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Ile Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 25

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 25

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Thr Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Ile Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 26

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 26

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Thr Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Ile Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Ala Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 27

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 27

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Arg Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Val Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 28

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<220>

<221> variants

<222> (354)..(354)

<223> "Xaa" is any amino acid except Arg

<400> 28

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Xaa Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Val Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 29

<211> 1026

<212> PRT

<213> Streptococcus thermophilus

<400> 29

Met Asn Met Thr Glu Lys Ile Gln Thr Tyr Leu Asn Asp Pro Lys Ile

1 5 10 15

Val Ser Val Asn Thr Val Asp Ala His Ser Asp His Lys Tyr Phe Glu

20 25 30

Ser Leu Glu Glu Phe Ser Glu Gly Glu Met Lys Leu Arg Gln Ser Leu

35 40 45

Asn Gly Lys Trp Lys Ile His Tyr Ala Gln Asn Thr Asn Gln Val Leu

50 55 60

Lys Asp Phe Tyr Lys Thr Glu Phe Asp Glu Thr Asp Leu Asn Phe Ile

65 70 75 80

Asn Val Pro Gly His Leu Glu Leu Gln Gly Phe Gly Ser Pro Gln Tyr

85 90 95

Val Asn Thr Gln Tyr Pro Trp Asp Gly Lys Glu Phe Leu Arg Pro Pro

100 105 110

Gln Val Pro Gln Glu Ser Asn Ala Val Ala Ser Tyr Val Lys His Phe

115 120 125

Thr Leu Asn Asp Ala Leu Lys Asp Lys Lys Val Phe Ile Ser Phe Gln

130 135 140

Gly Val Ala Thr Ser Ile Phe Val Trp Val Asn Gly Asn Phe Val Gly

145 150 155 160

Tyr Ser Glu Asp Ser Phe Thr Pro Ser Glu Phe Glu Ile Ser Asp Tyr

165 170 175

Leu Val Glu Gly Asp Asn Lys Leu Ala Val Ala Val Tyr Arg Tyr Ser

180 185 190

Thr Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Leu Tyr Gly Ile

195 200 205

Phe Arg Asp Val Tyr Leu Tyr Ala Ile Pro Lys Val His Val Gln Asp

210 215 220

Leu Phe Val Lys Gly Asp Tyr Asp Tyr Gln Thr Lys Ala Gly Gln Leu

225 230 235 240

Asp Ile Asp Leu Lys Thr Val Gly Asp Tyr Glu Asp Lys Lys Ile Lys

245 250 255

Tyr Val Leu Ser Asp Tyr Glu Gly Ile Val Thr Glu Gly Asp Ala Ser

260 265 270

Val Asn Gly Asp Gly Glu Leu Ser Val Ser Leu Glu Asn Leu Lys Ile

275 280 285

Lys Pro Trp Ser Ala Glu Ser Pro Lys Leu Tyr Asp Leu Ile Leu His

290 295 300

Val Leu Asp Asp Asp Gln Val Val Glu Val Val Pro Val Lys Val Gly

305 310 315 320

Phe Arg Arg Phe Glu Ile Lys Asp Lys Leu Met Leu Leu Asn Gly Lys

325 330 335

Arg Ile Val Phe Lys Gly Val Asn Arg His Glu Phe Asn Ala Arg Thr

340 345 350

Gly Cys Cys Ile Thr Glu Glu Asp Met Leu Trp Asp Ile Lys Val Met

355 360 365

Lys Gln His Asn Ile Asn Ala Val Arg Thr Ser His Tyr Pro Asn Gln

370 375 380

Thr Arg Trp Tyr Glu Leu Cys Asp Glu Tyr Gly Leu Tyr Val Ile Asp

385 390 395 400

Glu Ala Asn Leu Glu Thr His Gly Thr Trp Gln Lys Leu Gly Leu Cys

405 410 415

Glu Pro Ser Trp Asn Ile Pro Ala Ser Glu Pro Glu Trp Leu Pro Ala

420 425 430

Cys Leu Asp Arg Ala Asn Asn Met Phe Gln Arg Asp Lys Asn His Ala

435 440 445

Ser Val Ile Ile Trp Ser Cys Gly Asn Glu Ser Tyr Ala Gly Lys Asp

450 455 460

Ile Ala Asp Met Ala Asp Tyr Phe Arg Ser Val Asp Asn Thr Arg Pro

465 470 475 480

Val His Tyr Glu Gly Val Ala Trp Cys Arg Glu Phe Asp Tyr Ile Thr

485 490 495

Asp Ile Glu Ser Arg Met Tyr Ala Lys Pro Ala Asp Ile Glu Glu Tyr

500 505 510

Leu Thr Thr Gly Lys Leu Val Asp Leu Ser Ser Val Ser Asp Lys His

515 520 525

Phe Ala Ser Gly Asn Leu Thr Asn Lys Pro Gln Lys Pro Tyr Ile Ser

530 535 540

Cys Glu Tyr Met His Thr Met Gly Asn Ser Gly Gly Gly Leu Gln Leu

545 550 555 560

Tyr Thr Asp Leu Glu Lys Tyr Pro Glu Tyr Gln Gly Gly Phe Ile Trp

565 570 575

Asp Phe Ile Asp Gln Ala Ile Tyr Lys Thr Leu Pro Asn Gly Ser Glu

580 585 590

Phe Leu Ser Tyr Gly Gly Asp Trp His Asp Arg Pro Ser Asp Tyr Glu

595 600 605

Phe Cys Gly Asn Gly Ile Val Phe Ala Asp Arg Thr Leu Thr Pro Lys

610 615 620

Leu Gln Thr Val Lys His Leu Tyr Ser Asn Ile Lys Ile Ala Val Asp

625 630 635 640

Glu Lys Ser Val Thr Ile Lys Asn Asp Asn Leu Phe Glu Asp Leu Ser

645 650 655

Ala Tyr Thr Phe Leu Ala Arg Val Tyr Glu Asp Gly Arg Lys Val Ser

660 665 670

Glu Ser Glu Tyr His Phe Asp Val Lys Pro Gly Glu Glu Ala Thr Phe

675 680 685

Pro Val Asn Phe Val Val Glu Ala Ser Asn Ser Glu Gln Ile Tyr Glu

690 695 700

Val Ala Cys Val Leu Arg Glu Ala Thr Glu Trp Ala Pro Lys Gly His

705 710 715 720

Glu Ile Val Arg Gly Gln Tyr Val Val Glu Lys Ile Ser Thr Glu Thr

725 730 735

Pro Val Lys Ala Pro Leu Asn Val Val Glu Gly Asp Phe Asn Ile Gly

740 745 750

Ile Gln Gly Gln Asn Phe Ser Ile Leu Leu Ser Arg Ala Gln Asn Thr

755 760 765

Leu Val Ser Ala Lys Tyr Asn Gly Val Glu Phe Ile Glu Lys Gly Pro

770 775 780

Lys Leu Ser Phe Thr Arg Ala Tyr Thr Asp Asn Asp Arg Gly Ala Gly

785 790 795 800

Tyr Pro Phe Glu Met Ala Gly Trp Lys Val Ala Gly Asn Tyr Ser Lys

805 810 815

Val Thr Asp Thr Gln Ile Gln Ile Glu Asp Asp Ser Val Lys Val Thr

820 825 830

Tyr Val His Glu Leu Pro Gly Leu Ser Asp Val Glu Val Lys Val Thr

835 840 845

Tyr Gln Val Asp Tyr Lys Gly Arg Ile Phe Val Thr Ala Asn Tyr Asp

850 855 860

Gly Lys Ala Gly Leu Pro Asn Phe Pro Glu Phe Gly Leu Glu Phe Ala

865 870 875 880

Ile Gly Ser Gln Phe Thr Asn Leu Ser Tyr Tyr Gly Tyr Gly Ala Glu

885 890 895

Glu Ser Tyr Arg Asp Lys Leu Pro Gly Ala Tyr Leu Gly Arg Tyr Glu

900 905 910

Thr Ser Val Glu Lys Thr Phe Ala Pro Tyr Leu Met Pro Gln Glu Ser

915 920 925

Gly Asn His Tyr Gly Thr Arg Glu Phe Thr Val Ser Asp Asp Asn His

930 935 940

Asn Gly Leu Lys Phe Thr Ala Leu Asn Lys Val Phe Glu Phe Ser Ala

945 950 955 960

Leu Arg Asn Ser Thr Glu Gln Ile Glu Asn Ala Arg His Gln Tyr Glu

965 970 975

Leu Gln Glu Ser Asp Ala Thr Trp Ile Lys Val Leu Ala Ala Gln Met

980 985 990

Gly Val Gly Gly Asp Asp Thr Trp Gly Ala Pro Val His Asp Glu Phe

995 1000 1005

Leu Leu Ser Ser Ala Asp Ser Tyr Gln Leu Ser Phe Met Ile Glu

1010 1015 1020

Pro Leu Asn

1025

<210> 30

<211> 1905

<212> DNA

<213> Streptococcus thermophilus

<400> 30

atggaaaaat ctaaaggtca gatgaagtct cgtttatcct acgcagctgg tgcttttggt 60

aacgacgtct tctatgcaac cttgtcaaca tactttatca tgtttgtgac aactcacttg 120

tttaacacag gtgatccaaa gcaaaatagt cactacgtac tattaatcac taacattatc 180

tctattttgc gtatcttgga agtatttatc gatccattga tcggtaatat gattgataac 240

actaatacta agtatggtaa attcaaacca tgggtagttg gtggtggtat catcagttct 300

atcaccttgt tgcttctctt caccgattta ggtggtttga ataaaacaaa tcctttcttg 360

taccttgtac tttttggaat tatctacctt ataatggatg tcttctactc gattaaagat 420

atcggtttct ggtcaatgct tcctgccttg tctcttgaca gtcacgaacg tgaaaaaatg 480

gcaacttttg cccgtattgg ttctacgatt ggtgccaata ttgtaggtgt tgccatcatg 540

ccaatcgttt tgttcttctc tatgacgaac agtagtggct ctggagataa atctggatgg 600

ttctggtttg catttatcgt tgcactcatt ggtgtgatta catcaattgc tgttggtatt 660

ggtacacgtg aagttgagtc aaaacttcgt gataacaatg aaaaaactag ccttaaacaa 720

gtctttaaag ttcttggtaa aaacgaccaa ttgatgtggc tgtctctcgg atattggttc 780

tatggtcttg gtattaatac acttaatgct cttcaacttt attacttcac atttatcctt 840

ggtgattcag ggaaatactc aatcctttac ggattgaata cagttgttgg tttggtttca 900

gtttcactgt tccctagtct tgctggtaaa ttcaaccgta aacgtttgtt ctatggatgt 960

attgcagtaa tgctcggtgg tatcggaatc tttagtattg caggtacttc acttccaatg 1020

atcttgactg cagctgaact cttcttcatt ccacaacctc ttgtgttcct tgttatcctt 1080

atgattatct ctgactcagt agaatatggt caatggaaat tgggacaccg tgatgaatca 1140

cttactttgt cagttcgtcc acttgttgat aaacttggtg gtgcgatgtc aaactggctt 1200

gtttctacaa ttgccgtagc tgccggtatg acaacaggtg cctcagcatc aacaattaca 1260

acacatcaac agtctatttt taagcttagc atgtttggtt tcccagcagc agcaatgctt 1320

atcggtgcct tcattattgc tcgtaaaatc actttgactg aagcacgtca cgctaaaatt 1380

gttgaagaat tggaacatcg ctttagcgta gcaacttctg aaaatgaagt taaagctaac 1440

gtcgtatctc ttgtaacccc tacaactggt tatttggttg atctctcaag tgttaatgat 1500

gaacactttg cttcaggtag catgggtaaa ggtttcgcca ttaaacctac tgatggagct 1560

gtctttgcac caattagtgg taccattcgt caaattcttc ctactcgcca tgcagttggt 1620

attgaaagtg aagatggtgt cattgttctt atccacgttg gcatcggaac agttaaactt 1680

aatggtgaag gattcattag ttacgtagaa caaggtgatc gtgttgaagt tggacaaaaa 1740

cttcttgagt tctggtcacc aattattgag aaaaatggtc ttgatgacac agtacttgtc 1800

actgtaacta attcagaaaa attcagtgct ttccatcttg aacaaaaagt tggagaaaag 1860

gtagaagctt tgtctgaagt tattaccttc aaaaaaggag aataa 1905

<210> 31

<211> 634

<212> PRT

<213> Streptococcus thermophilus

<400> 31

Met Glu Lys Ser Lys Gly Gln Met Lys Ser Arg Leu Ser Tyr Ala Ala

1 5 10 15

Gly Ala Phe Gly Asn Asp Val Phe Tyr Ala Thr Leu Ser Thr Tyr Phe

20 25 30

Ile Met Phe Val Thr Thr His Leu Phe Asn Thr Gly Asp Pro Lys Gln

35 40 45

Asn Ser His Tyr Val Leu Leu Ile Thr Asn Ile Ile Ser Ile Leu Arg

50 55 60

Ile Leu Glu Val Phe Ile Asp Pro Leu Ile Gly Asn Met Ile Asp Asn

65 70 75 80

Thr Asn Thr Lys Tyr Gly Lys Phe Lys Pro Trp Val Val Gly Gly Gly

85 90 95

Ile Ile Ser Ser Ile Thr Leu Leu Leu Leu Phe Thr Asp Leu Gly Gly

100 105 110

Leu Asn Lys Thr Asn Pro Phe Leu Tyr Leu Val Leu Phe Gly Ile Ile

115 120 125

Tyr Leu Ile Met Asp Val Phe Tyr Ser Ile Lys Asp Ile Gly Phe Trp

130 135 140

Ser Met Leu Pro Ala Leu Ser Leu Asp Ser His Glu Arg Glu Lys Met

145 150 155 160

Ala Thr Phe Ala Arg Ile Gly Ser Thr Ile Gly Ala Asn Ile Val Gly

165 170 175

Val Ala Ile Met Pro Ile Val Leu Phe Phe Ser Met Thr Asn Ser Ser

180 185 190

Gly Ser Gly Asp Lys Ser Gly Trp Phe Trp Phe Ala Phe Ile Val Ala

195 200 205

Leu Ile Gly Val Ile Thr Ser Ile Ala Val Gly Ile Gly Thr Arg Glu

210 215 220

Val Glu Ser Lys Leu Arg Asp Asn Asn Glu Lys Thr Ser Leu Lys Gln

225 230 235 240

Val Phe Lys Val Leu Gly Lys Asn Asp Gln Leu Met Trp Leu Ser Leu

245 250 255

Gly Tyr Trp Phe Tyr Gly Leu Gly Ile Asn Thr Leu Asn Ala Leu Gln

260 265 270

Leu Tyr Tyr Phe Thr Phe Ile Leu Gly Asp Ser Gly Lys Tyr Ser Ile

275 280 285

Leu Tyr Gly Leu Asn Thr Val Val Gly Leu Val Ser Val Ser Leu Phe

290 295 300

Pro Ser Leu Ala Gly Lys Phe Asn Arg Lys Arg Leu Phe Tyr Gly Cys

305 310 315 320

Ile Ala Val Met Leu Gly Gly Ile Gly Ile Phe Ser Ile Ala Gly Thr

325 330 335

Ser Leu Pro Met Ile Leu Thr Ala Ala Glu Leu Phe Phe Ile Pro Gln

340 345 350

Pro Leu Val Phe Leu Val Ile Leu Met Ile Ile Ser Asp Ser Val Glu

355 360 365

Tyr Gly Gln Trp Lys Leu Gly His Arg Asp Glu Ser Leu Thr Leu Ser

370 375 380

Val Arg Pro Leu Val Asp Lys Leu Gly Gly Ala Met Ser Asn Trp Leu

385 390 395 400

Val Ser Thr Ile Ala Val Ala Ala Gly Met Thr Thr Gly Ala Ser Ala

405 410 415

Ser Thr Ile Thr Thr His Gln Gln Ser Ile Phe Lys Leu Ser Met Phe

420 425 430

Gly Phe Pro Ala Ala Ala Met Leu Ile Gly Ala Phe Ile Ile Ala Arg

435 440 445

Lys Ile Thr Leu Thr Glu Ala Arg His Ala Lys Ile Val Glu Glu Leu

450 455 460

Glu His Arg Phe Ser Val Ala Thr Ser Glu Asn Glu Val Lys Ala Asn

465 470 475 480

Val Val Ser Leu Val Thr Pro Thr Thr Gly Tyr Leu Val Asp Leu Ser

485 490 495

Ser Val Asn Asp Glu His Phe Ala Ser Gly Ser Met Gly Lys Gly Phe

500 505 510

Ala Ile Lys Pro Thr Asp Gly Ala Val Phe Ala Pro Ile Ser Gly Thr

515 520 525

Ile Arg Gln Ile Leu Pro Thr Arg His Ala Val Gly Ile Glu Ser Glu

530 535 540

Asp Gly Val Ile Val Leu Ile His Val Gly Ile Gly Thr Val Lys Leu

545 550 555 560

Asn Gly Glu Gly Phe Ile Ser Tyr Val Glu Gln Gly Asp Arg Val Glu

565 570 575

Val Gly Gln Lys Leu Leu Glu Phe Trp Ser Pro Ile Ile Glu Lys Asn

580 585 590

Gly Leu Asp Asp Thr Val Leu Val Thr Val Thr Asn Ser Glu Lys Phe

595 600 605

Ser Ala Phe His Leu Glu Gln Lys Val Gly Glu Lys Val Glu Ala Leu

610 615 620

Ser Glu Val Ile Thr Phe Lys Lys Gly Glu

625 630

<210> 32

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 32

cttgactgca gctgaactc 19

<210> 33

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 33

ctcgactaca aagttaactg g 21

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 34

cagagttacc cattgtgtgc 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 35

aggttggctt catcgataac 20

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 36

catcaccttc tgtaacgatg c 21

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 37

gtaacttcgt aggatacagt g 21

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400> 38

aggacgttgt atcactgaag 20

Claims

1. Encoding beta-galactosidase^FSDefined as the lacZ allele that converts the ratio of the activity of lactose import of LacS permease, calculated by assay a at ph4.5, to the activity of lactose hydrolysis of beta-galactosidase, calculated by assay B at ph4.5, in DGCC715 derivatives (LacS)_pH4.5：LacZ_pH4.5Ratio) to more than 8, said DGCC715 derivative being the strain DGCC715 (deposited at DSMZ with access number DSM33036 on 12/2 of 2019) in which its lacZ gene is encoded by said encoded beta-galactosidase^FSThe polynucleotide substitution of (3).

2. The polynucleotide of claim 1, wherein the lacS_pH4.5：LacZ_pH4.5The ratio is increased to greater than 10 or greater than 12.

3. The polynucleotide of claim 1 or 2, wherein the lactose hydrolysis activity of β -galactosidase (LacZ) is calculated by assay B at pH6 in DGCC 715-derived strain_pH6) Is at least 7.10^-8mol/(mg total protein extract. min).

4. The polynucleotide of any one of claims 1 to 3, encoding a β -galactosidase^FSSaid beta-galactosidase^FSComprising amino acid inhibition, amino acid addition, amino acid substitution or amino acid inhibition and addition with respect to a beta-galactosidase selected from the group consisting of:

b) beta-galactosidase variant comprising an amino acid sequence having at least 95% identity to SEQ ID NO 2 encoded by a lacZ variant allele that does not bind LacS in DGCC 715-derived strains_pH4.5：LacZ_pH4.5The ratio is increased to 5 or greater than 5, and the DGCC715 derivative is strain DGCC715 in which its lacZ gene is replaced by the lacZ variant allele.

5. The polynucleotide of any one of claims 1 to 4, wherein the beta-galactosidase enzyme^FSComprises or consists of an amino acid sequence having at least 95% identity to SEQ ID No. 2.

6. The polynucleotide of any one of claims 1 to 5, wherein the beta-galactosidase enzyme^FSDoes not comprise an arginine at position 354, in particular comprises a cysteine or equivalent amino acid thereof at position 354.

7. According toThe polynucleotide of any one of claims 1 to 6, wherein the beta-galactosidase enzyme^FSThe sequence of (a) comprises:

a) an amino acid sequence which is otherwise as defined in SEQ ID NO. 2, but which does not comprise arginine at position 354;

b) an amino acid sequence having at least 95% identity to SEQ ID No. 2 and comprising NO arginine at position 354;

c) an amino acid sequence as otherwise defined as one of the β -galactosidase variant proteins having at least 95% identity to SEQ ID No. 2, but which does not comprise an arginine at position 354.

8. The polynucleotide of any one of claims 1 to 7, wherein the beta-galactosidase enzyme^FSThe sequence of (a) comprises:

a) an amino acid sequence which is otherwise as defined in SEQ ID NO 2 but which comprises a cysteine or equivalent amino acid thereof at position 354;

b) an amino acid sequence having at least 95% identity to SEQ ID No. 2 and comprising a cysteine or equivalent amino acid thereof at position 354;

c) an amino acid sequence which is otherwise as defined as one of the β -galactosidase variant proteins having at least 95% identity to SEQ ID No. 2, but which comprises a cysteine or equivalent amino acid thereof at position 354.

9. A polynucleotide comprising a portion of at least 100 nucleotides of the polynucleotide according to any one of claims 5 to 8, wherein said nucleotide portion encompasses a nucleotide corresponding to said beta-galactosidase^FSCodon for residue 354.

10. Streptococcus thermophilus strain comprising an allele of the lacZ gene, which is encoding a beta-galactosidase according to any one of claims 1 to 8^FSlacZ of^FSAn allele.

11. The streptococcus thermophilus strain according to claim 10, which causes an acidification slope between pH6 and 5.3 of at least-0.005 UpH/min, in particular at least-0.01 UpH/min, when tested by assay C.

12. Streptococcus thermophilus strain according to claim 10 or 11, characterized in that the difference in the hydrolysis efficiency of the input lactose (Δ EH) is less than-0.5, calculated by the following formula (I):

13. A bacterial composition comprising the streptococcus thermophilus strain according to any one of claims 10 to 12, and optionally one or more further lactic acid bacteria selected from the group consisting of streptococcus, lactococcus, lactobacillus, leuconostoc, pediococcus, enterococcus, oenococcus and bifidobacterium.

14. Food or feed product, in particular a dairy product, meat or cereal food or feed product, in particular a fermented dairy food product, comprising a streptococcus thermophilus strain according to any of claims 10 to 12 or a bacterial composition according to claim 13.

15. A method of making a fermentation product, comprising:

a) inoculating a substrate with the streptococcus thermophilus strain of any one of claims 10 to 12 or the bacterial composition of claim 13; and

b) fermenting the inoculated substrate obtained from step a) to obtain a fermented product, preferably a fermented dairy product.

16. The method of claim 15 for making stirred style yogurt, the method comprising:

a) fermenting a milk substrate, in particular milk, inoculated with the streptococcus thermophilus strain of any one of claims 10 to 12 or the bacterial composition of claim 13 to obtain a stirred yoghurt, preferably having a pH of 4.2 to 4.7, more preferably 4.45 to 4.6;

b) cooling and stirring the yogurt;

c) packaging the stirred yoghurt; and

d) optionally, transferring the package of step c) into a cold storage compartment;

wherein the cooling and packaging temperature is at least 24 ℃, at least 25 ℃, at least 26 ℃, at least 27 ℃, at least 28 ℃, at least 29 ℃, at least 30 ℃, at least 31 ℃, at least 32 ℃, at least 33 ℃, at least 34 ℃, at least 35 ℃, at least 36 ℃, at least 37 ℃, at least 38 ℃, at least 39 ℃ and at least 40 ℃.

17. The method of claim 15 for making stirred style yogurt, the method comprising:

b) packaging the stirred yoghurt; and

d) optionally, transferring the package of step b) into a cold storage compartment;

wherein the process does not include any cooling step between the end of fermentation and packaging.

18. A method according to claim 15 for making set style yoghurt, the method comprising:

a) packaging a milk substrate, in particular milk, inoculated with a streptococcus thermophilus strain or a bacterial composition according to the invention into a package;

b) fermenting the inoculated milk base to obtain a set yoghurt, preferably having a pH of 4.2 to 4.7, more preferably 4.45 to 4.6;

c) optionally, the packaging of step b) is transferred directly into a cold storage,

wherein the process does not comprise a cooling step in the cooling chamber after the fermentation step b).

19. Use of a streptococcus thermophilus strain according to any one of claims 10 to 12 or a bacterial composition according to claim 13 for the manufacture of a food or feed product, preferably a fermented food product, more preferably a fermented dairy product.

20. Use of a polynucleotide according to any one of claims 1 to 9 for obtaining a streptococcus thermophilus strain having a complete STOP phenotype when used in fermented milk by assay C.

21. A method of making a streptococcus thermophilus strain with a complete STOP phenotype comprising:

a) streptococcus thermophilus strains are provided having a ratio of the activity of lactose import of LacS permease calculated by assay A at pH4.5 to the activity of lactose hydrolysis of beta-galactosidase calculated by assay B at pH4.5 (LacS) of less than 5_pH4.5：LacZ_pH4.5Ratio);

b) replacement of the Streptococcus thermophilus strain of step a) with a polynucleotide according to any one of claims 1 to 8lacZAlleles of genes or the replacement of the S.thermophilus strain of step a) with the corresponding polynucleotide according to claim 9lacZA part of an allele of a gene, or modifying the S.thermophilus strain of step a)lacZThe sequence of the gene to have the same sequence as the polynucleotide according to any one of claims 1 to 8lacZ ^FSAn allele; and

22. The method according to claim 21, wherein the streptococcus thermophilus strain of step a) is further characterized by its ability to result in an acidification slope between pH6 and 5.3 of at least-0.01 UpH/min when tested by assay C.

23. Identification of encoded beta-galactosidase^FSIs/are as followslacZ ^FSA method of alleles comprising:

a) inserted into a test objectlacZAllelic replacement of the strain DGCC715 (deposited at DSMZ at 12/2/2019 under the access number DSM33036)lacZ(ii) alleles of genes to obtain DGCC 715-derived strains; and

b) determination of the lactose import Activity of LacS permease by assay A at pH4.5 (LacS)_pH4.5) And the lactose hydrolysis activity of beta-galactosidase was determined by assay B at pH4.5 (LacZ)_pH4.5)；

24. The method of claim 23, further comprising determining the lactose hydrolysis activity of β -galactosidase by assay B at pH6 in DGCC 715-derivative (LacZ)_pH6) And wherein LacS_pH4.5：LacZ_pH4.5Ratio greater than 8 and LacZ_pH6Is at least 7.10^-8mol/(mg total protein extract. min.) indicateslacZThe allele is a gene encoding beta-galactosidase^FSIs/are as followslacZ ^FSAn allele.