WO1998013479A1 - Enzyme a activite cyclomaltodextrine glucanotransferase (cgtase) - Google Patents

Enzyme a activite cyclomaltodextrine glucanotransferase (cgtase) Download PDF

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Publication number
WO1998013479A1
WO1998013479A1 PCT/DK1997/000411 DK9700411W WO9813479A1 WO 1998013479 A1 WO1998013479 A1 WO 1998013479A1 DK 9700411 W DK9700411 W DK 9700411W WO 9813479 A1 WO9813479 A1 WO 9813479A1
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cgtase
enzyme
dna sequence
thermoalcalibacter
seq
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PCT/DK1997/000411
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English (en)
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Carsten SJØHOLM
Steffen Prowe
Garabed Antranikian
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Novo Nordisk A/S
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Priority to EP97941877A priority Critical patent/EP0956346A1/fr
Priority to AU43765/97A priority patent/AU4376597A/en
Publication of WO1998013479A1 publication Critical patent/WO1998013479A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes

Definitions

  • TITLE An enzyme with CYCLOMALTODEXTRIN GLUCANOTRANSFERASE (CGTase) activity
  • the present invention relates to a DNA sequence encoding a novel alkali CGTase, the novel stable alkaline CGTase, an enzyme composition comprising said CGTase, and the use of said enzyme and enzyme composition for a number of industrial applications .
  • Cyclo altodextrin glucanotransferase (E.C. 2.4.1.19), also designated cyclodextrin glucanotransferase or cyclodextrin glycosyltransferase, in the following termed CGTase, catalyses the conversion of starch and similar substrates into cyclomaltodextrins via an intramolecular transglycosylation reaction, thereby forming cyclomaltodextrins, in the following termed cyclodextrins (or CD) , of various sizes.
  • CGTase Cyclo altodextrin glucanotransferase
  • CGTase catalyses the conversion of starch and similar substrates into cyclomaltodextrins via an intramolecular transglycosylation reaction, thereby forming cyclomaltodextrins, in the following termed cyclodextrins (or CD) , of various sizes
  • cyclodextrins of 6, 7 and 8 glucose units which are termed ⁇ -, ⁇ - and ⁇ -cyclodextrins, respectively.
  • cyclodextrins of 9, 10, and 11 glucose units which are termed ⁇ -, ⁇ -, and ⁇ -cyclodextrins , respectively.
  • Cyclodextrins are thus cyclic glucose oligo ers with a hydrophobic internal cavity. They are able to form inclusion complexes with many small hydrophobic molecules in aqueous solutions, resulting in changes in physical properties, e.g. increased solubility and stability and decreased chemical reactivity and volatility. Cyclodextrins find applications particularly in the food, cosmetic, chemical and pharmaceutical industries .
  • CGTases have both starch-degrading activity and transglycosylation activity. Although some CGTases produce ⁇ - cyclodextrins and some CGTases produce mainly respective ⁇ - cyclodextrins or ⁇ -cyclodextrins, CGTases usually form a mixture of ⁇ -, ⁇ - and ⁇ -cyclodextrins. Selective precipitation steps with organic solvents may be used for the isolation of separate ⁇ -, ⁇ - and ⁇ -cyclodextrins. To avoid expensive and environmentally harmful procedures, the availability of CGTases capable of producing an increased ratio of one particular type of cyclodextrin is desirable.
  • CGTases are functionally related to ⁇ -amylases. CGTases and ⁇ -amylases both degrade starch by hydrolysis of the ⁇ -(l,4)- glycosidic bonds, but produce virtually exclusively cyclic and linear products, respectively.
  • CGTase family possess a high overall amino acid sequence identity, more than 60 % of CGTases. Further, relatively to ⁇ -amylases CGTases share about 30% amino acid sequence identity.
  • CGTases have the specific characteristics of the CGTase of the present invention, in particular the ability to mainly producing ⁇ -CD.
  • CGTases from different bacterial sources including CGTases obtained from Bacillus , Brevibacterium, Clostridium ,
  • EP 614971 describes a Brevibacterium CGTase, Haeckel & Bahl [Haeckel K, Bahl H; FEMS Microbiol. Lett. 1989 60 333-338] describe Clo ⁇ tridium thermosulfurogene ⁇ CGTase, Podkovyrov & Zeiku ⁇ [Podkovyrov S M, Zeiku ⁇ J G; J. Bacteriol. 1992 174 5400- 5405] describe a Clo ⁇ tridium thermohydro ⁇ ulfuricum CGTase, JP 7000183 describes a Corynebacterium CGTase, Binder et al .
  • the present inventors have surprisingly identified a
  • Thermoalcalibacter bogoriae which belong to the Clo ⁇ tridium /Bacillu ⁇ subphyllum.
  • the CGTase of the invention has been thoroughly characterized and shown that it is producing mainly ⁇ -cyclodextrin (relative to ⁇ and ⁇ - cyclodextrin) .
  • the invention relates to an isolated CGTase characterized by producing at least 75% ⁇ - cyclodextrin (relative to ⁇ and ⁇ - cyclodextrin) after 2 hours of incubation with amylopectin at 65°C, pH 8.0.
  • at least 75% ⁇ -cyclodextrin calculated on the basis of the total amount of cyclodextrin, i.e. ⁇ -, ⁇ - and ⁇ -cyclodextrin.
  • CGTases and CGTase variants either produce a minor fraction of ⁇ -cyclodextrin relatively to ⁇ or ⁇ -CD (WO 96/33267) , or when they are able to produce relatively high amounts of ⁇ -cyclodextrin they are not thermostable (i.e. not able to exhibit any substantial activity at 65°C) (Kitahata, S. and Okada, S. 1982. Comparison of
  • the present invention relates to an isolated extracellular CGTase obtained from a strain of
  • the invention in a third aspect relates to a method of producing a CGTase of the invention homologously, the method comprising culturing a strain of Thermoalcalibacter sp. under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
  • the invention relates to an enzyme or an enzyme composition and the use of such an enzyme or enzyme composition for various industrial applications.
  • the invention also relates to an isolated DNA sequence encoding an enzyme exhibiting CGTAse activity comprising the partial sequence shown in SEQ ID NO. 13; an expression vector comprising said CGTase encoding sequence comprising the sequence shown in SEQ ID No. 13; a host cell into which has been introduced an expression vector of the invention which cell expresses the CGTase encoded by the DNA sequence comprising the DNA sequence shown in SEQ ID No. 13.
  • Figure 1 SDS-PAGE of variaos purification steps.
  • Figure 1 shows electrophoretic separation of the proteins of
  • FIG. 2 shows the pH optimum of CGTase from
  • Thermoalcalibacter bogoriae 15 Thermoalcalibacter bogoriae .
  • pH optimum universal buffer (Britton & Robinson) pH 4.0-11.0 containing 0.5% (wt/vol) soluble starch was used at 65°C and 30 minutes incubation time.
  • the hydrolysis activity (J) or the cyclization activity (C) exhibited by the CGTase was determined.
  • Thermoalcalibacter bogoriae Incubation was done for 30 minutes 25 in 100 mM sodium phosphate buffer pH 8.0 containing 0.5% (wt/vol) soluble starch. 100% residual activity corresponds to 0.11 U/ml activity measured as conversion to oligosaccharides.
  • Figure 4 shows analysis of hydrolysis products by HPLC after incubation of CGTase from Thermoalcalibacter bogoriae with 30 soluble starch (A), and amylopectin (B) , at pH 9.0 for up to 16 hours.
  • Prepurified CGTase was incubated with various substrates pH 8.0 and 65°C for up to 16 hours.
  • Figure 5 shows the ratio of cyclodextrins produced by prepurified CGTase action.
  • a cloned DNA sequence refers to a DNA sequence cloned by standard cloning procedure used in genetic engineering to relocate a segment of DNA from its natural location to a different site where it will be reproduced. The cloning process involves excision and isolation of the desired DNA segment, alternatively its manufacture by PCR amplification, insertion of the piece of DNA into the vector molecule and incorporation of the recombinant vector into a cell where multiple copies or clones of the DNA segment will be replicated.
  • the "cloned DNA sequence” of the invention may alternatively be termed “DNA construct” or “isolated DNA sequence” .
  • the term “obtained from” as used herein in connection with a specific microbial source means that the enzyme is produced by the specific source, or by a cell in which a gene from the source have been inserted.
  • an isolated polypeptide As defined herein the term, "an isolated polypeptide” or “isolated CGTase”, as used about the CGTase of the invention, is a CGTase or CGTase part which is essentially free of other non-CGTase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
  • isolated polypeptide may alternatively be termed “purified polypeptide”.
  • homologous impurities means any impurity (e.g. another polypeptide than the enzyme of the invention) which originate from the homologous cell where the enzyme of the invention is originally obtained from.
  • the homologous cell may e . g . be a strain of Thermoalcalibacter bogoriae .
  • CGTase Cyclodextrin- glycosyltransferase. Cyclodextrin glucanotransferase degrades starch to cyclodextrins by formation of a 1,4-alpha-D- glucosidic bond.
  • amylolytic In the present context, the term “amylolytic” or “amylolytic activity” is intended to indicate that the enzyme in question has a starch-degrading capability.
  • Specific examples of enzymes having amylolytic activity i.e. amylolytic enzymes, includes ⁇ -amylases, pullulanases, neo-pullulanases, iso- amylases, beta-amylases, CTGases, maltogenases as well as G-4 and G-6 amylases.
  • Moderate ther o alkaliphile relates to a cell which is capable of surviving at relatively high temperatures, i.e. at a temperature above 55°C such as above 60°C or 65°C, and at relatively high pH levels, above 8.5 such as above 9 or 10.
  • extracellular refers to an enzyme which is exported out of the cell producing the enzyme, i.e. it is secreted by or diffused out of the cell.
  • an enzyme will generally comprise a signal-peptide to guide the secretion (i.e. exporting out of the cell) of the enzyme.
  • alignment used herein in connection with a alignment of a number of DNA and/or amino acid sequences means that the sequences of interest is aligned in order to identify mutual/common sequences of homology/identity between the sequences of interest. This procedure is used to identify common "conserved regions" between sequences of interest.
  • An alignment may suitably be determined by means of computer programs known in the art, such as ClusterW or PILEUP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443- 453) .
  • conserved region used herein in connection with a “conserved region” between DNA and/or amino acid sequences of interest means a mutual common sequence region of the sequences of interest, wherein there is a relatively high degree of se- quence identity between the sequences of interest.
  • a conserved sequence is preferably at least 10 base pairs (bp)/3 amino acids(a.a), more preferably at least 20 bp/7 a. a., and even more preferably at least 30 bp/10 a. a..
  • GAP Program Manual for the
  • the degree of DNA sequence identity within the conserved region is preferably of at least 80%, more preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%.
  • primer used herein especially in connection with a PCR reaction is an oligonucleotide (especially a "PCR-primer”) defined and constructed according to general standard specification known in the art ("PCR A practical approach” IRL Press, (1991)).
  • a primer directed to a sequence means that the primer (preferably to be used in a PCR reaction) is constructed so it exhibits at least 80% degree of sequence identity to the sequence part of interest, more preferably at least 90% degree of sequence identity to the sequence part of interest, which said primer consequently is "directed to” .
  • the primer is designed in order to specifically anneal at the region at a given temperature it is directed towards. Especially identity at the 3' end of the primer is essential for the function of the polymerase, i.e.
  • expression vector denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • the expression vector of the invention may be any expression vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which the vector it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the term "recombinant expressed” or “recombinantly expressed” used herein in connection with expression of a polypeptide or protein is defined according to the standard definition in the art. Reco binantly expression of a protein is generally performed by using an expression vector as described immediately above.
  • isolated when applied to a DNA, denotes that the DNA has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated DNA are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 1 and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985).
  • an isolated polynucleo- tide may alternatively be termed “a cloned polynucleotide” .
  • isolated indicates that the protein is found in a condition other than its native environment.
  • the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities” (see below)). It is preferred to provide the protein in a greater than 40% pure form, more preferably greater than 60% pure form.
  • the protein in a highly purified form, i.e., greater than 80% pure, more pref- erably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
  • isolated protein/polypeptide may alternatively be termed “purified protein/polypeptide” .
  • partial DNA sequence denotes a partial DNA sequence which is comprised in a longer DNA sequence, wherein said longer DNA sequence contains sufficient information to encode a polypeptide having the activity of interest.
  • partial polypeptide sequence denotes a partial polypeptide sequence which is comprised in a longer polypeptide sequence, wherein said longer polypeptide sequence is having the activity of interest.
  • homologous impurities means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originate from the homologous cell where the polypeptide of the invention is originally obtained from.
  • the term "obtained from” as used herein in connection with a specific microbial source means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell in which a gene from the source have been inserted.
  • operably linked when referring to DNA segments, denotes that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator
  • the enzyme was incubated with various substrates (e.g. amylopectin) at 65°C and pH 8.0 for 1, 2 and up to 16 hour.
  • substrates e.g. amylopectin
  • the substrate specificity was determined qualitatively measuring the cyclization activity and the ratio between the produced CDs was elucidated from a quantitative HPLC analysis.
  • the CGTase of the invention is capable of producing minor fractions of ⁇ or ⁇ - cyclodextrin.
  • the CGTase of the invention is preferably one which has a molecular mass of 67 ⁇ 10 kD (i.e. 57-
  • the CGTase of the invention is preferably one which has a temperature optimum of 65 ⁇ 10 °c
  • the CGTase of the invention or a DNA sequence encoding the CGTase of the invention may be obtained from bacteria 35 corresponding to the Thermoalcalibacter line within the Clostridium/Bacillu ⁇ subphyllum in particular a strain of Thermoalcalibacter bogoriae as described below:
  • Charateristic of Thermoalcalibacter bogoriae Cells are rod-shaped, 0.3-0.5 ⁇ m thick and 3-5 ⁇ m long. Colonies are 3-5 mm in diameter, pale-whitish, lense-shaped. Obligately anaerobic. Temperature range for growth from 30°C to
  • Thermoalcalibacter bogoriae represents a new line within the Clo ⁇ tridium /Bacillu ⁇ subphyllum.
  • the 16 rRNA sequencing analysis was done at Deutche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) .
  • the CGTase of the present invention may be produced by cultivation of a homologous strain e . g . the above mentioned deposited strain in a suitable medium resulting in conditions permitting the production of the enzyme.
  • the medium used to culture the strain may be any conventional medium suitable for growing the cells in question.
  • the secreted, into the culture medium, CGTase may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chro- matographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • a DNA sequence encoding an CGTase of the present invention can be cloned from a strain of Thermoalcalibacter bogoriae .
  • DNA sequence may be cloned by purifying the enzyme (e.g. as described in a working example herein (vide infra) ) , sequencing the amino acid sequence, and preparing a suitable probe or PCR primers based on this amino acid sequence.
  • DNA sequence the invention may also be cloned by any general method involving
  • Example 3 and 4 a partial DNA sequence (see SEQ ID No. 13 comprising SEQ ID No. 10) was isolated, cloned and sequenced. First 3 conserved regions of known CGTases were identified by aligning an number of known CGTases available in the public domain on the SWISSPROT database. From the knowledge to these conserved regions 3 primers were designed.
  • PCR amplification was then carried out on purified genomic DNA from T. bogoriae DSM No. 9380 providing a PCR product of 1.15 kb which was sequenced.
  • Said partial CGTase encoding DNA sequence is shown in SEQ ID NO. 10.
  • This partial sequence shown in SEQ ID No. 10 was then used as the starting point for determining a further part of the CGTase sequence.
  • SEQ ID No. 10 was extended to give the sequence shown in SEQ ID No. 13 by the use of Inverse PCR (See M.J. MCPherson et al. ("PCR A practical approach" Information Press Ltd. , Oxford England) .
  • a full length DNA sequence encoding the entire CGTase of the invention can easily be cloned by a person skilled in the art.
  • the entire gene may be cloned by PCR amplification procedures into suitable expression vectors, e.g. plasmids derived from pUBHO (Gryczan, et al. (1978), J. Bacteriol., 134,318-329), pE194 (Horinouchi et al. (1982), J. Bacteriol., 150, 815-825) or pC194 (Horinouchi et al. (1982), J. Bacteriol., 150, 804-814 for use in Bacillu ⁇ species.
  • suitable expression vectors e.g. plasmids derived from pUBHO (Gryczan, et al. (1978), J. Bacteriol., 134,318-329), pE194 (Horinouchi et al. (1982), J. Bacterio
  • an analogous DNA sequence is obtainable from other bacteria, such as a strain of the following genera: Bacillu ⁇ , Brevibacterium , Clo ⁇ tridium, Corynebacterium, JQejbsiella, Micro- coccu ⁇ , ThermoanaeroJacter and ThermoanaeroJbacteriujTi, such as the species mentioned above in the "Previously characterized CGTases" section.
  • DNA construct which comprises a DNA sequence, which DNA sequence comprises a) a CGTase encoding DNA sequence comprising the partial DNA sequence shown in SEQ ID No. 13, or b) an analogue of the DNA sequence defined in a) , which i) is at least 70% homologous with the DNA sequence defined in a) comprising the partial sequence shown in SEQ ID No. 13, or ii) hybridizes with the same oligonucleotide probe as the DNA sequence defined in a) comprising the partial sequence shown SEQ ID No.
  • iii) encodes a polypeptide which is at least 70% homologous with the polypeptide encoded by the DNA sequence defined in a) comprising the partial DNA sequence shown in SEQ ID NO. 13, or iv) encodes a polypeptide which is immunologically reactive with an antibody raised against the purified CGTase derived from T. bogoriae DSM no. 9380 encoded by the DNA sequence defined in a) , comprising the partial sequence shown in SEQ ID NO. 13.
  • ID No. 13 is intended to indicate an DNA sequence encoding polypeptides, which has the properties i)-iv) above.
  • the analogous DNA sequence is intended to indicate an DNA sequence encoding polypeptides, which has the properties i)-iv) above.
  • - is constructed on the basis of the defined DNA sequence comprising the partial sequence shown in SEQ ID No. 13, e.g. by introduction of nucleotide substitutions, which do not give rise to another amino acid sequence of the CGTase encoded by a DNA sequence comprising the partial sequence shown in SEQ ID NO. 13, but which correspond to the codon usage of the host organism intended for production of the enzyme (s) , or by introduction of nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a mutant with different properties than the native enzymes.
  • analogous DNA sequence may be a subsequence of the partial DNA sequence shown in SEQ ID No. 13.
  • the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology 48, p. 443-
  • the coding region of the DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%,
  • an analogous DNA sequence is highly homologous to
  • the DNA sequence such as at least 70% homologous to the above defined DNA sequence comprising the partial sequence shown in SEQ ID NO. 13, such as at least 80%, at least 85%, at least 90%, at least 95% or even at least 99% homologous to the defined DNA sequence comprising the partial sequence shown in SEQ ID No. 13.
  • the degree of homology referred to in iii) above is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known in the art.
  • the polypeptide encoded by an analogous DNA sequence exhibits a degree of homology of at least 70%, such as at least 80%, 85%, 90%, 95%, 99% with the enzyme encoded by the above defined DNA construct comprising a DNA sequence comprising the partial DNA sequence shown in SEQ ID No. 13.
  • the immunological reactivity may be determined by the method described in the Materials and Methods section below.
  • the DNA sequence defined above comprising the partial DNA sequence shown in SEQ ID No. 13 may subsequently be inserted into a recombinant expression vector.
  • This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding the CGTase of the invention should be operably connected to a suitable promoter and terminator sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Host cells which can be transformed with the DNA sequence encoding the CGTase of the invention, may be either eukaryotic or prokaryotic.
  • Suitable prokaryotic host cells are bacterial cells.
  • Examples of such bacterial host cells which, on cultivation, are capable of producing the novel enzyme of the invention are grampositive bacteria such as strains of Bacillu ⁇ , such as strains of B . ⁇ ubtili ⁇ , B . licheniformi ⁇ , B . lentu ⁇ , B . brevi ⁇ , B . ⁇ tearothermophilu ⁇ , B . alkalophilu ⁇ , B . amyloliquefacien ⁇ , B . coagulan , B . circulan ⁇ , B . lautu ⁇ , B . megaterium or B .
  • the transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al.,(1989), supra).
  • the polypeptide When expressing the CGTase in bacteria such as E. coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies) , or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.
  • sonication or osmotic shock to release the contents of the periplasmic space and recovering the polypeptide.
  • Suitable eukaryotic cells are, in particular fungal cells, such as a yeast or filamentous fungal cells.
  • yeasts cells include cells of Saccharomyce ⁇ spp. , in particular strains of Saccharomyce ⁇ cerevi ⁇ iae, Saccharomyce ⁇ kluyveri , Sacchromyce ⁇ uvarum, or Schizo ⁇ accharomyce ⁇ spp., such as Schizo ⁇ accharomyce ⁇ pombe .
  • Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in US 4,599,311, US 4,931,373, US 4,870,008, 5,037,743, and US 4,845,075, all of which are hereby incorporated by reference.
  • Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • a preferred vector for use in yeast is the POT1 vector disclosed in
  • inventions may be preceded by a signal sequence and optionally a leader sequence , e.g. as described above. Further examples of
  • suitable yeast cells are strains of Kluyveromyce ⁇ spp. , such as K. lacti ⁇ , or Han ⁇ enula spp., e.g. H. polymorpha , or Pichia spp., e.g. P. pastor is , Yarrowia spp., such as Yarrowia lipolytica (cf. Gleeson et al. , (1986), J. Gen. Microbiol. 132, p. 3459-3465; US 4,882,279) .
  • Examples of other fungal cells are cells of filamentous fungi, e.g. A ⁇ pergillu ⁇ spp., Neurospora spp., Fu ⁇ arium spp. or Trichoderma spp., in particular strains of A . oryzae , A. nidulan ⁇ or A. niger.
  • a ⁇ pergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023 and EP 184
  • F. oxy ⁇ porum may, for instance, be carried out as described by Malardier et al., (1989), Gene 78, p. 147-156.
  • a filamentous fungus When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveniently
  • the present invention relates to a
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) .
  • the expressed CGTase produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
  • a salt e.g. ammonium sulphate
  • the invention relates to a CGTase, which a) is encoded by a DNA construct of the invention, b) produced by the method of the invention, and/or c) is immunologically reactive with an antibody raised against a purified CGTase encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 13 derived from T. bogoriae DMS No. 9380.
  • the present invention relates to an enzyme composition, which comprises an homologously or heterologously expressed CGTase as described above.
  • the enzyme composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the enzyme composition may be in the form of a granulate or a microgranulate (US 4106991, US
  • the enzyme to be included in the composition may be stabilized in accordance with methods known in the art.
  • the dosage of the enzyme composi- tion of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.
  • the enzyme and/or the enzyme composition according to the invention may be useful for at least one of the following purposes.
  • the CGTase of the invention find application in processes for the manufacture of cyclodextrins for various industrial applications, particularly in the food, cosmetic, chemical, agrochemical and pharmaceutical industries. Therefore, in another aspect, the invention relates to the use, of a CGTase of the invention, in a process for the manufacture of cyclodextrins, in particular ⁇ -cyclodextrins.
  • the CGTase of the invention may also be used in a process for the manufacture of linear oligosaccharides, in particular linear oligosaccharides of 2 to 12 glucose units, preferably linear oligosaccharides of 2 to 9 glucose units.
  • the CGTase of the invention may be used for in situ generation of cyclodextrins.
  • the CGTase of the invention may be added to a substrate containing medium in which the enzyme is capable of forming the desired cyclodextrins.
  • This application is particularly well suited for being implemented in methods of producing baked products, in methods for stabilizing chemical products during their manufacture, and in detergent compositions. Certain cyclodextrins are known to improve the quality of baked products.
  • the CGTase of the invention therefore also may be used for implementation into bread-improving additives, e.g. dough compositions, dough additives, dough conditioners, pre- mixes, and similar preparations conventionally used for adding to the flour and/or the dough during processes for making bread or other baked products.
  • the invention relates to a bread- improving and/or a dough-improving composition, and further to the use of a CGTase of the invention in such compositions, and to a dough or baked product comprising a bread-improving and/or a dough-improving composition of the invention.
  • the terms "bread-improving composition” and “dough-improving composition” are intended to indicate compositions which, in addition to the enzyme component, may comprise other substances conventionally used in baking to improve the properties of dough and/or baked products. Examples of such components are given below.
  • the term "improved properties” is intended to indicate any property which may be improved by the action of a CGTase enzyme.
  • CGTase results in an increased volume and an improved crumb structure and softness of the baked product, as well as an increased strength, sta- bility and reduced stickiness and thereby improved machinability of the dough.
  • the effect on the dough has been found to be particularly good when a poor quality flour has been used.
  • the improved machinability is of particular importance in connection with dough which is to be processed industrially.
  • the improved properties are evaluated by comparison with dough and/or baked products prepared without addition of CGTase in accordance with the present invention.
  • the bread- and/or dough-improving composition of the invention may further comprise another enzyme.
  • Other en- zymes are a cellulase, a hemicellulase, a pentosanase (useful for the partial hydrolysis of pentosans which increases the extensibility of the dough) , a glucose oxidase (useful for strengthening the dough) , a lipase (useful for the modification of lipids present in the dough or dough constituents so as to soften the dough) , a peroxidase (useful for improving the dough consistency) , a protease (useful for gluten weakening, in particular when using hard wheat flour), a peptidase and/or an amylase, e.g. ⁇ -amylase (useful for providing sugars fermentable by yeast) .
  • the dough-improving and/or bread-improving composition may comprise a conventionally used baking agent, e.g. one or more of the following constituents:
  • a milk powder (providing crust colour) , gluten (to improve the gas retention power of weak flours) , an emulsifier (to i - prove dough extensibility and to some extent the consistency of the resulting bread) , granulated fat (for dough softening and consistency of bread) , an oxidant (added to strengthen the gluten structure; e.g. ascorbic acid, potassium bromate, azodicarbona- mide, calcium peroxide, potassium iodate or ammonium persulfate) , an amino acid (e.g. cysteine) , a sugar, and salt (e.g.
  • e ulsifiers are mono- and diglycerides, diacetyl tartaric acid esters of mono- and diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monogly- cerides, polyoxyethylene stearates, phospholipids, lecithin and lysolecithin.
  • baked product is intended to include any product prepared from dough or batter, either of a soft or a crisp character.
  • baked products whether of a white, light or dark type, which may advantageously be produced by the present invention are bread (in particular white, wholemeal, rye bread or mixtures) , typically in the form of loaves or rolls, French baguette-type bread, bagels, pita bread, tacos, tortillas, cakes, pan-cakes, pannetone, biscuits, pizza, crisp bread, steamed bread and the like.
  • the dough of the invention may be of any of the types discussed above, and may be fresh, par-baked or frozen.
  • the dough of the invention is normally a leavened dough or batter, or a dough or batter to be subjected to leavening.
  • the dough or batter may be leavened in various ways such as by chemical leavening agents, sour culture/dough, and/or yeast, but it is preferred to leaven the dough by adding a suitable yeast culture such as a culture of Saccharomyce ⁇ cerevi ⁇ iae (baker's yeast) . Any of the commercially available S . cereviciae strains may be employed. It is further contemplated that the invention may be advantageously used for the preparation of pasta dough, preferably prepared from durum flour or a flour of comparable quality.
  • the dough may be prepared by use of conventional techniques and the CGTase used in a manner similar to that described above. It is believed that when used in the preparation of pasta the CGTase results in a strengthening of the gluten structure and thus a reduction in the dough stickiness and an increased dough strength.
  • Cyclodextrins have an inclusion ability useful for stabilization, solubilization, etc.
  • cyclodextrins can make oxidizing and photolytic substances stable, volatile substances non-volatile, poorly-soluble substances soluble, and odoriferous substances odorless, etc. and thus are useful to encapsulate perfumes, vitamins, dyes, pharmaceuticals, pesticides and fungicides.
  • Cyclodextrins are also capable of binding lipophilic substances such as cholesterol, to remove them from egg yolk, butter, etc.
  • Cyclodextrins also find utilization in products and processes relating to plastics and rubber, where they have been used for different purposes in plastic laminates, films, membranes, etc.
  • cyclodextrins have been used for the manufacture of biodegradable plastics.
  • Deposited organisms Thermoalcalibacter bogoriae DSM No. 9380 comprising the CGTase of the invention.
  • Electrophoresis and molecular mass determination According to Laemmli (Laemmli et al.) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 11.5% polyacrylamide gels in a Mini Protean II electrophoresis system (Bio-Rad) at constant current of 24 mA and voltage high. Proteins were silver stained according to Blum et al (Blum et al) . In order to determine the molecular weight, a broad range molecular weight protein mixture (Bio-Rad) was used as standard.
  • Amylolytic protein bands were detected by incubating the gel for 10 min at 65°C in 100 mM sodium phosphate buffer pH 8.0 for CGTase, supplemented with 0.5% soluble starch (Merck) . Protein bands with amylolytic activity were visualized by staining the gel with a KJ-J 2 solution (3 g KJ, 2 g J 2 per liter aqua dest.), resulting in white activity bands within a brownish background.
  • Amylolytic assay The enzyme assay routinely used was carried out with enzyme solution using the respective prepurified enzyme and substrate solutions at 0.5% soluble starch (Merck, Darmstadt,
  • enzyme activity was calculated using a standard calibartion curve with 0-1% (wt/vol) maltose.
  • U amylolytic activity was defined as the amount of 1 ⁇ mol reducing sugars liberated by the enzyme per minute under standard conditions (pH 9.0; 65 °C) .
  • CGTase cyclodextrine glycosyltransferase assay: For determination of CGTase activity a specific assay for production of ⁇ -CD was used (Vikmon, 1982), measuring the cyclization activity. Enzyme solution containing the prepurified CGTase was 20 used with substrate solutions containing 0.5% starch (Merck) or 0.2% amylose or 0.2% amylopectine (each wt/vol) in 100 mM sodium phosphate buffer pH 8.0 to give a final assay volume of 0.1 ml.
  • CGTase activity catalyzes the formation of 1 ⁇ mol ⁇ -CD per minute under standard conditions (65°C, pH 8.0). Derived from HPLC analysis, also reducing sugars were detected as 30 products of CGTase action (see below) . Thus, CGTase hydrolysis activity could be elucidated from the amount of reducing sugars.
  • Protein concentrations were determined by the Lowry method. Microassays were performed and bovine serum 35 albumine was used as standard protein. Effect of pH and temperature: To study the influence of pH and temperature on amylase and CGTase activity, the prepurified enzyme solution was used. 10ml of the enzyme solution were mixed with 90 ml of a 0.5% (wt/vol) substrate solution (soluble starch; Merck) in 120 mM universal buffer (Britton & Robinsson) with pH 4.0 to 11.0. The changes in pH due to the mixture of the enzyme solution and substrate solution were measured. After a preincubation for 30 minutes of this mixture on ice, the enzyme assay was performed at 65°C for 30 minutes. The developed reducing sugars were plotted against the respective pH value.
  • Substrate specificity In order to determine the substrate specificity of the ⁇ -amylase or CGTase, the enzyme was incubated with substrate solution (each wt/vol) containing soluble starch (Merck) (0.5%), amylopectin (0.2%), amylose (0.2%), pullulan (0.2%), maltotriose, maltotetraose and maltopentose (each 0.1%). The assay was incubated for 30 min under standard conditions (65°C, pH 8.0). The enzyme activity was determined by measuring the amount of cyclodextrins produced by the enzymes action.
  • substrate solution each wt/vol
  • soluble starch containing soluble starch (Merck) (0.5%), amylopectin (0.2%), amylose (0.2%), pullulan (0.2%), maltotriose, maltotetraose and maltopentose (each 0.1%).
  • the assay was incubated for 30 min
  • hydrolysis products The hydrolysis pattern of amylase and CGTase action on different substrates were analyzed by high-performance liquid chromatography (HPLC) (Knauer GmbH, Berlin, Germany) with an Aminex-HPX-42 A column (300 by 7.8 mm; Bio-Rad, Hercules, Calif.). One part of the prepurified respective enzyme was incubated together with 9 parts of substrate solution pH 8.0, at 65°C for up to 16 hours. After incubation the samples were kept frozen at -20°C until they were analyzed.
  • HPLC high-performance liquid chromatography
  • Hybridization of Southern blots on nylon filters (Hybond-N, Amersham) with 32 P-labelled PCR probe is carried out following methods described by Sambrook et al., (1989), supra.
  • the membrane is placed in a plastic bag and pre-hybridized in 50% (v/v) formamide, 6xSSC, 0.05xBLOTTO, 1 mM EDTA at 42 °C for 1-2 hours.
  • the membrane is hybridized with the radiolabelled and denaturated DNA probe in 50% (v/v) formamide, 6xSSC, 0.5% (w/v) SDS, 1 mM EDTA at 42°C (which corresponds to a temperature of 68°C without formamide in the hybridization solution) , overnight.
  • the membrane is washed first in 2xSSC, 0.5% (w/v) SDS and then with O.lxSSC, 0.5% (w/v) SDS at 50°C. Then, the membrane is wrapped in Saran Wrap and exposed to X-ray Film at -70° for the requested period of time.
  • Antibodies to be used in determining immunological cross-reactivity may be prepared by use of a purified CGTase. More specifically, antiserum against the CGTase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) .
  • Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ((NH_ ⁇ ) 2 SO 4 ) , followed by dialysis and ion exchange chromatography, e.g. on DEAE-Sephadex.
  • Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (O. Ouchterlony in: Handbook of Experimental Immunology, (D.M. Weir, Ed.), Blackwell Scientific Publications, (1967) , p. 655-706) , by crossed immunoelectrophoresis (N. Axelsen et al., supra, Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2) .
  • Thermoalcalibacter bogoriae DSM No. 9380 comprises the CGTase of the invention was cultivated under anaerobic conditions in the following medium: (NH 4 ) 2 S ⁇ 4 , 1.0; NH C1, 0.4; Na 2 S 2 0 4 , 0.1; K 2 HP0 , 0.5; MgS0 4 , 0.1; CaCl 2 , 0.05; NaCl, 10.0; Trypton, 0.25; yeast extract, 0.25; FeCl 3 , 0.01; Resazurin, 0.001; NaHC0 3 , 2.2; Na 2 C0 3 , 2.2; Cystein, 0.5, Starch, 5.0 , all concentrations in grams per litre.
  • Large scale cultivation was done in a 19 liter fermentor (Bioengineering, Wald, Switzerland) under pH regulation at pH 9.0 and 50°C, the culture was stirred at 300 rpm and flushed with N 2 at 10 liters/hour. Inoculation of the fermentor was done with one liter of a preculture, grown for 8 hours at 50°C in a 2 liter flask without shaking.
  • the concentrated supernatant was applied to a PD-10 ion exchange column (Pharmacia) and eluated with 100 mM sodium phosphate buffer pH 9.0.
  • the eluate containing amylolytic activity was collected and concentrated 10-fold in an Amicon chamber (10 kD filter Amicon) .
  • Samples of this solution were applied to a Q- Sepharose anion exchange chromatography column (15 x 2.5 cm) (Pharmacia) preequilibrated with 100 mM sodium phosphate buffer pH 9.0. The column was washed with 90 ml of equilibrating buffer.
  • the enzyme solution was eluated with equilibration buffer containing 1 M NaCl, using a gradient of NaCl from 0 to 300 mM and 300 to 500 mM at a flow rate of 0.2 ml/min. Fractions were collected (2 ml per tube) and their amylolytic activity was determined as above in the "Materials and Methods" section above. The active fractions were collected, assembled and subsequently 10-fold concentrated in a Amicon chamber. Samples of this prepurified amylase were added to a Superose 75 gel filtration column (Pharmacia) preequilibrated with 50 mM sodium phosphate buffer pH 9.0.
  • the enzyme was eluated with the equilibration buffer at a flow rate of 0.1 ml/min.
  • the fractions were collected (1 ml/tube) and the active fractions were pooled and subsequently concentrated in an Amicon chamber with a 10 kDa membrane.
  • the specific activity of the amylase/CGTase in a 70-fold concentrated culture supernatant after cultivation was determined to 0.096 U/mg. Due to the production of H 2 S during fermentation, as previously described, a purification using a PD-10 ion exchange column was necessary in order to remove H 2 S, sulfides and other activity disturbing agents. After this treatment, the amount of detectable activity was raised to 0.48 U/mg. This effect was regardless to the used method for detection of reducing sugars (data not shown) .
  • the concentrated culture supernatant revealed three activity bands in an SDS-PAGE electrophoresis gel ( Figure 1, lane 2) by activity staining.
  • the lowest activity band with an apparent molecular weight of 57 ⁇ 3 kDa was shown to exhibit ⁇ -amylase activity.
  • Samples of the 10- fold concentrated PD-10 eluate were applied to a Q-Sepharose anion exchange chromatography column (Pharmacia, Sweden; 25 x 200 mm) and the column was run at 1.0 ml/min with the equilibration buffer (100 mM sodium phosphate pH 8.0) using the Bio-Rad Econo System.
  • the molecular mass of the CGTase was determined by activity stained SDS-PAGE gel and revealed to be 67 ⁇ 2 kDa ( Figure 1, lane 6+7) .
  • the substrate specificity was determined qualitativelly measuring the cyclization activity and the ratio between the produced CDs was elucidated from a quantitative HPLC analysis.
  • Both starch (Merck) and amylopectine (Figure 4) were hydrolyzed well by the CGTase of the present invention.
  • ⁇ -CD was detected as major product, as well as after 120 min incubation ( Figure 4 A) ; the same effect could be observed with amylopectin after 120 min incubation ( Figure 4 B) .
  • PI was designed from the knowledge to the identifyed conserved region: TDVIYQI (SEQ ID No. 1) .
  • PI is an N-terminal primer.
  • PI was designed to cover some differences in the CGTase sequence. This was done by incorporating either deoxy-inosine or degenerated bases. PI is read down-stream relative to the protein sequence.
  • P2 was designed from the knowledge to the identifyed con- served region: RWINNDV (SEQ ID NO. 2) . P2 is read up-strearns relative to the protein sequence. Also the P2 primer was degenerated to cover some degree of sequence diversity.
  • P3 was designed from the knowledge to the identifyed conserved region: TSYHGYWA (SEQ ID NO. 3) . P3 is like PI read down-stream relative to the protein sequence.
  • P4-P6 was designed on the basis of primes P1-P3.
  • PI 5 'ACNGAYGTGATITAYCARAT » 3 SEQ ID NO. 4
  • P2 5 'ACRTCRTTRTTDATCCANCG ' 3 SEQ ID NO. 5
  • P3 5 • CATCNTAYCAYGGNTAYTGGGC ' 3 SEQ ID NO. 6
  • PCR was carried out on genomic DNA from Thermoalcalibacter bogoriae purified as described in Example 4 (below) by using either the primer set Pi and P2 or the primer set P2 and P3.
  • Ampli-Taq DNA polymerase (Perkin Elmer inc.) was used under buffer conditions according to the manufactures instructions. 2 minutes of denaturation at 94°C, 5 cycles with an annealing temperature at 35°C and 2 minutes elongation time was followed by 25 cycles with an annealing temperature at 50°C and 2 minutes elongation.
  • SEQ ID NO. 10 shows the DNA sequence obtained from sequencing a PCR fragment of the internal of the Thermoalcalibacter bogoriae CGTase.
  • Example 3 the DNA sequence of a 1.15 kb fragment PCR ampli- fied from the T . bogoriae DSM No. 9380 CGTase encoding gene was determined. This sequence was used as the starting point for the determination of a further part of the CGTase gene sequence by an inverse PCR approach. Thus, the following two oligonu- cleotide primers, corresponding to a region near each end of the sequenced 1.15 kb DNA sequence, and reading away from each other, were prepared:
  • Chromosomal DNA was extracted from Thermoalcalibacter bogoriae cells (lysozyme treatment/phenol extractions) .
  • this DNA was digested with restriction enzyme Pstl, ligated after phenol extraction, and a portion of this sample subsequently digested with Bglll.
  • This material was used as template in a PCR amplification with primer 101304 and Primer 101305 (annealing temperature 49°C) . No amplification products were observed.
  • the ligation mixture was subsequently digested with en- zymes Pstl + BamHI + EcoRI + Hindlll + Nsil + Sail, and this digested sample ligated. After ligation, a Bglll digestion was performed. This material was used as template in the PCR reaction. This time, fragments of 1 kb, 1.4 kb and 2 kb was obtained. These fragments were gel purified and used as templates in new PCR reactions. Again, 1 kb and 1.4 kb fragments were obtained. These fragments were gel purified, and DNA sequenced, using the same primers for sequencing as was used for PCR amplification.
  • the deduced amino acid sequence SEQ ID No. 14, was compared to database sequences using the BLASTP and FASTA programs from the GCG version 9 program package. The same three sequences were identified as highest scoring, using either pro- gram.
  • the homology between SEQ ID 14 and these sequences was determined using the program gap in the GCG program package, version 8. The figures given below are percent identity.
  • Hayashi, T., T. Akiba, and K. Horokoshi. 1988 Production and purification of new maltohexaose-froming amylases from alkalophilic Bacillu ⁇ sp. H-167. Agric Biol Chem 52:443-448. Hofmann, B. E., H. Bender, and G. E. Schulze. 1989. Three- dimensional structure of cyclodextrin glycosyltransferase from Bacillu ⁇ circulan ⁇ at 3.4 A resolution. J Mol Biol 209: 793-800. Horikoshi, K.. 1991. General view of alkaliphiles and thermophiles, p. 3-14. In K. Horikoshi and W. D. Grant (ed.), Superbugs: Microorganisms in extreme environments. Springer Verlag, Berlin.
  • thermostable pullulanase from B. ⁇ taerothermophilu ⁇ G-82. Appl Biochem Biotechnol 33:193-203. Kanai, H., T. Kobayashi, R. Aono, and T. Kudo. 1995.
  • Natronococcus amylolyticu ⁇ sp. nov. a haloalkaliphilic archaeon.
  • Cyclodextrin glycosyltransferase may be the only starch-degrading enzyme in
  • thermostable pullulanase from Clo ⁇ tridium thermo ⁇ ulfurogene ⁇ EMI which hydrolyses both a-1,6 and a-1,4- glycosidic linkages.
  • Y T or C
  • N A, C, T or G
  • N A, C, T or G (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
  • MOLECULE TYPE other nucleic acid
  • FEATURE FEATURE
  • MOLECULE TYPE other nucleic acid
  • FEATURE (A) NAME/KEY: misc-feature
  • GCA ACT AAA AGA AAT GTA GAA ATT GGA TTA GCA TTT TTA CTA ACA TCA 1008

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Abstract

La présente invention concerne une nouvelle CGTase alcaline stable produisant principalement une alpha-cyclodextrine, une composition enzymatique comprenant la CGTase considérée, et enfin l'utilisation de cette enzyme et de la composition enzymatique considérée pour de nombreuses applications industrielles telles qu'un améliorant de boulangerie.
PCT/DK1997/000411 1996-09-26 1997-09-26 Enzyme a activite cyclomaltodextrine glucanotransferase (cgtase) WO1998013479A1 (fr)

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EP97941877A EP0956346A1 (fr) 1996-09-26 1997-09-26 Enzyme a activite cyclomaltodextrine glucanotransferase (cgtase)
AU43765/97A AU4376597A (en) 1996-09-26 1997-09-26 An enzyme with cyclomaltodextrin glucanotransferase (cgtase) activity

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6190883B1 (en) 1998-09-09 2001-02-20 Novo Nordisk A/S Method for the production of heterologous polypeptides in transformed yeast cells
WO2002006508A2 (fr) * 2000-07-19 2002-01-24 Novozymes A/S Cgtase et sequence d'adn codant celle-ci
WO2004054383A1 (fr) * 2002-12-13 2004-07-01 General Mills, Inc. Produits alimentaires contenant des cyclodextrines presentant des effets hypocholesterolemiques benefiques, et leur methode de fabrication
ES2215482A1 (es) * 2003-03-28 2004-10-01 Consejo Sup. De Invest. Cientificas Composicion mejoradora de masas para panaderia y pasteleria.
WO2011114251A1 (fr) * 2010-03-18 2011-09-22 Danisco A/S Produits alimentaires
USRE43135E1 (en) 2001-05-18 2012-01-24 Danisco A/S Method of improving dough and bread quality
US8889371B2 (en) 2004-07-16 2014-11-18 Dupont Nutrition Biosciences Aps Lipolytic enzyme: uses thereof in the food industry
CN105087513A (zh) * 2015-07-23 2015-11-25 中国水产科学研究院黄海水产研究所 由海洋微生物菌株Y112制备α-环糊精葡萄糖基转移酶的方法
WO2016113399A1 (fr) * 2015-01-16 2016-07-21 Novozymes A/S Procédé pour améliorer la tranchabilité de produits de boulangerie-pâtisserie
CN107254497A (zh) * 2017-06-30 2017-10-17 中国水产科学研究院黄海水产研究所 海洋微生物酶法制备α‑环糊精响应面设计方法及应用

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CN103993026B (zh) * 2014-04-15 2017-01-18 福建省农业科学院土壤肥料研究所 一种高温环糊精葡萄糖基转移酶基因及其重组表达
CN111534498B (zh) * 2020-05-28 2022-03-25 江南大学 歧化比活和aa-2g产量提高的环糊精葡萄糖基转移酶突变体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2169902A (en) * 1984-12-03 1986-07-23 Hayashibara Biochem Lab Polypeptide possessing cyclomaltodextrin glucanotransferase activity
WO1989001043A1 (fr) * 1987-07-28 1989-02-09 Genetics Institute, Inc. Procede et enzyme de preparation de cyclodextrines, en particulier des alpha-cyclodextrines
EP0687414A1 (fr) * 1994-06-17 1995-12-20 Gist-Brocades B.V. Composition d'amélioration du pain

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2169902A (en) * 1984-12-03 1986-07-23 Hayashibara Biochem Lab Polypeptide possessing cyclomaltodextrin glucanotransferase activity
WO1989001043A1 (fr) * 1987-07-28 1989-02-09 Genetics Institute, Inc. Procede et enzyme de preparation de cyclodextrines, en particulier des alpha-cyclodextrines
EP0687414A1 (fr) * 1994-06-17 1995-12-20 Gist-Brocades B.V. Composition d'amélioration du pain

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 357, DBA, Dialog Accession No. 077113, DBA Accession No. 88-07962, YU E.K.C. et al., "Specific Alpha-Cyclodextrin Production by a Novel Thermostable Cyclodextrin-Glycosyltransferase - from Bacillus Amyloliquefaciens"; & APPL. MICROBIOL. BIOTECHNOL., (28, 4-5, 377-79), *
FILE WPI, Derwent Accession No. 93-234161, AS URALS SECT BASHKIR BIOL INST et al., "Prepn. of Dough for Prodn. of Bread and Rolls - Involves Heating Mixt. of Part of Flour with Water, Adding Active Alpha-Amylase"; & SU,A,1 750 571, (30-07-92), DW9329. *
PATENT ABSTRACTS OF JAPAN, Vol. 14, No. 126; & JP,A,02 000 455 (RIKAGAKU KENKYUSHO), 5 January 1990. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6190883B1 (en) 1998-09-09 2001-02-20 Novo Nordisk A/S Method for the production of heterologous polypeptides in transformed yeast cells
WO2002006508A2 (fr) * 2000-07-19 2002-01-24 Novozymes A/S Cgtase et sequence d'adn codant celle-ci
WO2002006508A3 (fr) * 2000-07-19 2002-05-02 Novozymes As Cgtase et sequence d'adn codant celle-ci
US7700338B2 (en) 2000-07-19 2010-04-20 Novozymes A/S CGTase and DNA sequence encoding same
USRE43135E1 (en) 2001-05-18 2012-01-24 Danisco A/S Method of improving dough and bread quality
WO2004054383A1 (fr) * 2002-12-13 2004-07-01 General Mills, Inc. Produits alimentaires contenant des cyclodextrines presentant des effets hypocholesterolemiques benefiques, et leur methode de fabrication
AU2003290917B2 (en) * 2002-12-13 2009-12-24 General Mills, Inc. Food products containing cyclodextrins having beneficial hypocholesterolemic effects and method of making and communicating the benefits of such products
ES2215482A1 (es) * 2003-03-28 2004-10-01 Consejo Sup. De Invest. Cientificas Composicion mejoradora de masas para panaderia y pasteleria.
WO2004084638A1 (fr) * 2003-03-28 2004-10-07 Consejo Superior De Investigaciones Científicas Composition enzymatique permettant d'ameliorer la qualite des masses de boulangerie et de patisserie
US8889371B2 (en) 2004-07-16 2014-11-18 Dupont Nutrition Biosciences Aps Lipolytic enzyme: uses thereof in the food industry
WO2011114251A1 (fr) * 2010-03-18 2011-09-22 Danisco A/S Produits alimentaires
WO2016113399A1 (fr) * 2015-01-16 2016-07-21 Novozymes A/S Procédé pour améliorer la tranchabilité de produits de boulangerie-pâtisserie
CN107105677A (zh) * 2015-01-16 2017-08-29 诺维信公司 用来改进烘焙品的可切片性的方法
AU2016207959B2 (en) * 2015-01-16 2019-06-27 Novozymes A/S Method to improve sliceability of baked goods
CN105087513A (zh) * 2015-07-23 2015-11-25 中国水产科学研究院黄海水产研究所 由海洋微生物菌株Y112制备α-环糊精葡萄糖基转移酶的方法
CN107254497A (zh) * 2017-06-30 2017-10-17 中国水产科学研究院黄海水产研究所 海洋微生物酶法制备α‑环糊精响应面设计方法及应用

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CN1231691A (zh) 1999-10-13
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