WO2007007053A1 - Modified amylase from pseudomonas saccharophilia - Google Patents

Abstract

We describe a PS4 variant polypeptide derivable from a parent polypeptide having amylase activity selected from the group consisting of: (a) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334; (b) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 145, 146, 157, 178, 179, 223, 229, 272, 303, 307 and 334; (c) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334; and (d) a polypeptide comprising an amino acid mutation at each of positions 3, 33, 34, 70, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334; with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1 , uses of such a polypeptide as a food or feed additive, and nucleic acids encoding such.

Description

POLYPEPTIDE

Reference is made to US provisional applications serial nos. 60/485,413, 60/485,539 and 60/485,616 filed July 7, 2003. Reference is also made to international applications PCT/US2004/021723 and PCT/US2004/021739 filed July 7, 2004 and designating the US (applicant: Genencor International, Inc). Reference is also made to US utility applications serial numbers 10/886,905 and 10/866,903 all of which were also filed July 7, 2004.

Reference is also made to US provisional application serial number 60/608,919 (filed as US utility application serial number 10/887,056 on July 7, 2004 but converted to a provisional application on September 15, 2004). Reference is also made to US provisional application serial number 60/612,407 which was filed September 22, 2004.

Reference is additionally made to US application serial no. 60/485,539 filed July 7, 2003. Reference is also made to international application PCT/IB2004/002487 filed July 7, 2004 and designating the US (applicant: Danisco A/S). Reference is also made to US utility application serial number 10/886,023 filed July 7, 2004.

Reference is also made to US utility applications serial numbers 10/886,505, 10/886,527 and 10/886,504, all of which were filed July 7, 2004. Reference is also made to US utility application serial number 10/947,612 filed September 22^nd, 2004.

Reference is also made to International Patent Application serial number PCT/GB2005/002675 filed July 7, 2005 and designating the US (applicants: Danisco A/S and Genencor International, Inc, D Young & Co Attorney Reference: P020161WO). Reference is also made to US provisional application serial number 60/697,302 filed July 7^th, 2005.

The foregoing applications, and each document cited or referenced in each of the present and foregoing applications, including during the prosecution of each of the foregoing applications ("application and article cited documents"), and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the foregoing applications and articles and in any of the application and article cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or reference in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text or in any document hereby incorporated into this text, are hereby incorporated herein by reference. Documents incorporated by reference into this text or any teachings therein may be used in the practice of this invention. Documents incorporated by reference into this text are not admitted to be prior art. FIELD

This invention relates to polypeptides, specifically amylase polypeptides and nucleic acids encoding these, and their uses as non-maltogenic exoamylases in producing food products. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered exospecifity and/or altered thermostability. In particular, the polypeptides are derived from polypeptides having non-maltogenic exoamylase activity, in particular, glucan 1,4-alpha- maltotetrahydrolase (EC 3.2.1.60) activity.

BACKGROUND

Improved amylases can ameliorate problems inherent in certain processes, such as baking. Crystallisation of amylopectin takes place in starch granules days after baking, which leads to increased firmness of bread and causes bread staling. When bread stales, bread loses crumb softness and crumb moisture. As a result, crumbs become less elastic, and bread develops a leathery crust.

Enzymatic hydrolysis (by amylases, for example) of amylopectin side chains can reduce crystallization and increase anti-staling. Crystallization depends upon the length of amylopectin side chains: the longer the side chains, the greater the crystallization. Most starch granules are composed of a mixture of two polymers: amylopectin and amylose, of which about 75% is amylopectin. Amylopectin is a very large, branched molecule consisting of chains of α-D-glucopyranosyl units j oined by ( 1 -4) linkages, where the chains are attached by α-D-(l-6) linkages to form branches. Amylose is a linear chain of (1-4) linked α-D-glucopyranosyl units having few α-D-(l-6) branches.

Baking of farinaceous bread products such as white bread, bread made from bolted rye flour and wheat flour and rolls is accomplished by baking the bread dough at oven temperatures in the range of from 180 to 250°C for about 15 to 60 minutes. During the baking process a steep temperature gradient (200 → 120°C) prevails over the outer dough layers where the crust of the baked product is developed. However, due to steam, the temperature in the crumb is only about 100°C at the end of the baking process. Above temperatures of about 85°C, enzyme inactivation can take place and the enzyme will have no anti-staling properties. Only thermostable amylases, thus, are able to modify starch efficiently during baking.

Endoamylase activity can negatively affect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins. Exoamylase activity is preferred, because it accomplishes the desired modification of starch that leads to retardation of staling, with fewer of the negative effects associated with endo- amylase activity. Reduction of endoamylase activity can lead to greater exospecifity, which can reduce branched dextrins and produce a higher quality bread.

SUMMARY

We provide, according to the invention, a PS4 variant polypeptide as set out in the claims. We further provide for the use of such a PS4 variant polypeptide, including in and as food additives, food products, bakery products, improver compositions, feed products including animal feeds, etc as set out in the claims. We provide for nucleic acids which encode and which relate to PS4 variant polypeptides, as set out in the claims. Methods for producing such PS4 variant polypeptides, as well as other aspects of the invention, are also set out in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an example of a curve from a Texture Analyser.

Figure 2 shows an improved firmness effect, i.e. lower firmness, of bread treated with pSac-pMD229 versus bread treated pSac-D34 during storage time after baking. The figure shows the results of a baking trial in which firmness of bread treated with pSac- pMD229, pSac-D34 and untreated bread are tested. The X-axis shows the number of days, while the Y-axis shows firmness expressed as hPa. Diamond: 40,000 Betamyl units/kg of pSac-D34. Square: 40,000 Betamyl units/kg of pSac-pMD229. Cross: Control (no enzyme).

Figure 3 shows an improved resilience effect, i.e. higher resilience, of bread treated with pSac-pMD229 versus bread treated with pSac-D34 during storage time after baking. The figure shows the results of a baking trial in which resilience of bread treated with pSac-pMD229, pSac-D34 and untreated bread are tested. The X-axis shows the number of days, while the Y-axis shows resilience expressed as Resilience Units. Diamond: 40,000 Betamyl units/kg of pSac-D34. Square: 40,000 Betamyl units/kg of pSac-pMD229. Cross: Control (no enzyme).

Figure 4 shows an improved cohesiveness effect, i.e. higher cohesiveness, of bread treated with pSac-pMD229 versus bread treated with pSac-D34 during storage time after baking. The figure shows the results of a baking trial in which cohesiveness of bread treated with pSac-pMD229, pSac-D34 and untreated bread are tested. The X-axis shows the number of days, while the Y-axis shows cohesiveness expressed as Cohesiveness Units. Diamond: 40,000 Betamyl/kg of ρSac-D34. Square: 40,000 Betamyl/kg of pSac- pMD229. Cross: Control (no enzyme).

SEQUENCE LISTINGS

105 SEQ ID NO: 1 shows a PS4 reference sequence, derived from Pseudomonas saccharophila maltotetrahydrolase amino acid sequence. SEQ ID NO: 2 shows a pSac- D34 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with 11 substitutions and deletion of the starch binding domain. SEQ ID NO: 3 shows a pSac-D20 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid

110 sequence with 13 substitutions and deletion of the starch binding domain. SEQ ID NO: 4 shows a pSac-D14 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with 14 substitutions and deletion of the starch binding domain. SEQ ID NO: 5 shows & Pseudomonas saccharophila Glucan 1,4-alpha-maltotetrahydrolase precursor (EC 3.2.1.60) (G4-amylase) (Maltotetraose-forming amylase) (Exo-

115 maltotetraohydrolase) (Maltotetraose-forming exo-amylase). SWISS-PROT accession number P22963. SEQ ID NO: 6 shows a P. saccharophila mta gene encoding maltotetraohydrolase (EC number = 3.2.1.60). GenBank accession number Xl 6732. SEQ ID NO:7 shows a PS4 reference sequence, derived from Pseudomonas stutzeri maltotetrahydrolase amino acid sequence. SEQ ID NO: 8 shows a PStu-D34 sequence;

120 Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with 9 substitutions. SEQ ID NO: 9 shows a PStu-D20 sequence; Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with 11 substitutions. SEQ ID NO: 10 shows a PStu-D14 sequence; Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with 12 substitutions. SEQ ID NO: 11 shows & Pseudomonas stutzeri (Pseudomonas perfectomarina). Glucan

125 1,4-alpha-maltotetrahydrolase precursor (EC 3.2.1.60) (G4-amylase) (Maltotetraose- forming amylase) (Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase). SWISS-PROT accession number P13507. SEQ ID NO: 12 shows a P.stutzeri maltotetraose-forming amylase (amyP) gene, complete cds. GenBank accession number M24516.

130 SEQ ID NO: 13 shows a pSac-pMD229 amino acid sequence having mutations at

33Y, 34N, 121F, 134R, 141P, 146G₅ 157L, 161A, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P. SEQ ID NO: 14 shows a ρSac-ρMD229 nucleic acid sequence. SEQ ID NO: 15 shows apSac-pMD248 amino acid sequence having mutations at 33 Y, 34N, 121F, 134R, 141P, 145D, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L

135 and 334P. SEQ ID NO: 16 shows a ρSac-ρMD248 nucleic acid sequence. SEQ ID NO: 17 shows a ρSac-pMD253 amino acid sequence having mutations at 33Y, 34N₅ 121D, 134R, 141P, 146G₅ 157L, 178F, 179T₅ 223E₅ 229P₅ 272Q₅ 303E, 307L₅ 309P and 334P. SEQ ID NO: 18 shows apSac-pMD253 nucleic acid sequence. SEQ ID NO: 19 shows a pSac-pMD271 amino acid sequence having mutations at 3S, 33Y, 34N, 7OD, 121D, 134R, 140 141P₅ 146G, 157L, 178F, 179T, 223E₅ 229P₅ 272Q, 303E, 307L, 309P and 334P. SEQ ID NO: 20 shows apSac-pMD271 nucleic acid sequence.

DETAILED DESCRIPTION

hi the following description and examples, unless the context dictates otherwise, dosages of PS4 variant polypeptides are given in parts per million (micrograms per gram)

145 of flour. For example, "1 D34" indicates 1 part per million of pSac-D34 based on weight per weight. Preferably, enzyme quantities or amounts are determined based on activity assays as equivalents of pure enzyme protein measured with bovine serum albumin (BSA) as a standard, using the assay described in Bradford (1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-

150 dye binding. Anal. Biochem. 72:248-254).

In describing the different PS4 variant polypeptide variants produced or which are contemplated to be encompassed by this document, the following nomenclature will be adopted for ease of reference:

(i) where the substitution includes a number and a letter, e.g., 141P₅ then this refers 155 to [position according to the numbering system/substituted amino acid].

Accordingly, for example, the substitution of an amino acid to proline in position 141 is designated as 141 P;

(ii) where the substitution includes a letter, a number and a letter, e.g., A141P, then this refers to [original amino acid/position according to the numbering 160 system/substituted amino acid]. Accordingly, for example, the substitution of alanine with proline in position 141 is designated as A141P.

Where two or more possible substituents are possible at a particular position, this will be designated by contiguous letters, which may optionally be separated by slash marks "/", e.g., G303ED or G303E/D. Where the relevant amino acid at a position can be 165 substituted by any amino acid, this is designated by [position according to the numbering system/X], e.g., 121X.

Multiple mutations maybe designated by being separated by slash marks "/", e.g. A141P/G223A or commas ",", e.g., A141P, G223A representing mutations in position 141 and 223 substituting alanine with proline and glycine with alanine respectively. 170 Unless defined otherwise herein, 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 invention belongs. Singleton, et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of

175 skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid

180 sequences are written left to right in amino to carboxy orientation, respectively.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E.

185 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: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D.

190 McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; D. M. J. LiIl ey and J. E. Dahlberg, 1992, Methods ofEn∑ymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies : A Laboratory Manual : Portable Protocol NO. I by Edward Harlow, David

195 Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies : A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson "Immuno cytochemistry: Theory and Practice", CRC Press inc., Baca Raton, Florida, 1988, ISBN 0-8493-6078-1, John D. Pound (ed); "Immunochemical Protocols, vol 80", in the

200 series: "Methods in Molecular Biology", Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603-493-3, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory,

205 ISBN 0-87969-630-3. Each of these general texts is herein incorporated by reference. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

PS4 VARIANT POLYPEPTIDES

We provide a polypeptide having a substitution at one or more positions which 210 effect an altered property, preferably altered exospecificity or altered thermostability, or both, relative to the parent enzyme. Such variant polypeptides are referred to in this document for convenience as "PS4 variant polypeptides".

The PS4 variant polypeptides preferably exhibit enzyme activity. More preferably, the PS4 variant polypeptides comprise amylase activity, preferably exoamylase activity. In 215 highly preferred embodiments, the PS4 variant polypeptides exhibit non-maltogenic exoamylase activity.

We further provide for compositions, including food additives, food products, bakery products, improver compositions, feed products including animal feeds, etc comprising such altered PS4 variant polypeptides, preferably those which have non- 220 malto genie exoamylase activity, as well as methods of making and using such polypeptides and the compositions.

As noted above, the PS4 variant polypeptides may comprise one or more improved handling properties, preferably improved baking properties. Thus, the PS4 variant polypeptides are such that the food products so treated have one or more of (preferably all 225 of) a lower firmness, a higher resilience or a higher cohesiveness. Such improved handling or baking properties exhibited by the PS4 variant polypeptides are described in further detail below.

We provide for the treatment of food products, particularly doughs and bakery products with such polypeptides, and such that the food products exhibit the desired 230 qualities set out above.

We provide for other uses of such compositions such as in the preparation of detergents, as sweeteners, syrups, etc. The compositions include the polypeptide together with at least one other component. In particular, we provide for food or feed additives comprising the polypeptides.

235 Such polypeptides and nucleic acids vary from their parent sequences by including a number of mutations. In other words, the sequence of the PS4 variant polypeptide or nucleic acid is different from that of its parent at a number of positions or residues. In preferred embodiments, the mutations comprise amino acid substitutions, that is, a change of one amino acid residue for another. Thus, the PS4 variant polypeptides comprise a 240 number of changes in the nature of the amino acid residue at one or more positions of the parent sequence.

As used herein, the term "variant" should be taken to mean a molecule being derivable from a parent molecule. Variants include polypeptides as well as nucleic acids. Variants include deletions, insertions and substitutions at the amino acid level and 245 transversions, transitions and inversions at the nucleic acid level among other things, at one or more locations. Variants also include truncations. Variants include homologous and functional derivatives of parent molecules. Variants include sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein.

250 LOCATION OF MUTATIONS IN PS4 VARIANT POLYPEPTIDES

We provide for PS4 variant polypeptides with sequence alterations comprising amino acid substitutions in a amylase sequence, preferably an exoamylase activity, more preferably a non-maltogenic exoamylase sequence.

Specifically, we provide for a PS4 variant polypeptide derivable from a parent 255 polypeptide having non-maltogenic exoamylase activity comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334 with reference to the position numbering of a Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

We further provide for a PS4 variant polypeptide derivable from a parent 260 polypeptide having non-maltogenic exoamylase activity comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 145, 146, 157, 178, 179, 223, 229, 272, 303, 307 and 334 with reference to the position numbering of a. Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

We also provide for a PS4 variant polypeptide derivable from a parent polypeptide 265 having non-maltogenic exoamylase activity comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334 with reference to the position numbering of a Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

Finally, we provide for a PS4 variant polypeptide derivable from a parent 270 polypeptide having non-maltogenic exoamylase activity comprising an amino acid mutation at each of positions 3, 33, 34, 70, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334 with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1.

In preferred embodiments each of the amino acid mutations in these polypeptides 275 are independently selected from the group consisting of: 3S , 33Y, 34N, 7OD, 121D, 121F, 134R, 141P, 145D, 146G, 157L, 161A, 178F, 179T, 223E, 229P, 272Q, 303E₅ 307L, 309P and 334P.

In such preferred embodiments, each of the amino acid mutations in these polypeptides are preferably independently selected from the group of substitutions 280 consisting of: A3S, N33Y, D34N, G70D, G121D, G121F, G134R, A141P, N145D,

Y146G, I157L, S161A, L178F, A179T, G223E, S229P, H272Q, G303E, H307L, A309P and S334P.

Li highly preferred embodiments, the PS4 variant polypeptide a comprises the sequence pSac-pMD229 (SEQ ID NO: 13), pSac-pMD248 (SEQ ID NO: 15), pSac- 285 pMD253 (SEQ ID NO: 17) or pSac-pMD271 (SEQ ID NO: 19).

The PS4 variant polypeptides may comprise mutations at other sites, as described in further detail below.

Such variant polypeptides, and others as described in this document, are referred to in this document as "PS4 variant polypeptides". Nucleic acids encoding such variant 290 polypeptides are also disclosed and will be referred to for convenience as "PS4 variant nucleic acids". PS4 variant polypeptides and nucleic acids will be described in further detail below.

The "parent" sequences, i.e., the sequences on which the PS4 variant polypeptides and nucleic acids are based, preferably are polypeptides having non-maltogenic 295 exoamylase activity. The terms "parent enzymes" and "parent polypeptides" should be interpreted accordingly, and taken to mean the enzymes and polypeptides on which the PS4 variant polypeptides are based. They are described in further detail below.

The mutations and amino acid changes may be made on any suitable polypeptide backbone or background, wild type or mutated, as described in further detail below.

300 In particularly preferred embodiments, the parent sequences are non-maltogenic exoamylase enzymes, preferably bacterial non-maltogenic exoamylase enzymes. In highly preferred embodiments, the parent sequence comprises a glucan 1,4-alpha- maltotetrahydrolase (EC 3.2.1.60). Preferably, the parent sequence is derivable from Pseudomonas species, for example Pseudomonas saccharophilia or Pseudomonas stutzeri. 105 In some embodiments, the parent polypeptide comprises, or is homologous to, a wild type non-maltogenic exoamylase sequence, e.g., from Pseudomonas spp.

Thus, the parent polypeptide may comprise a Pseudomonas saccharophilia non- maltogenic exoamylase having a sequence shown as SEQ ID NO: 1. In other preferred embodiments, the parent polypeptide comprises a non-maltogenic exoamylase from 110 Pseudomonas stutzeri having a sequence shown as SEQ ID NO: 11, or a Pseudomonas stutzeri non-maltogenic exoamylase having SWISS-PROT accession number Pl 3507.

On the other hand, the parent polypeptide may be a variant of any of the wild type sequences, that is to say, the parent polypeptide may itself be engineered, or comprise a PS4 variant polypeptide.

515 In preferred embodiments, the mutations and changes are made on a PS4 sequence which is already mutated, preferably pSac-D34 (e.g., SEQ ID NO: 2).

However, it will be clear to the skilled reader that although the PS4 variant polypeptides may be derivable by mutating already mutated sequences, it is possible to construct such variant polypeptides by starting from a wild type sequence (or indeed any 520 suitable sequence), identifying the differences between the starting sequence and the desired variant, and introducing the required mutations into the starting sequence in order to achieve the desired variant.

Proteins and nucleic acids related to, preferably having sequence or functional homology with Pseudomonas saccharophilia non-maltogenic exoamylase sequence shown 525 as SEQ ID NO: 1 or a Pseudomonas stutzeri non-maltogenic exoamylase having a sequence shown as SEQ ID NO: 11 are referred to in this document as members of the "PS4 family". Examples of "PS4 family" non-maltogenic exoamylase enzymes suitable for use in generating the PS4 variant polypeptides and nucleic acids are disclosed in further detail below.

530 The PS4 variant polypeptides described in this document preferably retain the features of the parent polypeptides, and additionally preferably have additional beneficial properties, for example, enhanced activity or thermostability, or pH resistance, or any combination (preferably all). This is described in further detail below.

The PS4 substitution mutants described here may be used for any suitable purpose. 535 They may preferably be used for purposes for which the parent enzyme is suitable. In particular, they may be used in any application for which exo-maltotetraohydrolase is used. In highly preferred embodiments, they have the added advantage of higher thermostability, or higher exoamylase activity or higher pH stability, or any combination. Examples of suitable uses for the PS4 variant polypeptides and nucleic acids include food 340 production, in particular baking, as well as production of foodstuffs; further examples are set out in detail below.

The PS4 variant polypeptides may comprise one or more further mutations in addition to those positions set out above. There may be one, two, three, four, five, six, seven or more mutations preferably substitutions in addition to those already set out. Other

345 mutations, such as deletions, insertions and substitutions at the amino acid level and transversions, transitions and inversions at the nucleic acid level, at one or more other locations, may also be included, as described below. In addition, the PS4 variants need not have all the substitutions at the positions listed. Indeed, they may have one, two, three, four, or five substitutions missing, i.e., the wild type amino acid residue is present at such

350 positions.

PREFERRED SUBSTITUTIONS

The substitution at position 3, where present, may comprise 3S, preferably, A3S.

The substitution at position 33, where present, may comprise 33 Y, preferably,

N33Y.

355 The substitution at position 34 may comprise any of 34N, 34G, 34A, 34S or 34T, preferably 34N, D34G, D34A, D34S or D34T. In highly preferred embodiments, the substitution at position 34 comprises 34N, prefereably D34N.

The substitution at position 70, where present, may comprise 7OD, preferably, G70D.

360 The substitution at position 121 may comprise any of 121 F, 121 Y, 121 W, 121 H,

121A, 121M, 121G, 121S, 121T, 121D, 121E, 121L, 121K, 121V, preferably G 12 IF, G121Y, G121W, G121H, G121A, G121M, G121G, G121S, G121T, G121D, G121E, G121L, G121K, G121 V. In highly preferred embodiments, the substitution at position 121 comprises 121D or 121F, preferably G121D or G121F.

365 The substitution at position 134 may comprise 134R, preferably Gl 34R.

The substitution at position 141 may comprise 141P, preferably A141P.

The substitution at position 145, where present, may comprise 145D, preferably N145D. The substitution at position 146 may comprise any of 146M, 146G, preferably 370 Y146M, Y146G. In highly preferred embodiments, the substitution at position 146 comprises 146G, preferably Yl 46G.

The substitution at position 157 may comprise any of 157L, 57M, 157V, 157N, 157L, preferably I157L, I157M, I157V, I157N, I157L. In highly preferred embodiments, the substitution at position 157 comprises 157L, preferably I157L.

375 The substitution at position 161, where present, may comprise 161 A, preferably

S161A.

The substitution at position 178 may comprise 178F, preferably L178F.

The substitution at position 179 may comprise any of 179T, 179V, preferably A179T, Al 79V. In highly preferred embodiments, the substitution at position 179 380 comprises 179T, preferably Al 79T.

The substitution at position 223 may comprise any of 223 A, 223 E, 223K, G223L, 2231, 223S, 223T, 223V, 223R, 223P, 223D, preferably G223A, G223E, G223K, G223L, G223I, G223S, G223T, G223V, G223R, G223P, G223D. In highly preferred embodiments, the substitution at position 223 comprises 223E, preferably G223E.

385 The substitution at position 229 may comprise 229P, preferably S229P.

The substitution at position 272 may comprise 272Q, preferably H272Q.

The substitution at position 303 may comprise any of 303E, 303D G303E, G303D. In highly preferred embodiments, the substitution at position 303 comprises 303E, preferably G303E.

390 The substitution at position 307 may comprise 307L, preferably H307L.

The substitution at position 309, where present, may comprise 309P, preferably A309P.

The substitution at position 334 may comprise 334P, preferably S334P.

FURTHER SUBSTITUTIONS

395 A mutation at 160 may also be present, preferably 160D, more preferably El 6OD.

One or more other mutations as set out in the table below may further be present.

OTHER PS4 VARIANT POLYPEPTIDE SEQUENCES

We specifically provide for a PS4 variant polypeptide derivable from a parent polypeptide having non-maltogenic exoamylase activity, in which the PS4 variant polypeptide comprises a mutation at each of the following positions 33, 34, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307 and 334, with reference to the position numbering of a Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

The position 33 mutation may comprise 33Y, preferably N33Y. The position 34 mutation may comprise 34N, preferably D34N. The position 121 mutation may comprise 121F, preferably G121F. The position 134 mutation may comprise 134R, preferably G134R. The position 141 mutation may comprise 141P, preferably A141P. The position 146 mutation may comprise 146G, preferably Y146G. The position 157 mutation may comprise 157L, preferably I157L. The position 178 mutation may comprise 178F, preferably L178F. The position 179 mutation may comprise 179T, preferably A179T. The position 223 mutation may comprise 223E, preferably G223E. The position 229 mutation may comprise 229P, preferably S229P. The position 272 mutation may comprise 272Q, preferably H272Q. The position 303 mutation may comprise 303 E, preferably G303E. The position 307 mutation may comprise 307L, preferably H307L. The position 334 mutation may comprise 334P, preferably S334P. Preferably, the PS4 variant polypeptide comprises each of the following substitutions 33Y, 34N, 121F, 134R, 141P, 146G, 157L₅ 178F, 179T, 223E, 229P, 272Q, 303E, 307L and 334P, preferably N33 Y, D34N, G121F, G134R, A141P, Y146G, I157L, L178F, A179T, G223E, S229P, H272Q, G303E, H307L and S334P.

420 In a preferred embodiment, the PS4 variant polypeptide comprises further mutations at positions 161 and 309. The position 161 mutation may comprise 161 A, preferably S161 A. Furthermore, the position 309 mutation may comprise 309P, preferably A309P. Preferably,^" the PS4 variant polypeptide comrpises the sequence pSac-pMD229 (SEQ ID NO: 13).

425 In another preferred embodiment, the PS4 variant polypeptide comprises a further mutation at position 145. The position 145 mutation may comprise 145D, preferably N145D. Preferably, the PS4 variant polypeptide comprises the sequence pSac-pMD248 (SEQ ID NO: 15).

hi a further preferred embodiment, the PS4 variant polypeptide comprises a further 430 mutation at position 309. The position 309 mutation may comprise 309P, preferably

A309P. Preferably, the PS4 variant polypeptide comprises the sequence pSac-pMD253 (SEQ ID NO: 17).

In yet a further preferred embodiment, the PS4 variant polypeptids comprises further mutations at positions 3, 70 and 309. The position 3 mutation may comprises 3S, 435 preferably A3 S. The position 70 mutation may comprise 7OD, preferably G70D. The position 309 mutation may comprise 309P, preferably A309P. Preferably, the PS4 variant polypeptide comprises the sequence pSac-pMD271 (SEQ ID NO: 19).

PS4 VARIANT NUCLEIC ACIDS

We also describe PS4 nucleic acids having sequences which correspond to or 440 encode the alterations in the PS4 variant polypeptide sequences, for use in producing such polypeptides for the purposes described here. Thus, we provide nucleic acids capable of encoding any polypeptide sequence set out in this document.

The skilled person will be aware of the relationship between nucleic acid sequence and polypeptide sequence, in particular, the genetic code and the degeneracy of this code, 445 and will be able to construct such PS4 nucleic acids without difficulty. For example, he will be aware that for each amino acid substitution in the PS4 variant polypeptide sequence, there may be one or more codons which encode the substitute amino acid. Accordingly, it will be evident that, depending on the degeneracy of the genetic code with respect to that particular amino acid residue, one or more PS4 nucleic acid sequences may 450 be generated corresponding to that PS4 variant polypeptide sequence. Furthermore, where the PS4 variant polypeptide comprises more than one substitution, for example A141P/G223A, the corresponding PS4 nucleic acids may comprise pairwise combinations of the codons which encode respectively the two amino acid changes.

The PS4 variant nucleic acid sequences may be derivable from parent nucleic acids 455 which encode any of the parent polypeptides described above. In particular, parent nucleic acids may comprise wild type sequences, e.g., SEQ ID NO: 6 or SEQ ID NO: 12. The PS4 variant nucleic acids may therefore comprise nucleic acids encoding wild type non- maltogenic exoamylases, but which encode another amino acid at the relevant position instead of the wild type amino acid residue. The PS4 variant nucleic acid sequences may 460 also comprise wild type sequences with one or more mutations, e.g., which encode parent polypeptides described above under "Combinations".

It will be understood that nucleic acid sequences which are not identical to the particular PS4 variant nucleic acid sequences, but are related to these, will also be useful for the methods and compositions described here, such as a variant, homologue, derivative 465 or fragment of a PS4 variant nucleic acid sequence, or a complement or a sequence capable of hybridising thereof. Unless the context dictates otherwise, the term "PS4 variant nucleic acid" should be taken to include each of these entities listed above.

Mutations in amino acid sequence and nucleic acid sequence may be made by any of a number of techniques, as known in the art. Variant sequences may easily be made 470 using any of the known mutagenesis techniques, for example, site directed mutagenesis using PCR with appropriate oligonucleotide primers, 5' add-on mutagenesis, mismatched primer mutagenesis, etc. Alternatively, or in addition, the PS4 variant nucleic acid sequences may be made de novo.

hi particularly preferred embodiments, the mutations are introduced into parent 475 sequences by means of PCR (polymerase chain reaction) using appropriate primers, as illustrated in the Examples. It is therefore possible to alter the sequence of a polypeptide by introducing any desired amino acid substitutions into a parent polypeptide, preferably having non-maltogenic exoamylase activity, such as into a Pseudomonas saccharophilia or a Pseudomonas stutzeri exoamylase sequence at amino acid or nucleic acid level, as 480 described. We describe a method in which the sequence of a non-maltogenic exoamylase is altered by altering the sequence of a nucleic acid which encodes the non-maltogenic exoamylase.

However, it will of course be appreciated that the PS4 variant polypeptide does not need in fact to be actually derived from a wild type polypeptide or nucleic acid sequence 485 by, for example, step by step mutation. Rather, once the sequence of the PS4 variant polypeptide -is established, the skilled person can easily make that sequence from the wild type with all the mutations, via means known in the art, for example, using appropriate oligonucleotide primers and PCR. In fact, the PS4 variant polypeptide can be made de novo with all its mutations, through, for example, peptide synthesis methodology.

490 In general, however, the PS4 variant polypeptides and/or nucleic acids are derived or derivable from a "precursor" sequence. The term "precursor" as used herein means an enzyme that precedes the enzyme which is modified according to the methods and compositions described here. A precursor therefore includes an enzyme used to produce a modified enzyme. Thus, the precursor may be an enzyme that is modified by mutagenesis

495 as described elsewhere in this document. Likewise, the precursor may be a wild type enzyme, a variant wild type enzyme or an already mutated enzyme.

The PS4 variant polypeptides and nucleic acids may be produced by any means known in the art. Specifically, they may be expressed from expression systems, which may be in vitro or in vivo in nature. Specifically, we describe plasmids and expression 500 vectors comprising PS4 nucleic acid sequences, preferably capable of expressing PS4 variant polypeptides. Cells and host cells which comprise and are preferably transformed with such PS4 nucleic acids, plasmids and vectors are also disclosed, and it should be made clear that these are also encompassed in this document.

In preferred embodiments, the PS4 variant polypeptide sequence is used as a food 505 additive in an isolated form. The term "isolated" means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature. In one aspect, preferably the sequence is in a purified form. The term "purified" means that the sequence is in a relatively pure state - e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.

510 The PS4 variant polypeptides may for example be made using site directed mutagenesis using PCR with appropriate oligonucleotide primers, 5' add-on mutagenesis, mismatched primer mutagenesis, etc as described in the Examples. In order to produce PS4 variant polypeptides with the relevant mutations, for example, a nucleic acid sequence corresponding to a pSac-D34 sequence (SEQ ID NO: 2) may be made and the relevant

515 changes introduced. The skilled reader will be aware, however, that any suitable starting sequence can be used, and indeed that it is possible to start from a wild type exoamylase sequence to get to the desired variant polypeptide either in a single step, or via other intermediate sequences. In highly preferred embodiments, the nucleic acid sequence comprises the 520 sequence pSac-pMD229 (SEQ ID NO: 14), pSac-pMD248 (SEQ ID NO: 16), pSac- pMD253 (SEQ ID NO: 18) or pSac-pMD271 (SEQ ID NO: 20).

POSITION NUMBERING

All positions referred to in the present document by numbering refer to the numbering of a Pseudomonas saccharophilia exoamylase reference sequence shown 525 below (SEQ ID NO: 1):

1 DQAGKSPAGV RYHGGDEHL QGFHWNWRE APNDWYNILR QQASTIAADG FSAIWMPVPW

61 RDFSSWTDGG KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDWPNHMNR

121 GYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFIGGE SDLNTGHPQI YGMFRDELAN

181 LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WKGPSEYPSW DWRNTASWQQ

530 241 IIKDWSDRAK CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQHHWAL QDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRTAGVRAD

361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG SFSEAVNASN GQVRVWRSGS

421 GDGGGNDGGE GGLVNVNFRC DNGVTQMGDS VYAVGNVSQL GNWSPASAVR LTDTSSYPTW

481 KGSIALPDGQ NVEWKCLIRN EADATLVRQW QSGGNNQVQA AAGASTSGSF

535 The reference sequence is derived from the Pseudomonas saccharophilia sequence having SWISS-PROT accession number P22963, but without the signal sequence

MSHI LRAAVLAAVLLPFPALA.

The C-terminal starch binding domain EGGLVNVNFR CDNGVTQMGD SVYAVGNVSQ

LGNWSPASAV RLTDTSSYPT WKGSIALPDG QNVEWKCLIR NEADATLVRQ WQSGGNNQVQ

540 AAAGASTSGS F may optionally be deleted or disregarded. Alternatively, it may be included in the PS4 variant polypeptide sequence.

In the context of the present description a specific numbering of amino acid residue positions in PS4 exoamylase enzymes is employed. In this respect, by alignment of the amino acid sequences of various known exoamylases it is possible to unambiguously allot

545 a exoamylase amino acid position number to any amino acid residue position in any exoamylase enzyme, the amino acid sequence of which is known. Using this numbering system originating from for example the amino acid sequence of the exoamylase obtained from Pseudomonas saccharophilia, aligned with amino acid sequences of a number of other known exoamylase, it is possible to indicate the position of an amino acid residue in

550 a exoamylase unambiguously.

Therefore, the numbering system, even though it may use a specific sequence as a base reference point, is also applicable to all relevant homologous sequences. For example, the position numbering may be applied to homologous sequences from other Pseudomonas species, or homologous sequences from other bacteria. Preferably, such 555 homologous have 60% or greater homology, for example 70% or more, 80% or more, 90% or more or 95% or more homology, with the reference sequence SEQ ID NO: 1 above, or the sequences having SWISS-PROT accession numbers P22963 or P13507, preferably with all these sequences. Sequence homology between proteins may be ascertained using well known alignment programs and hybridisation techniques described herein. Such 560 homologous sequences, as well as the functional equivalents described below, will be referred to in this document as the "PS4 Family".

Furthermore, and as noted above, the numbering system used in this document makes reference to a reference sequence SEQ ID NO: 1, which is derived from the Pseudomonas saccharophilia sequence having SWISS-PROT accession number P22963,

565 but without the signal sequence MSHi LRAAVLAAVLLPF PALA. This signal sequence is located N terminal of the reference sequence and consists of 21 amino acid residues. Accordingly, it will be trivial to identify the particular residues to be mutated or substituted in corresponding sequences comprising the signal sequence, or indeed, corresponding sequences comprising any other N- or C- terminal extensions or deletions.

570 In relation to N- terminal additions or deletions, all that is required is to offset the position numbering by the number of residues inserted or deleted. For example, position 1 in SEQ ID NO: 1 corresponds to position 22 in a sequence with the signal sequence.

PARENT ENZYME / POLYPEPTIDE

The PS4 variant polypeptides are derived from, or are variants of, another 575 sequence, known as a "parent enzyme", a "parent polypeptide" or a "parent sequence".

The term "parent enzyme" as used in this document means the enzyme that has a close, preferably the closest, chemical structure to the resultant variant, i.e., the PS4 variant polypeptide or nucleic acid. The parent enzyme may be a precursor enzyme (i.e. the enzyme that is actually mutated) or it may be prepared de novo. The parent enzyme 580 may be a wild type enzyme, or it may be a wild type enzyme comprising one or more mutations.

The term "precursor" as used herein means an enzyme that precedes the enzyme which is modified to produce the enzyme. Thus, the precursor may be an enzyme that is modified by mutagenesis. Likewise, the precursor may be a wild type enzyme, a variant 585 wild type enzyme or an already mutated enzyme.

The term "wild type" is a term of the art understood by skilled persons and means a phenotype that is characteristic of most of the members of a species occurring naturally and contrasting with the phenotype of a mutant. Thus, in the present context, the wild type enzyme is a form of the enzyme naturally found in most members of the relevant species. 590 Generally, the relevant wild type enzyme in relation to the variant polypeptides described here is the most closely related corresponding wild type enzyme in terms of sequence homology. However, where a particular wild type sequence has been used as the basis for producing a variant PS4 polypeptide as described here, this will be the corresponding wild type sequence regardless of the existence of another wild type sequence that is more 595 closely related in terms of amino acid sequence homology.

The parent enzyme or polypeptide can be any suitable starting polypeptide. It may preferably have some enzymatic activity. Preferably, this enzymatic activity is an amylase activity. More preferably, the parent polypeptide comprises exoamylase activity.

The parent enzyme is preferably a polypeptide which preferably exhibits non- 600 maltogenic exoamylase activity. Preferably, the parent enzyme is a non-maltogenic exoamylase itself. For example, the parent enzyme may be a Pseudomonas saccharophila non-maltogenic exoamylase, such as a polypeptide having SWISS-PROT accession number P22963, or & Pseudomonas stutzeri non-maltogenic exoamylase, such as a polypeptide having SWISS-PROT accession number P13507.

605 Other members of the PS4 family may be used as parent enzymes; such "PS4 family members" will generally be similar to, homologous to, or functionally equivalent to either of these two enzymes, and may be identified by standard methods, such as hybridisation screening of a suitable library using probes, or by genome sequence analysis.

In particular, functional equivalents of either of these two enzymes, as well as 610 other members of the "PS4 family" may also be used as starting points or parent polypeptides for the generation of PS4 variant polypeptides as described here.

A "functional equivalent" of a protein means something that shares one or more, preferably substantially all, of the functions of that protein. Preferably, such functions are biological functions, preferably enzymatic functions, such as amylase activity, preferably 615 non-maltogenic exoamylase activity. Such functions may include any property of the protein, including exo-specificity, thermostability, and improved handling such as firmness, resilience and cohesiveness (as described below).

In relation to a parent enzyme, the term "functional equivalent" preferably means a molecule having similar or identical function to a parent molecule. The parent molecule 620 may be a Pseudomonas saccharophila non-maltogenic exoamylase or a Pseudomonas stutzeri non-maltogenic exoamylase or a polypeptide obtained from other sources.

The term "functional equivalent" in relation to a parent enzyme being a Pseudomonas saccharophila non-maltogenic exoamylase, such as a polypeptide having SWISS-PROT accession number P22963, or a Pseudomonas stutzeri non-maltogenic 625 exoamylase, such as a polypeptide having SWISS-PROT accession number P13507 means that the functional equivalent could be obtained from other sources. The functionally equivalent enzyme may have a different amino acid sequence but will have non- maltogenic exoamylase activity. Examples of assays to determine functionality are described herein and are known to one skilled in the art.

530 In highly preferred embodiments, the functional equivalent will have sequence homology to either of the Pseudomonas saccharophila and Pseudomonas stutzeri non- maltogenic exoamylases mentioned above, preferably both. The functional equivalent may also have sequence homology with any of the sequences set out as SEQ ID NOs: 1 to 14, preferably SEQ ID NO: 1 or SEQ ID NO: 7 or both. Sequence homology between such

635 sequences is preferably at least 60%, preferably 65% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more. Such sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA₅ etc. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit

640 package (University of Wisconsin, U.S. A; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al, 1999 ibid- Chapter 18), FASTA (Atschul et al, 1990, J. MoI. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching

645 (see Ausubel et al, 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

In other embodiments, the functional equivalents will be capable of specifically hybridising to any of the sequences set out above. Methods of determining whether one sequence is capable of hybridising to another are known in the art, and are for example 650 described in Sambrook, et al (supra) and Ausubel, F. M. et al. (supra). In highly preferred embodiments, the functional equivalents will be capable of hybridising under stringent conditions, e.g. 65°C and O.lxSSC {lxSSC = 0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0}.

For example, functional equivalents which have sequence homology to Pseudomonas saccharophila and Pseudomonas stutzeri non-maltogenic exoamylases are 655 suitable for use as parent enzymes. Such sequences may differ from the Pseudomonas saccharophila sequence at any one or more positions. Furthermore, non-maltogenic exoamylases from other strains of Pseudomonas spp, such as ATCCl 7686, may also be used as a parent polypeptide. The PS4 variant polypeptide residues may be inserted into any of these parent sequences to generate the variant PS4 polypeptide sequences.

660 It will be understood that where it is desired for PS4 variant polypeptides to additionally comprise one or more mutations, as set out above, corresponding mutations may be made in the nucleic acid sequences of the functional equivalents of Pseudomonas spp non-maltogenic exoamylase, as well as other members of the "PS4 family", in order that they may be used as starting points or parent polypeptides for the generation of PS4 665 variant polypeptides as described here.

Specifically included within the term "PS4 variant polypeptides" are the polypeptides disclosed in:

US 60/485,413, 60/485,539 and 60/485,616; PCT/US2004/021723 and PCT/US2004/021739; US 10/886,905 and 10/866,903; US 60/608,919; US 60/612,407; 670 US 60/485,539; PCT/IB2004/002487; US 10/886,023; US 10/886,505, US 10/886,527 and US 10/886,504; US 10/947,612. These documents however are not admitted to be prior art.

Such polypeptides are suitable for use in the applications described herein, in particular, as food additives, to treat starch as described, to prepare a food product, to 675 make a bakery product, for the formulation of improver compositions, for the formulation of combinations, etc.

Modification of Parent Sequences

The parent enzymes may be modified at the amino acid level or the nucleic acid level to generate the PS4 variant sequences described here. Therefore, we provide for the 680 generation of PS4 variant polypeptides by introducing one or more corresponding codon changes in the nucleotide sequence encoding a non-maltogenic exoamylase polypeptide.

The nucleic acid numbering should preferably be with reference to the position numbering of a Pseudomonas saccharophilia exoamylase nucleotide sequence shown as SEQ ID NO: 6. Alternatively, or in addition, reference may be made to the sequence with

685 GenBank accession number Xl 6732. In preferred embodiments, the nucleic acid numbering should be with reference to the nucleotide sequence shown as SEQ ID NO: 6. However, as with amino acid residue numbering, the residue numbering of this sequence is to be used only for reference purposes only. In particular, it will be appreciated that the above codon changes can be made in any PS4 family nucleic acid sequence. For example,

690 sequence changes can be made to a Pseudomonas saccharophila or a Pseudomonas stutzeri non-maltogenic exoamylase nucleic acid sequence (e.g., Xl 6732, SEQ ID NO: 6 or M24516, SEQ ID NO: 12).

The parent enzyme may comprise the "complete" enzyme, i.e., in its entire length as it occurs in nature (or as mutated), or it may comprise a truncated form thereof. The 695 PS4 variant derived from such may accordingly be so truncated, or be "full-length". The truncation may be at the N-terminal end, or the C-terminal end, preferably the C-terminal end. The parent enzyme or PS4 variant may lack one or more portions, such as subsequences, signal sequences, domains or moieties, whether active or not etc. For example, the parent enzyme or the PS4 variant polypeptide may lack a signal sequence, as described 700 above. Alternatively, or in addition, the parent enzyme or the PS4 variant may lack one or more catalytic or binding domains.

In highly preferred embodiments, the parent enzyme or PS4 variant may lack one or more of the domains present in non-maltogenic exoamylases, such as the starch binding domain. For example, the PS4 polypeptides may have only sequence up to position 429, 705 relative to the numbering of a Pseudomonas saccharophilia non-maltogenic exoamylase shown as SEQ ID NO: 1. It is to be noted that this is the case for the PS4 variants pSac- d34, pSac-D20 and pSac-D14.

In other embodiments, the parent enzyme or PS4 variant may comprise a "complete" enzyme, i.e., in its entire length as it occurs in nature (or as mutated), together 710 with one or more additional amino acid sequences at the N terminus or C terminus. For example, the parent enzyme or PS4 variant polypeptide may comprise a single extra amino acid residue at the C terminus or N terminus, e.g., M, A, G, etc. Preferably, the additional amino acid residue is present at the N terminus. Where one or more additional residues is included, the position numbering will be offset by the length of the addition.

715 AMYLASE

The PS4 variant polypeptides generally comprise amylase activity.

The term "amylase" is used in its normal sense - e.g. an enzyme that is inter alia capable of catalysing the degradation of starch. In particular they are hydrolases which are capable of cleaving α-D-(l— »4) O-glycosidic linkages in starch.

720 Amylases are starch-degrading enzymes, classified as hydrolases, which cleave α-

D-(l->4) O-glycosidic linkages in starch. Generally, α-amylases (E.G. 3.2.1.1, α-D- (1— >4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving α-D-(l->4) O-glycosidic linkages within the starch molecule in a random fashion. In contrast, the exo- acting amylolytic enzymes, such as β-amylases (E.G. 3.2.1.2, α-D-(l— »4)-glucan

725 maltohydrolase), and some product-specific amylases like maltogenic alpha-amylase (E.G. 3.2.1.133) cleave the starch molecule from the non-reducing end of the substrate, β- Amylases, α-glucosidases (E.G. 3.2.1.20, α-D-glucoside glucohydrolase), glucoamylase (E.C. 3.2.1.3, α-D-(l— >4)-glucan glucohydrolase), and product-specific amylases can produce malto-oligosaccharides of a specific length from starch. 730 NON-MALTOGENIC EXOAMYLASE

The PS4 variant polypeptides described in this document are derived from (or variants of) polypeptides which preferably exhibit non-maltogenic exoamylase activity. Preferably, these parent enzymes are non-maltogenic exoamylases themselves. The PS4 variant polypeptides themselves in highly preferred embodiments also exhibit non- 735 maltogenic exoamylase activity.

In highly preferred embodiments, the term "non-maltogenic exoamylase enzyme" as used in this document should be taken to mean that the enzyme does not initially degrade starch to substantial amounts of maltose as analysed in accordance with the product determination procedure as described in this document.

740 In highly preferred embodiments, the non-maltogenic exoamylase comprises an exo-maltotetraohydrolase. Exo-maltotetraohydrolase (E.C.3.2.1.60) is more formally known as glucan 1,4-alpha-maltotetrahydrolase. This enzyme hydrolyses 1,4-alpha-D- glucosidic linkages in amylaceous polysaccharides so as to remove successive maltotetraose residues from the non-reducing chain ends.

745 Non-maltogenic exoamylases are described in detail in US Patent number

6,667,065, hereby incorporated by reference.

ASSAYS FOR NON-MALTOGENIC EXOAMYLASE ACTIVITY

The following system is used to characterize polypeptides having non-maltogenic exoamylase activity which are suitable for use according to the methods and compositions 750 described here. This system may for example be used to characterise the PS4 parent or variant polypeptides described here.

By way of initial background information, waxy maize amylopectin (obtainable as WAXILYS 200 from Roquette, France) is a starch with a very high amylopectin content (above 90%). 20 mg/ml of waxy maize starch is boiled for 3 min. in a buffer of 50 mM 755 MES (2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 and subsequently incubated at 5O⁰C and used within half an hour.

One unit of the non-maltogenic exoamylase is defined as the amount of enzyme which releases hydrolysis products equivalent to 1 μmol of reducing sugar per min. when incubated at 50 degrees C in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 760 mM MES, 2 mM calcium chloride, pH 6.0 prepared as described above. Reducing sugars are measured using maltose as standard and using the dinitrosalicylic acid method of Bernfeld, Methods Enzymol, (1954), 1, 149-158 or another method known in the art for quantifying reducing sugars.

The hydrolysis product pattern of the non-malto genie exoamylase is determined by 765 incubating 0.7 units of non-maltogenic exoamylase for 15 or 300 min. at 50⁰C in a test tube with 4 ml of 10 mg/ml waxy maize starch in the buffer prepared as described above. The reaction is stopped by immersing the test tube for 3 min. in a boiling water bath.

The hydrolysis products are analyzed and quantified by anion exchange HPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxide and water as 770 eluents, with pulsed amperometric detection and with known linear maltooligosaccharides of from glucose to maltoheptaose as standards. The response factor used for maltooctaose to maltodecaose is the response factor found for maltoheptaose.

Preferably, the PS4 variant polypeptides have non-maltogenic exoamylase activity such that if an amount of 0.7 units of said non-maltogenic exoamylase were to incubated

775 for 15 minutes at a temperature of 5O⁰C at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maize starch per ml buffered solution containing 50 mM 2-(N- morpholino)ethane sulfonic acid and 2 mM calcium chloride then the enzyme would yield hydrolysis ρroduct(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%,

780 preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis products would consist of linear maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.

For ease of reference, and for the present purposes, the feature of incubating an ^■ 785 amount of 0.7 units of the non-maltogenic exoamylase for 15 minutes at a temperature of 50°C at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maize starch per ml buffered solution containing 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calcium chloride, may be referred to as the "Waxy Maize Starch Incubation Test".

Thus, alternatively expressed, preferred PS4 variant polypeptides which are non- 790 maltogenic exoamylases are characterised as having the ability in the waxy maize starch incubation test to yield hydrolysis product(s) that would consist of one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear 795 maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units. The hydrolysis products in the waxy maize starch incubation test may include one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose. The hydrolysis products in the waxy maize starch incubation test may

800 also include other hydrolytic products. Nevertheless, the % weight amounts of linear maltooligosaccharides of from three to ten D-glucopyranosyl units are based on the amount of the hydrolysis product that consists of one or more linear maltooligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose. In other words, the % weight amounts of linear maltooligosaccharides of from three to ten D-

805 glucopyranosyl units are not based on the amount of hydrolysis products other than one or more linear malto-oligosaccharides of from two to ten D-glucopyranosyl units and glucose.

The hydrolysis products can be analysed by any suitable means. For example, the hydrolysis products may be analysed by anion exchange HPLC using a Dionex PA 100 810 column with pulsed amperometric detection and with, for example, known linear maltooligosaccharides of from glucose to maltoheptaose as standards.

For ease of reference, and for the present purposes, the feature of analysing the hydrolysis product(s) using anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with known linear maltooligosaccharides of from 815 glucose to maltoheptaose used as standards, can be referred to as "analysing by anion exchange". Of course, and as just indicated, other analytical techniques would suffice, as well as other specific anion exchange techniques.

Thus, alternatively expressed, a preferred PS4 variant polypeptide is one which has non-maltogenic exoamylase such that it has the ability in a waxy maize starch incubation

820 test to yield hydrolysis product(s) that would consist of one or more linear maltooligosaccharides of from two to ten D-glucopyranosyl units and optionally glucose, said hydrolysis products being capable of being analysed by anion exchange; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the said hydrolysis product(s) would consist of linear

825 maltooligosaccharides of from three to ten D-glucopyranosyl units, preferably of linear maltooligosaccharides consisting of from four to eight D-glucopyranosyl units.

As used herein, the term "linear malto-oligosaccharide" is used in the normal sense as meaning 2-10 units of α-D-glucopyranose linked by an α-(l->4) bond.

In highly preferred embodiments, the PS4 polypeptides described here have 830 improved exoamylase activity, preferably non-maltogenic exoamylase activity, when compared to the parent polypeptide, preferably when tested under the same conditions, hi particular, in highly preferred embodiments, the PS4 variant polypeptides have 10% or more, preferably 20% or more, preferably 50% or more, exoamylase activity compared to their parents, preferably when measured in a waxy maize starch test.

835 The hydrolysis products can be analysed by any suitable means. For example, the hydrolysis products may be analysed by anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with, for example, known linear maltooligosaccharides of from glucose to maltoheptaose as standards.

As used herein, the term "linear malto-oligosaccharide" is used in the normal sense 840 as meaning 2-20 units of α-D-glucopyranose linked by an α-(l — >4) bond.

IMPROVED HANDLING PROPERTIES

The PS4 variants described here preferably have improved properties when compared to their parent enzymes, such as any one or more of improved thermostability, improved pH stability, or improved exo-specificity. The PS4 variants described here 845 preferably also have improved handling properties, such that a food product treated with a the PS4 variant polypeptide has any one or all of lower firmness, higher resilience or higher cohesiveness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

Without wishing to be bound by any particular theory, we believe that the 850 mutations at the particular positions have individual and cumulative effects on the properties of a polypeptide comprising such mutations.

THERMOSTABILITY AND PH STABILITY

Preferably, the PS4 variant polypeptide is thermostable; preferably, it has higher thermostability than its parent enzyme.

855 hi wheat and other cereals the external side chains in amylopectin are in the range of DP 12-19. Thus, enzymatic hydrolysis of the amylopectin side chains, for example, by PS4 variant polypeptides as described having non-maltogenic exoamylase activity, can markedly reduce their crystallisation tendencies.

Starch in wheat and other cereals used for baking purposes is present in the form of 860 starch granules which generally are resistant to enzymatic attack by amylases. Thus starch modification is mainly limited to damaged starch and is progressing very slowly during dough processing and initial baking until gelatinisation starts at about 6OC. As a consequence hereof only amylases with a high degree of thermostability are able to modify starch efficiently during baking. And generally the efficiency of amylases is 865 increased with increasing thermostability. That is because the more thermostable the enzyme is the longer time it can be active during baking and thus the more antistaling effect it will provide.

Accordingly, the use of PS4 variant polypeptides as described here when added to the starch at any stage of its processing into a food product, e.g., before during or after 870 baking into bread can retard or impede or slow down the retrogradation. Such use is described in further detail below.

As used herein the term "thermostable" relates to the ability of the enzyme to retain activity after exposure to elevated temperatures. Preferably, the PS4 variant polypeptide is capable of degrading starch at temperatures of from about 55°C to about 875 80⁰C or more. Suitably, the enzyme retains its activity after exposure to temperatures of up to about 95⁰C.

The thermostability of an enzyme such as a non-maltogenic exoamylase is measured by its half life. Thus, the PS4 variant polypeptides described here have half lives extended relative to the parent enzyme by preferably 10%, 20%, 30%, 40%, 50%, 60%, 880 70%, 80%, 90%, 100%, 200% or more, preferably at elevated temperatures of from 55°C to about 95⁰C or more, preferably at about 8O⁰C.

As used here, the half life (tl/2) is the time (in minutes) during which half the enzyme activity is inactivated under defined heat conditions. In preferred embodiments, the half life is assayed at 80 degrees C. Preferably, the sample is heated for 1-10 minutes 885 at 80°C or higher. The half life value is then calculated by measuring the residual amylase activity, by any of the methods described here. Preferably, a half life assay is conducted as described in more detail in the Examples.

Preferably, the PS4 variants described here are active during baking and hydrolyse starch during and after the gelatinization of the starch granules which starts at tempera-

890 tares of about 55°C. The more thermostable the non-maltogenic exoamylase is the longer time it can be active and thus the more antistaling effect it will provide. However, during baking above temperatures of about 85⁰C, enzyme inactivation can take place. If this happens, the non-maltogenic exoamylase may be gradually inactivated so that there is substantially no activity after the baking process in the final bread. Therefore

895 preferentially the non-maltogenic exoamylases suitable for use as described have an optimum temperature above 50⁰C and below 98⁰C.

The thermostability of the PS4 variants described here can be improved by using protein engineering to become more thermostable and thus better suited for the uses described here; we therefore encompass the use of PS4 variants modified to become more 00 thermostable by protein engineering.

Preferably, the PS4 variant polypeptide is pH stable; more preferably, it has a higher pH stability than its cognate parent polypeptide. As used herein the term "pH stable" relates to the ability of the enzyme to retain activity over a wide range of pHs. Preferably, the PS4 variant polypeptide is capable of degrading starch at a pH of from 05 about 5 to about 10.5. In one embodiment, the degree of pH stability may be assayed by measuring the half life of the enzyme in specific pH conditions. In another embodiment, the degree of pH stability may be assayed by measuring the activity or specific activity of the enzyme in specific pH conditions. The specific pH conditions may be any pH from pH5 to pH10.5.

10 Thus, the PS4 variant polypeptide may have a longer half life, or a higher activity

(depending on the assay) when compared to the parent polypeptide under identical conditions. The PS4 variant polypeptides may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longer half life when compared to their parent polypeptides under identical pH conditions. Alternatively, or in addition, they may have

15 such higher activity when compared to the parent polypeptide under identical pH conditions.

EXO-SPECIFICITY

It is known that some non-maltogenic exoamylases can have some degree of endoamylase activity. In some cases, this type of activity may need to be reduced or 20 eliminated since endoamylase activity can possibly negatively effect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins.

Exo-specifϊcity can usefully be measured by determining the ratio of total amylase activity to the total endoamylase activity. This ratio is referred to in this document as a

'25 "Exo-specificity index". In preferred embodiments, an enzyme is considered an exoamylase if it has a exo-specificity index of 20 or more, i.e., its total amylase activity (including exo-amylase activity) is 20 times or more greater than its endoamylase activity. In highly preferred embodiments, the exo-specificity index of exoamylases is 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more.

'30 In highly preferred embodiments, the exo-specificity index is 150 or more, 200 or more, 300 or more, 400 or more, 500 or more or 600 or more.

The total amylase activity and the endoamylase activity may be measured by any means known in the art. For example, the total amylase activity may be measured by assaying the total number of reducing ends released from a starch substrate. Alternatively , 935 the use of a Betamyl assay is described in further detail in the Examples, and for convenience, amylase activity as assayed in the Examples is described in terms of "Betamyl Units" in the Tables.

Endoamylase activity may be assayed by use of a Phadebas Kit (Pharmacia and Upjohn). This makes use of a blue labelled crosslinked starch (labelled with an azo dye); 940 only internal cuts in the starch molecule release label, while external cuts do not do so. Release of dye may be measured by spectrophotometry. Accordingly, the Phadebas Kit measures endoamylase activity, and for convenience, the results of such an assay (described in the Examples) are referred to in this document as "Phadebas units".

hi a highly preferred embodiment, therefore, the exo-specificity index is expressed 945 in terms of Betamyl Units / Phadebas Units, also referred to as "B/Phad".

Exo-specificity may also be assayed according to the methods described in the prior art, for example, in our International Patent Publication Number WO99/50399. This measures exo-specificity by way of a ratio between the endoamylase activity to the exoamylase activity. Thus, in a preferred aspect, the PS4 variants described here will have 950 less than 0.5 endoamylase units (EAU) per unit of exoamylase activity. Preferably the non-maltogenic exoamylases which are suitable for use according to the present invention have less than 0.05 EAU per unit of exoamylase activity and more preferably less than 0.01 EAU per unit of exoamylase activity.

The PS4 variants described here will preferably have exospeciflcity, for example 955 measured by exo-specificity indices, as described above, consistent with their being exoamylases. Moreoever, they preferably have higher or increased exospeciflcity when compared to the parent enzymes or polypeptides from which they are derived. Thus, for example, the PS4 variant polypeptides may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher exo-specificity index when compared to their parent 960 polypeptides, preferably under identical conditions. They may have 1.5x or higher, 2x or higher, 5 x or higher, 10 x or higher, 50 x or higher, 100 x or higher, when compared to their parent polypeptides, preferably under identical conditions.

IMPROVED HANDLING PROPERTIES

The PS4 variants described here preferably comprise one or more improved 965 handling properties compared to a parent polypeptide or a wild type polypeptide. The improved handling properties may in preferred embodiments comprise improved baking properties. Thus, the PS4 variants are such that a food product treated with the PS4 variant polypeptide an improved handling or preferably baking property compared to a food 970 product which has been treated with a parent polypeptide or a wild type polypeptide. The handling or baking property may be selected from the group consisting of: firmness, resilience and cohesiveness.

These handling properties may may be tested by any means known in the art. For example, firmness, resilience and cohesiveness may be determined by analysing bread 975 slices by Texture Profile Analysis using for example a Texture Analyser, as described in the Examples.

Firmness

The PS4 variants described here are preferably such that a food product treated with the PS4 variant polypeptide lower firmness compared to a food product which has 980 been treated with a parent polypeptide or a wild type polypeptide.

The firmness is in preferred embodiments inversely correlated with the softness of the food product; thus, a higher softness may reflect a lower firmness, and vice versa.

Firmness is preferably measured by the "Firmness Evaluation Protocol" set out in in Example 12.

985 Thus, the PS4 variants described here are preferably such that a food product treated with the PS4 variant polypeptide has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more lower firmness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide. A food product treated with the PS4 variant polypeptide may have a 1. Ix, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 1 Ox

990 or more lower firmness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

Resilience

The PS4 variants described here are preferably such that a food product treated with the PS4 variant polypeptide higher resilience compared to a food product which has 995 been treated with a parent polypeptide or a wild type polypeptide.

Resilience is preferably measured by the "Resilience Evaluation Protocol" set out in Example 13.

Thus, the PS4 variants described here are preferably such that a food product treated with the PS4 variant polypeptide has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more higher resilience compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide. A food product treated with the PS4 variant polypeptide may have a l.lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 1Ox or more higher resilience compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

Cohesiveness

The PS4 variants described here are preferably such that a food product treated with the PS4 variant polypeptide higher cohesiveness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

Cohesiveness is preferably measured by the "Cohesiveness Evaluation Protocol" set out in Examples 14.

Thus, the PS4 variants described here are preferably such that a food product treated with the PS4 variant polypeptide has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more higher cohesiveness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide. A food product treated with the PS4 variant polypeptide may have a l.lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more higher cohesiveness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

USES OF PS4 VARIANT POLYPEPTIDES AND NUCLEIC ACIDS

ThePS4 variant polypeptides, nucleic acids, host cells, expression vectors, etc, may be used in any application for which an amylase may be used. In particular, they may be used to substitute for any non-maltogenic exoamylase. They may be used to supplement amylase or non-maltogenic exoamylase activity, whether alone or in combination with other known amylases or non-maltogenic exoamylases.

The PS4 variant sequences described here maybe used in various applications in the food industry - such as in bakery and drink products, they may also be used in other applications such as a pharmaceutical composition, or even in- the chemical industry. In particular, the PS4 variant polypeptides and nucleic acids are useful for various industrial applications including baking (as disclosed in WO 99/50399) and flour standardisation (volume enhancement or improvement). They may be used to produce maltotetraose from starch and other substrates.

We therefore describe a method for preparing a food product, the method comprising: (a) obtaining a non-maltogenic exoamylase; (b) introducing a mutation at any one or more of the positions cof the non-maltogenic exoamylase as set out in this document; (c) admixing the resulting polypeptide with a food ingredient.

The PS4 variant polypeptides may be used to enhance the volume of bakery products such as bread. While not wishing to be bound by any particular theory, we believe that this results from the reduction in viscosity of the dough during heating (such as baking) as a result of the exoamylase shortening amylose molecules. This enables the carbon dioxide generated by fermentation to increase the size of the bread with less hindrance.

Thus, food products comprising or treated with PS4 variant polypeptides are expanded in volume when compared to products which have not been so treated, or treated with parent polypeptides. In other words, the food products have a larger volume of air per volume of food product. Alternatively, or in addition, the food products treated with PS4 variant polypeptides have a lower density, or weight (or mass) per volume ratio. In particularly preferred embodiments, the PS4 variant polypeptides are used to enhance the volume of bread. Volume enhancement or expansion is beneficial because it reduces the gumminess or starchiness of foods. Light foods are preferred by consumers, and the customer experience is enhanced. In preferred embodiments, the use of PS4 variant polypeptides enhances the volume by 10%, 20%, 30% 40%, 50% or more.

The use of PS4 variant polypeptides to increase the volume of foods is described in detail in the Examples.

FOOD USES

The PS4 variant polypeptides and nucleic acids described here may be used as - or , in the preparation of - a food. In particular, they may be added to a food, i.e., as a food additive. The term "food" is intended to include both prepared food, as well as an . ingredient for a food, such as a flour. In a preferred aspect, the food is for human consumption. The food may be in the from of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

The PS4 variant polypeptides and nucleic acids may be used as a food ingredient.

As used herein the term "food ingredient" includes a formulation, which is or can be added to functional foods or foodstuffs and includes formulations which can be used at low levels in a wide variety of products that require, for example, acidifying or emulsifying. The food ingredient may be in the from of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration. The PS4 variant polypeptides and nucleic acids disclosed here may be - or may be added to - food supplements. The PS4 variant polypeptides and nucleic acids disclosed here may be — or may be added to - functional foods. As used herein, the term "functional food" means food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a further beneficial effect to consumer. Although there is no legal definition of a functional food, most of the parties with an interest in this area agree that they are foods marketed as having specific health effects.

The PS4 variant polypeptides may also be used in the manufacture of a food product or a foodstuff. Typical foodstuffs include dairy products, meat products, poultry products, fish products and dough products. The dough product may be any processed dough product, including fried, deep fried, roasted, baked, steamed and boiled doughs, such as steamed bread and rice cakes. In highly preferred embodiments, the food product is a bakery product.

Preferably, the foodstuff is a bakery product. Typical bakery (baked) products include bread - such as loaves, rolls, buns, pizza bases etc. pastry, pretzels, tortillas, cakes, cookies, biscuits, krackers etc.

The food products preferably benefit from one or more of the improved handling or baking properties of the PS4 variant polypeptides described here. The improved handling or baking property may be selected from the group consisting of: improved firmness, improved resilience and improved cohesiveness.

We therefore describe a method of modifying a food additive comprising a non- maltogenic exoamylase, the method comprising introducing a mutation at any one or more of the positions of the non-maltogenic exoamylase as set out in this document. The same method can be used to modify a food ingredient, or a food supplement, a food product, or a foodstuff.

RETROGRADATION / STALING

We describe the use of PS4 variant proteins that are capable of retarding the staling of starch media, such as starch gels. The PS4 variant polypeptides are especially capable of retarding the detrimental retrogradation of starch.

Most starch granules are composed of a mixture of two polymers: an essentially linear amylose and a highly branched amylopectin. Amylopectin is a very large, branched molecule consisting of chains of α-D-glucopyranosyl units joined by (1-4) linkages, wherein said chains are attached by α-D-(l-6) linkages to form branches. Amylopectin is present in all natural starches, constituting about 75% of most common starches. Amylose 1100 is essentially a linear chain of (1-4) linked α -D-glucopyranosyl units having few α-D-(l- 6) branches. Most starches contain about 25% amylose.

Starch granules heated in the presence of water undergo an order-disorder phase transition called gelatinization, where liquid is taken up by the swelling granules. Gelatinization temperatures vary for different starches. Upon cooling of freshly baked

1105 bread the amylose fraction, within hours, retrogrades to develop a network. This process is beneficial in that it creates a desirable crumb structure with a low degree of firmness and improved slicing properties. More gradually crystallisation of amylopectin takes place within the gelatinised starch granules during the days after baking. In this process amylopectin is believed to reinforce the amylose network in which the starch granules are

1110 embedded. This reinforcement leads to increased firmness of the bread crumb. This reinforcement is one of the main causes of bread staling.

It is known that the quality of baked products gradually deteriorates during storage As a consequence of starch recystallisation (also called retrogradation), the water-holding capacity of the crumb is changed with important implications on the organoleptic and L 115 dietary properties. The crumb loses softness and elasticity and becomes firm and crumbly. The increase in crumb firmness is often used as a measure of the staling process of bread.

The rate of detrimental retrogradation of amylopectin depends on the length of the side chains of amylopectin. Thus, enzymatic hydrolysis of the amylopectin side chains, for example, by PS4 variant polypeptides having non-maltogenic exoamylase activity, can 1120 markedly reduce their crystallisation tendencies.

Accordingly, the use of PS4 variant polypeptides as described here when added to the starch at any stage of its processing into a food product, e.g., before during or after baking into bread can retard or impede or slow down the retrogradation. Such use is described in further detail below.

1125 We therefore describe a method of improving the ability of a non-maltogenic exoamylase to prevent staling, preferably detrimental retrogradation, of a dough product, the method comprising introducing a mutation at any one or more of the positions of the non-maltogenic exoamylase as set out in this document.

ASSAYS FOR MEASUREMENT OF RETROGRADATION (INC. STALING)

L 130 For evaluation of the antistaling effect of the PS4 variant polypeptides having non- maltogenic exoamylase activity described here, the crumb firmness can be measured 1, 3 and 7 days after baking by means of an Instron 4301 Universal Food Texture Analyzer or similar equipment known in the art. Another method used traditionally in the art and which is used to evaluate the 135 effect on starch retrogradation of a PS4 variant polypeptide having non-maltogenic exoamylase activity is based on DSC (differential scanning calorimetry). Here, the melting enthalpy of retrograded amylopectin in bread crumb or crumb from a model system dough baked with or without enzymes (control) is measured. The DSC equipment applied in the described examples is a Mettler-Toledo DSC 820 run with a temperature gradient of 10°C 140 per min. from 20 to 95°C. For preparation of the samples 10-20 mg of crumb are weighed and transferred into Mettler-Toledo aluminium pans which then are hermetically sealed.

The model system doughs used in the described examples contain standard wheat flour and optimal amounts of water or buffer with or without the non-maltogenic PS4 variant exoamylase. They are mixed in a 10 or 50 g Brabender Farinograph for 6 or 7 145 min., respectively. Samples of the doughs are placed in glass test tubes (15*0.8 cm) with a lid. These test tubes are subjected to a baking process in a water bath starting with 30 min. incubation at 33°C followed by heating from 33 to 95°C with a gradient of 1.1°C per min. and finally a 5 min. incubation at 95°C. Subsequently, the tubes are stored in a thermostat at 20°C prior to DSC analysis.

[ 150 In preferred embodiments, the PS4 variants described here have a reduced melting enthalpy, compared to the control, hi highly preferred embodiments, the PS4 variants have a 10% or more reduced melting enthalpy. Preferably, they have a 20% or more, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduced melting enthalpy when compared to the control.

1155 Table 2

The above Table 2 shows DSC values of model dough systems prepared with different doses of pSac-D34 after 7 days of storage. 0.5, 1 and 2 parts per million (or microgram per gram) of flour are tested. PREPARATION OF STARCH PRODUCTS

160 We provide the use of PS4 variant polypeptides in the preparation of food products, in particular, starch products. The method comprises forming the starch product by adding a non-maltogenic exoamylase enzyme such as a PS4 variant polypeptide, to a starch medium. If the starch medium is a dough, then the dough is prepared by mixing together flour, water, the non-maltogenic exoamylase which is a PS4 variant polypeptide

165 and optionally other possible ingredients and additives.

The term "starch" should be taken to mean starchier se or a component thereof, especially amylopectin. The term "starch medium" means any suitable medium comprising starch. The term "starch product" means any product that contains or is based on or is derived from starch. Preferably, the starch product contains or is based on or is

170 derived from starch obtained from wheat flour. The term "flour" as used herein is a synonym for the finely-ground meal of wheat or other grain. Preferably, however, the term means flour obtained from wheat per se and not from another grain. Thus, and unless otherwise expressed, references to "wheat flour" as used herein preferably mean references to wheat flour per se as well as to wheat flour when present in a medium, such

175 as a dough.

A preferred flour is wheat flour or rye flour or mixtures of wheat and rye flour. However, dough comprising flour derived from other types of cereals such as for example from rice, maize, barley, and durra are also contemplated. Preferably, the starch product is a bakery product. More preferably, the starch product is a bread product. Even more 180 preferably, the starch product is a baked farinaceous bread product. The term "baked farinaceous bread product " refers to any baked product based on a dough obtainable by mixing flour, water, and a leavening agent under dough forming conditions. Further components can of course be added to the dough mixture.

Thus, if the starch product is a baked farinaceous bread product, then the process 185 comprises mixing - in any suitable order - flour, water, and a leavening agent under dough forming conditions and further adding a PS4 variant polypeptide, optionally in the form of a premix. The leavening agent may be a chemical leavening agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker's Yeast).

The PS4 variant non-maltogenic exoamylase can be added together with any dough 190 ingredient including the water or dough ingredient mixture or with any additive or additive mixture. The dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry making flour dough based products. Baking of farinaceous bread products such as for example white bread, bread made 1195 from bolted rye flour and wheat flour, rolls and the like is typically accomplished by baking the bread dough at oven temperatures in the range of from 180 to 250°C for about 15 to 60 minutes. During the baking process a steep temperature gradient (200 -» 12O⁰C) is prevailing in the outer dough layers where the characteristic crust of the baked product is developed. However, owing to heat consumption due to steam generation, the 1200 temperature in the crumb is only close to 100°C at the end of the baking process.

We therefore describe a process for making a bread product comprising: (a) providing a starch medium; (b) adding to the starch medium a PS4 variant polypeptide as described in this document; and (c) applying heat to the starch medium during or after step (b) to produce a bread product. We also describe a process for making a bread product 1205 comprising adding to a starch medium a PS4 variant polypeptide as described.

The non-maltogenic exoamylase PS4 variant polypeptide can be added as a liquid preparation or as a dry pulverulent composition either comprising the enzyme as the sole active component or in admixture with one or more additional dough ingredient or dough additive.

1210 IMPROVING COMPOSITION

We describe improver compositions, which include bread improving compositions and dough improving compositions. These comprise a PS4 variant polypeptide, optionally together with a further ingredient, or a further enzyme, or both.

We also provide for the use of such a bread and dough improving compositions in 1215 baking, hi a further aspect, we provide a baked product or dough obtained from the bread improving composition or dough improving composition. In another aspect, we describe a baked product or dough obtained from the use of a bread improving composition or a dough improving composition.

DOUGH PREPARATION

1220 A dough may be prepared by admixing flour, water, a dough improving composition comprising PS4 variant polypeptide (as described above) and optionally other ingredients and additives.

The dough improving composition can be added together with any dough ingredient including the flour, water or optional other ingredients or additives. The dough 1225 improving composition can be added before the flour or water or optional other ingredients and additives. The dough improving composition can be added after the flour or water, or optional other ingredients and additives. The dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry making flour dough based products.

.230 The dough improving composition can be added as a liquid preparation or in the form of a dry powder composition either comprising the composition as the sole active component or in admixture with one or more other dough ingredients or additive.

The amount of the PS4 variant polypeptide non-maltogenic exoamylase that is added is normally in an amount which results in the presence in the finished dough of 50 .235 to 100,000 units per kg of flour, preferably 100 to 50,000 units per kg of flour. Preferably, the amount is in the range of 200 to 20,000 units per kg of flour. Alternatively, the PS4 variant polypeptide non-maltogenic exoamylase is added in an amount which results in the presence in the finished dough of 0.02 - 50 ppm of enzyme based on flour (0.02 - 50 mg enzyme per kg of flour), preferably 0.2 - 10 ppm.

[240 In the present context, 1 unit of the non-maltogenic exoamylase is defined as the amount of enzyme which releases hydrolysis products equivalent to 1 μmol of reducing sugar per min. when incubated at 50 degrees C in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter.

The dough as described here generally comprises wheat meal or wheat flour and/or 1245 other types of meal, flour or starch such as corn flour, corn starch, maize flour, rice flour, rye meal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch. The dough may be fresh, frozen, or part-baked.

The dough may be a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways, such as by adding chemical leavening agents, 1250 e.g., sodium bicarbonate or by adding a leaven (fermenting dough), but it is preferred to leaven the dough by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), e.g. a commercially available strain of S. cerevisiae.

The dough may comprise fat such as granulated fat or shortening. The dough may further comprise a further emulsifier such as mono- or diglycerides, sugar esters of fatty 1255 acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxethylene stearates, or lysolecithin.

We also describe a pre-mix comprising flour together with the combination as described herein. The pre-mix may contain other dough-improving and/or bread- improving additives, e.g. any of the additives, including enzymes, mentioned herein. 260 FURTHER DOUGH ADDITIVES OR INGREDIENTS

In order to improve further the properties of the baked product and impart distinctive qualities to the baked product further dough ingredients and/or dough additives may be incorporated into the dough. Typically, such further added components may include dough ingredients such as salt, grains, fats and oils, sugar or sweeteber, dietary .265 fibres, protein sources such as milk powder, gluten soy or eggs and dough additives such as emulsifiers, other enzymes, hydrocolloids, flavouring agents, oxidising agents, minerals and vitamins

The emulsifiers are useful as dough strengtheners and crumb softeners. As dough strengtheners, the emulsifiers can provide tolerance with regard to resting time and 1270 tolerance to shock during the proofing. Furthermore, dough strengtheners will improve the tolerance of a given dough to variations in the fermentation time. Most dough strengtheners also improve on the oven spring which means the increase in volume from the proofed to the baked goods. Lastly, dough strengtheners will emulsify any fats present in the recipe mixture.

1275 Suitable emulsifiers include lecithin, polyoxyethylene stearat, mono- and diglycerides of edible fatty acids, acetic acid esters of mono- and diglycerides of edible fatty acids, lactic acid esters of mono- and diglycerides of edible fatty acids, citric acid esters of mono- and diglycerides of edible fatty acids, diacetyl tartaric acid esters of mono- and diglycerides of edible fatty acids, sucrose esters of edible fatty acids, sodium stearoyl-

1280 2-lactylate, and calcium stearoyl-2-lactylate.

The further dough additive or ingredient can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or the dough improving composition. The further dough additive or ingredient can be added before the flour, water, optional other ingredients and additives or the dough improving 1285 composition. The further dough additive or ingredient can be added after the flour, water, optional other ingredients and additives or the dough improving composition.

The further dough additive or ingredient may conveniently be a liquid preparation. However, the further dough additive or ingredient may be conveniently in the form of a dry composition.

1290 Preferably the further dough additive or ingredient is at least 1% the weight of the flour component of dough. More preferably, the further dough additive or ingredient is at least 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 6%. If the additive is a fat, then typically the fat may be present in an amount of from 1 to 5%, typically 1 to 3%, more typically about 2%. 1295 FURTHER ENZYME

In addition to the PS4 variant polypeptides, one or more further enzymes may be used, for example added to the food, dough preparation, foodstuff or starch composition.

Further enzymes that may be added to the dough include oxidoreductases, hydrolases, such as lipases and esterases as well as glycosidases like α-amylase,

1300 pullulanase, and xylanase. Oxidoreductases, such as for example glucose oxidase and hexose oxidase, can be used for dough strengthening and control of volume of the baked products and xylanases and other hemicellulases may be added to improve dough handling properties, crumb firmness and bread volume. Lipases are useful as dough strengtheners and crumb softeners and α-amylases and other amylolytic enzymes may be incorporated

1305 into the dough to control bread volume and further reduce crumb firmness.

Further enzymes that may be used may be selected from the group consisting of a cellulase, a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase.

Examples of useful oxidoreductases include oxidises sush as maltose oxidising enzyme, a glucose oxidase (EC 1.1.3.4), carbohydrate oxidase, glycerol oxidase, pyranose 1310 oxidase, galactose oxidase (EC 1.1.3.10) and hexose oxidase (EC 1.1.3.5).

Among starch degrading enzymes, amylases are particularly useful as dough improving additives, α-amylase breaks downs starch into dextrins which are further broken down by β-amylase to maltose. Other useful starch degrading enzymes which may be added to a dough composition include glucoamylases and pullulanases.

1315 Preferably, the further enzyme is at least a xylanase and/or at least an amylase. The term "xylanase" as used herein refers to xylanases (EC 3.2.1.32) which hydrolyse xylosidic linkages. A lipase may also be added.

The term "amylase" as used herein refers to amylases such as α-amylases (EC 3.2.1.1), β-amylases (EC 3.2.1.2) and γ-amylases (EC 3.2.1.3.

1320 The further enzyme can be added together with any dough ingredient including the flour, water or optional other ingredients or additives, or the dough improving composition. The further enzyme can be added before the flour, water, and optionally other ingredients and additives or the dough improving composition. The further enzyme can be added after the flour, water, and optionally other ingredients and additives or the

1325 dough improving composition. The further enzyme may conveniently be a liquid preparation. However, the composition may be conveniently in the form of a dry composition. Some enzymes of the dough improving composition are capable of interacting with each other under the dough conditions to an extent where the effect on improvement of the 330 theological and/or machineability properties of a flour dough and/or the quality of the product made from dough by the enzymes is not only additive, but the effect is synergistic.

In relation to improvement of the product made from dough (finished product), it may be found that the combination results in a substantial synergistic effect in respect to crumb structure. Also, with respect to the specific volume of baked product a synergistic 335 effect may be found.

The further enzyme may be a lipase (EC 3.1.1) capable of hydrolysing carboxylic ester bonds to release carboxylate. Examples of Upases include but are not limited to triacylglycerol lipase (EC 3.1.1.3), galactolipase (EC 3.1.1.26), phospholipase Al (EC 3.1.1.32, phospholipase A2 (EC 3.1.1.4) and lipoprotein lipase A2 (EC 3.1.1.34).

[340 OTHER USES

The PS4 variants are suitable for the production of maltose and high maltose syrups. Such products are of considerable interest in the production of certain confectioneries because of the low hygroscoposity, low viscosity, good heat stability and mild, not too sweet taste of maltose. The industrial process of producing maltose syrups 1345 comprises liquefying starch, then saccharification with a maltose producing enzyme, and optionally with an enzyme cleaving the 1.6- branching points in amylopectin, for instance an .alpha.-1.6- amyloglucosidase.

The PS4 variants described here may be added to and thus become a component of a detergent composition. The detergent composition may for example be formulated as a

1350 hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations. In a specific aspect, we describe a detergent additive comprising the PS4 variant. The detergent

1355 additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase. In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other

1360 enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts. The PS4 variant may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where amylases can facilitate the

.365 disintegration of the waste material through degradation of the reinforcing starch. The PS4 variants may especially be useful in a process for producing a papermaking pulp from starch-coated printed paper. The process may be performed as described in WO 95/14807, comprising the following steps: a) disintegrating the paper to produce a pulp, b) treating with a starch-degrading enzyme before, during or after step a), and c) separating ink

.370 particles from the pulp after steps a) and b). The PS4 variant may also be very useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the PS4 variants described here it becomes possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process. A PS4 variant may also be very useful in textile

[375 desizing. In the textile processing industry, amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is import-ant to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch break-down is preferred

1380 because it does not involve any harmful effect on the fiber material. The PS4 variant may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.

The PS4 variant may also be an amylase of choice for production of sweeteners from starch A "traditional" process for conversion of starch to fructose syrups normally

1385 consists of three consecutive enzymatic processes, viz., a liquefaction process followed by a saccharification process and an isomerization process. During the liquefaction process, starch is degraded to dextrins by an amylase at pH values between 5.5 and 6.2 and at temperatures of 95-160° C. for a period of approx. 2 hours. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium

1390 ions). After the liquefaction process the dextrins are converted into dextrose by addition of a glucoamylase and a debranching enzyme, such as an isoamylase or a pullulanase . Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95° C), and the liquefying .amylase activity is denatured. The temperature is lowered to 60° C, and glucoamylase and debranching enzyme are added. The

1395 saccharification process proceeds for 24-72 hours.

BIOETHANOL PRODUCTION

The PS4 variant polypeptide of the invention may in general be used to convert starch into sugars that can then be processed into ethanol or other value-added products such as high fructose corn sweetener. Thus, we disclose the use of PS4 variant 1-00 polypeptides in the production of bioethanol, which in this document should be regarded as any ethanol produced by biomass fermentation

The ethanol so produced may be used as a fuel or beverage or may be used in a fermentation process for producing organic compounds, such as citric acid, ascorbic acid, lysine, glutamic acid.

405 Ethanol (or ethyl alcohol) is best known as being the basis of alcoholic beverages like spirits, beer and wine. In addition, ethanol has many uses in the production of industrial chemicals, pharmaceuticals and as a transportation fuel.

Ethanol can be produced from almost any raw material containing sugar or carbohydrates. As such, ethanol can be made from a wide variety of biological material. 410 The 3 major types of biomass feedstocks used to produce ethanol include sugar crops, such as sugar cane; starch crops, including wheat and corn, and cellulosic materials, such as crop residues (straw, etc.), and forestry waste. Ethanol production from readily available sources of cellulose provides a stable, renewable fuel source.

The processing technology most frequently used is dry grain milling. In this L 415 process, the grain is first milled to a grain meal consistency. The meal is then mixed with water and amylase and passed through cookers where the starch in the grain is liquefied. Under the addition of gluco-amylase the liquefied starch is converted into fermentable sugars. Yeast is then added to the mash to ferment the sugars to ethanol. After fermentation, the mash goes through a distillation and dehydration process where the 1420 alcohol is removed from the solids and the water. In practice about two thirds of each tonne of grain is converted to fuel ethanol. The remaining by-products - thin stillage and wet distillers grain - are a high protein livestock feed which is particularly well suited for animals such as cattle or sheep.

Ethanol may also be made from cellulose containing sources, such as wood pulp. 1425 Cellulose-based feedstocks are comprised of agricultural wastes, grasses and woods and other low- value biomass such as municipal waste (e.g., recycled paper, yard clippings, etc.). Ethanol may be produced from the fermentation of any of these cellulosic feedstocks. However, the cellulose must first be converted to sugars before there can be conversion to ethanol, by treatment with a suitable enzyme such as cellulase.

1430 Once ethanol leaves the processing plant, it can theoretically be used as an automotive fuel by itself or it can be mixed with gasoline at a ratio of 85 to 15 to form what is called "neat ethanol fuel". However, most commonly, ethanol is blended with gasoline at concentrations of 7 to 10 % by volume. The ethanol may be used as an octane enhancer. Ethanol as a fuel source is more environmentally friendly than petroleum 435 derived products. It is known that the use of ethanol will improve air quality and possibly reduce local ozone levels and smog. Moreover, utilization of ethanol in lieu of gasoline can be of strategic importance in buffering the impact of sudden shifts in non-renewable energy and petro-chemical supplies.

FEED APPLICATIONS

440 In one embodiment, the PS4 variant polypeptide is capable of degrading resistant starch.

As used herein the term 'degrading' relates to the partial or complete hydrolysis or degradation of resistant starch to glucose and/or oligosaccharides - such as maltose and/or dextrins.

445 The PS4 variant polypeptide may degrade residual resistant starch that has not been completely degraded by an animals amylase. By way of example, the PS4 variant polypeptide may be used to assist an animal's amylase (eg. pancreatic amylase) in improving the degradation of resistant starch. Pancreatic α-amylase is excreted in the digestive system by animals. Pancreatic α-amylase degrades starch in the feed. However, a

450 part of the starch, the resistant starch, is not degraded fully by the pancreatic α-amylase and is therefore not absorbed in the small intestine (see definition of resistant starch). The PS4 variant polypeptide in some embodiments is able to assist the pancreatic α-amylase in degrading starch in the digestive system and thereby increase the utilisation of starch by the animal.

1455 The ability of an enzyme to degrade resistant starch may be analysed for example by a method developed and disclosed by Megazyme International Ireland Ltd. for the measurement of resistant starch, solubilised starch and total starch content of a sample (Resistant Starch Assay Procedure, AOAC Method 2002.02, AACC Method 32-40).

Accordingly, the PS4 variant polypeptides may be ingested by an animal for 1460 beneficial purposes, and may therefore be incorporated into animal feeds.

We therefore disclose the use of a PS4 variant polypeptide as a component for use in a feed comprising starch, or for use in a feed improvement composition, in which the PS4 variant polypeptide is capable of degrading resistant starch. We also disclose a feed comprising a starch and a PS4 variant polypeptide. We further disclose a method of 1465 degrading resistant starch in a feed comprising contacting said resistant starch with a PS4 variant polypeptide. We further describe the use of a PS4 variant polypeptide in the preparation of a feed comprising a starch, to degrade resistant starch. Furthermore, we disclose the use of a PS4 variant polypeptide in the preparation of a feed to improve the calorific value of said 470 feed. We disclose the use of an enzyme in the preparation of a feed to improve animal performance. In a further embodiment, we describe a process for preparing a feed comprising admixing a starch and a PS4 variant polypeptide enzyme.

By way of example, use of a component comprising PS4 variant polypeptides and which is capable of degrading resistant starch is advantageous because there is a marked

475 increase in the degradation of starch and/or starch degradation products in an animal. Furthermore, such use is advantageous because there is a marked increase in the digestibility of starch and/or starch degradation products by an animal. Furthermore, such use is advantageous because it provides a means of enhancing the efficiency of deriving energy from a feed by an animal. Furthermore, such use is advantageous because it

480 provides a means to enhance the bioavailability of resistant starch.

ANIMAL FEEDS

Animal feeds for which the PS4 variant polypeptides are suitable for use may be formulated to meet the specific needs of particular animal groups and to provide the necessary carbohydrate, fat, protein and other nutrients in a form that can be metabolised 485 by the animal.

Preferably, the animal feed is a feed for swine or poultry.

As used herein the term 'swine' relates to non-ruminant omnivores such as pigs, hogs or boars. Typically, swine feed includes about 50 percent carbohydrate, about 20 percent protein and about 5% fat. An example of a high energy swine feed is based on

1490 corn which is often combined with feed supplements for example, protein, minerals, vitamins and amino acids such as lysine and tryptophan. Examples of swine feeds include animal protein products, marine products, milk products, grain products and plant protein products, all of which may further comprise natural flavourings, artificial flavourings, micro and macro minerals, animal fats, vegetable fats, vitamins, preservatives or

1495 medications such as antibiotics.

It is to be understood that where reference is made in the present specification, including the accompanying claims, to 'swine feed' such reference is meant to include "transition" or "starter" feeds (used to wean young swine) and "finishing" or "grower" feeds (used following the transition stage for growth of swine to an age and/or size 1500 suitable for market). As used herein the term 'poultry' relates to fowl such as chickens, broilers, hens, roosters, capons, turkeys, ducks, game fowl, pullets or chicks. Poultry feeds may be referred to as "complete" feeds because they contain all the protein, energy, vitamins, minerals, and other nutrients necessary for proper growth, egg production, and health of 505 the birds. However, poultry feeds may further comprise vitamins, minerals or medications such as coccidiostats (for example Monensin sodium, Lasalocid, Amprolium, Salinomycin, and Sulfaquinoxaline) and/or antibiotics (for example Penicillin, Bacitracin, Chlortetracycline, and Oxytetracycline).

Young chickens or broilers, turkeys and ducks kept for meat production are fed 510 differently from pullets saved for egg production. Broilers, ducks and turkeys have larger bodies and gain weight more rapidly than do the egg-producing types of chickens. Therefore, these birds are fed diets with higher protein and energy levels.

It is to be understood that where reference is made in the present specification, including the accompanying claims, to 'poultry feed' such reference is meant to include 1515 "starter" feeds (post-hatching), "finisher", "grower" or "developer" feeds (from 6-8 weeks of age until slaughter size reached) and "layer" feeds (fed during egg production).

Animal feeds may be formulated to meet the animal's nutritional needs with respect to, for example, meat production, milk production, egg production, reproduction and response to stress. In addition, the animal feeds are formulated to improve manure 1520 quality.

In a preferred aspect the animal feed contains a raw material such as a legume, for example pea or soy or a cereal, for example wheat, corn (maize), rye or barley. Suitably, the raw material may be potato.

FEED STUFFS

1525 The PS4 variant polypeptides may be used in feeds for animal consumption by the indirect or direct application of the PS4 variant polypeptides to the feed, whether alone or in combination with other ingredients, such as food ingredients.

Typical food ingredients may include any one or more of an additive such as an animal or vegetable fat, a natural or synthetic seasoning, antioxidant, viscosity modifier, 1530 essential oil, and/or flavour, dye and/or colorant, vitamin, mineral, natural and/or non- natural amino acid, nutrient, additional enzyme (including genetically manipulated enzymes), a binding agent such as guar gum or xanthum gum, buffer, emulsifier, lubricant, adjuvant, suspending agent, preservative, coating agent or solubilising agent and the like. Examples of the application methods include, hut are not limited to, coating the 35 feed in a material comprising the PS4 variant polypeptide, direct application by mixing the PS4 variant polypeptide with the feed, spraying the PS4 variant polypeptide onto the feed surface or dipping the feed into a preparation of the PS4 variant polypeptide.

The PS4 variant polypeptide is preferably applied by mixing it with a feed or by spraying onto feed particles for animal consumption. Alternatively, the PS4 variant 540 polypeptide may be included in the emulsion of a feed, or the interior of solid products by injection or tumbling.

The PS4 variant polypeptide may be applied to intersperse, coat and/or impregnate a feed. Mixtures with other ingredients may also be used and may be applied separately, simultaneously or sequentially. Chelating agents, binding agents, emulsifiers and other 545 additives such as micro and macro minerals, amino acids, vitamins, animal fats, vegetable fats, preservatives, flavourings, colourings, may be similarly applied to the feed simultaneously (either in mixture or separately) or applied sequentially.

Amount ofPS4 Variant Polypeptide

The optimum amount of the PS4 variant polypeptide to be used will depend on the 550 feed to be treated and/or the method of contacting the feed with the PS4 variant polypeptide and/or the intended use for the same. The amount of PS4 variant polypeptide should be in a sufficient amount to be effective to substantially degrade resistant starch following ingestion and during digestion of the feed.

Advantageously, the PS4 variant polypeptide would remain effective following [555 ingestion of a feed for animal consumption and during digestion of the feed until a more complete digestion of the feed is obtained, i.e. an increased calorific value of the feed is released.

AMYLASE COMBINATIONS

We disclose in particular combinations of PS4 variant polypeptides with amylases, 1560 in particular, maltogenic amylases. Maltogenic alpha-amylase (glucan 1 ,4-a- maltohydrolase, E.G. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration.

A maltogenic alpha-amylase from Bacillus (EP 120 693) is commercially available under the trade name Novamyl (Novo Nordisk A/S, Denmark) and is widely used in the 1565 baking industry as an anti-staling agent due to its ability to reduce retrogradation of starch. Novamyl is described in detail in International Patent Publication WO 91/04669. The maltogenic alpha-amylase Novamyl shares several characteristics with cyclodextrin glucanotransferases (CGTases), including sequence homology (Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) and formation of transglycosylation products 70 (Christophersen, C, et al, 1997, Starch, vol. 50, No. 1, 39-45).

In highly preferred embodiments, we disclose combinations comprising PS4 variant polypeptides together with Novamyl or any of its variants. Such combinations are useful for food production such as baking. The Novamyl may in particular comprise Novamyl 1500 MG.

575 Other documents describing Novamyl and its uses include Christophersen, C,

Pedersen, S., and Christensen, T., (1993) Method for production of maltose an a limit dextrin, the limit dextrin, and use of the limit dextrin. Denmark, and WO 95/10627. It is further described in U.S. Pat. No. 4,598,048 and U.S. Pat. No. 4,604,355. Each of these documents is hereby incorporated by reference, and any of the Novamyl polypeptides

580 described therein may be used in combinations with any of the PS4 variant polypeptides described here.

Variants, homologues, and mutants of Novamyl may be used for the combinations, provided they retain alpha amylase activity. For example, any of the Novamyl variants disclosed in US Patent Number 6,162,628, the entire disclosure of which is hereby

585 incorporated by reference, may be used in combination with the PS4 variant polypeptides described here. In particular, any of the polypeptides described in that document, specifically variants of SEQ ID NO:1 of US 6,162,628 at any one or more positions corresponding to Q13, 116, D17, N26, N28, P29, A30, S32, Y33, G34, L35, K40, M45, P73, V74, D76 N77, D79, N86, R95, N99, 1100, H103, Ql 19, N120, N131, S141, T142,

590 A148, N152, A163, H169, N171, G172, 1174, N176, N187, F188, A192, Q201, N203, H220, N234, G236, Q247, K249, D261, N266, L268, R272, N275, N276, V279, N280, V281, D285, N287, F297, Q299, N305, K316, N320, L321, N327, A341, N342, A348, Q365, N371, N375, M378, G397, A381, F389, N401, A403, K425, N436, S442, N454, N468, N474, S479, A483, A486, V487, S493, T494, S495, A496, S497, A498, Q500,

1595 N507, 1510, N513, K520, Q526, A555, A564, S573, N575, Q581, S583, F586, K589, N595, G618, N621, Q624, A629, F636, K645, N664 and/or T681 may be used.

AMINO ACΪD SEQUENCES

The invention makes use of a PS4 variant nucleic acid, and the amino acid sequences of such PS4 variant nucleic acids are encompassed by the methods and 1600 compositions described here.

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

605 The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

The PS4 variant enzyme described here may be used in conjunction with other enzymes. Thus we further disclose a combination of enzymes wherein the combination comprises a PS4 variant polypeptide enzyme described here and another enzyme, which itself 610 may be another PS4 variant polypeptide enzyme.

PS4 VARIANTNUCLEOTIDE SEQUENCE

As noted above, we disclose nucleotide sequences encoding the PS4 variant enzymes having the specific properties described.

The term "nucleotide sequence" or "nucleic acid sequence" as used herein refers to 1615 an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.

The term "nucleotide sequence" as used in this document includes genomic DNA, 1620 cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for a PS4 variant polypeptide.

Typically, the PS4 variant nucleotide sequence is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods 1625 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).

PREPARATION OF NUCLEIC ACID SEQUENCES

A nucleotide sequence encoding either an enzyme which has the specific properties as defined herein (e.g., a PS4 variant polypeptide) or an enzyme which is suitable for

1630 modification, such as a parent enzyme, may be identified and/or isolated and/or purified from any cell or organism producing said enzyme. Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or

1635 purified. By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme or a part of the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and 1640 used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.

1645 Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming enzyme- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for enzyme (i.e. maltose), thereby allowing clones expressing the enzyme to be identified.

1650 In a yet further alternative, the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al, (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al, (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA

1655 synthesiser, purified, annealed, ligated and cloned in appropriate vectors.

The nucleotide sequence maybe of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire 1660 nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al, {Science (1988) 239, pp 487-491).

VARIANTS/HOMOLOGUES/DERIVATIVES

We further describe the use of variants, homologues and derivatives of any amino 1665 acid sequence of an enzyme or of any nucleotide sequence encoding such an enzyme, such as a PS4 variant polypeptide or a PS4 variant nucleic acid. Unless the context dictates otherwise, the term "PS4 variant nucleic acid" should be taken to include each of the nucleic acid entities described below, and the term "PS4 variant polypeptide" should likewise be taken to include each of the polypeptide or amino acid entities described below. [670 Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include an amino acid sequence which maybe at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 1675 98 or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of this document it is preferred to express homology in terms of sequence identity.

1680 In the present context, an homologous sequence is taken to include a nucleotide sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding a PS4 variant polypeptide enzyme (such as a PS4 variant nucleic acid). Typically, the homologues will comprise the same sequences that code for the active sites etc as the subject sequence. Although homology

1685 can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of this document it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer 1690 programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only 1695 over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is 1700 performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology. However, these more complex methods assign "gap penalties" to each gap that 1705 occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affϊne gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used 1710 gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

1715 Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see

1720 Ausubel et al, 1999 Short Protocols in Molecular Biology, 4^th Ed - Chapter 18), FASTA (Altschul et al, 1990 J. MoI. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfit program. A 1725 new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison.

1730 Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for

1735 further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to 740 CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The sequences may also have deletions, insertions or substitutions of amino acid

.745 residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based on the properties of their

1750 side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone CD. and Barton GJ. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" ComputAppl Biosci. 9: 745-756)(Taylor W.R. (1986) "The classification of

1755 amino acid conservation" J.Theor.Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.

We further disclose sequences comprising homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid

1760 residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 765 O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β- alanine residues. A further form of variation, involves the presence of one or more amino

770 acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α- carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al, PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends

775 Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences described here, and suitable for use in the methods and compositions described here (such as PS4 variant nucleic acids) may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and 780 phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of this document, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences.

785 We further describe the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the PS4 variant sequences may 790 be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be 795 obtained by probing cDNA libraries made from or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences described here.

800 Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the

805 GCG Wisconsin PiIeUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides maybe obtained by site directed mutagenesis of [810 characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes maybe desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

815 The polynucleotides (nucleotide sequences) such as the PS4 variant nucleic acids described in this document may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15,

820 preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides.

Polynucleotides such as DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques, m general, primers will be produced by synthetic 825 means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. The primers may be 830 designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Preferably, the variant sequences etc. are at least as biologically active as the sequences presented herein. As used herein "biologically active" refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but 535 not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.

HYBRIDISATION

We further describe sequences that are complementary to the nucleic acid sequences of PS4 variants or sequences that are capable of hybridising either to the PS4 840 variant sequences or to sequences that are complementary thereto.

The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies. Therefore, we disclose the use of nucleotide sequences that are capable of 845 hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.

The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein.

Preferably, the term "variant" encompasses sequences that are complementary to 850 sequences that are capable of hybridising under stringent conditions (e.g. 5O⁰C and 0.2xSSC {lxSSC = 0.15 M NaCl, 0.015 M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein. More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e.g. 65⁰C and 0.IxSSC {lxSSC = 0.15 M NaCl, 0.015 M Na₃citrate pH 7.0}) 1855 to the nucleotide sequences presented herein.

We further disclose nucleotide sequences that can hybridise to the nucleotide sequences of PS4 variants (including complementary sequences of those presented herein), as well as nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of PS4 variants (including complementary sequences of those 1860 presented herein). We further describe polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.

In a preferred aspect, we disclose nucleotide sequences that can hybridise to the nucleotide sequence of a PS4 variant nucleic acid, or the complement thereof, under 1865 stringent conditions (e.g. 5O⁰C and 0.2xSSC). More preferably, the nucleotide sequences can hybridise to the nucleotide sequence of a PS4 variant, or the complement thereof, under high stringent conditions (e.g. 65⁰C and 0. IxSSC).

SITE-DIRECTED MUTAGENESIS

Once an enzyme-encoding nucleotide sequence has been isolated, or a putative 870 enzyme-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare an enzyme. Accordingly, a PS4 variant sequence may be prepared from a parent sequence. Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.

875 A suitable method is disclosed in Morinaga et al, {Biotechnology (1984) 2, p646-

649). Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147- 151). A further method is described in Sarkar and Sommer (Biotechniques (1990), 8, p404-407 - "The megaprimer method of site directed mutagenesis").

880 In one aspect the sequence for use in the methods and compositions described here is a recombinant sequence — i.e. a sequence that has been prepared using recombinant DNA techniques. These recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A

885 Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.

In one aspect the sequence for use in the methods and compositions described here is a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.

890 The nucleotide sequence for use in the methods and compositions described here may be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in enzyme form, in and/or from a compatible host cell. Expression may be controlled using control sequences eg. regulatory sequences. The enzyme produced by a host recombinant cell by expression of the

895 nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences may be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane. EXPRESSION OF PS4 NUCLEIC ACIDS AND POLYPEPTIDES

500 The PS4 polynucleotides and nucleic acids may include DNA and RNA of both synthetic and natural origin which DNA or RNA may contain modified or unmodified deoxy- or dideoxy- nucleotides or ribonucleotides or analogs thereof. The PS4 nucleic acid may exist as single- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNA copolymer, wherein the term "copolymer" refers to a single nucleic acid strand

905 that comprises both ribonucleotides and deoxyribonucleotides. The PS4 nucleic acid may even be codon optimised to further increase expression.

The term "synthetic", as used herein, is defined as that which is produced by in vitro chemical or enzymatic synthesis. It includes but is not limited to PS4 nucleic acids made with optimal codon usage for host organisms such as the the methylotrophic yeasts 910 Pichia and Hansenula.

Polynucleotides, for example variant PS4 polynucleotides described here, can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. The vector comprising the polynucleotide sequence may be transformed into a suitable host cell. Suitable hosts may include bacterial, yeast, 915 insect and fungal cells.

The term "transformed cell" includes cells that have been transformed by use of recombinant DNA techniques. The transformation typically occurs by insertion of one or more nucleotide sequences into a cell that is to be transformed. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e. is a sequence that is not natural 920 to the cell that is to be transformed. In addition, or in the alternative, the inserted nucleotide sequence may be an homologous nucleotide sequence (i.e. is a sequence that is natural to the cell that is to be transformed) - so that the cell receives one or more extra copies of a nucleotide sequence already present in it.

Thus in a further embodiment, we provide a method of making PS4 variant 925 polypeptides and polynucleotides by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector maybe recovered from the host cell.

EXPRESSION CONSTRUCTS

930 The PS4 nucleic acid may be operatively linked to transcriptional and translational regulatory elements active in a host cell of interest. The PS4 nucleic acid may also encode a fusion protein comprising signal sequences such as, for example, those derived from the glucoamylase gene from Schwanniomyces occidentalis, α-factor mating type gene from Saccharomyces cerevisiae and the TAKA-amylase from Aspergillus oryzae. Alternatively, ?35 the PS4 nucleic acid may encode a fusion protein comprising a membrane binding domain.

Expression Vector

The PS4 nucleic acid may be expressed at the desired levels in a host organism using an expression vector.

?40 An expression vector comprising a PS4 nucleic acid can be any vector which is capable of expressing the gene encoding PS4 nucleic acid in the selected host organism, and the choice of vector will depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomously replicating vector, i.e. a vector that exists as an episomal entity, the replication of which is independent of chromosomal replication, such

945 as, for example, a plasmid, a bacteriophage or an episomal element, a minichromosome or an artificial chromosome. Alternatively, 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.

Components of the Expression Vector

950 The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences encoding a promoter, operator, ribosome binding site, translation initiation signal and

955 optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the PS4 variant polypeptide to a host cell organelle such as a peroxisome or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence SKL. In the present context, the term 'expression signal" includes any of the

960 above control sequences, repressor or activator sequences. For expression under the direction of control sequences, the nucleic acid sequence the PS4 variant polypeptide is operably linked to the control sequences in proper manner with respect to expression.

Preferably, a polynucleotide in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. 965 the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

>70 The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.

Promoter

)75 In the vector, the nucleic acid sequence encoding for the variant PS4 polypeptide is operably combined with a suitable promoter sequence. The promoter can be any DNA sequence having transcription activity in the host organism of choice and can be derived from genes that are homologous or heterologous to the host organism.

Bacterial Promoters

980 Examples of suitable promoters for directing the transcription of the modified nucleotide sequence, such as PS4 nucleic acids, in a bacterial host include the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the

985 Bacillus amyloliquefaciens α-amylase gene (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes, the promoter of the Bacillus subtilis aprE gene and a promoter derived from a Lactococcus sp.-derived promoter including the P170 promoter. When the gene encoding the PS4 variant polypeptide is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter

990 including a T7 promoter and a phage lambda promoter.

Fungal Promoters

For transcription in a fungal species, examples of useful promoters are those derived from the genes encoding the, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, A. niger acid stable α- 995 amylase, A. niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase. Yeast Promoters

Examples of suitable promoters for the expression in a yeast species include but )00 are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOXl or A0X2 promoters.

HOST ORGANISMS

(I) Bacterial Host Organisms

Examples of suitable bacterial host organisms are gram positive bacterial species 305 such as Bacillaceae including Bacillus clausii, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis, Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp. 010 including Lactobacillus reuteri, Leuconostoc spp., Pediococcus spp. and Streptococcus spp. Alternatively, strains of a gram-negative bacterial species belonging to Enterobacteriace&e including E. coli, or to Pseudomonadaceae can be selected as the host organism.

(II) Yeast Host Organisms

015 A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp or Kluyveromyces, Yarrowinia species or a species of Saccharomyces including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyce such as, for example, S. Pombe species.

020 Preferably a strain of the methylotrophic yeast species Pichia pastoris is used as the host organism. Preferably the host organism is a Hansenula species.

(III) Fungal Host Organisms

Suitable host organisms among filamentous fungi include species of Aspergillus, e.g. Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori or 025 Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g. Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species.

Suitable fungal host organisms may also include Trichoderma spp (especially Trichoderma reesei formerly Trichoderma longibrachiatum; also known as Hypocrea ,030 jecorina). PROTEIN EXPRESSION AND PURIFICATION

Host cells comprising polynucleotides may be used to express polypeptides, such as variant PS4 polypeptides, fragments, homologues, variants or derivatives thereof. Host cells may be cultured under suitable conditions which allow expression of the proteins. 335 Expression of the polypeptides may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.

Polypeptides can be extracted from host cells by a variety of techniques known in 040 the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.

Polypeptides may also be produced recombinantly in an in vitro cell-free system, such as the TnT™ (Promega) rabbit reticulocyte system.

EXAMPLES

Example 1. Cloning of PS4

045 Cloning of Ps eudomonas sacharophila non-maltogenic exoamylase PS4 and the generation of plasmids pCSmta and pCSmta-SBD is described in WO 2005/003339, particularly Example 1.

Site directed mutagenesis (SDM) may be conducted using the methods described in WO 2005/003339, particularly in Example 2 of that document.

050 Example 2. Multi SDM

The PS4 variants were generated using a QuikChange^® Multi Site Directed Mutagenesis Kit (Stratagene) according to the manufactures protocol with some modifications as described.

Step 1: Mutant Strand Synthesis Reaction (PCR)

055 Inoculate 3ml. LB (22g/l Lennox L Broth Base, Sigma) + antibiotics (0,05 μg/ml kanamycin, Sigma) in a 10ml Falcon tube

- Incubate o/n 37°C, ca. 200 rpm.

- Spin down the cells by centrifugation (5000 rpm/5 min) Poor off the medium

,060 - Prepare ds-DNA template using QIAGEN Plasmid Mini Purification Protocol

1. The mutant strand synthesis reaction for thermal cycling was prepared as follow: PCR Mix:

)65 2,5 μl 1 OX QuickChange^® Multi reaction buffer

0,75 μl QuickSolution

X μl Primers primer length 28-35 bp -^ 10 pmol^" primer length 24-27 bp -> 7 pmol

370 primer length 20-23 bp -^ 5 pmol

1 μl dNTP mix

X μl ds-DNA template (200 ng)

1 μl QuickChange^® Multi enzyme blend (2,5 U/μl) (PfuTurbo^® DNA polymerase)

375 X μl dH₂O (to a final volume of 25 μl)

Mix all components by pipetting and briefly spin down the reaction mixtures.

2. Cycle the reactions using the following parameters: 080 35 cycles of denaturation (96°C/lmin) primer annealing (62,8°C/lmin) elongation (65°C/15min) then hold at 4°C

Preheat the lid of the PCR machine to 105°C and the plate to 95⁰C before the PCR 085 tubes are placed in the machine (eppendorf thermal cycler).

Step 2: Dpn /Digestion

1. Add 2 μl Dpn I restriction enzyme (10 U/μl) to each amplification reaction, mix by 090 pipetting and spin down mixture.

2. Incubate at 37°C for ~3 hr.

Step 3: Transformation of XLIO-Gold^® Ultracompetent Cells

095 1. Thaw XLl 0-Gold cells on ice. Aliquot 45 μl cells per mutagenesis reaction to prechilled Falcon tubes.

2. Turn on the waterbath (42°C) and place a tube with NZY⁺ broth in the bath to preheat.

3. Add 2 μl β-mercaptoethanol mix to each tube. Swirl and tap gently and incubate 100 10 min on ice, swirling every 2 min.

4. Add 1 ,5 μl Dpn /-treated DNA to each aliquot of cells, swirl to mix and incubate on ice for 30 min.

5. Heat-pulse the tubes in 42⁰C waterbath for 30 s and place on ice for 2 min.

6. Add 0.5 ml preheated NZY⁺ broth to each tube and incubate at 37⁰C for lhr with ,105 shaking at 225-250 rpm.

7. Plate 200 μl of each transformation reaction on LB plates (33,6 g/1 Lennox L Agar, Sigma) containing 1% starch and 0,05 μg/ml kanamycin

8. Incubate the transformation plates at 37⁰C overnight. [ 10 "SDM" primers may be used to modify the specified positions using a method described in Example 2 of WO 2005/003339. "MSDM" -primers may be used with the method described in Example 3 herein.

115 Example 3. Transformation into Bacillus subtilis (Protoplast Transformation)

Bacillus subtilis (strain DB104A; Smith et al. 1988; Gene 70, 351-361) is transformed with the mutated plasmids according to the following protocol.

A. Media for protoplasting and transformation

120 2 x SMM per litre: 342 g sucrose (1 M); 4.72 g sodium maleate (0.04 M); 8:12 g MgCl₂,6H₂0 (0.04 M); pH 6.5 with concentrated NaOH. Distribute in 50-ml portions and autoclave for 10 min.

:125 4 x YT (1/2 NaCl) 2 g Yeast extract + 3.2 g Tryptone + 0.5 g NaCl per 100 ml.

SMMP mix equal volumes of 2 x SMM and 4 x YT.

PEG 10 g polyethyleneglycol 6000 (BDH) or 8000 (Sigma) in 25 ml 1 x SMM (autoclave for 10 min.).

:i30 B. Media for plating/regeneration

agar 4% Difco minimal agar. Autoclave for 15 min. sodium succinate 270 g/1 (1 M), pH 7.3 with HCl. Autoclave for 15 min.

»135 phosphate buffer 3.5 g K₂HPO₄ + 1.5 g KH₂PO₄ per 100ml. Autoclave for 15 min.

MgCl₂ 20.3 g MgCl₂, 6H₂O per 100 ml (1 M).

^l 40 casamino acids 5% (w/v) solution. Autoclave for 15 min. yeast extract 1O g per 100 ml, autoclave for 15 min. glucose 20% (w/v) solution. Autoclave for 10 min.

DM3 regeneration medium: mix at 60 C (waterbath; 500-ml bottle): »145

250 ml sodium succinate

50 ml casamino acids

25 ml yeast extract

50 ml phosphate buffer >150 15 ml glucose

1O mI MgCl₂

100 ml molten agar

Add appropriate antibiotics: chloramphenicol and tetracycline, 5 ug/ml; Zl 55 erythromycin, 1 ug/ ml. Selection on kanamycin is problematic in DM3 medium: concentrations of 250 ug/ml may be required.

C. Preparation of protoplasts

1. Use detergent-free plastic or glassware throughout.

2160 2. Inoculate 10 ml of 2 x YT medium in a 100-ml flask from a single colony.

Grow an overnight culture at 25-30 C in a shaker (200 rev/min).

3. Dilute the overnight culture 20 fold into 100 ml of fresh 2 x YT medium (250-ml flask) and grow until OD₆₀₀ = 0.4-0.5 (approx. 2h) at 37C in a shaker (200-250 rev/min). 2165 4. Harvest the cells by centrifugation (900Og, 20 min, 4 C).

5. Remove the supernatant with pipette and resuspend the cells in 5 ml of SMMP + 5 mg lysozyme, sterile filtered.

6. Incubate at 37 C in a waterbath shaker (100 rev/min).

After 30 min and thereafter at 15 min intervals, examine 25 ul samples by 2170 microscopy. Continue incubation until 99% of the cells are protoplasted (globular appearance). Harvest the protoplasts by centrifugation (4000g, 20 min, RT) and pipet off the supernatant. Resuspend the pellet gently in 1-2 ml of SMMP.

The protoplasts are now ready for use. (Portions (e.g. 0.15 ml) can be frozen at -80 C for future use (glycerol addition is not required). Although this may result in some 2175 reduction of transformability, 106 transformants per ug of DNA can be obtained with frozen protoplasts). D. Transformation

1. Transfer 450 ul of PEG to a microtube.

2. Mix 1-10 ul of DNA (0.2 ug) with 150 ul of protoplasts and add the

:i80 mixture to the microtube with PEG. Mix immediately, but gently.

3. Leave for 2 min at RT, and then add 1.5 ml of SMMP and mix.

4. Harvest protoplasts by microfuging (10 min, 13.000 rev/min (10-12.000 g)) and pour off the supernatant. Remove the remaining droplets with a tissue.

Add 300 ul of SMMP (do not vortex) and incubate for 60-90 min at 37 C in a

»185 waterbath shaker (100 rev/min) to allow for expression of antibiotic resistance markers. (The protoplasts become sufficiently resuspended through the shaking action of the waterbath.). Make appropriate dilutions in 1 x SSM and plate 0.1 ml on DM3 plates

Example 4. Fermentation of PS4 Variants in Shake Flasks

The shake flask substrate is prepared as follows:

2190 The substrate is adjusted to pH 6.8 with 4N sulfuric acid or sodium hydroxide before autoclaving. 100 ml of substrate is placed in a 500 ml flask with one baffle and autoclaved for 30 minutes. Subsequently, 6 ml of sterile dextrose syrup is added.The dextrose syrup is prepared by mixing one volume of 50% w/v dextrose with one volume of water followed by autoclaving for 20 minutes.

2195 The shake flasks are inoculated with the variants and incubated for 24 hours at 35°C/180rpm in an incubator. After incubation cells are separated from broth by centrifugation (10.000 x g in 10 minutes) and finally, the supernatant is made cell free by microfiltration at 0,2μm. The cell free supernatant is used for assays and application tests.

Example 5. Amylase Assays

2200 Betamyl assay

One Betamyl unit is defined as activity degrading 0,0351 mmole per 1 min. of PNP-coupled maltopentaose so that 0,0351 mmole PNP per 1 min. can be released by excess a-glucosidase in the assay mix. The assay mix contains 50 ul 50 mM Na-citrate, 5 mM CaC12, pH 6,5 with 25 ul enzyme sample and 25 ul Betamyl substrate (Glc5-PNP and 205 a-glucosidase) from Megazyme, Ireland (1 vial dissolved in 10 ml water). The assay mix is incubated for 30 min. at 4OC and then stopped by adding 150 ul 4% Tris. Absorbance at 420 nm is measured using an ELISA-reader and the Betamyl activity is calculate based on Activity = A420 * d in Betamyl units/ml of enzyme sample assayed.

Endo-amylase assay

210 The endo-amylase assay is identical to the Phadebas assay run according to manufacturer (Pharmacia & Upjohn Diagnostics AB).

Exo-specificity

The ratio of exo-amylase activity to Phadebas activity was used to evaluate exo- specificity.

:215 Specific activity

For the pSac-D14, pSac-D20 and pSac-D34 variants we find an average specific activity of 10 Betamyl units per microgram of purified protein measured according to Bradford (1976; Anal. Biochem. 72, 248). This specific activity is used for based on activity to calculate the dosages used in the application trials.

»220 Example 6. Half-life Determination

tl/2 is defined as the time (in minutes) during which half the enzyme activity is inactivated under defined heat conditions. In order to determine the half life of the enzyme, the sample is heated for 1-40 minutes at constant temperatures of 60°C to 90°C. The half life is calculated based on the residual Betamyl assay.

1225 Procedure: In an Eppendorf vial, 1000 μl buffer is preheated for at least 10 minutes at 60°C or higher. The heat treatment of the sample is started addition of 100 μl of the sample to the preheated buffer under continuous mixing (800 rpm) of the Eppendorf vial in an heat incubator (Termomixer comfort from Eppendorf). After 0, 2, 4, 6, 8 and 9 minutes of incubation, the treatment is stopped by transferring 45 μl of the sample to 1000

2230 μl of the buffer equilibrated at 2O⁰C and incubating for one minute at 1500 rpm and at 20°C. The residual activity is measured with the Betamyl assay.

Calculation: Calculation of tl/2 is based on the slope of loglO (the base-10 logarithm) of the residual Betamyl activity versus the incubation time, tl/2 is calculated as Slope/0.301=tl/2. 235 Example 7. Model System Baking Tests

The doughs are made in the Farinograph at 30.0°C. 10.00 g reformed flour is weighed out and added in the Farinograph; after 1 min. mixing the reference/sample (reference = buffer or water, sample = enzyme+ buffer or water) is added with a sterile pipette through the holes of the kneading vat. After 30 sec. the flour is scraped off the 1240 edges - also through the holes of the kneading vat. The sample is kneaded for 7 min.

A test with buffer or water is performed on the Farinograph before the final reference is run. FU should be 400 on the reference, if it is not, this should be adjusted with, for example, the quantity of liquid. The reference/sample is removed with a spatula and placed in the hand (with a disposable glove on it), before it is filled into small glass Ϊ245 tubes (of approx. 4.5 em's length) that are put in NMR tubes and corked up. 7 tubes per dough are made.

When all the samples have been prepared, the tubes are placed in a (programmable) water bath at 33°C (without corks) for 25 min. and hereafter the water bath is set to stay for 5 min. at 33⁰C, then to heated to 98°C over 56 min. (1.1⁰C per Z250 minute) and finally to stay for 5 min. at 96°C.

The tubes are stored at 20.0⁰C in a thermo cupboard. The solid content of the crumb was measured by proton NMR using a Bruker NMS 120 Minispec NMR analyser at day 1, 3 and 7 as shown for crumb samples prepared with 0, 05, 1 abnd 2 ppm pSac- D34 in Fig. 2. The lower increase in solid content over time represents the reduction in 2255 amylopectin retrogradation. After 7 days of storage at 20.0⁰C in a thermo cupboard 10-20 mg samples of crumb weighed out and placed in 40 μl aluminium standard DSC capsules and kept at 20⁰C.

The capsules are used for Differential Scanning Calorimetry on a Mettler Toledo DSC 820 instrument. As parameters are used a heating cycle of 20-95⁰C with 10⁰C per 2260 min. heating and Gas/flow: N₂/80 ml per min. The results are analysed and the enthalpy for melting of retrograded amylopectin is calculated in J/g.

Example 8. Antistaling Effects

Model bread crumbs are prepared and measured according to Example 7. PS4 variants show a strong reduction of the amylopectin retrogradation after baking as 2265 measured by Differential Scanning Calorimetry in comparison to the control. The PS4 variants show a clear dosage effect. Example 9. Recipe for Baking Trials

Baking trials were carried out with a standard white bread sponge and dough recipe for US toast. The sponge dough is prepared from 1400 g of flour "Gold Medal" from :270 General Mills, USA, 800 g of water, 40 g of rape seed oil, 7,5 g GRINDSTED™ SSL P55 Veg, 1O g emulsifier DIMOD AN™ PH200 and 60 g of compressed yeast. The sponge is mixed for 1 min. at low speed and subsequently 3 min. at speed 2 on a Hobart spiral mixer. The sponge is subsequently fermented for 3 hours at 25°C, 85% RH.

Thereafter, 600 g of "Gold Medal" flour, 18 g of compressed yeast, 5 g of calcium .275 propionate, 16O g of sucrose, 5 g of calcium propionate, 432 g of water and ascorbic acid (60 ppm final concentration) and ADA (azodicarbonamide; 40 ppm final concentration) are added to the sponge. The resulting dough is mixed for 1 min. at low speed and then 2 min. on high speed on a Diosna mixer. Then 30 g of salt is added to the dough.

The dough is rested for 5 min. at ambient temperature, and then 550 g dough 2280 pieces are scaled, moulded on Glimek sheeter with the settings 1 :4, 2:4, 3 : 15, 4: 12 and width 8 on both sides and transferred to a baking form. After 65 min. proofing at 43 °C at 95% RH the doughs are baked for 26 min. at 200⁰C in an MIWE oven.

Example 10. Control of Volume of Danish Rolls

Danish Rolls are prepared from a dough based on 2000 g Danish reform flour 2285 (from Cerealia), 12O g compressed yeast, 32 g salt, and 32 g sucrose. Water is added to the dough according to prior water optimisation.

The dough is mixed on a Diosna mixer (2 min. at low speed and 5 min. at high speed). The dough temperature after mixing is kept at 26°C. 1350 g dough is scaled and rested for 10 min. in a heating cabinet at 3O⁰C. The rolls are moulded on a Fortuna molder 2290 and proofed for 45 min. at 34°C and at 85% relative humidity. Subsequently the rolls are baked in a Bago 2 oven for 18 min. at 250⁰C with steam in the first 13 seconds. After baking the rolls are cooled for 25 min. before weighing and measuring of volume.

The rolls are evaluated regarding crust appearance, crumb homogeneity, capping of the crust, ausbund and specific volume (measuring the volume with the rape seed 2295 displacement method).

Based on these criteria it is found that the PS4 variants increase the specific volume and improve the quality parameters of Danish rolls. Thus PS4 variants are able to control the volume of baked products. Example 11. Protocol for Evaluation of Firmness, Resilience and Cohesiveness

300 Texture Profile Analysis of Bread

Firmness, resilience and cohesiveness are determined by analysing bread slices by Texture Profile Analysis using a Texture Analyser From Stable Micro Systems, UK. Calculation of firmness and resilience is done according to preset standard supplied by Stable Micro System, UK. The probe used is aluminium 50 mm round.

305 Bread is sliced with the width of 12.5 mm. The slices are stamped out to a circular piece with a diameter of 45 mm and measured individually.

The following settings are used:

Pre Test Speed: 2 mrn/s

Test Speed: 2 mm/s

•310 Post Test Speed: 10 mm/s

Rupture Test Distance: 1%

Distance: 40%

Force: 0.098 N

Time: 5.00 sec

>315 Count: 5

Load Cell: 5 kg

Trigger Type: Auto - 0.01 N

The mode of compression is a modification to the one used in Standard method AACC 74-09. The sample is compressed twice in the test. Figure 1 shows an example of a 2320 curve from the Texture Analyser.

Example 12. Protocol for Evaluation of Firmness

Firmness is determined at 40% compression during the first compression. The figure is the force needed to compress the slice to 40% of the total thickness. The lower the value, the softer the bread. The firmness is expressed as a pressure, for example, in 2325 hPa. This assay may be referred to as the "Firmness Evaluation Protocol".

Example 13. Protocol for Evaluation of Resilience

Area under the curve is a measure of work applied during the test. The area under the curve in the compression part (Al) and the withdrawal part (A2) during the first '330 compression are shown in Figure 1.

The ratio between Al and A2 is defined as the resilience of the sample, and is expressed as Resilience Units. True elastic material will give a symmetric curve, as the force applied during the first part will be equal to the force in the second part. For bread and bread-like material, A2 is normally smaller than A2 due to disturbance of the structure .335 during compression. Hence, resilience is always lower than 1.

This assay may be referred to as the "Resilience Evaluation Protocol".

Example 14. Protocol for Evaluation of Cohesiveness

The cohesiveness is defined as the ratio between the area under second compression to the area under first compression (A3/A1+A2), and is expressed as 2.340 Cohesiveness Units. It is a measure of the decay of the sample during compression. The higher the ability of the sample to regain its shape after first compression the closer the value will be to 1. For bread and bread-like material cohesiveness is always lower than 1.

This assay may be referred to as the "Cohesiveness Evaluation Protocol".

Example 15. Enhanced Exo-specificity of PS4 Variant Polypeptide with Mutation

2345 272Q

A PS4 variant polypeptide designated pMD229 having amino acid mutations at N33Y D34N G121F G134R A141P Y146G I157L S161A L178F A179T G223E S229P H272Q G303E H307L A309P S334P is tested for exo-specificity. This polypeptide displays improved exo-specificity as shown in the table below.

:350 The half-life t'/2-85 is determined according to Example 6, after gel-filtration of the samples with PD-IO columns (from Amersham Biosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 16: Firmness Effects of pMD229, 248, 253 and 271 in Baking Trials

355 Baking trials are carried out with a standard white bread sponge and dough recipe for US toast as described in Example 10. Samples of pMD229, 248, 253 and 271 were applied in dosages of the interval 0.1 to 20 mg/kg of flour. The enzyme samples are added to the dough after sponge fermentation together with the remaining ingredients.

Firmness measurements show that the enzymes significantly reduce the firmness 5360 development from day 1 to day 7 and show a higher effect with increasing enzyme dosage.

Example 17. Improved Handling Properties of Food Products Treated with PS4 Variant Polypeptides: Firmness

Bread is baked with 40,000 Betamyl units/kg of pSac-pMD229 and the firmness of the bread is tested according to the protocol set out in Example 12 at various times after 1365 baking. Bread is also baked with 40,000 Betamyl units/kg of pSac-D34 / pMD3 (SEQ ID NO: 2) . The firmness of the bread is tested. As a control, firmness of bread baked without any enzyme is also measured.

Figure 2 shows the results of a baking trial in which firmness of bread treated with pSac-pMD229 is compared to firmness of bread treated with pSac-D34.

2370 Example 18. Improved Handling Properties of Food Products Treated with PS4 Variant Polypeptides: Resilience

Bread is baked with 40,000 Betamyl units/kg of pSac-pMD229 and the resilience of the bread is tested according to the protocol set out in Example 13 at various times after baking. Bread is also baked with 40,000 Betamyl units/kg of pSac-D34 / pMD3 (SEQ ID 2375 NO: 2) . The resilience of the bread is tested. As a control, resilience of bread baked without any enzyme is also measured.

Figure 3 shows the results of a baking trial in which resilience of bread treated with pSac-pMD229 is compared to resilience of bread treated with pSac-D34. Example 19. Improved Handling Properties of Food Products Treated with PS4 1380 Variant Polypeptides: Cohesiveness

Bread is baked with 40,000 Betamyl units/kg of pSac-ρMD229 and the cohesiveness of the bread is tested according to the protocol set out in Example 14 at various times after baking. Bread is also baked with 40,000 Betamyl units/kg of pSac-D34 / pMD3 (SEQ ID NO: 2) . The cohesiveness of the bread is tested. As a control, !385 cohesiveness of bread baked without any enzyme is also measured.

Figure 4 shows the results of a baking trial in which cohesiveness of bread treated with pSac-pMD229 is compared to cohesiveness of bread treated with pSac-D34.

REFERENCES

Penninga, D., van der Veen, B.A., Knegtel, R.M., van Hijum, S.A., Rozeboom, »390 HJ., KaIk, K.H., Dijkstra, B.W., Dijkhuizen, L. (1996). The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. J.Biol. Chem. 271, 32777-32784.

Sambrook J, F.E.M.T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory, Cold Spring Harbor NY.

.395 Zhou,J.H.₅ Baba,T., Takano,T., Kobayashi,S., Arai,Y. (1989). Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila. FEBS Lett. 255, 37- 41.

Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including

Z400 during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any

2405 manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with 2410 specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes "for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

415 SEQUENCE LISTINGS

SEQ ID NO: 1

PS4 reference sequence, derived from Pseudomonas saccharophila maltotetrahydrolase amino acid sequence.

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APJMOTYNILR QQASTIAADG FSAIWMPVPW

420 61 RDFSSWTDGG KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDWPNHMNR

121 GYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFJGGE SDLNTGHPQI YGMFRDELAN

181 LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WKGPSEYPSW DWRNTASWQQ

241 IIKDWSDRAK CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQHHWAL QDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRTAGVRAD

425 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG SFSEAVNASN GQVRVWRSGS

421 GDGGGNDGGE GGLVNVNFRC DNGVTQMGDS VYAVGNVSQL GNWSPASAVR LTDTSSYPTW

481 KGSIALPDGQ NVEWKCLIRN EADATLVRQW QSGGNNQVQA AAGASTSGSF

SEQ ID NO: 2 pSac-D34 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid ,430 sequence with 11 substitutions and deletion of the starch binding domain. pSac-D34 (also known as pMD3) comprises mutations N33Y, D34N,G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L, S334P relative to wild type non-maltogenic exoamylase.

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APIfWWYNILR QQASTIAADG FSAIWMPVPW

1435 61 RDFSSWTDPG KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDWPNHMNR

121 PYPDKEINLP AGQiSFWRNDC PDPGNYPNDC DDGDRFLGGE SDLNTGHPQI YGMFRDEFTN

181 LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WKAPSEYPSW DWRNTASWQQ

241 IIKDWSDRAK CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQKLWAL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ VRRTAGVRAD

1440 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG SFSEAVNASN GQVRVWRSGS

421 GDGGGNDGG

SEQ ID NO: 3 pSac-D20 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with 13 substitutions and deletion of the starch binding domain.

>445 1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APKMWYNILR QQASTIAADG FSAIWMPVPW

61 RDFSSWTDPG ΛSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDWPNHMNR

121 .DYPDKEINLP AGQJRFWRNDC PDPGNYPNDC DDGDRFXGGE SDLNTGHPQI YGMFRDEFTN

181 LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WKAPSEYPSW DWRNTASWQQ

241 IIKDWSDRAK CPVFDFALKE RMQNGSVADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

.450 301 QNGGQHIWAL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGiEFIRQLIQ VRRTAGVRAD

361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVAS¹G SFSEAVNASN GQVRVWRSGS

421 GDGGGNDGG

SEQ IDNO: 4 pSac-D14 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid 2455 sequence with 14 substitutions and deletion of the starch binding domain.

1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APMWYNILR QQASTIAADG FSAIWMPVPW 61 RDFSSWTDPG KSGGGEGYFW HDFNKNSRYG SDAQLRQAAG ALGGAGVKVL YDWPNHMNR 121 I7YPDKEINLP AGQHFWRNDC FDPGNYPNDC DDGDRFXGGE SDLNTGHPQI YGMFRDEFTN 181 LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WKAPSEYPSW DWRNTASWQQ 460 241 IIKDWSDRAK CPVFDFALKE RMQNGSVΛDW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHI-WAL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGEFIRQLIQ VRRTAGVRAD 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG SFSEAVNASN GQVRVWRSGS 421 GDGGGNDGG

SEQ ID NO: 5

465 Pseudomonas saccharophila Glucan 1,4-alpha-maltotetrahydrolase precursor (EC

3.2.1.60) (G4-amylase) (Maltotetraose-forming amylase) (Exo-maltotetraohydrolase) (Maltotetraose-forming exo-amylase). SWISS-PROT accession number P22963.

MSHILRAAVL AAVLLPFPAL ADQAGKSPAG VRYHGGDEII LQGFHWNWR EAPNDWYNIL RQQASTIAAD GFSAIWMPVP WRDFSSWTDG GKSGGGEGYF WHDFNKNGRY GSDAQLRQAA

:470 GALGGAGVKV LYDWPNHMN RGYPDKEINL PAGQGFWRND CADPGNYPND CDDGDRFIGG ESDLNTGHPQ IYGMFRDELA NLRSGYGAGG FRFDFVRGYA PERVDSWMSD SADSSFCVGE LWKGPSEYPS WDWRNTASWQ QIIKDWSDRA KCPVFDFALK ERMQNGSVAD WKHGLNGNPD PRWREVAVTF VDNHDTGYSP GQNGGQHHWA LQDGLIRQAY AYILTSPGTP WYWSHMYDW GYGDFIRQLI QVRRTAGVRA DSAISFHSGY SGLVATVSGS QQTLWALNS DLANPGQVAS

"Al5 GSFSEAVNAS NGQVRVWRSG SGDGGGNDGG EGGLVNVNFR CDNGVTQMGD SVYAVGNVSQ LGNWSPASAV RLTDTSSYPT WKGSIALPDG QNVEWKCLIR NEADATLVRQ WQSGGNNQVQ AAAGASTSGS F

SEQ ID NO: 6

P. saccharophilamta gene encodingmaltotetraohydrolase (EC number= 3.2.1.60). 1480 GenBank accessionnumberXl6732. gatcggcgta ggtttcgcat tcgttgccca ggcgatattt cgccggtgcg ccagcagcct ggaagcaggc ctggtcgccg ccgccggccg tggcgccgac gcccgaacgc agatagccgt ggaaatcgac cgccagggcc gggccgccga ccagcagggc ggcaagcagg caggcgggtt ttaggacgaa cagggggtgc gcggtgtgct tcatgacgag gtccttgttt ttcttgttaa

1485 tgccgaatcg atcacgcctt cgctgcgtgt cgcagggcgc agctcggtgg cgaaagcctc ggggatggct ccgctggcgg catcctcccg accagagatt tcgctggcgc agctcgaggg cgtaatcagg atgagtgcgg cgtaatccct ggggtggggc tacgcccggc agggcgcaga tgattgccag gggccttcgg cctggccact acgccgcctg caactgggcg ggggaggttg gtggtcgggg cgtgcagggg cagcctgcgg gtgccggtcg aagacccggc cggcgttcat

1490 cctcgtccgg cggccttgcc gtaggatacc cgaacaagca caagaaccgg agtattgcga tgagccacat cctgcgtgcc gccgtattgg cggcggtcct gctgccgttt cccgcactgg ccgatcaggc cggcaagagc ccggccgggg tgcgctacca cggcggcgac gaaatcatcc tccagggctt ccactggaac gtcgtccgcg aagcgcccaa cgactggtac aacatcctcc gccaacaggc ctcgacgatc gcggccgacg gcttctcggc aatctggatg ccggtgccct

.495 ggcgtgactt ctccagctgg accgacggcg gcaagtccgg cggcggcgaa ggctacttct ggcacgactt caacaagaac ggccgctacg gcagcgacgc ccagctgcgc caggccgccg gcgcactcgg tggcgccggg gtgaaggtgc tctacgatg-t ggtgcccaat cacatgaacc gcggctaccc ggacaaggag atcaacctgc cggccggcca gggcttctgg cgcaacgact gcgccgaccc gggcaactac cccaacgact gcgacgacgg tgaccgcttc atcggcggcg

£500 agtcggacct gaacaccggc catccgcaga tttacggcat gtttcgcgac gagcttgcca acctgcgcag cggctacggc gccggcggct tccgcttcga cttcgttcgc ggctatgcgc ccgagcgggt cgacagctgg atgagcgaca gcgccgacag cagcttctgc gttggcgagc tgtggaaagg cccttctgaa tatccgagct gggactggcg caacacggcg agctggcagc agatcatcaa ggactggtcc gaccgggcca agtgcccggt gttcgacttc gctctcaagg

2505 agcgcatgca gaacggctcg gtcgccgact ggaagcatgg cctcaatggc aaccccgacc cgcgctggcg cgaggtggcg gtgaccttcg tcgacaacca cgacaccggc tattcgcccg ggcagaacgg cggccagcac cactgggcgc tgcaggacgg gctgatccgc caggcctacg cctacatcct caccagcccg ggcacgccgg tggtgtactg gtcgcacatg tacgactggg gctacggcga cttcatccgc cagctgatcc aggtgcggcg caccgccggc gtgcgcgccg

1510 attcggcgat cagcttccat agcggctaca gcggtctggt cgctaccgtc agcggcagcc agcagaccct ggtggtggcg ctcaactccg atctggccaa ccccggccag gttgccagcg gcagcttcag cgaggcggtc aacgccagca acggccaggt gcgcgtctgg cgcagcggta gcggcgatgg cggcgggaat gacggcggcg agggtggctt ggtcaatgtg aactttcgct gcgacaacgg cgtgacgcag atgggcgaca gcgtctacgc ggtgggcaac gtcagccagc

'515 tcggcaactg gagcccggcc tccgcggtac ggctgaccga caccagcagc tatccgacct ggaagggcag catcgccctg cctgacggtc agaacgtgga atggaagtgc ctgatccgca acgaggcgga cgcgacgctg gtgcgtcagt ggcaatcggg cggcaacaac caggtccagg ccgccgccgg cgcgagcacc agcggctcgt tctgacgaca tgcccgcccg gcctcggcta cgcctacgcc gggcggctcc tcccgaccca gggtgggcag ggaggaggcc ggcgacgggc

'520 cgggccgccg atgctggcac gacaaccata aaagccttcg cgctgcgctg tcgtatcagg agctgttcat gttggcccag acccgctcga cccctttccg gcttggcttc ctggcccggc tgtacctgct gatcgccgca ctggtggcct tgctgatgct ggtagccggc accagcctgg ttgccatcgg ccgcctgcaa ggcaatgccg agcaaatctc gtcgaccgcg tcgcgtctgc tggtcagcga gagcttcttc ggtacgttgc agagcctgac gcagaacctg tccgacgccc

Ϊ525 tggccgagga ccggcctgac cagctcgacg gctatgtcgg ccggcatcgc acgctgcagg accaggccct cgagctgttc gcccagctgg agcgggtgac gccggcacat gccgagacca agcaagcctg gcggcgctgt tgccggagct cgaccgccgc agcctggcgc tgatcgatgc gcacgcgacc tgctcgcgcg tggggcgcaa cgccgtcgcc tgcgcgatct gcagctgcag ttctcgcggc tcaagcagga cctgctgcag gcgcagttcg tgacgggcga cgagctggtc

.530 gcctattcca tcaagcagtt catcatcccg ctcgagcagg tcgagcgctg ctgttcgatg ccatcggcgt gtcttcgatc aaggcactcg atgaagcggg tgcgcagatc

SEQ ID NO:7

PS4 reference sequence, derived from Pseudomonas stutzeri maltotetrahydrolase amino acid sequence.

Z535 1 DQAGKSPNAV RYHGGDEIIL QGFHWNWRE APiMIWYNILR QQAATIAADG FSAIWMPVPW

61 RDFSSWSDGS KSGGGEGYFW HDFNKNGRYG SDAQLRQAAS ALGGAGVKVL YDWPNHMNR

121 GfYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFXGGD ADLNTGHPQV YGMFRDEFTN

181 LRSQYGAGGF RFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKGPSEYPNW DWRNTASWQQ

241 IIKDWSDRAK CPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

2540 301 QNGGQHHWAL QDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRAAGVRAD 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LGNPGQVASG SFSEAVNASN GQVRVWRSGT 421 GSGGGEPGAL VSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTD TSGYPTWKGS 481 IALPAGQNEE WKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL

SEQ ID NO: 8

2545 PStu-D34 sequence; Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with 9 substitutions.

1 DQAGKSPNAV RYHGGDEIIL QGFHWNWRE APΪ-VWYNILR QQAATIAADG FSAIWMPVPW

61 RDFSSWSDPS KSGGGEGYFW HDFNKNGRYG SDAQLRQAAS ALGGAGVKVL YDWPNHMNR

121 27YPDKEINLP AGQΛFWRNDC PDPGNYPNDC DDGDRFLGGD ADLNTGHPQV YGMFRDEFTN

2550 181 LRSQYGAGGF RFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKAPSEYPNW DWRNTASWQQ

241 IIKDWSDRAK CPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQHIIWAL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGDFIRQLIQ VRRAAGVRAD

361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LGNPGQVASG SFSEAVNASN GQVRVWRSGT

421 GSGGGEPGAL VSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTD TSGYPTWKGS

2555 481 IALPAGQNEE WKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL SEQ ID NO: 9

PStu-D20 sequence; Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with 11 substitutions.

1 DQAGKSPNAV RYHGGDEIIL QGFHWNWRE APYWWYNILR QQAATIAADG FSAIWMPVPW

1560 61 RDFSSWSDPS JRSGGGEGYFW HDFNKNGRYG SDAQLRQAAS ALGGAGVKVL YDWPNHMNR

121 J3YPDKEINLP AGQKFWRNDC PDPGNYPNDC DDGDRFiGGD ADLNTGHPQV YGMFRDEFTN

181 LRSQYGAGGF RFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKAPSEYPNW DWRNTASWQQ

241 IIKDWSDRAK CPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQHIrWAL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGEFIRQLIQ VRRAAGVRAD

»565 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LGNPGQVASG SFSEAVNASN GQVRVWRSGT

421 GSGGGEPGAL VSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTD TSGYPTWKGS

481 IALPAGQNEE WKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL

SEQ ID NO: 10

PStu-D14 sequence; Pseudomonas stutzeri maltotetrahydrolase amino acid sequence »570 with 12 substitutions.

1 DQAGKSPNAV RYHGGDEIIL QGFHWNWRE APYNWYNILR QQAATIAADG FSAIWMPVPW

61 RDFSSWSDPS JRSGGGEGYFW HDFNKNSRYG SDAQLRQAAS ALGGAGVKVL YDWPNHMNR

121 DYPDKEINLP AGQKFWRNDC PDPGNYPNDC DDGDRFJCX3GD ADLNTGHPQV YGMFRDEFTN

181 LRSQYGAGGF RFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKAPSEYPNW DWRNTASWQQ

..575 241 IIKDWSDRAK CPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG

301 QNGGQHIIWAL QDGLIRQAYA YILTSPGTPV VYWPHMYDWG YGEFIRQLIQ VRRAAGVRAD

361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LGNPGQVASG SFSEAVNASN GQVRVWRSGT

421 GSGGGEPGAL VSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTD TSGYPTWKGS

481 IALPAGQNEE WKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL

2580 SEQ ID NO: 11

Pseudomonas stutzeri (Pseudomonas perfectomarina). Glucan 1,4-alpha- maltotetrahydrolase precursor (EC 3.2.1.60) (G4-amylase) (Maltotetraose-forming amylase) (Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase). SWISS-PROT accession number P 13507.

2585 MSHILRAAVL AAMLLPLPSM ADQAGKSPNA VRYHGGDEII LQGFHWNWR EAPNDWYNIL RQQAATIAAD GFSAIWMPVP WRDFSSWSDG SKSGGGEGYF WHDFNKNGRY GSDAQLRQAA SALGGAGVKV LYDWPNHMN RGYPDKEINL PAGQGFWRND CADPGNYPND CDDGDRFIGG DADLNTGHPQ VYGMFRDEFT NLRSQYGAGG FRFDFVRGYA PERVNSWMTD SADNSFCVGE LWKGPSEYPN WDWRNTASWQ QIIKDWSDRA KCPVFDFALK ERMQNGSIAD WKHGLNGNPD

2590 PRWREVAVTF VDNHDTGYSP GQNGGQHHWA LQDGLIRQAY AYILTSPGTP WYWSHMYDW GYGDFIRQLI QVRRAAGVRA DSAISFHSGY SGLVATVSGS QQTLWALNS DLGNPGQVAS GSFSEAVNAS NGQVRVWRSG TGSGGGEPGA LVSVSFRCDN GATQMGDSVY AVGNVSQLGN WSPAAALRLT DTSGYPTWKG SIALPAGQNE EWKCLIRNEA NATQVRQWQG GANNSLTPSE GATTVGRL

2595 SEQ ID NO: 12

P. stutzeri maltotetraose-forming amylase (amyP) gene, complete cds. GenBank accession number M24516.

1 gatcggcctt tacggaaagt gatagagctt ctcttccggc aaactttgtt ccccagtgac 61 agagggttag tatcggatcg cttcctcttt gggtttggta gatcaggagc gccgagagca 2600 121 ggatgaaatc ctgcggccag aaggtcgcgc cgaagatgtg gaactgctgc tggccgagat 181 ccggccggcg ttcatcctcg tccggcggcc ttgccgccag ctacccgaac aagcacaaga 241 accggagtat tgcgatgagc cacatcctgc gagccgccgt attggcggcg atgctgttgc 301 cgttgccgtc catggccgat caggccggca agagccccaa cgctgtgcgc taccacggcg 361 gcgacgaaat cattctccag ggctttcact ggaacgtcgt ccgcgaagcg cccaacgact

1605 421 ggtacaacat cctgcgccag caggccgcga ccatcgccgc cgacggcttc tcggcgatct 481 ggatgccggt gccctggcgc gacttctcca gctggagcga cggcagcaag tccggcggcg 541 gtgaaggcta cttctggcac gacttcaaca agaacggccg ctatggcagt gacgcccagc 601 tgcgtcaggc cgccagcgcg ctcggtggcg ccggcgtgaa agtgctttac gacgtggtgc 661 ccaaccacat gaaccgtggc tatccggaca aggagatcaa cctcccggcc ggccagggct

.610 721 tctggcgcaa cgactgcgcc gacccgggca actaccccaa tgattgcgac gacggcgacc 781 gcttcatcgg cggcgatgcg gacctcaaca ccggccaccc gcaggtctac ggcatgttcc 841 gcgatgaatt caccaacctg cgcagtcagt acggtgccgg cggcttccgc ttcgactttg 901 ttcggggcta tgcgccggag cgggtcaaca gctggatgac cgatagcgcc gacaacagct 961 tctgcgtcgg cgaactgtgg aaaggcccct ctgagtaccc gaactgggac tggcgcaaca

..615 1021 ccgccagctg gcagcagatc atcaaggact ggtccgaccg ggccaagtgc ccggtgttcg 1081 acttcgccct caaggaacgc atgcagaacg ctcgatcgcc gactggaagc acgcctgaac 1141 ggcaatcccg acccgcgtgg cgcgaggtgg cggtgacctt cgtcgacaac cacgacaccg 1201 gctactcgcc cgggcagaac ggtgggcagc accactgggc tctgcaggac gggctgatcc 1261 gccaggccta cgcctacatc ctcaccagcc ccggtacgcc ggtggtgtac tggtcgcaca

2620 1321 tgtacgactg gggttacggc gacttcatcc gtcagctgat ccaggtgcgt cgcgccgccg 1381 gcgtgcgcgc cgattcggcg atcagcttcc acagcggcta cagcggtctg gtcgccaccg 1441 tcagcggcag ccagcagacc ctggtggtgg cgctcaactc cgacctgggc aatcccggcc 1501 aggtggccag cggcagcttc agcgaggcgg tcaacgccag caacggccag gtgcgcgtgt 1561 ggcgtagcgg cacgggcagc ggtggcggtg aacccggcgc tctggtcagt gtgagtttcc

2625 1621 gctgcgacaa cggcgcgacg cagatgggcg acagcgtcta cgcggtcggc aacgtcagcc 1681 agctcggtaa ctggagcccg gccgcggcgt tgcgcctgac cgacaccagc ggctacccga 1741 cctggaaggg cagcattgcc ttgcctgccg gccagaacga ggaatggaaa tgcctgatcc 1801 gcaacgaggc caacgccacc caggtgcggc aatggcaggg cggggcaaac aacagcctga 1861 cgccgagcga gggcgccacc accgtcggcc ggctctagcc cgggcggcaa ctcggccgtc

2630 1921 tcgcggatgt gaggcggctg gtctcggcgg cggtatcgtt gcgctggggg cggggccgcc 1981 gttcacgcgc cctgctatcg ctagttttcg gcgctccgcg catcggccag ttgccagcga 2041 atcgcctgcg cttcggcctg gtgcaggtcg tcgagcagcg ct

SEQ ID NO: 13

pSac-pMD229 sequence; Pseudomonas saccharophila maltotetrahydrolase amino 2635 acid sequence with 17 substitutions and deletion of the starch binding domain.

pSac-pMD229 comprises mutations N33Y, D34N, G121F, G134R, A141P, Y146G, I157L, S161A, L178F, A179T, G223E, S229P, H272Q, G303E, H307L, A309P, S334P relative to wild type non-maltogenic exoamylase.

MDQAGKSPAGVRYHGGDEIILQGFHW]WVREAPYIWYNILRQQASTIAADGFSAIWMPVPWRDFSS

2640 WTDGGKSGGGEGYFWHDFNKNGRYGSDAQLRQAAGALGGAGVΈVLYDVVPNHMNRFYPDKEINLPA GQRFWRNDCPDPGNGPNDCDDGDRFLGGEADLNTGHPQIYGMFRDEFTNLRSGYGAGGFRFDFVRG YAPERVDSWMSDSADSSFCVGELWKEPSEYPPWDWRNTASWQQIIKDWSDRAKCPVFDFALKERMQ NGSVADWKQGLNGNPDPRWREVAVTFVDNHDTGYSPGQNEGQHLWPLQDGLIRQAYAYILTSPGTP WYWPHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPG

2645 QVASGSFSEAVNASNGQVRVWRSGSGDGGGNDGG-

SEQ ID NO: 14

pSac-pMD229 sequence; Pseudomonas saccharophila maltotetrahydrolase nucleotide sequence with 17 substitutions and deletion of the starch binding domain.

1 atggatcagg ccggcaagag cccggccggg gtgcgctacc acggcggcga cgaaatcatc

2650 61 ctccagggct tccactggaa cgtcgtccgc gaagcgccct acaactggta caacatcctc

121 cgccaacagg cctcgacgat cgcggccgac ggcttctcgg caatctggat gccagtgccc 181 tggcgtgact tctccagctg gaccgacggc ggcaagtccg gcggcggcga aggctacttc

241 tggcacgact tcaacaagaa cggccgctac ggcagcgacg cccagctgcg ccaggccgcc

301 ggcgcactcg gtggcgccgg ggtgaaggtg ctctacgatg tggtgcccaa tcacatgaac

,655 361 cgcttctacc cggacaagga gatcaacctg ccggccggcc agcgcttctg gcgcaacgac

421 tgcccggatc cgggcaacgg ccccaacgac tgcgacgacg gtgaccgctt cctgggcggc

481 gaggcggacc tgaacaccgg ccatccgcag atttacggca tgtttcgcga cgagtttacc

541 aacctgcgca gcggctacgg cgccggcggc ttccgcttcg acttcgttcg cggctatgcg

601 cccgagcggg tcgacagctg gatgagcgac agcgccgaca gcagcttctg cgttggcgag

£660 661 ctgtggaaag agccttctga atatccgccg tgggactggc gcaacacggc gagctggcag

^■721 cagatcatca aggactggtc cgaccgggcc aagtgcccgg tgttcgactt cgctctcaag

781 gagcgcatgc agaacggctc ggtcgccgac tggaagcagg gcctcaatgg caaccccgac

841 ccgcgctggc gcgaggtggc ggtgaccttc gtcgacaacc acgacaccgg ctattcgccc

901 gggcagaacg aaggccagca cctgtggccg ctgcaggacg ggctgatccg ccaggcctac

.665 961 gcctacatcc tcaccagccc gggcacgccg gtggtgtact ggccgcacat gtacgactgg

1021 ggctacggcg acttcatccg ccagctgatc caggtgcggc gcaccgccgg cgtgcgcgcc

1081 gattcggcga tcagcttcca tagcggctac agcggtctgg tcgctaccgt cagcggcagc

1141 cagcagaccc tggtggtggc gctcaactcc gatctggcca accccggcca ggttgccagc

1201 ggcagcttca gcgaggcggt caacgccagc aacggccagg tgcgcgtctg gcgcagcggt

2670 1261 agcggcgatg gcggcgggaa tgacggcggc tga

SEQ IDNO: 15

pSac— pMD248 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with 16 substitutions and deletion of the starch binding domain.

MDQAGKSPAGWYHGGDEIILQGFiHWNVVREAPYNWYNILRQQASTIAADGFSAIWMPVPWRDFSS

2675 WTDGGKSGGGEGYFWHDFNKNGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRFYPDKEINLPA

GQRFWRNDCPDPGDGPNDCDDGDRFLGGESDLNTGHPQIYGMFRDEFTNLRSGYGAGGFRFDFVRG

YAPERVDSWMSDSADSSFCVGELWKEPSEYPPWDWRNTASWQQIIKDWSDRAKCPVFDFALKERMQ

NGSVADWKQGLNGNPDPRWREVAVTFVDNHDTGYSPGQNEGQHLWALQDGLIRQAYAYILTSPGTP

WYWPHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPG

2680 QVASGSFSEAVNASNGQVRVWRSGSGDGGGNDGG

SEQ ID NO: 16

pSac-pMD248 sequence; Pseudomonas saccharophila maltotetrahydrolase nucleotide sequence with 16 substitutions and deletion of the starch binding domain.

1 atggatcagg ccggcaagag cccggccggg gtgcgctacc acggcggcga cgaaatcatc

2685 61 ctccagggct tccactggaa cgtcgtccgc gaagcgccct acaactggta caacatcctc

121 cgccaacagg cctcgacgat cgcggccgac ggcttctcgg caatctggat gccagtgccc

181 tggcgtgact tctccagctg gaccgacggc ggcaagtccg gcggcggcga aggctacttc

241 tggcacgact tcaacaagaa cggccgctac ggcagcgacg cccagctgcg ccaggccgcc

301 ggcgcactcg gtggcgccgg ggtgaaggtg ctctacgatg tggtgcccaa tcacatgaac

2690 361 cgcttctacc cggacaagga gatcaacctg ccggccggcc agcgcttctg gcgcaacgac

421 tgcccggacc cgggcgacgg ccccaacgac tgcgacgacg gtgaccgctt cctgggcggc

481 gagtcggacc tgaacaccgg ccatccgcag atttacggca tgtttcgcga cgagtttacc

541 aacctgcgca gcggctacgg cgccggcggc ttccgcttcg acttcgttcg cggctatgcg

601 cccgagcggg tcgacagctg gatgagcgac agcgccgaca gcagcttctg cgttggcgag

2695 661 ctgtggaaag agccttctga atatccgccg tgggactggc gcaacacggc gagctggcag

721 cagatcatca aggactggtc cgaccgggcc aagtgcccgg tgttcgactt cgctctcaag

781 gagcgcatgc agaacggctc ggtcgccgac tggaagcagg gcctcaatgg caaccccgac

841 ccgcgctggc gcgaggtggc ggtgaccttc gtcgacaacc acgacaccgg ctattcgccc

901 gggcagaacg aaggccagca cctgtgggcg ctgcaggacg ggctgatccg ccaggcctac

2700 961 gcctacatcc tcaccagccc gggcacgccg gtggtgtact ggccgcacat gtacgactgg 1021 ggctacggcg acttcatccg ccagctgatc caggtgcggc gcaccgccgg cgtgcgcgcc 1081 gattcggcga tcagcttcca tagcggctac agcggtctgg tcgctaccgt cagcggcagc 1141 cagcagaccc tggtggtggc gctcaactcc gatctggcca accccggcca ggttgccagc 1201 ggcagcttca gcgaggcggt caacgccagc aacggccagg tgcgcgtctg gcgcagcggt 705 1261 agcggcgatg gcggcgggaa tgacggcggc tga

SEQ ID NO: 17

pSac-pMD253 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with 16 substitutions and deletion of the starch binding domain.

MDQAGKSPAGWYHGGDEIILQGFHWNVWEAPYNWYNILRQQASTIAADGFSAIWMPVPWRDFSS

,710 WTDGGKSGGGEGYFWHDFNKNGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRDYPDKEINLPA

GQRFWRNDCPDPGNGPNDCDDGDRFLGGESDLNTGHPQIYGMFRDEFTNLRSGYGAGGFRFDFVRG

YAPERVDSWMSDSADSSFCVGELWKEPSEYPPWDWRNTASWQQIIKDWSDRAKCPVFDFALKERMQ

NGSVADWKQGLNGNPDPRWREVAVTFVDNHDTGYSPGQNEGQHLWPLQDGLIRQAYAYILTSPGTP

WYWPHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPG

715 QVASGSF SEAVNASNGQVRVWRSGSGDGGGNDGG

SEQ ID NO: 18

pSac-pMD253 sequence; Pseudomonas saccharophila maltotetrahydrolase nucleotide sequence with 16 substitutions and deletion of the starch binding domain.

1 atggatcagg ccggcaagag cccggccggg gtgcgctacc acggcggcga cgaaatcatc

.720 61 ctccagggct tccactggaa cgtcgtccgc gaagcgccct acaactggta caacatcctc

121 cgccaacagg cctcgacgat cgcggccgac ggcttctcgg caatctggat gccagtgccc

181 tggcgtgact tctccagctg gaccgacggc ggcaagtccg gcggcggcga aggctacttc

241 tggcacgact tcaacaagaa cggccgctac ggcagcgacg cccagctgcg ccaggccgcc

301 ggcgcactcg gtggcgccgg ggtgaaggtg ctctacgatg tggtgcccaa tcacatgaac

2725 361 cgcgactacc cggacaagga gatcaacctg ccggccggcc agcgcttctg gcgcaacgac

421 tgcccggacc cgggcaacgg ccccaacgac tgcgacgacg gtgaccgctt cctgggcggc

481 gagtcggacc tgaacaccgg ccatccgcag atttacggca tgtttcgcga cgagtttacc

541 aacctgcgca gcggctacgg cgccggcggc ttccgcttcg acttcgttcg cggctatgcg

601 cccgagcggg tcgacagctg gatgagcgac agcgccgaca gcagcttctg cgttggcgag

2730 661 ctgtggaaag agccttctga atatccgccg tgggactggc gcaacacggc gagctggcag

721 cagatcatca aggactggtc cgaccgggcc aagtgcccgg tgttcgactt cgctctcaag

781 gagcgcatgc agaacggctc ggtcgccgac tggaagcagg gcctcaatgg caaccccgac

841 ccgcgctggc gcgaggtggc ggtgaccttc gtcgacaacc acgacaccgg ctattcgccc

901 gggcagaacg aaggccagca cctgtggccg ctgcaggacg ggctgatccg ccaggcctac

2735 961 gcctacatcc tcaccagccc gggcacgccg gtggtgtact ggccgcacat gtacgactgg

1021 ggctacggcg acttcatccg ccagctgatc caggtgcggc gcaccgccgg cgtgcgcgcc

1081 gattcggcga tcagcttcca tagcggctac agcggtctgg tcgctaccgt cagcggcagc

1141 cagcagaccc tggtggtggc gctcaactcc gatctggcca accccggcca ggttgccagc

1201 ggcagcttca gcgaggcggt caacgccagc aacggccagg tgcgcgtctg gcgcagcggt

2740 1261 agcggcgatg gcggcgggaa tgacggcggc tga

SEQ ID NO: 19

pSac-pMD271 sequence; Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with 18 substitutions and deletion of the starch binding domain.

MDQSGKSPAGWYHGGDEIILQGFHWNVVREAPYNWYNILRQQASTIAADGFSAIWMPVPWRDFSS

2745 WTDGDKSGGGEGYFWHDFNKNGRYGSDAQLRQAAGALGGAGVKVLYDWPNHMNRDYPDKEINLPA

GQRFWRNDCPDPGNGPNDCDDGDRFLGGESDLNTGHPQIYGMFRDEFTNLRSGYGAGGFRFDFVRG YAPERVDSWMSDSADSSFCVGELWKEPSEYPPWDWRNTASWQQIIKDWSDRAKCPWDFALKERMQ NGSVADWKQGLNGNPDPRWREVAVTFVDNHDTGYSPGQNEGQHLWPLQDGLIRQAYAYILTSPGTP VVYWPHMYDWGYGDFIRQLIQWRTAGVRADSAISFHSGYSGLVATVSGSQQTLWALNSDLANPG 750 QVASGSFSEAVNASNGQVRVWRSGSGDGGGNDGG

SEQ ID NO: 20

pSac-pMD271 sequence; Pseudomonas saccharophila maltotetrahydrolase nucleotide sequence with 18 substitutions and deletion of the starch binding domain.

1 atggatcaga gcggcaagag cccggccggg gtgcgctacc acggcggcga cgaaatcatc

755 61 ctccagggct tccactggaa cgtcgtccgc gaagcgccct acaactggta caacatcctc

121 cgccaacagg cctcgacgat cgcggccgac ggcttctcgg caatctggat gccagtgccc

181 tggcgtgact tctccagctg gaccgacggc gacaagtccg gcggcggcga aggctacttc

241 tggcacgact tcaacaagaa cggccgctac ggcagcgacg cccagctgcg ccaggccgcc

301 ggcgcactcg gtggcgccgg ggtgaaggtg ctctacgatg tggtgcccaa tcacatgaac

760 361 cgcgactacc cggacaagga gatcaacctg ccggccggcc agcgcttctg gcgcaacgac

421 tgcccggacc cgggcaacgg ccccaacgac tgcgacgacg gtgaccgctt cctgggcggc

481 gagtcggacc tgaacaccgg ccatccgcag atttacggca tgtttcgcga cgagtttacc

541 aacctgcgca gcggctacgg cgccggcggc ttccgcttcg acttcgttcg cggctatgcg

601 cccgagcggg tcgacagctg gatgagcgac agcgccgaca gcagcttctg cgttggcgag

765 661 ctgtggaaag agccttctga atatccgccg tgggactggc gcaacacggc gagctggcag

721 cagatcatca aggactggtc cgaccgggcc aagtgcccgg tgttcgactt cgctctcaag

781 gagcgcatgc agaacggctc ggtcgccgac tggaagcagg gcctcaatgg caaccccgac

841 ccgcgctggc gcgaggtggc ggtgaccttc gtcgacaacc acgacaccgg ctattcgccc

901 gggcagaacg aaggccagca cctgtggccg ctgcaggacg ggctgatccg ccaggcctac

:770 961 gcctacatcc tcaccagccc gggcacgccg gtggtgtact ggccgcacat gtacgactgg

1021 ggctacggcg acttcatccg ccagctgatc caggtgcggc gcaccgccgg cgtgcgcgcc

1081 gattcggcga tcagcttcca tagcggctac agcggtctgg tcgctaccgt cagcggcagc

1141 cagcagaccc tggtggtggc gctcaactcc gatctggcca accccggcca ggttgccagc

1201 ggcagcttca gcgaggcggt caacgccagc aacggccagg tgcgcgtctg gcgcagcggt

1775 1261 agcggcgatg gcggcgggaa tgacggcggc tga

Claims

1. A PS4 variant polypeptide derivable from a parent polypeptide having amylase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 780 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334;

(b) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 145, 146, 157, 178, 179, 223, 229, 272, 303, 307 and 334;

(c) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334;

'785 (d) a polypeptide comprising an amino acid mutation at each of positions 3, 33, 34,

70, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334;

with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1.

2. A PS4 variant polypeptide according to Claim 1 , in which each of the amino acid .790 mutations in polypeptide (a) are independently selected from the group consisting of: 33 Y,

34N, 121F, 134R₅ 141P, 146G, 157L, 161A, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P, preferably N33 Y, D34N, G121F, G134R, A141P, Y146G, I157L, S161A, L178F, A179T, G223E, S229P, H272Q, G3O3E, H307L, A309P and S334P.

3. A PS4 variant polypeptide according to Claim 1 or 2 which comprises the 2795 sequence pSac-pMD229 (SEQ ID NO: 13).

4. A PS4 variant polypeptide according to Claim 1, in which each of the amino acid mutations in polypeptide (b) are independently selected from the group consisting of: 33 Y, 34N, 121F, 134R, 141P, 145D, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L and 334P, preferably N33 Y, D34N, G121F, G134R, A141P, N145D, Y146G, I157L,

2800 L178F, A179T, G223E, S229P, H272Q, G3O3E, H307L and S334P.

5. A PS4 variant polypeptide according to Claim 1 or 4 which comprises the sequence ρSac-ρMD248 (SEQ ID NO: 15).

6. A PS4 variant polypeptide according to Claim 1, in which each of the amino acid mutations in polypeptide (c) are independently selected from the group consisting of: 33Y, 1805 34N, 121D, 134R, 141P, 146G, 157L₅ 178F, 179T, 223E, 229P, 272Q₅ 303E, 307L₅ 309P and 334P, preferably N33 Y₅ D34N₅ G121D, G134R, A141P, Y146G, 1157L₅ L178F, Al 79T₅ G223E, S229P, H272Q, G303E, H307L, A309P and S334P.

7. A PS4 variant polypeptide according to Claim 1 or 6 which comprises the sequence pSac-pMD253 (SEQ ID NO: 17).

1810 8. A PS4 variant polypeptide according to Claim 1 , in which each of the amino acid mutations in polypeptide (d) are independently selected from the group consisting of: 3 S, 33Y₅ 34N₅ 7OD, 121D₅ 134R₅ 141P₅ 146G₅ 157L₅ 178F₅ 179T₅ 223E₅ 229P₅ 272Q₅ 303E₅ 307L₅ 309P and 334P₅ preferably A3 S₅ N33Y, D34N, G70D, G121D, G134R, A141P, Y146G, 1157L₅ Ll 78F₅ Al 79T₅ G223E, S229P, H272Q, G303E, H307L, A309P and

»815 S334P.

9. A PS4 variant polypeptide according to Claim 1 or 8 which comprises the sequence ρSac-ρMD271 (SEQ ID NO: 19).

10. A PS4 variant polypeptide according to any preceding claim, in which the parent polypeptide comprises exoamylase activity, preferably a non-maltogenic exoamylase,

Ϊ820 more preferably a glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60).

11. A PS4 variant polypeptide according to any preceding claim, in which the parent polypeptide is or is derivable from Pseudomonas species, preferably Pseudomonas saccharophilia or Pseudomonas stutzeri.

12. A PS4 variant polypeptide according to any preceding claim, in which the parent Z825 polypeptide is a non-maltogenic exoamylase from Pseudomonas saccharophilia exoamylase having a sequence shown as SEQ ID NO: 1 or SEQ ID NO: 5.

13. A PS4 variant polypeptide according to any preceding claim having an amino acid sequence which at least 75% identical to SEQ ID NO: 1 or SEQ ID NO: 5.

14. A PS4 variant polypeptide according to any of Claims 1 to 10, in which the parent 2830 polypeptide is a non-maltogenic exoamylase from Pseudomonas stutzeri having a sequence shown as SEQ ID NO: 7 or SEQ ID NO: 11.

15. A PS4 variant polypeptide according to according to any of Claims 1 to 10 or 14 having an amino acid sequence which at least 75% identical to SEQ ID NO: 7 or SEQ ID NO: 11.

1835 16. A PS4 variant polypeptide according to any preceding claim, in which the PS4 variant polypeptide has a higher thermostability compared to the parent polypeptide or a wild type polypeptide when tested under the same conditions.

17. A PS4 variant polypeptide according to any preceding claim, in which the half life (tl/2), preferably at 60 degrees C, is increased by 15% or more, preferably 50% or more,

1840 most preferably 100% or more, relative to the parent polypeptide or the wild type polypeptide.

18. A PS4 variant polypeptide according to any preceding claim, in which the PS4 variant polypeptide has a higher exo-specificity compared to the parent polypeptide or a wild type polypeptide when tested under the same conditions.

1845 19. A PS4 variant polypeptide according to any preceding claim, in which the PS4 variant polypeptide has 10% or more, preferably 20% or more, preferably 50% or more, exo-specificity compared to the parent polypeptide or the wild type polypeptide.

20. A PS4 variant polypeptide according to any preceding claim, in which a food product treated with a the PS4 variant polypeptide has any one or more, preferably all of

1850 the following properties: (a) lower firmness; (b) higher resilience; and (c) higher cohesiveness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

21. A PS4 variant polypeptide according to Claim 20, in which the resilience or cohesiveness of the food product is independently increased by 15% or more, preferably

1855 50% or more, most preferably 100% or more, relative to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

22. A PS4 variant polypeptide according to Claim 20 or 21 , in which each of resilience and cohesiveness of a food product treated with a the PS4 variant polypeptide is increased compared to a food product which has been treated with a parent polypeptide or a wild

1860 type polypeptide.

23. A PS4 variant polypeptide according to Claim 20, in which the firmness of the food product is independently decreased by 15% or more, preferably 50% or more, most preferably 100% or more, relative to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

1865 24. A PS4 variant polypeptide according to Claim 20 or 23, in which the firmness of a food product treated with a the PS4 variant polypeptide is increased compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

25. A polypeptide derivable from a PS4 variant polypeptide according to any preceding claim by mutation at one or more residues of the PS4 variant polypeptide

£870 sequence, in which the polypeptide has a higher thermostability or a higher exo- specificity, or both, compared to the parent polypeptide of the PS4 variant polypeptide or a wild type polypeptide, or in which a food product treated with a the PS4 variant polypeptide has any one or more, preferably all of the following properties: (a) lower firmness; (b) higher resilience; or (c) higher cohesiveness as compared to a food product

2875 which has been treated with a parent polypeptide or a wild type polypeptide.

26. A polypeptide comprising a fragment of at least 20 residues of a PS4 variant polypeptide according to any preceding claim, in which the polypeptide has non- maltogenic exoamylase activity.

27. A polypeptide derivable from a polypeptide according to any preceding claim by 2880 mutation at one or more residues of the PS4 variant polypeptide sequence, in which the polypeptide has a higher thermostability or a higher exo-specificity, or both, compared to the parent polypeptide of the PS4 variant polypeptide or a wild type polypeptide.

28. Use of a polypeptide as set out in any preceding claim as a food or feed additive.

29. A process for treating a starch comprising contacting the starch with a polypeptide 2885 as set out in any of Claims 1 to 25 and allowing the polypeptide to generate from the starch one or more linear products.

30. Use of a polypeptide as set out in any of Claims 1 to 25 in preparing a food or feed product.

31. A process of preparing a food or feed product comprising admixing a polypeptide 2890 as set out in any of Claims 1 to 25 with a food or feed ingredient.

32. Use according to Claim 30, or a process according to Claim 31, in which the food product comprises a dough or a dough product, preferably a processed dough product.

33. A use or process according to any of Claims 28 to 32, in which the food product is a bakery product.

2895 34. A process for making a bakery product comprising: (a) providing a starch medium; (b) adding to the starch medium a polypeptide as set out in any of Claims 1 to 25; and (c) applying heat to the starch medium during or after step (b) to produce a bakery product.

35. A food product, feed product, dough product or a bakery product obtained or obtainable by a process according to any of Claims 28 to 34.

1900 36. An improver composition for a dough, in which the improver composition comprises a polypeptide as set out in any of Claims 1 to 25, and at least one further dough ingredient or dough additive.

37. A composition comprising a flour and a polypeptide as set out in any of Claims 1 to 25.

.905 38. Use of a polypeptide as set out in any of Claims 1 to 25, in a dough product to retard or reduce staling, preferably detrimental retrogradation, of the dough product.

39. Use of a PS4 variant polypeptide as set out in any preceding claim, in a dough product to improve any one or more of firmness, resilience or cohesiveness of the dough product.

.910 40. A combination of a PS4 variant polypeptide as set out in any preceding claim, together with Novamyl, or a variant, homologue, or mutants thereof which has malto genie alpha-amylase activity.

41. Use of a combination according to Claim 40 for an application according to any preceding claim.

Z915 42. A food or feed product produced by treatment with a combination according to Claim 40.

43. A nucleic acid capable of encoding a polypeptide according to any of Claims 1 to

25.

44. A nucleic acid according to Claim 43 having a nucleic acid sequence which at least 2920 75% identical to SEQ ID NO: 6 or SEQ ID NO: 12.

45. A nucleic acid comprising a fragment of at least 60 residues of a nucleic acid according to Claim 43 or 44 which is capable of encoding a polypeptide having non- maltogenic exoamylase activity.

46. A nucleic acid sequence derivable from a parent sequence, the parent sequence 2925 capable of encoding an amylase, which nucleic acid sequence comprises a substitution at one or more residues such that the nucleic acid encodes one or more of the following mutations at the positions specified: (a) 33Y, 34N₅ 121F, 134R, 141P, 146G, 157L, 161 A, 178F, 179T, 223E, 229P, 272Q₃ 303E, 307L, 309P and 334P; (b) 33Y, 34N, 121F, 134R, 141P, 145D, 146G, 157L, 178F, 179T, 223E, 229P, 272Q₅ 303E, 307L and 334P (c) 33Y, 2930 34N₅ 121D₅ 134R₅ 141P₅ 146G, 157L₅ 178F, 179T, 223E, 229P, 272Q, 303E, 307L₅ 309P and 334P; (d) 3S, 33Y, 34N, 7OD, 121D, 134R₅ 141P₅ 146G₅ 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P; with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1.

47. A nucleic acid sequence according to any of Claims 43 to 46, which is derived '935 from a parent sequence encoding a non-maltogenic exoamylase by substitution of one or more nucleotide residues.

48. A nucleic acid sequence according to any of Claims 43 to 47, selected from the group consisting of: pSac-ρMD229 (SEQ ID NO: 14), ρSac-ρMD248 (SEQ ID NO: 16), pSac-pMD253 (SEQ ID NO: 18) and pSac-pMD271 (SEQ ID NO: 20).

Ϊ940 49. A plasmid comprising a PS4 nucleic acid according to any of Claims 43 to 48.

50. An expression vector comprising a PS4 nucleic acid according to any of Claims 43 to 49, or capable of expressing a polypeptide according to any of Claims 1 to 25.

51. A host cell comprising, preferably transformed with, a plasmid according to Claim 49 or an expression vector according to Claim 50.

.945 52. A cell capable of expressing a polypeptide according to any of Claims 1 to 25.

53. A host cell according to Claim 51 , or a cell according to Claim 52, which is a bacterial, fungal or yeast cell.

54. A method of expressing a PS4 variant polypeptide, the method comprising obtaining a host cell or a cell according to Claim 51, 52 or 53 and expressing the

2950 polypeptide from the cell or host cell, and optionally purifying the polypeptide.

55. A method of altering the sequence of a polypeptide by introducing an amino acid substitution selected from the group consisting of: (a) 33Y, 34N, 121F, 134R, 141P, I46G, 157L, 161A, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P; (b) 33Y, 34N, 121F, 134R, 141P, 145D, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L and

2955 334P (c) 33Y, 34N, 121D, 134R, 141P, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P; (d) 3S, 33Y, 34N, 7OD, 121D, 134R, 141P, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P (with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1), into a parent polypeptide having amylase activity.

2960 56. A method of altering the sequence of a non-maltogenic exoamylase by introducing a substitution selected from the group consisting of: (a) 33Y, 34N, 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P; (b) 33Y, 34N, 121F, 134R, 141P, 145D, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L and 334P (c) 33Y, 34N, 121D, 134R, 141P₅ 146G, 157L, 178F₃ 179T, 223E, 229P, 272Q, :965 303E, 307L, 309P and 334P; (d) 3S₅ 33Y, 34N, 7OD, 121D, 134R, 141P, 146G, 157L₅

178F₅ 179T, 223E₅ 229P₅ 272Q₅ 303E₅ 307L₅ 309P and 334P with reference to the position numbering of a Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

57. A method according to Claim 55 or 56, in which the sequence of the non-

'970 maltogenic exoamylase is altered by altering the sequence of a nucleic acid which encodes the non-maltogenic exoamylase.

58. A method of producing a PS4 polypeptide variant, the method comprising introducing an amino acid substitution into a parent polypeptide having amylase activity, the amino acid substitution being selected from the group consisting of: (a) 33Y, 34N₅

»975 121F₅ 134R₅ 141P, 146G, 157L₅ 161A₅ 178F₅ 179T, 223E₅ 229P₅ 272Q₅ 303E₅ 307L₅ 309P and 334P; (b) 33Y, 34N₅ 121F₅ 134R₅ 141P₅ 145D, 146G₅ 157L₅ 178F₅ 179T₅ 223E₅ 229P₅ 272Q₅ 303E₅ 307L and 334P (c) 33Y₅ 34N₅ 121D₅ 134R₅ 141P₅ 146G, 157L₅ 178F₅ 179T₅ 223E₅ 229P₅ 272Q₅ 303E, 307L, 309P and 334P; (d) 3S₅ 33Y, 34N₅ 70D₅ 121D₅ 134R, 141P₅ 146G₅ 157L₅ 178F, 179T, 223E, 229P, 272Q₅ 3O3E, 307L₅ 309P and 334P with

Ϊ980 reference to the position numbering of a Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

59. A method according to Claim 55 or 56, in which the sequence of a nucleic acid encoding the parent polypeptide is altered to introduce the amino acid substitution.

60. A method of altering the sequence of a nucleic acid encoding a non-maltogenic 2985 exoamylase, the method comprising introducing into the sequence a codon which encodes an amino acid residue selected from the group consisting of: (a) 33Y₅ 34N₅ 121 F, 134R₅ 141P₅ 146G₅ 157L₅ 161A₅ 178F, 179T₅ 223E, 229P, 272Q, 303E, 307L₅ 309P and 334P; (b) 33Y₅ 34N, 121F, 134R, 141P₅ 145D, 146G₅ 157L₅ 178F₅ 179T₅ 223E₅ 229P₅ 272Q₅ 303E₅ 307L and 334P (c) 33Y₅ 34N, 121D₅ 134R, 141P₅ 146G₅ 157L₅ 178F₅ 179T₅ 223E₅ 2990 229P₅ 272Q, 303E, 307L, 309P and 334P; (d) 3S, 33Y₅ 34N₅ 7OD, 121D, 134R₅ 141P₅

146G₅ 157L₅ 178F₅ 179T₅ 223E₅ 229P, 272Q, 3O3E, 307L, 309P and 334P₅ with reference to the position numbering of a Pseudomonαs sαcchαrophiliα exoamylase sequence shown as SEQ ID NO: 1.

61. A method of increasing the thermostability, or the exo-specificity, or both, of a 2995 polypeptide, the method comprising the steps as set out in any of Claims 55 to 60.

62. A method according to any of Claims 55 to 61 , in which the polypeptide is isolated or purified, or both.

63. A polypeptide obtainable by a method according to any of Claims 55 to 62.

64. A polypeptide obtained by a method according to any of Claims 55 to 62.

58. A PS4 variant polypeptide, use, process, food product, feed product, dough product, bakery product, improver composition, composition, nucleic acid, vector or host cell substantially as hereinbefore described with reference to and as shown in the accompanying drawings.