WO2003056002A1 - Improvement in amylase stability - Google Patents

Abstract

The invention relates to an enzyme and a process for producing an enzyme capable of cleaving α-1,4-glucosidic bonds of starch. The enzyme comprises an amylase which has been modified by having an amine-containing group linked to a side chain of an amino acid residue of the amylase and/or to a terminal amino acid residue of the amylase. The enzyme functions at an elevated temperature and/or has an extended half-life compared to the corresponding unmodified amylase.

Description

Improvement in Amylase Stability

TECHNICAL FIELD OF THE INVENTION

The invention relates to improvement in the stability of an enzyme by chemical modification. The invention also provides a stabilised enzyme produced by this method.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Starch is the major nutritional reservoir in plants and consequently constitutes a significant proportion of plant matter. Starch is relatively insoluble in water and as a result of its abundance causes complications during processing of plant derived material. One mechanism by which starch is removed is by digestion with industrial enzymes. Starch is a polymer of glucose residues joined by α-1,4 linkages. The α-1,4 linkages are hydrolysed by the enzyme amylase to yield the soluble products maltose, maltotriose and α-dextrin. Different amylases are capable of cleaving the starch molecule at different positions in the glucose polymer.

For example, α-amylase is an endoglucanase which cleaves starch at internal α-1, 4-linkages to produce oligo- saccharides, and gluco-amylase (also known as amyloglucosidase) is an exoglucanase which cleaves the terminal α-1,4-linkage to release β-D-glucose residues.

Amylases are used in many industrial processes including for example paper manufacture, textile manufacture, food processing, brewing and baking and is used as an additive in detergents.

A significant problem with the industrial application of amylases is that many processes in which amylases would be useful are carried out at temperatures above that at which amylases are functional, or at temperatures at which amylases are rapidly inactivated. Thus, a significant limitation applies to the use of amylases in many industrial applications.

It is generally believed that a combination of a number of features including hydrophobic interactions, compact packing, salt bridges, reduction of conformational strain, reduction of the entropy of unfolding, α-helix stabilization, hydrogen bonding, disulfide bridges, metal binding, surface loop stabilization and resistance to covalent degradation, contribute to enzyme thermo- stability and activity at elevated temperature.

The inventors have found that by linking an amine- containing group to a side chain of an amino acid residue, and/or to a terminal amino acid residue of an amylase, the thermostability of the enzyme is improved.

SUMMARY OF THE INVENTION In a first aspect, the invention provides an enzyme capable of cleaving α-l,4-glucosidic bonds of starch comprising an amylase which has been modified by having an amine-containing group linked: (a) to a side chain of an amino acid residue of the amylase; and/or (b) to a terminal amino acid residue of the amylase. Preferably, the enzyme functions at an elevated temperature and/or has an extended half-life compared to the corresponding unmodified amylase.

The enzyme optionally also comprises a further amine- containing group linked to a side chain of an amino acid residue of the enzyme, or to a terminal amino acid residue of the enzyme. In one embodiment, the amine-containing group is linked to a carboxyl group of a side chain of an amino acid or to the carboxy terminal amino acid.

In a preferred embodiment, the amine-containing group is linked to the side chain of an aspartate residue, a glutamate residue and/or to a carboxy terminal amino acid residue .

In another embodiment, the amine-containing group is linked to an amino group of a side chain of an amino acid or to the amino terminal amino acid. In a preferred embodiment, the amine-containing group is linked to the side chain of a lysine residue of the enzyme.

Preferably, the amine-containing group is linked to the side chain of an amino acid and/or to a terminal amino acid by an amide bond. In yet another embodiment, the amine-containing group is linked to a hydroxyl group of a side chain of an amino acid. Preferably, the amine-containing group is linked to a hydroxyl group of a tyrosine residue. The amine-containing group may be any amine- containing group that improves the capacity of the enzyme to cleave α-1, 4-glucosidic bonds of starch at elevated temperature, and/or extends the half-life of the enzyme. The amine-containing group may, for example, be an aromatic group. In one embodiment, the aromatic group is a derivative of benzene. Preferably, the derivative of benzene is benzylamine, 2-guanidino-benzimidazole or tryptophan methyl ester. In another embodiment the aromatic group is a heterocyclic amine. Preferably, the heterocyclic amine is selected from the group consisting of adenosine, adenine, cytosine, cytidine or pyridine.

In another embodiment, the amine-containing group is an amine containing carbohydrate such as glucosamine or chitosan. Preferably, the chitosan is oligo-chitosan. More preferably, the oligo-chitosan has a molecular weight of 5000. Even more preferably, the oligo-chitosan is 75%- 85% deacetylated. In another embodiment, the amine-containing group is an aliphatic amine-containing group. In a preferred embodiment, the aliphatic amine-containing group is selected from the group consisting of arginine methyl ester, argininamide, arginine ethyl ester, glycinamide, methylamine, ethylenediamine, dimethylamine and trimethylamine .

In other embodiments :

(a) the amine-containing group is an amino group; and/or

(b) the amine-containing group is an amidino group. In one embodiment, the enzyme further comprises an aromatic group. The aromatic group may be selected from the group consisting of benzoic acid, pyromellitic acid, mellitic acid, trimellitic acid, phthalic acid, cis aconitic acid, 3, 3', 4, 4' benzophenone tetracarboxylic acid and 2, 3 pyridine carboxylic acid. Preferably, the further aromatic group is phthalic acid.

The enzyme may have an amino acid sequence of an amylase of an organism. In one embodiment, the organism is selected from the group consisting of vertebrates, invertebrates, angiosperms, fungi, yeast, bacteria, archeae and algae. In a preferred embodiment, the organism is a psychrophilic, a mesophilic or a thermophilic microorganism. More preferably, the microorganism is a fungus. Even more preferably the fungus is Aspergillus sp., Scopulariopsis sp., Rhizopus sp. or Trichoderma sp. Most preferably the microorganism is Aspergillus oryzae or Rhizopus sp.. The enzyme may have an amino acid sequence of an amylase encoded by a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may be obtained from an organism selected from the group consisting of vertebrates, invertebrates, angiosperms, fungi, yeast, bacteria, archeae and algae. In a preferred embodiment, the recombinant nucleic acid is obtained from a psychrophilic, a mesophilic or a thermophilic microorganism. More preferably, the microorganism is a fungus. Even more preferably the fungus is Aspergillus sp., Scopulariopsis sp., Rhizopus sp. or Trichoderma sp.

Most preferably the microorganism is Aspergillus oryzae or Rhizopus sp.

Preferably, the amylase is α-amylase or glucoamylase . It is particularly contemplated that the α-amylase is that of Aspergillus oryzae . It is particularly contemplated that the gluco- amylase is that of Rhizopus sp.

In a second aspect, the invention provides a process for producing an enzyme of the first aspect of the invention, the process comprising the step of contacting an enzyme capable of cleaving α-l,4-glucosidic bonds of starch with a compound which comprises an amine-containing group in conditions sufficient for linking the amine- containing group to a side chain of an amino acid residue of the enzyme, and/or to a terminal amino acid residue of the enzyme .

In one embodiment, the amine-containing group is linked to a carboxyl group of a side chain of an amino acid or to the carboxy terminal amino acid. Preferably, the amine-containing group is linked to the side chain of an aspartate residue, a glutamate residue and/or to a carboxy terminal residue of the enzyme.

In another embodiment, the amine-containing group is linked to an amino group of a side chain of an amino acid and/or to an amino terminal amino acid. Preferably, the amine-containing group is linked to the side chain of a lysine residue.

In one embodiment, the process comprises activating carboxyl groups of amino acid residues of the enzyme in the presence of a compound which comprises an amine- containing group. In a preferred embodiment, the compound is a nucleophile. Preferably, the nucleophile is selected from the group consisting of aromatic nucleophiles, carbohydrate nucleophiles, aliphatic amine nucleophiles, heterocyclic amine nucleophiles, cytidine nucleophiles and amino nucleophiles.

In one embodiment, the aliphatic amine nucleophile is selected from the group consisting of argininamide dihydrochloride, arginine methyl ester dihydrochloride, arginine ethyl ester dihydrochloride, glycinamide hydrochloride, methylamine hydrochloride, dimethylamine hydrochloride, ethylenediamine dihydrochloride and trimethylamine hydrochloride.

In one embodiment, the aromatic nucleophile is selected from the group consisting of benzylamine hydrochloride, tryptophan methyl ester hydrochloride and 2 -guanidino-benzimidazole dihydrochloride . In one embodiment, the heterocyclic amine nucleophile is selected from the group consisting of adenine hydrochloride, adenosine hydrochloride, pyridine hydrochloride, cytidine and cytosine.

In one embodiment, the carbohydrate nucleophile is selected from the group consisting of glucosamine and chitosan. Preferably, the chitosan is an oligo-chitosan molecule. Preferably, the oligo-chitosan molecule has a molecular weight of 5000. Preferably, the chitosan molecule comprises N-acetyl-glucosamine and glucosamine residues.

In one embodiment, the amino nucleophile is ethylenediamine dihydrochloride or trimethylamine.

The carboxyl groups may be activated by any compound that provides sufficient conditions for an amine- containing group to be linked to the side chain of an amino acid residue of the enzyme, and/or linked to the carboxy terminal amino residue acid of the enzyme. In one embodiment, carboxyl groups are activated by carbodiimide. In a preferred embodiment, the carboxyl groups of the enzyme are activated by l-ethyl-3 (3-dimethylaminopropyl) carbodiimide or 1- (3-dimethylaminopropyl) -3 -ethyl carbodiimide methiodide. In another embodiment, the process comprises contacting the enzyme with a compound selected from the group consisting of O-methyl isourea and 3,5- dimethylpyrazole-1-carboxamidine nitrate (guanyl -3,5- dimethyl pyrazole) under conditions sufficient to permit conversion of a lysine residue of the enzyme to a homoarginine residue.

The process optionally also comprises the step of contacting the enzyme with an agent for controlling the linkage of the amine-containing group to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme. Preferably the agent is an inhibitor of the enzyme. Preferably the inhibitor is maltose or cyclodextrin. Alternatively the agent is a substrate of the enzyme. The substrate may be any oligomer of α-1,4 linked glucose residues. Preferably, the substrate is selected from the group maltodextrose, maltotriose and starch.

In one embodiment, the process comprises the further step of contacting the enzyme in the presence of an aromatic anhydride in conditions sufficient for linking the aromatic group of the aromatic anhydride to an amino group of a basic amino acid residue of the enzyme, and/or to the amino terminal amino acid residue of the enzyme. The aromatic anhydride may be any aromatic containing anhydride. Preferably, the aromatic anhydride is selected from the group consisting of benzoic anhydride, pyromellitic dianhydride, mellitic trianhydride, trimellitic anhydride, phthalic anhydride, cis aconitic anhydride, 3, 3', 4, 4' benzophenone tetracarboxylic dianhydride and 2, 3 pyridine carboxylic anhydride. Preferably, the aromatic anhydride is phthalic anhydride. In a third aspect, the invention provides an enzyme produced by the process of the second aspect of the invention.

In a fourth aspect, the invention provides a composition comprising an enzyme according to the first or third aspects of the invention and a suitable carrier. Suitable carriers are known in the art, and the skilled person will readily be able to select the most suitable carrier for a given use. In a fifth aspect, the invention provides a method of cleaving the α-l,4-glucosidic bonds of starch, comprising the step of exposing a compound or composition comprising starch to an enzyme according to the invention.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention employs, unless otherwise indicated, conventional chemistry, protein chemistry, molecular, biological and enzymological techniques within the skill in the art. Such techniques are well known to the skilled worker, and are explained fully in the literature See, for example, Coligan, Dunn,

Floegh, Speicher and Wingfield "Current protocols in Protein Science" (1999) Volume I and II (John Wiley & Sons Inc.); Sambrook and Russel "Molecular Cloning: A Laboratory Manual" (2001); Cloning: A Practical Approach," Volumes I and II; (D.N. Glover, ed., 1985); Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986; Glazer, AN; DeLange, RJ; Sigman, DS (1975) Chemical modification of proteins. North Holland Publishing Company, Amsterdam; Lundblad, RL (1995) Techniques in protein modification. CRC Press, Inc. Boca Raton, FI . USA; Hirs, CHW; Tamasheff, SN, Eds. (1972) Methods in Enzymology, Vol XXV. Academic Press, New York.

Before the present methods are described, it is understood that this invention is not limited to the particular materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, and a reference to "an amino acid" is a reference to one or more amino acids. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

All publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The present invention relates to an enzyme that comprises a modified amylase for cleaving the α-1,4- glucosidic bonds of starch at elevated temperature and/or having an extended half-life. As described herein, amine- containing groups were linked to the side chains of amino acids of amylase, and/or to a terminal amino acid residue and the resulting enzyme was then able to function at elevated temperature and/or had an extended half-life. As used herein, the expression "elevated temperature" refers to a temperature above that at which the corresponding amylase not having an amine-containing group linked to the side chain of an amino acid of the enzyme and/or to a terminal amino acid exhibits maximum activity. For example, an amylase not having an amine-containing group linked to the side chain of an amino acid and/or to a terminal amino acid residue would lose activity rapidly at 50°C while the same enzyme having an amine-containing group linked to the side chain of an amino acid and/or to a terminal amino acid residue will lose activity at 50°C at a slower rate, or may retain activity. As used herein, an enzyme that has an "extended half-life" has a half-life that is longer than that of the corresponding amylase that does not have an amine-containing group linked to a side chain of an amino acid or to a terminal amino acid. For example, as described herein, linkage of a pyridine group to the side chain of an amino acid of α-amylase from Aspergillus oryzae resulted in up to a 3.7 fold increase in the half-life of the enzyme at 50°C when compared to the native α-amylase. The expression "corresponding unmodified amylase" refers to an amylase having the same amino acid sequence as the enzyme but not having an amine-containing group linked to the side chain of an amino acid or to a terminal amino acid of the enzyme. As used herein, the term "α-1, 4-glucosidic bonds" refers to the α-1,4 bonds formed between glucose residues in a molecule of starch.

The first step in preparing the enzyme of the invention involves selecting the amylase to which the amine-containing group is to be linked. The amylase may be any enzyme that is capable of cleaving the α-1, 4-glycosidic bonds of starch. The amylase may be, for example, α-amylase or gluco-amylase. An α-amylase is an amylase that cleaves internal α-1, 4-glucosidic bonds of starch, and gluco- amylase is an amylase that cleaves the terminal residues from starch. It will be understood by those skilled in the art that amylases are classified according to their ability to cleave starch. While amylase may be isolated from different organisms and therefore have slightly different activities and/or properties, the overall classification is the same. In other words, an amylase isolated from one organism will have very similar properties to an amylase isolated from a different organism.

Accordingly, the "unmodified" amylase can be "wild-type", "naturally-occurring" or "recombinant" amylase or variant thereof obtained from any suitable origin, such as vertebrate, invertebrate, angiosperm, fungus, yeast, prokaryotes including bacteria, archeaebacteria and eubacteria, or a mesophilic organism. Origin can further be psychrotolerant, psychrotrophic, mesophilic or extre ophilic (psychrophilic, psychrotrophic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes may be used. Also included by definition and described herein, are mutants of wild-type amylases. Mutants can be obtained eg. by protein and/or genetic engineering, chemical and/or physical modifications of wild-type enzymes. Common practice as well is the expression of the amylase via host organisms in which the genetic material responsible for the production of the amylase has been cloned. Examples of organisms from which the amylase may be obtained include species such as Humicola, Coprinuc, Thielavia,

Myceliopthora , Fusarium, Mycelioph thora , Acremonium, Cephalosporium, Scytalidium, Penici Ilium, Aspergillus,

Trichoderma , Bacill us , Streptomyces , Scopuloropsis or Rhizopus sp.. Examples of particular organisms and strains from which amylases may be isolated include Humicola insolens, Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila , Meripilus giganteus, Thielavia terrestris, Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinker toniae , Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Arachniotus sp. , Cephelosporium sp., Trichoderma viride, Trichoderma reesei , Trichoderma koningii , Bacillus sp. and

■Sftreptomyces sp. Preferably, the organism from which the amylase is isolated is a fungus. More preferably, the amylase is isolated from an organism selected from the group consisting of Aspergillus sp., Trichoderma sp., Bacillus sp., Microtetraspora sp., Scopulariopsis sp . , Actinomodura sp., Cryptococcus alhidis, Thermonospora fucsa, Butyribrio fibrisolvens, Lactobacillus plant arum, Streptomyces sp., Rhizopus sp. Even more preferably, the organism is Aspergillus oryzae . While it will be appreciated by those skilled in the art that the "unmodified" or "wild-type" amylase per se may be isolated de novo , or obtained through commercial means as described below, unmodified amylase may also be obtained by recombinant means. Moreover, it is common practice these days to modify wild-type enzymes via protein/genetic engineering techniques in order to optimise their performance efficiency.

In particular, amino acids sensitive to oxidation or amino acids that affect the surface charges are of interest. The isoelectric point of such amylases may also be modified by the substitution of some charged amino acids, eg. an increase in isoelectric point may help to improve compatibility with anionic surfactants. The stability of the amylases may be further enhanced by the creation of eg. additional salt bridges and enforcing metal binding sites to increase chelant stability.

The term "amino acid" as used herein refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers) , synthetic analogs and derivatives thereof. α-Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain." α-Amino acids also comprise a carbon atom to which is bonded an amino group, a carboxyl group, and two distinctive groups (which can be the same group or can be different groups) , in which case the amino acid has two side chains. The side chains of naturally occurring amino acids are well known in the art and include, for example, hydrogen (eg., as in glycine) , alkyl (eg., as in alanine, valine, leucine, isoleucine), substituted alkyl (eg., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (eg., as in phenylalanine), substituted arylalkyl (eg., as in tyrosine), selenocysteine, pyrolysine and heteroarylalkyl (eg., as in histidine and tryptophan). [See, eg., Harper et al . (1977) "Review of Physiological Chemistry", 16th Ed., Lange Medical

Publications, pp. 21-24] . One of skill in the art will appreciate that the term "amino acid" also includes β-γ-, δ-, and ω-amino acids, and the like, and α-imino acids such as proline. As used herein, "amino acids" includes proline. Non-naturally occurring amino acids are also known in the art, as set forth in, for example, Williams (ed.), "Synthesis of Optically Active α-Amino Acids", Pergamon Press, 1989; Evans et al . (1990) J. Amer. Chem. Soc, 112:4011-4030; Pu et al . (1991) J. Amer. Chem. Soc. 56:1280-1283; and Williams et al . (1991) J. Amer. Chem. Soc. 113:9276-9286; and all references cited therein.

Techniques for producing mutant or variant enzymes or producing "wild- type" or "unmodified enzymes" recombinantly are well known in the art. For example, PCT Publication Nos. WO 95/10615 and WO 91/06637, which are hereby incorporated by reference, provide alternative means of introducing unnatural amino acids into proteins to site-directed mutagenesis or chemical modification. Mutant or variant enzymes of the type in question, as well as detailed descriptions of the preparation and purification thereof are also disclosed in, for example, WO 90/00609, WO 94/24158 and WO 95/16782, as well as Greenwood et al . , Biotechnology and Bioengineering 44 (1994) pp. 1295-1305. All of these reference are hereby incorporated by reference.

However, briefly amino acid sequence mutants or variants of the unmodified amylase encompassed in the present invention may be prepared by introducing appropriate nucleotide changes into the DNA or cDNA of the unmodified amylase and thereafter expressing the resulting modified DNA or cDNA in a host cell, or by in vi tro synthesis. Such mutant and/or variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the amino acid sequence of the unmodified amylase. Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of the unmodified amylase, provided that such variant possesses che desired characteristics described herein.

The nucleotide sequence of nucleic acid molecules which encode amylase that would be particularly useful in the present invention are part of the public domain.

Nucleic acid molecules which encode amylases may be found, for example, in the Genbank database

(www.ncbi .nlm. gov/entrez) under Genbank accession number E09410, E09409, E09025, AB078768, AB078767, V00101, AB077387, AF504065, AF504064, AF504063, X12727, X12725 or X12726.

There are two principal variables in the construction of amino acid sequence variants of the unmodified amylase: the location of the mutation site and the nature of the mutation. These are variants from the amino acid sequence of the unmodified amylase, and may represent naturally occurring allelic forms of the unmodified amylase, or predetermined mutant forms of the unmodified amylase made by mutating the unmodified amylase DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the unmodified amylase characteristic to be modified.

For example, due to the degeneracy of nucleotide coding sequences, mutations can be made in the unmodified amylase nucleotide sequence without affecting the amino acid sequence of the unmodified amylase encoded thereby. Other mutations can be made that will result in the unmodified amylase having an amino acid sequence that is very different, but which is functionally active. Such functionally active amino acid sequence variants of the unmodified amylase are selected, for example, by substituting one or more amino acid residues with other amino acid residues of a similar or different polarity or charge .

Insertional, deletional, and substitutional changes in the amino acid sequence of the unmodified amylase may be made to improve the stability of the unmodified amylase before it is used in the present invention. For example, trypsin or other protease cleavage sites are identified by inspection of the encoded amino acid sequence for an arginyl or lysinyl residue. These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue. Also, any cysteine residues not involved in maintaining the proper conformation of the unmodified enzyme for functional activity may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

Cysteinyl residues most commonly are reacted with α- haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethy1 derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3 -nitro- 2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides.

Derivatization with these agents has the effect of reversing the charge of the lysinyl residues . Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0- methylisourea; 2,4-pentanedione; and transaminase- catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2 , 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK_a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N- terminal amine, and amidation of any C- terminal carboxyl group. Creighton, Proteins: Structure and Molecular Properties, pp.79-86 (W.H. Freeman & Co., 1983). The specific techniques used to create the mutant or variant amylase would depend upon the nature of the amylase and the mutation or variation required. However, a number of the techniques used to produce such mutants or variants are described in detail in publications such as Sambrook and Russell "Molecular Cloning: A Laboratory Manual" (2001); Frohman, et al . , Proc . Nat . Acad . Sci . USA 85:8998-9002 (1988); Saiki, et al . , Science 239:487-492 (1988); Mullis, et al . , Meth. Enzymol . 155:335-350 (1987); Zoller, et al . , Meth . Enz . 100:4668-500 (1983); Zoller, et al . , Meth . Enz . 154:329-350 (1987); Carter, Meth . Enz . 154:382-403 (1987); Horwitz, et al . , Λfefch. Enz. 185:599- 611 (1990); Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990); Vallette, et al . , Nuc . Acids Res . 17:723-733 (1989); Wagner, et al . , in PCR Topics, pp.69-71 (Springer-Verlag, 1991); Wells et al . , Gene, 34:315-323 (1985) all of which are incorporated herein by reference. Once a mutant or variant of the unmodified amylase has been created, or the DΝA from a wild-type amylase has been isolated, the DΝA is usually subcloned into a plasmid or other expression vector. "Plasmids" are DΝA molecules that are capable of replicating within a host cell, either extrachromosomally or as part of the host cell chromosome (s) , and are designated by a lower case "p" preceded and/or followed by capital letters and/or numbers .

Construction of suitable vectors containing the nucleotide sequence encoding the mutant, variant or wild- type amylase of interest and appropriate control sequences employs standard recombinant DΝA methods. DΝA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required. Normally it is desirable to add a signal sequence which provides for secretion of the enzyme. Typical examples of useful genes are: 1) Signal sequence-- (pro-peptide) - -carbohydrate- binding domain- -linker- - amylase sequence of interest, or 2) Signal sequence-- (pro-peptide)-- amylase sequence of interest- -linker-- carbohydrate-binding domain, in which the pro-peptide sequence normally contains 5-100, eg. 5- 25, amino acid residues.

Preparation of plasmids or vectors capable of expressing enzymes having the amino acid sequences derived from fragments of more than one polypeptide is well known in the art (see, for example, WO 90/00609 and WO 95/16782) . The DNA of the amylase of interest may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome. The DNA may be introduced) into the host in accordance with known techniques such as transformation, transfection, microinjection or the like.

Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype. The culture conditions, such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent to those skilled in the art.

Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, vertebrate, and mammalian host cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli , Bacillus species such as B. subtilis , Pseudomonas species such as P. aeruginosa , Salmonella typhimurium, or Serratia marcescens .

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for enzyme-encoding vectors. Saccharomyces cerevisiae , or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Beach and Nurse, Nature 290:140-142 (1981), Pichia pastoris , Cregg, et al . , Bio/Technology 5:479-485 (1987); Sreekrishna, et al . , Biochemistry 28:4117-4125 (1989), Neurospora crassa , Case, et al . , Proc . Natl . Acad. Sci . USA 76:5259-5263 (1979), and Aspergillus hosts such as A. nidulans, Ballance, et al . , Biochem. Biophys . Res . Commun . 112:284-289 (1983); Tilburn, et al . , Gene 26:205-221 (1983); Yelton, et al . , Proc . Natl . Acad. Sci . USA 81:1470-1474 (1984), and A. niger, Kelly, et al . , EMBO J. 4:475-479 (1985).

Suitable host cells for the expression of mutant, variant or wild-type amylases are also derived from ulticellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture. It will be appreciated, however, that because of the species-, tissue-, and cell-specificity of glycosylation, Rademacher, et al . , Ann . Rev. Biochem . 57:785-838 (1988), the extent or pattern of glycosylation of an amylase of interest in a foreign host cell typically will differ from that of the amylase obtained from a cell in which it is naturally expressed.

Examples of invertebrate cells include insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruitfly) , and Bombyx mori host cells have been identified. Luckow, et al . , Bio/Technology 6:47-55 (1988); Miller, et al . , in Genetic Engineering, vol. 8, pp.277 -279 (Plenum Publishing, 1986); Maeda, et al . , Nature 315:592-594 (1985).

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium AgrroJacterium tumefaciens, which has been previously altered to contain mutant, variant or wild-type enzyme DΝA. During incubation of the plant cells with A . tumefaciens, the DNA encoding the mutant, variant or wild-type enzyme is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the mutant, variant or wild-type enzyme. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences, and the ribulose biphosphate carboxylase promoter. Depicker, et al . , J. Mol. Appl. Gen. 1:561-573 (1982). Herrera-

Estrella, et al . , Nature 310:115-120 (1984). In addition, DΝA segments isolated from the upstream region of the T- DΝA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DΝA-containing plant tissue. European Pat. Pub. No. EP 321,196 (published June 21, 1989).

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Kruse & Patterson, eds., Tissue Culture (Academic Press, 1973). Examples of useful mammalian host cells are the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line 293 (or 293 cells subcloned for growth in suspension culture) , Graham, et al . , J. Gen Virol . 36:59-72 (1977); baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells (including DHFR-deficient CHO cells, Urlaub, et al . , Proc . Natl . Acad . Sci . USA 77:4216-4220 (1980); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather, et al . , Annals N. Y. Acad. Sci . 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .

Once the mutant, variant or "wild type" amylase gene has been introduced into the appropriate host, the host may be grown to express the amylase. One particularly preferred system of expression useful in this invention involves fermentation in which the mutant, variant or wild-type amylase of interest is introduced into a bacterial or yeast host as described above and then cultured in the presence of nutrient media containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art such as that described in Bennett, J.W. and LaSure(Eds.) "More Gene Manipulations in Fungi", Academic Press, CA, (1991) . Temperature ranges and other conditions suitable for growth and production of enzymes are also known in the art and are described in for example, Bailey, J.E. and Ollis, D.F., "Biochemical Engineering Fundamentals", McGraw-Hill Book Company, NY, 1986.

As used herein, the term "fermentation" refers to any growth condition which results in production of an enzyme by an organism(s) . It will be understood by persons skilled in the art that fermentation can refer to small or large scale fermentation and includes, for example, shake- flask cultivation, continuous, batch, fed-batch and solid state fermentation in laboratory or industrial fermenters. The mutant, variant or wild-type amylase may be isolated by any method that is suitable for isolating active amylase from organisms and/or growth media.

Suitable methods known in the art include, for example, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic- interaction chromatography (HIC) , affinity chromatography or the like, and combinations thereof. An example of an isolation method is provided in US Patent No. 5,434,067.

An example of an isolation method is as follows: fermentation broth is separated from the culture medium by centrifugation at δOOOrpm. Amylase is precipitated from the supernatant using a 65% saturated solution of ammonium sulphate. The precipitate is subsequently dissolved in 25mM phosphate buffer pH 7, 5mM EDTA. The solution is then applied to a Q-Sepharose FF (diameter 5cm, length

10cm) Anion Exchange column. The column is subsequently washed with 25mM phosphate buffer pH 7, 5mM EDTA until an absorbancy of 0.2 Absorbance Units at 280nm is attained. A gradient of 0 to 0.5M NaCl in 25mM phosphate buffer pH 7,

5mM EDTA is applied to the column in 80 minutes followed by a gradient from 0.5 to IM NaCl in 10 minutes. Elution may be performed in the first gradient.

Preferably, the amylase is an α-amylase or a gluco- amylase.

The amylase for use in the method of the invention may be a single isolated amylase or a mixture of amylases from different sources. For example, commercially available amylases BAN, TERMAMYL™, AMG, FUNGAMYL^R™, and PROMOZYME™, which are supplied by Novo Nordisk, and

Diazyme L-200, a product of Solvay Enzyme Products.

Preferably, the amylase is used as a single isolated amylase. However, the amylase may represent part of a mixture of different enzymes or other compounds. For example, it is envisaged that the amylase may be used in a crude form with contaminating compounds including other enzymes and proteins. In this case, the amylase may not be the only enzyme to which an amine-containing group is linked, however the resulting mixture will retain the ability to cleave starch at elevated temperature because of the presence in the mixture of amylase having an amine- containing group linked to a side chain of an amino acid residue of the enzyme or to a carboxy-terminal amino acid residue of the enzyme. Once the amylase is obtained as described above, an amine-containing group is contacted with the amino acid side chain. As used herein, the term "contacted" refers to sufficient contact between the amino acid side chain and the amine-containing group which permits the amine- containing group to be linked to the amino acid side chain in conditions sufficient for linking the amine-containing group to an amino acid side chain and/or to a carboxy- terminal amino acid of the enzyme. As used herein, the term "amine-containing group" means any compound that is amine containing and which improves the capacity of the enzyme to cleave α-1, 4-glucosidic bonds at elevated temperature and/or extends the half-life of the enzyme.

For example, the amine-containing group may be an aromatic group such as benzylamine, tryptophan methyl ester or 2- guanidino-benzimidazole, a heterocyclic group such as adenosine, adenine, cytosine, cytidine or pyridine, an amine containing carbohydrate such as glucosamine, an amine containing carbohydrate polymer such as chitosan, an aliphatic amine-containing group such as arginine methyl ester, argininamide, arginine ethyl ester, glycinamide, methamine, ethylenediamine, dimethylamine or trimethylamine, or other amine-containing group such as amidino. It will be understood by those skilled in the art that "chitosan" refers to a carbohydrate polymer - comprising N-acetyl-glucosamine and/or glucosamine residues. The term "linked" refers to any linkage formed between a portion of the amino acid side chain and the amine-containing group. It will be appreciated by those skilled in the art that following linkage of the amine- containing group to the amino acid side chain, the amino acid side chain to which the amine-containing group is linked will be altered and will differ from the amino acid side chains common to many proteins owing to the presence of the amine-containing group linked to the side chain of the amino acid. The amino acid side chains "common to many proteins" will be understood by those skilled in the art to mean the side chains belonging to the amino acids alanine, asparagine, aspartate, arginine, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, lysine, leucine, methionine, phenylalanine, proline, serine, tyrosine, tryptophan, threonine and valine. The amine-containing group may be linked to the amino acid side chain in any manner. In one embodiment, the amine- containing group is linked to the amino acid side chain through one or more nitrogen atoms. Preferably, the amine-containing group is linked to the amino acid side through an amide bond. It is envisaged that the amine group of the amine-containing group may form part of the linkage to the amino acid side chain. In another embodiment, the amine-containing group may be linked to the amino acid side chain through a linker. As used herein, a "linker" is a molecule which is not part of the amine-containing group nor part of the amino acid side chain, but serves to link the amine-containing group to the side chain of the amino acid.

The "conditions sufficient" for linking the amine- containing group to a side chain of an amino acid residue or a terminal amino acid residue may be any conditions which allow a reaction to occur between the amino acid side chain and the amine-containing group which results in linkage of the amine-containing group to the amino acid side chain.

In one embodiment, the conditions sufficient for linking the amine-containing group to a side chain of an amino acid residue and/or to a terminal amino acid residue comprise activating the carboxyl groups of the amylase at a temperature preferably between 18°C and 50°C, more preferably between 20°C and 40°C, even more preferably between 20°C and 28°C, and a pH preferably between 3.0 and 7.0, more preferably between 4.5 and pH7.0, and contacting the activated carboxyl groups with an amine-containing group containing nucleophile. Preferably, the carboxyl groups are on the side chains of aspartate and/or glutamate residues and/or on the carboxy-terminal amino acid.

As used herein, the term "activated" refers to a modification of an existing functional group to generate or introduce a new reactive functional group from the prior existing functional group, wherein the new reactive functional group is capable of undergoing reaction with another functional group to form a covalent bond. For example, a component containing carboxylic acid (-C00H) groups can be activated by reaction with N-hydroxy- succinimide or N-hydroxysulfosuccinimide using known procedures, to form an activated carboxylate (which is a reactive electrophilic group), ie., an N- hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester, respectively.

Activation of carboxylic acids may be accomplished in a variety of ways and by using a number of different reagents as described in Larock, "Comprehensive Organic Transformations", VCH Publishers, New York, 1989, all of which are incorporated herein by reference. However, activation often involves reaction with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) . For example, a carboxylic acid can be reacted with an alkoxy- substituted N-hydroxy- succinimide or N-hydroxysulfosuccinimide in the presence of DCC to form reactive electrophilic groups, the N- hydroxysuccinimide ester and the N-hydroxysulfosuccinimide ester, respectively. Carboxylic acids may also be activated by reaction with an acyl halide such as an acyl chloride (eg., acetyl chloride), again using known procedures, to provide an activated electrophilic group in the form of a reactive anhydride group. In a further example, a carboxylic acid may be converted to an acid chloride group using, eg., thionyl chloride or an acyl chloride capable of an exchange reaction. Specific reagents and procedures used to carry out such activation reactions will be known to those of ordinary skill in the art and are described in the pertinent texts and literature. In the present context, the term "activated carboxyl groups" means the rendering of one or more the carboxyl groups of the side chains of an amino acid of an enzyme reactive with a nucleophile. The terms "nucleophile" and "nucleophilic" refer to a functional group that is electron rich, has an unshared pair of electrons acting as a reactive site, and reacts with a positively charged or electron-deficient site, generally present on another molecule . The terms "electrophile" and "electrophilic" refer to a functional group that is susceptible to nucleophilic attack, ie., susceptible to reaction with an incoming nucleophilic group. Electrophilic groups herein are positively charged or electron-deficient, typically electron-deficient.

In one embodiment of the present invention, carboxyl groups of the enzyme are activated by incubating the enzyme with a carbodiimide, preferably utilising the carbodiimide condensation method described by Sheehan and Hess, and Khorana [Sheehan and Hess, J. Am. Chem. Soc. 77:1067, 1955; Khorana, Chem.Ind. 1087, 1995].

In another preferred embodiment of the present invention, carboxyl groups of the enzyme are activated by incubating the enzyme with Woodwards reagent. The difference between carbodiimide and Woodwards Reagent activation of carboxyl group is that in case of carbodiimide the carboxyl group must be protonated (COOH) , whereas in case of Woodwards reagent the carboxyl group may be ionised (COO^") . This means that carbodiimide activation works at low pH (4-6) whereas that of Woodward reagent works at high pH (6-8) . Some enzymes are precipitated at low pH, therefore for these enzymes the Woodward chemistry is better.

In one particularly preferred embodiment, the reaction is a condensation of the carboxyl with a substituted carbodiimide to form an active O-acylourea intermediate. Nucleophilic substitution with the amine containing group forms a stable amide with elimination of the substituted urea. The carbodiimide may be, for example, l-ethyl-3 (3- dimethylaminopropyl) carbodiimide or l-(3- dimethylaminopropyl) -3 -ethyl carbodiimide methiodide. Methods for the use of carbodiimide in the activation of carboxyl groups are provided in, for example, Carraway, K.L. and Koshland, D.E. Jr, Carbodiimide modification of proteins. In: Methods in Enzymology (Hirs, C.H.W. and Timasheff, S.N., Eds.) Academic Press, New York, 1972, XXV, 616-623.

The term "amine-containing group containing nucleophile" refers to any nucleophile comprising an amine-containing group. Amine-containing group containing nucleophiles may include, for example, benzylamine hydrochloride, pyridine hydrochloride, cytosine, adenosine hydrochloride, adenine hydrochloride, 2-guanidino- benzimidazole dihydrochloride, tryptophan methyl ester hydrochloride, glucosamine, chitosan, cytidine, arginine methyl ester dihydrochloride, ethylenediamine dihydrochloride, trimethylamine hydrochloride.

The carboxyl groups of the amino acid may be activated with carbodiimide prior to adding the amine- containing group containing nucleophile to the reaction. Preferably, the carboxyl groups of the amino acid side chains are activated with carbodiimide in the presence of the amine-containing group containing nucleophile. Preferably, the nucleophile is dissolved in an appropriate buffer such as, for example, K₂HP0₄/KH₂P0₄ buffer at a pH of preferably between 3.0 and 7.0, more preferably between 4.0 and 6.0. The buffer may optionally contain a amylase inhibitor. Suitable inhibitors may be, for example, maltose, cyclodextrin, maltotriose, maltodextrose, or any other substrate of amylase which is capable of protecting the active site of amylase from linkage of an amine- containing group to the active site residues. Amylase is added to the solution either as a dried preparation or as a solution. The reaction is initiated by the addition of carbodiimide to a final concentration of preferably between 30mM and 200mM, more preferably between 40mM and lOOmM.

It will be appreciated by persons skilled in the art that optimum times for allowing the reaction to proceed will vary depending on factors such as the concentration of reagents, the source of reagents, temperature conditions etc, and may be determined empirically.

Preferably, the enzyme is further purified using techniques known in the art such as, for example, dialysis, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic-interaction chromatography, affinity chromatography or the like, or combinations thereof.

In various embodiments, the modified amylase comprises :

(1) a pyridine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(2) the amino acid sequence of an α-amylase having a pyridine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (3) a benzylamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(4) an amino acid sequence of an α-amylase having a benzylamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(5) an adenine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (6) an amino acid sequence of an α-amylase having an adenine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (7) an adenosine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(8) an amino acid sequence of an α-amylase having an adenosine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(9) a glucosamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (10) an amino acid sequence of a α-amylase having a glucosamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(11) a 2-guanidino-benzimidazole group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(12) an amino acid sequence of a α-amylase having a 2- guanidino-benzimidazole group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(13) a cytidine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(14) an amino acid sequence of an α-amylase having a cytidine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(15) a cytosine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(16) an amino acid sequence of an α-amylase having a cytosine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (17) an arginine methyl ester group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(18) an amino acid sequence of an α-amylase having an arginine methyl ester group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(19) an ethylenediamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (20) an amino acid sequence of an α-amylase having an ethylenediamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(21) a trimethylamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid;

(22) an amino acid sequence of an α-amylase having a trimethylamine group linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid; (23) a chitosan molecule linked to the side chain of at least one amino acid and/or at least one tyrosine residue and/or to the carboxy-terminal amino acid of the enzyme; (24) a chitosan molecule linked to the side chain of at least one aspartate residue and/or at least one glutamate residue and/or at least one tyrosine residue and/or to the carboxy terminal amino acid; (25) an amino acid sequence of an α-amylase having a chitosan molecule linked to the side chain of at least one aspartate residue and/or at least one glutamate residue and/or at least one tyrosine residue and/or to the carboxy terminal amino acid.

In another embodiment, the amine-containing group may be linked by converting a lysine residue into a homoarginine residue. It will be understood by those skilled in the art that conversion of a lysine residue into a homoarginine residue results when an amidino group is linked to the side chain of a lysine residue. Preferably, the lysine residue is converted to a homoarginine residue by contacting the lysine residue of the enzyme with a compound selected from the group consisting of 0-methyl isourea and 3 , 5-dimethylpyrazole-l- carboxamidine nitrate (guanyl -3 , 5 -dimethyl pyrazole) under conditions sufficient to permit conversion of a lysine residue of the enzyme to a homoarginine residue. Thus, in various embodiments, the modified amylase comprises :

(1) an amidino group linked to the side chain of a lysine residue whereby the lysine residue is converted to a homoarginine residue; (2) an amino acid sequence of an α-amylase having an amidino group linked to the side chain of a lysine residue whereby the lysine residue is converted to a homoarginine residue; (3) an amino acid sequence of a gluco-amylase having an amidino group linked to the side chain of a lysine residue whereby the lysine residue is converted to a homoarginine residue.

Also contemplated are enzymes comprising two or more different amine-containing groups, or an amine-containing group and a different group linked to side chains of amino acids of the enzyme. In preparing these enzymes, the amine-containing group may be linked, for example, by incubating the enzyme with carbodiimide in the presence of two or more different amine-containing group containing nucleophiles. Alternatively, one type of amine-containing group may be linked to the amino acid side chain of the enzyme, and a second group, for example an aromatic group, subsequently linked to a side chain of an amino acid of the enzyme.

In various embodiments, the modified amylase comprises:

(1) an arginine methyl ester and a phthalic acid group, the arginine methyl ester group being linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid, and the phthalic acid group being linked to the side chain of a lysine residue or to an amino terminal amino acid;

(2) an amino acid sequence of an α-amylase having an arginine methyl ester and a phthalic acid group, the arginine methyl ester group being linked to the side chain of an aspartate residue, a glutamate residue, tyrosine residue and/or to a carboxy terminal amino acid, and the phthalic acid group being linked to the side chain of a lysine residue and/or to an amino terminal amino acid. While the modified amylase may be used directly after the amine-containing group has been linked to the enzyme, in one preferred embodiment the modified amylase is purified using a conventional enzyme purification method. For example, the modified amylase of the present invention may be purified by salting out with ammonium sulfate or other salts, gel filtration, dialysis, ion exchange chromatography, hydrophobic chromatography, crystallization, or by using a solvent such as acetone or an alcohol or the like. All of these methods are disclosed in well known literature such as Inman, "Methods in Enzymology", Vol. 34, "Affinity Techniques, Enzyme Purification"; Part B, Jacoby and Wichek (eds) Academic Press, New York, P. 30, 1974; R. Scriban, Biotechnology, (Technique et Documentation Lavoisier), 1982, pp. 267-276; and Wilcheck and Bayer, "The avidin-Biotin Complex" in Bioanalytical Applications Anal. Biochem. 171:1-32, 1988. All of these references are hereby expressly incorporated by reference in their entirety. In order to demonstrate that one or more amine- containing groups have been linked to the side chain of an amino acid residue and/or terminal amino acid of the enzyme, assays well known in the art may be employed. For example, the linking of an amine-containing group may be readily "observed" using techniques such as For example, structures from X-ray crystallographic techniques, NMR techniques, de novo modelling, homology modelling, PAGE, amino acid analysis, et cetera.

Also contemplated are compositions comprising the modified amylase of the present invention. In one embodiment, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a deoxyribonuclease, an esterase, an α-galactosidase, a β-galactosidase, an α-glucosidase, a β-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme, a ribonuclease, or a xylanase.

The composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the composition may be in the form of a granulate or a microgranulate . The additional enzymes to be included in the composition may be stabilized in accordance with methods known in the art. For example, see U.S. Pat. No. 4,238,345 issued Dec. 9,

1980; U.S. Pat. No. 4,243,543 issued Jun. 6, 1981 and U.S. Pat. No. 6,197,739 issued Mar. 6, 2001 incorporated herein by reference.

The dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art depending upon the application.

The modified amylase according to the present invention and compositions comprising the enzyme may be applied in industrial processes. For example, the modified amylase of the present invention can be formulated into powdered or liquid detergents. These detergent cleaning compositions or additives can also include other enzymes, such as known proteases, xylanases, cellulases, lipases, or endoglycosidases , as well as builders and stabilizers .

The modified amylase of the present invention is useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the modified enzyme of the present invention. These include non-ionic, anionic, cationic, anionic, or zwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 to Anderson and U.S. Pat. No. 4,261,868 to Flora et al . , which are hereby incorporated by reference. A suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015 to Caldwell et al . , which is hereby incorporated by reference. The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the modified amylase of the present invention may be used for any purpose that native or wild-type amylases are used. Thus, the modified amylase can be used, for example, in bar or liquid soap applications, dish-care formulations, contact lens cleaning solutions or products, peptide synthesis, feed applications such as feed additives or preparation of feed additives, waste treatment, textile applications such as the treatment of fabrics, and as fusion-cleavage enzymes in protein production. The modified amylase of the present invention may achieve improved wash performance in a detergent composition (as compared to the unmodified enzyme) . As used herein, "improved wash performance" in a detergent is defined as increasing cleaning of certain enzyme-sensitive stains such as grass or blood, as determined by light reflectance evaluation after a standard wash cycle.

The addition of the modified amylase of the present invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within a suitable range and the temperature is below the described modified amylase' s denaturing temperature. In addition, the modified amylase in accordance with the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

The laundry detergent and/or fabric care compositions of the invention may also contain additional detergent and/or fabric care components. The precise nature of these additional components, and levels of incorporation thereof will depend on the physical form of the composition, and the nature of the cleaning operation for which it is to be used.

The laundry detergent and/or fabric care compositions of the present invention preferably further comprise a detergent ingredient selected from cationic surfactants, proteolytic enzymes, bleaching agents, builders-in particular zeolite A and sodium tripolyphosphate-and/or clays . These laundry detergent and/or fabric care compositions achieve improved overall cleaning including stain removal and whitening maintenance, while preventing any negative effect on the fabric. These compositions further provide improved fabric care, including anti- bobbling, depilling, colour appearance, fabric softness and fabric anti-wear properties and benefits, while preventing any negative effect on the fabric. The laundry detergent and/or fabric care compositions according to the invention can be liquid, paste, gels, bars, tablets, spray, foam, powder or granular forms. Granular compositions can also be in "compact" form, the liquid compositions can also be in a "concentrated" form. The compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pre- treatment of stained fabrics, rinse added fabric softener compositions. Pre-or post treatment of fabric include gel, spray and liquid fabric care compositions. A rinse cycle with or without the presence of softening agents is also contemplated.

When formulated as compositions suitable for use in a laundry machine washing method, the compositions of the invention preferably contain both a surfactant and a builder compound and addition one or more detergent components preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors. Laundry compositions can also contain softening agents, as additional detergent components.

The laundry detergent and/or fabric care compositions according to the present invention comprise a surfactant system wherein the surfactant can be selected from non- ionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants.

The surfactant is typically present at a level of from 0.1% to 60% by weight. More preferred levels of incorporation are 1% to 35% by weight, most preferably from 1% to 30% by weight of laundry detergent and/or fabric care compositions in accord with the invention.

The surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated such that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.

Cationic detersive surfactants suitable for use in the laundry detergent and/or fabric care compositions of the present invention are those having one long-chain hydrocarbyl group. Examples of such cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides and quaternary ammonium surfactants such as coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C₁₂-₁₅ dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide. Other cationic surfactants useful herein are also described in U. S. Patent 4,228,044, Cambre, issued October 14,1980 and in European Patent Application EP 000,224.

Typical cationic fabric softening components include the water-insoluble quaternary-ammonium fabric softening actives or their corresponding amine precursor, the most commonly used having been di-long alkyl chain ammonium chloride or methyl sulfate. Preferred cationic softeners among these include the following: 1) ditallow dimethylammonium chloride (DTDMAC) ; 2) dihydrogenated tallow dimethylammonium chloride; 3) dihydrogenated tallow dimethylammonium methylsulfate; 4) distearyl dimethylammonium chloride; 5) dioleyl dimethylammonium chloride; 6) dipal ityl hydroxyethyl methylammonium chloride; 7) stearyl benzyl dimethylammonium chloride; 8) tallow trimethylammonium chloride; 9) hydrogenated tallow trimethylammonium chloride; 10) Cι_2-ι₄ alkyl hydroxyethyl dimethylammonium chloride; 11) Cι_2-ι₈ alkyl dihydroxyethyl methylammonium chloride; 12) di (stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC) ; 13) di ( tallow-oxy-ethyl) dimethylammonium chloride; 14) ditallow imidazolinium methylsulfate; 15) 1- (2 - tallowylamidoethyl) -2-tallowyl imidazolinium methylsulfate.

Biodegradable quaternary ammonium compounds have been presented as alternatives to the traditionally used di- long alkyl chain ammonium chlorides and methyl sulfates. Such quaternary ammonium compounds contain long chain_. alkyl, alkenyl, alkyne or groups interrupted by functional groups such as carboxy groups. Said materials and fabric softening compositions containing them are disclosed in numerous publications such as EP-A-0, 040, 562 , and EP-A- 0,239,910. Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight- chain or branched-chain configuration with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include Igepal™ CO-630, marketed by the GAF Corporation; and Triton™ X-45, X-114, X-100 and X-102, all marketed by the Rohm S_ Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (eg., alkyl phenol ethoxylates) .

The condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide are suitable for use as the non-ionic surfactant of the non-ionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Preferred are the condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol. About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available non- ionic surfactants of this type include Tergitol™ 15-S-9 (the condensation product of Cn-Cι₅ linear alcohol with 9 moles ethylene oxide) , Tergitol™ 24-

L-6 NMW (the condensation product of Cι₂-C₁₄ primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; Neodol™ 45-9 (the condensation product of C14-Cls linear alcohol with 9 moles of ethylene oxide), Neodol™ 23-3 (the condensation product of Cι₂-Cι₃ linear alcohol with 3.0 moles of ethylene oxide) , Neodol™ 45-7 (the condensation product of C14-Cls linear alcohol with 7 moles of ethylene oxide) , Neodol™ 45-5 (the condensation product of C14-Cls linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, Kyro™ EOB (the condensation product of Cι₃-Cι alcohol with 9 moles ethylene oxide) , marketed by The Procter & Gamble Company, and Genapol LA 030 or 050 (the condensation product of Cι₂-Cι₄ alcohol with 3 or 5 moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB in these products is from 8-11 and most preferred from 8-10.

Also useful as the non-ionic surfactant of the surfactant systems of the present invention are the alkylpolysaccharides disclosed in US Patent 4,565,647, Llenado, issued January 21,1986, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, eg. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms can be used, eg., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside) . The intersaccharide bonds can be, eg. , between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6 -positions on the preceding saccharide units. The laundry detergent and/or fabric care compositions of the present invention may also contain ampholytic, zwitterionic, and semi-polar surfactants, as well as the non- ionic and/or anionic surfactants other than those already described herein. Ampholytic surfactants are also suitable for use in the laundry detergent and/or fabric care compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight-or branched-chain. One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e. g. carboxy, sulfonate, sulfate. See U. S. Patent No. 3,929,678 to Laughlin et al . , issued December 30,1975 at column 19, lines 18-35, for examples of ampholytic surfactants .

When included therein, the laundry detergent and/or fabric care compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants .

Zwitterionic surfactants are also suitable for use in laundry detergent and/or fabric care compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US Patent No. 3,929,678 to Laughlin et al . , issued December 30,1975 at column 19, line 38 through column 22, line 48, for examples of zwitterionic surfactants.

When included therein, the laundry detergent and/or fabric care compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1 % to about 10% by weight of such zwitterionic surfactants .

Semi-polar non-ionic surfactants are a special category of non-ionic surfactants which include water- soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms . In another aspect of the present invention, the modified enzymes are used in the preparation of an animal feed, for example, a cereal-based feed. The cereal can be at least one of wheat, barley, maize, sorghum, rye, oats, triticale, and rice. Although the cereal component of a cereal-based feed constitutes a source of protein, it is usually necessary to include sources of supplementary protein in the feed such as those derived from fish-meal, meat-meat, or vegetables. Sources of vegetable proteins include at least one of full fat soybeans, rapeseeds, canola, soybean-meal, rapeseed-meal, and canola-meal.

The inclusion of a modified amylase of the present invention in an animal feed can enable the crude protein value and/or digestibility and/or amino acid content and/or digestibility coefficients of the feed to be increased, which permits a reduction in the amounts of alternative protein sources and/or amino acids supplements which had previously been necessary ingredients of animal feeds .

The feed provided by the present invention may also include other enzyme supplements such as one or more of β- glucanase, mannanase, α-galactosidase, phytase, lipase, α- arabinofuranosidase, xylanase, esterase, oxidase, oxido- reductase, and pectinase. It is particularly preferred to include a xylanase as a further enzyme supplement such as a subtilisin derived from the genus Bacillus. Such xylanases are, for example, described in detail in PCT Patent Application No. WO 97/20920, which is hereby incorporated by reference

The invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES

Example 1. Chemical modification of α-amylase from Aspergi llus oryzae . To link amine-containing groups to side chains of amino acid residues arranged on the surface of the enzyme, carboxyl groups were activated using l-ethyl-3 (3- di ethylaminopropyl) carbodiimide (EDC) in the presence of the nucleophiles containing amines listed in Table 1 with and without maltose (as indicated) as competitive inhibitor for the protection of active-site residues. Nucleophile (amount as indicated in Table 1) was added to 1-5 ml of 40 mM K₂HP0₄/KH₂P0₄, pH 5.25 (final pH as indicated in table 1) buffer with or without (as indicate in Table 1) 50 mM maltose or 10 mM cyclodextrin as competitive inhibitors, the pH was readjusted with 2 M NaOH. Dialyzed α-amylase in water was added to the above mentioned nucleophilic solution at a concentration of 0.5- 1 mg ml^"1 and allowed to equilibrate at room temperature for 30 min. The coupling reaction was initiated by adding solid EDC to a final concentration of 50-100 mM. Modified amylase was subjected to dialysis for the removal of excess reagents and subsequently characterized.

The results of assays are summarised in table 1.

Example 2. Double modification of α-amylase.

a. Carboxyl Group Modification Arginine methyl ester solution was made (IM) in 40mM

KH₂PO4/K2HPO4, pH 5.2 buffer containing 5mM NaCl. The pH was adjusted to 5.2 with 2M KOH. To the above 1ml solution was added maltose (50mM) and lOOμl dialysed enzyme was added.

The reaction was initiated by the addition of O.Olg EDC/ml (carbodiimide) [50mM] . The reaction was stopped after 60 min with 1ml of lOOmM sodium acetate, pH 7 buffer.

The modification enzyme was repeatedly dialysed to remove reagents against water.

b. Amino Group Modification (Phthalic anhydride)

2ml of arginine methyl ester modified amylase was mixed with an equal amount of 0.2M K₂HPO₄/KH₂PO₄, pH8.4 buffer containing 200mM sodium acetate. IM phthalic anhydride solution was made in DMSO (dimethyl sulfoxide) .

25μl of phthalic anhydride solution was added.

The double modified enzyme was put for repeated dialysis against 50mM K₂HP0₄/citric acid, pH5 and 5mM NaCl.

The results of assays are summarised in Table 1.

Example 3. Guanidation of α-amylase and Gluco-amylase

0.5 M of 2-3 dimethyl pyrazole- 1-carboxamidine nitrate was made in water and pH adjusted to 9.5 with 2 M NaOH. 0.5-1 mg amylase was added to 1 ml of reagent and incubated at 2 °C for 72 h (gluco-amylase) and 120 h (α-amylase) . After the reaction, the guanidinated enzymes were dialyzed against 50 mM sodium acetate/acetic acid or KH₂P0₄/citric acid, pH 5 buffers. α-Amylase was also guanidinated using O-methyl isourea. 0.5 M O-methylisourea was made in water and pH adjusted to 10.4 with 2 M NaOH. 10 mM cyclodextrin was added to the above solution. 0.5-1 mg α-Amylase/ml was added to above reagent and incubated at 2 °C for 72 h. After 72 h the guanidinated enzyme was dialyzed against KH₂P0₄/citric acid, pH 5 buffer and characterized.

The results of assays of reagents are summarised in table 1.

Example 4: Chemical modification of α-amylase from Aspergillus oryzae with ethyhlenediamine using Woodwards reagent .

To 1.9 ml of 0.2M K₂HPO₄/H₃PO₄, pH7.5 buffer containing 50mM maltose and ImM NaCl, 16 μl of Woodwards Reagent K (from a stock of 0.47M in dilute HCl,pH 3) was added per 2ml to give a final concentration of 3.8mM (Activation of COO^" results in formation of an Enol-ester which absorbs strongly at 340 nm) . Following 5-10min incubation at room temperature (the A₃₄₀ has been increased to -0.3-.5), 500μl of IM ethylenediamine hydrochloride pH7.5 was added to the mixture to give a final concentration of ethylenediamine of 200mM. (A decrease in A340 is indicative of the fact that nucleophile [ethylenediamine .2 HCl] has reacted with enol-ester intermediate) After 15 min, the mixture was dialysed against K₂HP0₄/citric acid pH5 buffer.

The resulting enzyme was assayed and the results of the assays are summarised in Table 1. Table 1. Results of chemical modification of α-amylase from Aspergillus oryzae (1-30 and 34) and Gluco-amylase from Rhizopus sp. (31-33) .

I = inhibitor (50 mM Maltose) . BCA = Bicinchonic acid protein estimation method, BF = Bradford protein estimation method. The specific activity is determined as Activity Absorbance units/BCA or BF Absorbance units using same amount of enzyme. The specific activity of native amylases is taken as 100% and the specific activities of all modified lipases are calculated relative to the native enzyme. Woodwards Reagent K = N-ethyl-5-

10 phenylisooxazolium-3 "-sulfonate.

The enzyme activity is determined by Reducing Sugar Assay using dinitrosalicylic acid Reagent. Appropriate amounts of amylase solution (20-100 μl) were added to 1 ml of 2% (w/v) Starch solution in 50 mM Na₂HP0₄/citric acid, pH 5 + 10 mM NaCl buffer and incubated at 40 °C. After 10 min the reaction was stopped by adding 1 ml of Dinitrosalicylic acid reagent and boiled for 5 min. The mixture is cooled and A₅₄₀ is determined against reagent blank.

The assay for gluco-amylase was done as described above for α-amylase except that the temperature of the assay was 45 °C and buffer was 50 mM sodium acetate, pH 5.

Half-lives (irreversible thermal denaturation) were determined by heating (20-100 μl) of amylases at a certain temperature (50 and/or 60 °C) . Aliquots were taken at various time intervals, cooled in ice and residual activity determined by assaying the enzyme.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

Claims

CLAIMS :

1. An enzyme capable of cleaving α-1, 4-glucosidic bonds of starch comprising an amylase which has been modified by having an amine-containing group linked: (c) to a side chain of an amino acid residue of the amylase; or (d) to a terminal amino acid residue of the amylase, wherein the enzyme functions at an elevated temperature and/or has an extended half-life compared to the corresponding unmodified amylase.

2. The enzyme of claim 1 comprising a further amine- containing group linked to a side chain of an amino acid residue of the enzyme, or to a terminal amino acid residue of the enzyme.

3. The enzyme of claim 1 or 2 wherein the amine-containing group is linked to a carboxyl group of a side chain of an amino acid or to the carboxy terminal amino acid.

4. The enzyme of any one of claims 1 to 3 wherein the amine-containing group is linked to the side chain of an aspartate residue, a glutamate residue and/or to a carboxy terminal amino acid residue.

5. The enzyme of any one of claims 1 to 3 wherein the amine-containing group is linked to an amino group of a side chain of an amino acid or to the amino terminal amino acid.

6. The enzyme of claim 5 wherein the amine-containing group is linked to the side chain of a lysine residue of the enzyme .

7. The enzyme of any one of claims 1 to 6 wherein the amine-containing group is linked to the side chain of an amino acid or to a terminal amino acid by an amide bond.

8. The enzyme of any one of claims 1 to 7 wherein the amine-containing group is an aromatic group.

9. The enzyme of claim 8 wherein the aromatic group is a derivative of benzene.

10. The enzyme of claim 9 wherein the derivative of benzene is benzylamine, 2-guanidino-benzimidazole or tryptophan methyl ester.

11. The enzyme of claim 9 wherein the aromatic group is a heterocyclic amine.

12. The enzyme of claim 11 wherein the heterocyclic amine is selected from the group consisting of adenosine, adenine, cytosine, cytidine or pyridine.

13. The enzyme of any one of claims 1 to 7 wherein the amine-containing group is an amine containing carbohydrate .

14. The enzyme of claim 13 wherein the amine-containing carbohydrate is glucosamine or chitosan.

15. The enzyme of any one of claims 1 to 7 wherein the amine-containing group is an aliphatic amine-containing group .

16. The enzyme of claim 15 wherein the aliphatic amine- containing group is selected from the group consisting of arginine methyl ester, argininamide, arginine ethyl ester, glycinamide, methylamine, ethylenediamine, dimethylamine and trimethylamine.

17. The enzyme of any one of claims 1 to 7 wherein the amine-containing group is selected from the group consisting of an amino group and an amidino group.

18. The enzyme of any one of claims 1 to 18 wherein the enzyme has the amino acid sequence of an amylase from an organism selected from the group comprising Aspergillus sp., Scopulariopsis sp., Rhizopus sp. or Trichoderma sp. Most preferably the microorganism is Aspergillus oryzae or Rhizopus sp.

19. The enzyme of any one of claims 1 to 18 wherein the amylase is an α-amylase or a glucoamylase .

20. The enzyme of claim 19 wherein the amylase is α-amylase is that of Aspergillus oryzae .

21. The enzyme of claim 19 wherein the amylase is gluco- amylase is that of Rhizopus sp.

22. A process for producing the enzyme of claim 1, the process comprising the step of contacting an enzyme capable of cleaving α-1, 4-glucosidic bonds of starch with a compound which comprises an amine-containing group in conditions sufficient for linking the amine- containing group to a side chain of an amino acid residue of the enzyme, or to a terminal amino acid residue of the enzyme.

23. The process of claim 22 wherein the process comprises activating carboxyl groups of amino acid residues of the enzyme in the presence of a compound which comprises an amine-containing group.

24. The process of claim 23 wherein the compound is a nucleophile selected from the group consisting of aromatic nucleophile, carbohydrate nucleophile, aliphatic amine nucleophile, heterocyclic amine nucleophile and amino nucleophile.

25. The process of claim 24 wherein the aliphatic amine nucleophile is selected from the group consisting of argininamide dihydrochloride, arginine methyl ester dihydrochloride, arginine ethyl ester dihydrochloride, glycinamide hydrochloride, methylamine hydrochloride, dimethylamine hydrochloride, ethylenediamine dihydrochloride and trimethylamine hydrochloride.

26. The process of claim 24 wherein the aromatic nucleophile is selected from the group consisting of benzylamine hydrochloride, tryptophan methyl ester hydrochloride and 2-guanidino-benzimidazole dihydrochloride .

27. The process of claim 24 wherein the heterocyclic nucleophile is selected from the group consisting of adenine hydrochloride, adenosine hydrochloride, pyridine hydrochloride, cytidine and cytosine.

28. The process of claim 24 wherein the carbohydrate nucleophile is selected from the group consisting of glucosamine and chitosan.

29. The process of claim 24 wherein the amino nucleophile is ethylenediamine dihydrochloride or trimethylamine.

30. The process of any one of claims 23 to 29 wherein carboxyl groups are activated by carbodiimide.

31. The process of claim 22 wherein the amylase is contacted with a compound selected from the group consisting of 0-methyl isourea and 3 , 5-dimethylpyrazole- 1-carboxamidine nitrate (guanyl -3 , 5-dimethyl pyrazole) under conditions sufficient to permit conversion of a lysine residue of the enzyme to a homoarginine residue.

32. An enzyme produced by the process of any one of claims 22 to 31.

33. A composition comprising an enzyme according to any one of claims 1 to 21 or 32 and a suitable carrier.

34. A method of cleaving the α-1, 4-glucosidic bonds of starch, comprising the step of exposing a compound or composition comprising starch to an enzyme according to any one of claims 1 to 21 or 32.