Oxidatively stable alpha-amylase

FIELD OF THE INVENTION

The present invention relates to novel alpha-amylase mutants having an amino acid sequence not found in nature, such mutants having an amino acid sequence wherein one or more amino acid residue(s) of a precursor alpha-amylase, specifically any oxidizable amino acid, have been substituted with a different amino acid. The mutant enzymes of the present invention exhibit altered stability/activity profiles including but not limited to altered oxidative stability, altered pH performance profile, altered specific activity and/or altered thermostability.

BACKGROUND OF THE INVENTION

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolase, EC3.2.1.1) hydrolyze internal alpha-1,4-glucosidic linkages in starch largely at random, to produce smaller molecular weight malto-dextrins. Alpha-amylases are of considerable commercial value, being used in the initial stages (liquefaction) of starch processing; in alcohol production; as cleaning agents in detergent matrices; and in the textile industry for starch desizing. Alpha-amylases are produced by a wide variety of microorganisms including Bacillus and Aspergillus, with most commercial amylases being produced from bacterial sources such as

B. licheniformis, B. amyloliquefaciens, B. subtilis

, or

B. stearothermophilus

. In recent years the preferred enzymes in commercial use have been those from

B. licheniformis

because of their heat stability and performance, at least at neutral and mildly alkaline pH's.

Previously there have been studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases (Vihinen, M. et al. (1990) J. Bichem. 107:267-272; Holm, L. et al. (1990) Protein Engineering 3:181-191; Takase, K. et al. (1992) Biochemica et Biophysica Acta, 1120:281-288; Matsui, I. et al. (1992) Febs Letters Vol. 310, No. 3, pp. 216-218); which residues are important for thermal stability (Suzuki, Y. et al. (1989) J. Biol. Chem. 264:18933-18938); and one group has used such methods to introduce mutations at various histidine residues in a

B. licheniformis

amylase, the rationale for making substitutions at histidine residues was that

B. licheniformis

amylase (known to be thermostable) when compared to other similar Bacillus amylases, has an excess of histidines and, therefore, it was suggested that replacing a histidine could affect the thermostability of the enzyme (Declerck, N. et al. (1990) J. Biol. Chem. 265:15481-15488; FR 2 665 178-A1; Joyet, P. et al. (1992) Bio/Technology 10:1579-1583).

It has been found that alpha-amylase is inactivated by hydrogen peroxide and other oxidants at pH's between 4 and 10.5 as described in the examples herein. Commercially, alpha-amylase enzymes can be used under dramatically different conditions such as both high and low pH conditions, depending on the commercial application. For example, alpha-amylases may be used in the liquefaction of starch, a process preferably performed at a low pH (pH <5.5). On the other hand, amylases may be used in commercial dish care or laundry detergents, which often contain oxidants such as bleach or peracids, and which are used in much more alkaline conditions.

In order to alter the stability or activity profile of amylase enzymes under varying conditions, it has been found that selective replacement, substitution or deletion of oxidizable amino acids, such as a methionine, tryptophan, tyrosine, histidine or cysteine, results in an altered profile of the variant enzyme as compared to its precursor. Because currently commercially available amylases are not acceptable (stable) under various conditions, there is a need for an amylase having an altered stability and/or activity profile. This altered stability (oxidative, thermal or pH performance profile) can be achieved while maintaining adequate enzymatic activity, as compared to the wild-type or precursor enzyme. The characteristic affected by introducing such mutations may be a change in oxidative stability while maintaining thermal stability or vice versa. Additionally, the substitution of different amino acids for an oxidizable amino acids in the alpha-amylase precursor sequence or the deletion of one or more oxidizable amino acid(s) may result in altered enzymatic activity at a pH other than that which is considered optimal for the precursor alpha-amylase. In other words, the mutant enzymes of the present invention may also have altered pH performance profiles, which may be due to the enhanced oxidative stability of the enzyme.

SUMMARY OF THE INVENTION

The present invention relates to novel alpha-amylase mutants that are the expression product of a mutated DNA sequence encoding an alpha-amylase, the mutated DNA sequence being derived from a precursor alpha-amylase by the deletion or substitution (replacement) of one or more oxidizable amino acid. In one preferred embodiment of the present invention the mutant result from substituting a different amino acid for one or more methionine residue(s) in the precursor alpha-amylase. In another embodiment of the present invention the mutants comprise a substitution of one or more tryptophan residue alone or in combination with the substitution of one or more methionine residue in the precursor alpha-amylase. Such mutant alpha-amylases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding a naturally occurring or recombinant alpha-amylase to encode the substitution or deletion of one or more amino acid residues in a precursor amino acid sequence.

Preferably the substitution or deletion of one or more amino acid in the amino acid sequence is due to the replacement or deletion of one or more methionine, tryptophan, cysteine, histidine or tyrosine residues in such sequence, most preferably the residue which is changed is a methionine residue. The oxidizable amino acid residues may be replaced by any of the other 20 naturally occurring amino acids. If the desired effect is to alter the oxidative stability of the precursor, the amino acid residue may be substituted with a non-oxidizable amino acid (such as alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, or valine) or another oxidizable amino acid (such as cysteine, methionine, tryptophan, tyrosine or histidine, listed in order of most easily oxidizable to less readily oxidizable). Likewise, if the desired effect is to alter thermostability, any of the other 20 naturally occurring amino acids may be substituted (i.e., cysteine may be substituted for methionine).

Preferred mutants comprise the substitution of a methionine residue equivalent to any of the methionine residues found in

B. licheniformis

alpha-amylase (+8, +15, +197, +256, +304, +366 and +438). Most preferably the methionine to be replaced is a methionine at a position equivalent to position +197 or +15 in

B. licheniformis

alpha-amylase. Preferred substitute amino acids to replace the methionine at position +197 are alanine (A), isoleucine (I), threonine (T) or cysteine (C). The preferred substitute amino acids at position +15 are leucine (L), threonine (T), asparagine (N), aspartate (D), serine (S), valine (V) and isoleucine (I), although other substitute amino acids not specified above may be useful. Two specifically preferred mutants of the present invention are M197T and M15L.

Another embodiment of this invention relates to mutants comprising the substitution of a tryptophan residue equivalent to any of the tryptophan residues found in

B. licheniformis

alpha-amylase (see FIG.

2

). Preferably the tryptophan to be replaced is at a position equivalent to +138 in

B. licheniformis

alpha-amylase. A mutation (substitution) at a tryptophan residue may be made alone or in combination with mutations at other oxidizable amino acid residues. Specifically, it may be advantageous to modify by substitution at least one tryptophan in combination with at least one methionine (for example, the double mutant +138/+197).

The alpha-amylase mutants of the present invention, in general, exhibit altered oxidative stability in the presence of hydrogen peroxide and other oxidants such as bleach or peracids, or, more specific, milder oxidants such as chloramine-T. Mutant enzymes having enhanced oxidative stability will be useful in extending the shelf life and bleach, perborate, percarbonate or peracid compatibility of amylases used in cleaning products. Similarly, reduced oxidative stability may be useful in industrial processes that require the rapid and efficient quenching of enzymatic activity. The mutant enzymes of the present invention may also demonstrate a broadened pH performance profile whereby mutants such as M15L show stability for low pH starch liquefaction and mutants such as M197T show stability at high pH cleaning product conditions. The mutants of the present invention may also have altered thermal stability whereby the mutant may have enhanced stability at either high or low temperatures. It is understood that any change (increase or decrease) in the mutant's enzymatic characteristic(s), as compared to its precursor, may be beneficial depending on the desired end use of the mutant alpha-amylase .

In addition to starch processing and cleaning applications, variant amylases of the present invention may be used in any application in which known amylases are used, for example, variant amylases can be used in textile processing, food processing, etc. Specifically, it is contemplated that a variant enzyme such as M197C, which is easily inactivated by oxidation, would be useful in a process where it is desirable to completely remove amylase activity at the end of the process, for example, in frozen food processing applications.

The preferred alpha-amylase mutants of the present invention are derived from a Bacillus strain such as

B. licheniformis, B. amyloliquefaciens

, and

B. stearothermophilus

, and most preferably from

Bacillus licheniformis.

In another aspect of the present invention there is provided a novel form of the alpha-amylase normally produced by

B. licheniformis

. This novel form, designated as the A4 form, has an additional four alanine residues at the N-terminus of the secreted amylase. (

FIG. 4

b

.) Derivatives or mutants of the A4 form of alpha-amylase are encompassed within the present invention. By derivatives or mutants of the A4 form, it is meant that the present invention comprises the A4 form alpha-amylase containing one or more additional mutations such as, for example, mutation (substitution, replacement or deletion) of one or more oxidizable amino acid(s).

In a composition embodiment of the present invention there are provided detergent compositions, liquid, gel or granular, comprising the alpha-amylase mutants described herein. Particularly preferred are detergent compositions comprising a +197 position mutant either alone or in combination with other enzymes such as endoglycosidases, cellulases, proteases, lipases or other amylase enzymes. Additionally, it is contemplated that the compositions of the present invention may include an alpha-amylase mutant having more than one site-specific mutation.

In yet another composition embodiment of the present invention there are provided compositions useful in starch processing and particularly starch liquefaction. The starch liquefaction compositions of the present invention preferably comprise an alpha-amylase mutant having a substitution or deletion at position M15. Additionally, it is contemplated that such compositions may comprise additional components as known to those skilled in the art, including, for example, antioxidants, calcium, ions, etc.

In a process aspect of the present invention there are provided methods for liquefying starch, and particularly granular starch slurries, from either a wet or dry milled process. Generally, in the first step of the starch degradation process, the starch slurry is gelatinized by heating at a relatively high temperature (up to about 110° C.). After the starch slurry is gelatinized it is liquefied and dextrinized using an alpha-amylase. The conditions for such liquefaction are described in commonly assigned U.S. patent applications Ser. Nos. 07/785,624 and 07/785,623 and U.S. Pat. No. 5,180,669, the disclosure of which are incorporated herein by reference. The present method for liquefying starch comprises adding to a starch slurry an effective amount of an alpha-amylase of the present invention, alone or in combination with additional excipients such as an antioxidant, and reacting the slurry for an appropriate time and temperature to liquefy the starch.

A further aspect of the present invention comprises the DNA encoding the mutant alpha-amylases of the present invention (including A4 form and mutants thereof) and expression vectors encoding the DNA as well as host cells transformed with such expression vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C

show the DNA sequence of the gene for alpha-amylase from

B. licheniformis

(NCIB8061), Seq ID No 31, and deduced translation product as described in Gray, G. et al. (1986) J. Bacter. 166:635-643.

FIG. 2

shows the amino acid sequence of the mature alpha-amylase enzyme from

B. licheniformis

(NCIB8061), Seq ID No 32.

FIGS. 3A-3B

show an alignment of primary structures of Bacillus alpha-amylases. The

B. licheniformis

amylase (Am-Lich), Seq ID No 33, is described by Gray, G. et al. (1986) J. Bact. 166:635-643; the

B. amyloliquefaciens

amylase (Am-Amylo), Seq ID No 34, is described by Takkinen, K. et al. (1983) J. Biol. Chem. 258:1007-1013; and the

B. stearothermophilus

(Am-Stearo), Seq ID No 35, is described by Ihara, H. et al. (1985) J. Biochem. 98:95-103.

FIG. 4

a

shows the amino acid sequence of the mature alpha-amylase variant M197T, Seq ID No 36.

FIG. 4

b

shows the amino acid sequence of the A4 form of alpha-amylase from

B. licheniformis

NCIB8061, Seq ID No 37. Numbering is from the N-terminus, starting with the four additional alanines.

FIG. 5

shows plasmid pA4BL wherein BLAA refers to

B. licheniformis

alpha-amylase gene, PstI to SstI; AmP

R

refers to the ampicillin-resistant gene from pBR322; and CAT refers to the Chloramphenicol-resistant gene from pC194.

FIG. 6

shows the signal sequence-mature protein junctions for

B. licheniformis

(Seq ID No 38),

B. subtilis

(Seq ID No 39),

B. licheniformis

in pA4BL (Seq ID No 40) and

B. licheniformis

in pBLapr (Seq ID No 41).

FIG. 7

a

shows inactivation of certain alpha-amylases (Spezyme® AA20 and M197L (A4 form) with 0.88M H

2

O

2

at pH 5.0, 25° C.

FIG. 7

b

shows inactivation of certain alpha-amylases (Spezyme® AA20, M197T) with 0.88M H

2

O

2

at pH 10.0, 25° C.

FIG. 7

c

shows inactivation of certain alpha-amylases (Spezyme® AA20, M15L) with 0.88M H

2

O

2

at pH 5.0, 25° C.

FIG. 8

shows a schematic for the production of M197X cassette mutants.

FIG. 9

shows expression of M197X variants.

FIG. 10

shows thermal stability of M197X variants at pH 5.0, 5 mM CaCl

2

at 95° C. for 5 mins.

FIGS. 11

a

and

11

b

show inactivation of certain amylases in automatic dish care detergents.

FIG. 11

a

shows the stability of certain amylases in Cascade™ (a commercially available dish care product) at 65° C. in the presence or absence of starch.

Fig. 11

b

shows the stability of certain amylases in Sunlight™ (a commercially available dish care product) at 65° C. in the presence or absence of starch.

FIG. 12

shows a schematic for the production of M15X cassette mutants.

FIG. 13

shows expression of M15X variants.

FIG. 14

shows specific activity of M15X variants on soluble starch.

FIG. 15

shows heat stability of M15X variants at 90° C., pH 5.0, 5 mM CaCl

2

, 5 mins.

FIG. 16

shows specific activity on starch and soluble substrate, and performance in jet liquefaction at pH 5.5, of M15 variants as a function of percent activity of

B. licheniformis

wild-type.

FIG. 17

shows the inactivation of

B. licheniformis

alpha-amylase (AA20 at 0.65 mg/ml) with chloramine-T at pH 8.0 as compared to variants M197A (1.7 mg/ml) and M197L (1.7 mg/ml).

FIG. 18

shows the inactivation of

B. licheniformis

alpha-amylase (AA20 at 0.22 mg/ml) with chloramine-T at pH 4.0 as compared to variants M197A (4.3 mg/ml) and M197L (0.53 mg/ml).

FIG. 19

shows the reaction of

B. licheniformis

alpha-amylase (AA20 at 0.75 mg/ml) with chloramine-T at pH 5.0 as compared to double variants M197T/W138F (0.64 mg/ml) and M197T/W138Y (0.60 mg/ml).

DETAILED DESCRIPTION OF THE INVENTION

It is believed that amylases used in starch liquefaction may be subject to some form of inactivation due to some activity present in the starch slurry (see commonly owned U.S. applications Ser. Nos. 07/785,624 and 07/785,623 and U.S. Pat. No. 5,180,669, issued Jan. 19, 1993, incorporated herein by reference). Furthermore, use of an amylase in the presence of oxidants, such as in bleach or peracid containing detergents, may result in partial or complete inactivation of the amylase. Therefore, the present invention focuses on altering the oxidative sensitivity of amylases. The mutant enzymes of the present invention may also have an altered pH profile and/or altered thermal stability which may be due to the enhanced oxidative stability of the enzyme at low or high pH's.

Alpha-amylase as used herein includes naturally occurring amylases as well as recombinant amylases. Preferred amylases in the present invention are alpha-amylases derived from

B. licheniformis

or

B. stearothermophilus

, including the A4 form of alpha-amylase derived from

B. lichenifromis

as described herein, as well as fungal alpha-amylases such as those derived from Aspergillus (i.e.,

A. oryzae

and

A. niger

).

Recombinant alpha-amylases refers to an alpha-amylase in which the DNA sequence encoding the naturally occurring alpha-amylase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the alpha-amylase sequence. Suitable modification methods are disclosed herein, and also in commonly owned U.S. Pat. Nos. 4,760,025 and 5,185,258, the disclosure of which are incorporated herein by reference.

Homologies have been found between almost all endo-amylases sequenced to date, ranging from plants, mammals, and bacteria (Nakajima, R. T. et al. (1986) Appl. Microbiol. Biotechnol. 23:355-360; Rogers, J. C. (1985) Biochem. Biophys. Res. Commun. 128:470-476). There are four areas of particularly high homology in certain Bacillus amylases, as shown in

FIG. 3

, wherein the underlined sections designate the areas of high homology. Further, sequence alignments have been used to map the relationship between Bacillus endo-amylases (Feng, D. F. and Doolittle, R. F. (1987) J. Molec. Evol. 35:351-360). The relative sequence homology between

B. stearothermophilus

and

B. licheniformis

amylase is about 66%, as determined by Holm, L. et al. (1990) Protein Engineering 3 (3) pp.181-191. The sequence homology between

B. licheniformis

and

B. amyloliquefaciens

amylases is about 81%, as per Holm, L. et al., supra. While sequence homology is important, it is generally recognized that structural homology is also important in comparing amylases or other enzymes. For example, structural homology between fungal amylases and bacterial (Bacillus) amylase have been suggested and, therefore, fungal amylases are encompassed within the present invention.

An alpha-amylase mutant has an amino acid sequence which is derived from the amino acid sequence of a precursor alpha-amylase. The precursor alpha-amylases include naturally occurring alpha-amylases and recombinant alpha-amylases (as defined). The amino acid sequence of the alpha-amylase mutant is derived from the precursor alpha-amylase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the precursor DNA sequence which encodes the amino acid sequence of the precursor alpha-amylase rather than manipulation of the precursor alpha-amylase enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in commonly owned U.S. Pat. Nos. 4,760,025 and 5,185,258.

Specific residues corresponding to positions M197, M15 and W138 of

Bacillus licheniformis

alpha-amylase are identified herein for substitution or deletion, as are all methionine, histidine, tryptophan, cysteine and tyrosine positions. The amino acid position number (i.e., +197) refers to the number assigned to the mature

Bacillus licheniformis

alpha-amylase sequence presented in FIG.

2

. The invention, however, is not limited to the mutation of this particular mature alpha-amylase (

B. licheniformis

) but extends to precursor alpha-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in

B. licheniformis

alpha-amylase. A residue (amino acid) of a precursor alpha-amylase is equivalent to a residue of

B. licheniformis

alpha-amylase if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in

B. licheniformis

alpha-amylase (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally).

In order to establish homology to primary structure, the amino acid sequence of a precursor alpha-amylase is directly compared to the

B. licheniformis

alpha-amylase primary sequence and particularly to a set of residues known to be invariant to all alpha-amylases for which sequence is known, as seen in FIG.

3

. It is possible also to determine equivalent residues by tertiary structure: crystal structures have been reported for porcine pancreatic alpha-amylase (Buisson, G. et al. (1987) EMBO J. 6:3909-3916); Taka-amylase A from

Aspergillus oryzae

(Matsuura, Y. et al. (1984) J. Biochem. (Tokyo) 95:697-702); and an acid alpha-amylase from

A. niger

(Boel, E. et al. (1990) Biochemistry 29:6244-6249), with the former two structures being similar. There are no published structures for Bacillus alpha-amylases, although there are predicted to be common super-secondary structures between glucanases (MacGregor, E. A. & Svensson, B. (1989) Biochem. J. 259:145-152) and a structure for the

B. stearothermophilus

enzyme has been modeled on that of Taka-amylase A (Holm, L. et al. (1990) Protein Engineering 3:181-191). The four highly conserved regions shown in

FIG. 3

contain many residues thought to be part of the active-site (Matsuura, Y. et al. (1984) J. Biochem. (Tokyo) 95:697-702; Buisson, G. et al. (1987) EMBO J. 6:3909-3916; Vihinen, M. et al. (1990) J. Biochem. 107:267-272) including, in the

licheniformis

numbering, His105; Arg229; Asp231; His235; Glu261 and Asp328.

Expression vector as used herein refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome-binding sites, and sequences which control termination of transcription and translation. A preferred promoter is the

B. subtilis

aprE promoter. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

Host strains (or cells) useful in the present invention generally are procaryotic or eucaryotic hosts and include any transformable microorganism in which the expression of alpha-amylase can be achieved. Specifically, host strains of the same species or genus from which the alpha-amylase is derived are suitable, such as a Bacillus strain. Preferably an alpha-amylase negative Bacillus strain (genes deleted) and/or an alpha-amylase and protease deleted Bacillus strain such as

Bacillus subtilis

strain BG2473 (ΔamyE,Δapr,Δnpr) is used. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the alpha-amylase and its variants (mutants) or expressing the desired alpha-amylase.

Preferably the mutants of the present invention are secreted into the culture medium during fermentation. Any suitable signal sequence, such as the aprE signal peptide, can be used to achieve secretion.

Many of the alpha-amylase mutants of the present invention are useful in formulating various detergent compositions, particularly certain dish care cleaning compositions, especially those cleaning compositions containing known oxidants. Alpha-amylase mutants of the invention can be formulated into known powdered, liquid or gel detergents having pH between 6.5 to 12.0. Suitable granular composition may be made as described in commonly owned U.S. patent applications Ser. Nos. 07/429,881, 07/533,721 and 07/957,973, all of which are incorporated herein by reference. These detergent cleaning compositions can also contain other enzymes, such as known proteases, lipases, cellulases, endoglycosidases or other amylases, as well as builders, stabilizers or other excipients known to those skilled in the art. These enzymes can be present as co-granules or as blended mixes or in any other manner known to those skilled in the art. Furthermore, it is contemplated by the present invention that multiple mutants may be useful in cleaning or other applications. For example, a mutant enzyme having changes at both +15 and +197 may exhibit enhanced performance useful in a cleaning product or a multiple mutant comprising changes at +197 and +138 may have improved performance.

As described previously, alpha-amylase mutants of the present invention may also be useful in the liquefaction of starch. Starch liquefaction, particularly granular starch slurry liquefaction, is typically carried out at near neutral pH's and high temperatures. As described in commonly owned U.S. applications Ser. Nos. 07/788,624 and 07/785,623 and U.S. Pat. No. 5,180,669, it appears that an oxidizing agent or inactivating agent of some sort is also present in typical liquefaction processes, which may affect the enzyme activity; thus, in these related patent applications an antioxidant is added to the process to protect the enzyme.

Based on the conditions of a preferred liquefaction process, as described in commonly owned U.S. applications Ser. Nos. 07/788,624 and 07/785,623 and U.S. Pat. No. 5,180,669, namely low pH, high temperature and potential oxidation conditions, preferred mutants of the present invention for use in liquefaction processes comprise mutants exhibiting altered pH performance profiles (i.e., low pH profile, pH <6 and preferably pH <5.5), and/or altered thermal stability (i.e., high temperature, about 90°-110° C.), and/or altered oxidative stability (i.e., enhanced oxidative stability).

Thus, an improved method for liquefying starch is taught by the present invention, the method comprising liquefying a granular starch slurry from either a wet or dry milling process at a pH from about 4 to 6 by adding an effective amount of an alpha-amylase mutant of the present invention to the starch slurry; optionally adding an effective amount of an antioxidant or other excipient to the slurry; and reacting the slurry for an appropriate time and temperature to liquefy the starch.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims. Abbreviations used herein, particularly three letter or one letter notations for amino acids are described in Dale, J. W., Molecular Genetics of Bacteria, John Wiley & Sons, (1989) Appendix B.

Experimental

EXAMPLE 1

Substitutions for the Methionine Residues in

B. licheniformis

Alpha-Amvlase

The alpha-amylase gene (

FIG. 1

) was cloned from

B. licheniformis

NCIB8061 obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (Gray, G. et al. (1986) J. Bacteriology 166:635-643). The 1.72 kb PstI-SstI fragment, encoding the last three residues of the signal sequence; the entire mature protein and the terminator region was subcloned into M13MP18. A synthetic terminator was added between the BcII and SstI sites using a synthetic oligonucleotide cassette of the form:

BclI SstI

5′ GATCAAAACATAAAAAACCGGCCTTGGCCCCGCCGGTTTTTTATTATTTTTGAGCT 3′

Seq ID No 1

3′ TTTTGTATTTTTTGGCCGGAACCGGGGCGGCCAAAAAATAATAAAAAC 5′

designed to contain the

B. amyloliquefaciens

subtilisin transcriptional terminator (Wells et al. (1983) Nucleic Acid Research 11:7911-7925).

Site-directed mutagenesis by oligonucleotides used essentially the protocol of Zoller, M. et al. (1983) Meth. Enzymol. 100:468-500: briefly, 5′-phosphorylated oligonucleotide primers were used to introduce the desired mutations on the M13 single-stranded DNA template using the oligonucleotides listed in Table I to substitute for each of the seven methionines found in

B. licheniformis

alpha-amylase. Each mutagenic oligonucleotide also introduced a restriction endonuclease site to use as a screen for the linked mutation.

TABLE I

Mutagenic Oligonucleotides for the Substitution of the

Methionine Residues in

B. licheniformis

Alpha-Amylase

M8A

5′-T GGG ACG CTG GCG C

AG TA

C

T

TT GAA TGG TGG T-3′

Seq ID No 2

ScaI+

M15L

5′-TG ATG C

AG TA

C

T

TT GAA T

GG TAC

C

TG CCC AAT GA-3′

Seq ID No 3

ScaI+ KpnI+

M197L

5′-GAT TAT TTG

T

TG TAT GCC

GA

T

ATC

GA

C

TAT GAC CAT-3′

Seq ID No 4

EcoRV+

M256A

5′-CG GGG AAG G

A

G GCC

T

TT ACG GTA GCT-3′

Seq ID No 5

StuI+

M304L

5′-GC GGC TAT GA

C T

T

A

AG

G AAA TTG C-3′

Seq ID No 6

AfIII+

M366A

5′-C TAC GGG G

AT

GCA

T

AC GGG ACG A-3′

Seq ID No 7

NsiI+

M366Y

5′-C TAC GGG GAT

TAC

TAC GGG A

C

C

AA

G

G

GA GAC TCC C-3′

Seq ID No 8

StyI+

M438A

5′-CC GGT GG

G GC

C

AAG CG

G GCC

TAT GT

T

GGC CGG CAA A-3′

Seq ID No 9

SfiI+

Bold letter indicate base changes introduced by oligonucleotide.

Codon changes indicated in the form M8A, where methionine (M) at position +8 has been changed to alanine (A).

Underlining indicates restriction endonuclease site introduced by oligonucleotide.

The heteroduplex was used to transfect

E. coli

mutL cells (Kramer et al. (1984) Cell 38:879) and, after plaque-purification, clones were analyzed by restriction analysis of the RF1's. Positives were confirmed by dideoxy sequencing (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and the PstI-SstI fragments for each subcloned into an

E. coli

vector, plasmid pA4BL.

Plasmid PA4BL

Following the methods described in U.S. application 860,468 (Power et al.), which is incorporated herein by reference, a silent Pstl site was introduced at codon +1 (the first amino-acid following the signal cleavage site) of the aprE gene from pS168-1 (Stahl, M. L. and Ferrari, E. (1984) J. Bacter. 158:411-418). The aprE promoter and signal peptide region was then cloned out of a pJH101 plasmid (Ferrari, F. A. et al. (1983) J. Bacter. 154:1513-1515) as a HindIII-PstI fragment and subcloned into the pUC18-derived plasmid JM102 (Ferrari, E. and Hoch, J. A. (1989) Bacillus, ed. C. R. Harwood, Plenum Pub., pp. 57-72). Addition of the PstI-SstI fragment from

B. licheniformis

alpha-amylase gave pA4BL (

FIG. 5

) having the resulting aprE signal peptide-amylase junction as shown in FIG.

6

.

Transformation Into

B. subtilis

pA4BL is a plasmid able to replicate in

E. coli

and integrate into the

B. subtilis

chromosome. Plasmids containing different variants were transformed into

B. subtilis

(Anagnostopoulos, C. and Spizizen, J. (1961) J. Bacter. 81:741-746) and integrated into the chromosome at the aprE locus by a Campbell-type mechanism (Young, M. (1984) J. Gen. Microbiol. 130:1613-1621). The

Bacillus subtilis

strain BG2473 was a derivative of I168 which had been deleted for amylase (ΔamyE) and two proteases (Δapr, Δnpr) (Stahl, M. L. and Ferrari, E., J. Bacter. 158:411-418 and U.S. Pat. No. 5,264,366, incorporated herein by reference). After transformation the sacU32(Hy) (Henner, D. J. et al. (1988) J. Bacter. 170:296-300) mutation was introduced by PBS-1 mediated transduction (Hoch, J. A. (1983) 154:1513-1515).

N-terminal analysis of the amylase expressed from pA4BL in

B. subtilis

showed it to be processed having four extra alanines at the N-terminus of the secreted amylase protein (“A4 form”). These extra residues had no significant, deleterious effect on the activity or thermal stability of the A4 form and in some applications may enhance performance. In subsequent experiments the correctly processed forms of the

licheniformis

amylase and the variant M197T were made from a very similar construction (see FIG.

6

). Specifically, the 5′ end of the A4 construction was subcloned on an EcoRI-SstII fragment, from pA4BL (

FIG. 5

) into M13BM20 (Boehringer Mannheim) in order to obtain a coding-strand template for the mutagenic oligonucleotide below:

5′-CAT CAG CGT CCC ATT AAG ATT TGC AGC CTG CGC AGA CAT GTT GCT-3′ Seq ID No 10

This primer eliminated the codons for the extra four N-terminal alanines, correct forms being screened for by the absence of the Pstl site. Subcloning the EcoRI-SstII fragment back into the pA4BL vector (

FIG. 5

) gave plasmid pBLapr. The M197T substitution could then be moved, on a SstII-SstI fragment, out of pA4BL (M197T) into the complementary pBLapr vector to give plasmid pBLapr (M197T). N-terminal analysis of the amylase expressed from pBLapr in

B. subtilis

showed it to be processed with the same N-terminus found in

B. licheniformis

alpha-amylase.

EXAMPLE 2

Oxidative Sensitivity of Methionine Variants

B. licheniformis

alpha-amylase, such as Spezyme® AA20 (commercially available from Genencor International, Inc.), is inactivated rapidly in the presence of hydrogen peroxide (FIG.

7

). Various methionine variants were expressed in shake-flask cultures of

B. subtilis

and the crude supernatants purified by ammonium sulphate cuts. The amylase was precipitated from a 20% saturated ammonium sulphate supernatant by raising the ammonium sulphate to 70% saturated, and then resuspended. The variants were then exposed to 0.88M hydrogen peroxide at pH 5.0, at 25° C. Variants at six of the methionine positions in

B. licheniformis

alpha-amylase were still subject to oxidation by peroxide while the substitution at position +197 (M197L) showed resistance to peroxide oxidation. (See

FIG. 7.

) However, subsequent analysis described in further detail below showed that while a variant may be susceptible to oxidation at pH 5.0, 25° C., it may exhibit altered/enhanced properties under different conditions (i.e., liquefaction).

EXAMPLE 3

Construction of All Possible Variants at Position 197

All of the M197 variants (M197X) were produced in the A4 form by cassette mutagenesis, as outlined in FIG.

8

:

1) Site directed mutagenesis (via primer extension in M13) was used to make M197A using the mutagenic oligonucleotide below:

M197A

5′-GAT TAT TTG‘GCG TAT GCC

GAT ATC

GAC TAT GAC CAT-3′

Seq ID No 11

EcoRV+

ClaI

-

which also inserted an EcoRV site (codons 200-201) to replace the ClaI site (codons 201-202).

2) Then primer LAAM12 (Table II) was used to introduce another silent restriction site (BstBI) over codons 186-188.

3) The resultant M197A (BstBI+, EcoRV+) variant was then subcloned (PstI-Sstl fragment) into plasmid pA4BL and the resultant plasmid digested with BstBI and EcoRV and the large vector-containing fragment isolated by electroelution from agarose gel.

4) Synthetic primers LAAM14-30 (Table II) were each annealed with the largely complementary common primer LAAM13 (Table II). The resulting cassettes encoded for all the remaining naturally occurring amino acids at position +197 and were ligated, individually, into the vector fragment prepared above.

TABLE II

Synthetic Oligonucleotides Used for Cassette Mutagenesis

to Produce M197X Variants

LAAM12

GG GAA GT

T TCG AA

T GAA AAC G

Seq ID No 12

LAAM13

X197bs

Seq ID No 13

(EcoRV)

G

TC GGC AT

A TG CAT

ATA ATC‘ATA GTT GCC GTT TTC ATT (BstBI)

LAAM14

I197

Seq ID No 14

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

ATC

TAT GCC GA

C

(EcoRV-)

LAAM15

F197

Seq ID No 15

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

TTC

TAT GCC GA

C

(EcoRV-)

LAAM16

V197

Seq ID No 16

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

GTT

TAT GCC GA

C

(EcoRV-)

LAAM17

S197

Seq ID No 17

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

AGC

TAT GCC GA

C

(EcoRV-)

LAAM18

P197

Seq ID No 18

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

CCT

TAT GCC GA

C

(EcoRV-)

LAAM19

T197

Seq ID No 19

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

ACA

TAT GCC GA

C

(EcoRV-)

LAAM20

Y197

Seq ID No 20

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

TAC

TAT GCC GA

C

(EcoRV-)

LAAM21

H197

Seq ID No 21

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

CAC

TAT GCC GA

C

(EcoRV-)

LAAM22

G197

Seq ID No 22

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

GGC

TAT GCC GA

C

(EcoRV-)

LAAM23

Q197

Seq ID No 23

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

CAA

TAT GCC GA

C

(EcoRV-)

LAAM24

N197

Seq ID No 24

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

AAC

TAT GCC GA

C

(EcoRV-)

LAAM25

K197

Seq ID No 25

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

AAA

TAT GCC GA

C

(EcoRV-)

LAAM26

D197

Seq ID No 26

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

GAT

TAT GCC GA

C

(EcoRV-)

LAAM27

E197

Seq ID No 27

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

GAA

TAT GCC GA

C

(EcoRV-)

LAAM28

C197

Seq ID No 28

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

TGT

TAT GCC GA

C

(EcoRV-)

LAAM29

W197

Seq ID No 29

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

TGG

TAT GCC GA

C

(EcoRV-)

LAAM30

R197

Seq ID No 30

(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG

AGA

TAT GCC GA

C

(EcoRV-)

The cassettes were designed to destroy the EcoRV site upon ligation, thus plasmids from

E. coli

transformants were screened for loss of this unique site. In addition, the common bottom strand of the cassette contained a frame-shift and encoded a NsiI site, thus transformants derived from this strand could be eliminated by screening for the presence of the unique NsiI site and would not be expected, in any case, to lead to expression of active amylase.

Positives by restriction analysis were confirmed by sequencing and transformed in

B. subtilis

for expression in shake-flask cultures (FIG.

9

). The specific activity of certain of the M197X mutants was then determined using a soluble substrate assay. The data generated using the following assay methods are presented below in Table III.

Soluble Substrate Assay

A rate assay was developed based on an end-point assay kit supplied by Megazyme (Aust.) Pty. Ltd.: Each vial of substrate (p-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10 ml of sterile water, followed by a 1 to 4 dilution in assay buffer (50 mM maleate buffer, pH 6.7, 5 mM calcium chloride, 0.002% Tween20). Assays were performed by adding 10 μl of amylase to 790 μl of the substrate in a cuvette at 25° C. Rates of hydrolysis were measured as the rate of change of absorbance at 410 nm, after a delay of 75 seconds. The assay was linear up to rates of 0.4 absorption units/min.

The amylase protein concentration was measured using the standard Bio-Rad assay (Bio-Rad Laboratories) based on the method of Bradford, M. (1976) Anal. Biochem. 72:248) using bovine serum albumin standards.

Starch Hydrolysis Assay

The standard method for assaying the alpha-amylase activity of Spezyme® AA20 was used. This method is described in detail in Example 1 of U.S. Ser. No. 07/785,624, incorporated herein by reference. Native starch forms a blue color with iodine but fails to do so when it is hydrolyzed into shorter dextrin molecules. The substrate is soluble Lintner starch 5 gm/liter in phosphate buffer, pH 6.2 (42.5 gm/liter potassium dihydrogen phosphate, 3.16 gm/liter sodium hydroxide). The sample is added in 25 mM calcium chloride and activity is measured as the time taken to give a negative iodine test upon incubation at 30° C. Activity is recorded in liquefons per gram or ml (LU) calculated according to the formula:

LU / ml or LU / g = \frac{570}{V \times t} \times D

Where LU=liquefon unit

V=volume of sample (5 ml)

t=dextrinization time (minutes)

D=dilution factor=dilution volume/ml or g of added enzyme.

TABLE III

SPECIFIC ACTIVITY

(as % of AA20 value) on:

ALPHA-AMYLASE

Soluble Substrate

Starch

Spezyme ® AA20

100

100

A4 form

105

115

M15L (A4 form)

93

94

M15L

85

103

M197T (A4 form)

75

83

M197T

62

81

M197A (A4 form)

88

89

M197C

85

85

M197L (A4 form)

51

17

EXAMPLE 4

Characterization of Variant M15L

Variant M15L made as per the prior examples did not show increased amylase activity (Table III) and was still inactivated by hydrogen peroxide (FIG.

7

). It did, however, show significantly increased performance in jet-liquefaction of starch, especially at low pH as shown in Table IV below.

Starch liquefaction was typically performed using a Hydroheater M 103-M steam jet equipped with a 2.5 liter delay coil behind the mixing chamber and a terminal back pressure valve. Starch was fed to the jet by a Moyno pump and steam was supplied by a 150 psi steam line, reduced to 90-100 psi. Temperature probes were installed just after the Hydroheater jet and just before the back pressure valve.

Starch slurry was obtained from a corn wet miller and used within two days. The starch was diluted to the desired solids level with deionized water and the pH of the starch was adjusted with 2% NaOH or saturated Na

2

CO

3

. Typical liquefaction conditions were:

Starch

32%-35% solids

Calcium

40-50 ppm (30 ppm added)

pH

5.0-6.0

Alpha-amylase

12-14 LU/g starch dry basis

Starch was introduced into the jet at about 350 ml/min. The jet temperature was held at 105°-107° C. Samples of starch were transferred from the jet cooker to a 95° C. second stage liquefaction and held for 90 minutes.

The degree of starch liquefaction was measured immediately after the second stage liquefaction by determining the dextrose equivalence (DE) of the sample and by testing for the presence of raw starch, both according to the methods described in the

Standard Analytical Methods of the Member Companies of the Corn Refiners Association, Inc.

, sixth edition. Starch, when treated generally under the conditions given above and at pH 6.0, will yield a liquefied starch with a DE of about 10 and with no raw starch. Results of starch liquefaction tests using mutants of the present invention are provided in Table IV.

TABLE IV

Performance of Variants M15L (A4 form)

and M15L in Starch Liquefaction

pH

DE after 90 Mins.

Spezyme ® AA20

5.9

9.9

M15L (A4 form)

5.9

10.4

Spezyme ® AA20

5.2

1.2

M15L (A4 form)

5.2

2.2

Spezyme ® AA20

5.9

9.3*

M15L

5.9

11.3*

Spezyme ® AA20

5.5

3.25**

M15L

5.5

6.7**

Spezyme ® AA20

5.2

0.7**

M15L

5.2

3.65**

*average of three experiments

**average of two experiments

EXAMPLE 5

Construction of M15X Variants

Following generally the processes described in Example 3 above, all variants at M15 (M15X) were produced in native

B. licheniformis

by cassette mutagenesis, as outlined in FIG.

12

:

1) Site directed mutagenesis (via primer extension in M13) was used to introduce unique restriction sites flanking the M15 codon to facilitate insertion of a mutagenesis cassette. Specifically, a BstB1 site at codons 11-13 and a Msc1 site at codons 18-20 were introduced using the two oligonucleotides shown below.

M15XBstB1

5′-G ATG CAG TAT

TTC GAA

CTGG TAT A-3′

Seq ID No 48

BstB1

M15CMsc1

5′-TG CCC AAT GA

T GGC CA

A CAT TGG AAG-3′

Seq ID No 49

Msc1

2) The vector for M15X cassette mutagenesis was then constructed by subcloning the Sfi1-SstII fragment from the mutagenized amylase (BstB1+, Msc1+) into plasmid pBLapr. The resulting plasmid was then digested with BstB1and Msc1 and the large vector fragment isolated by electroelution from a polyacrylamide gel.

3) Mutagenesis cassettes were created as with the M197X variants. Synthetic oligomers, each encoding a substitution at codon 15, were annealed to a common bottom primer. Upon proper ligation of the cassette to the vector, the Msc1 is destroyed allowing for screening of positive transformants by loss of this site. The bottom primer contains an unique SnaB1site allowing for the transformants derived from the bottom strand to be eliminated by screening for the SnaB1site. This primer also contains a frameshift which would also eliminate amylase expression for the mutants derived from the common bottom strand.

The synthetic cassettes are listed in Table V and the general cassette mutagenesis strategy is illustrated in FIG.

12

.

TABLE V

Synthetic Oligonucleotides Used for Cassette Mutagenesis

to Produce M15X Variants

M15A (BstB1) C GAA TGG TAT

GCT

CCC AAT GAC GG (Msc1) Seq ID No 50

M15R (BstB1) C GAA TGG TAT

CGC

CCC AAT GAC GG (Msc1) Seq ID No 51

M15N (BstB1) C GAA TGG TAT

AAT

CCC AAT GAC GG (Msc1) Seq ID No 52

M15D (BstB1) C GAA TGG TAT

GAT

CCC AAT GAC GG (Msc1) Seq ID No 53

M15H (BstB1) C GAA TGG TAT

CAC

CCC AAT GAC GG (Msc1) Seq ID No 54

M15K (BstB1) C GAA TGG TAT

AAA

CCC AAT GAC GG (Msc1) Seq ID No 55

M15P (BstB1) C GAA TGG TAT

CCG

CCC AAT GAC GG (Msc1) Seq ID No 56

M15S (BstB1) C GAA TGG TAT

TCT

CCC AAT GAC GG (Msc1) Seq ID No 57

M15T (BstB1) C GAA TGG TAT

ACT

CCC AAT GAC GG (Msc1) Seq ID No 58

M15V (BstB1) C GAA TGG TAT

GTT

CCC AAT GAC GG (Msc1) Seq ID No 59

M15C (BstB1) C GAA TGG TAT

TGT

CCC AAT GAC GG (Msc1) Seq ID No 60

M15Q (BstB1) C GAA TGG TAT

CAA

CCC AAT GAC GG (Msc1) Seq ID No 61

M15E (BstB1) C GAA TGG TAT

GAA

CCC AAT GAC GG (Msc1) Seq ID No 62

M15G (BstB1) C GAA TGG TAT

GGT

CCC AAT GAC GG (Msc1) Seq ID No 63

M15I (BstB1) C GAA TGG TAT

ATT

CCC AAT GAC GG (Msc1) Seq ID No 64

M15F (BstB1) C GAA TGG TAT

TTT

CCC AAT GAC GG (Msc1) Seq ID No 65

M15W (BstB1) C GAA TGG TAT

TGG

CCC AAT GAC GG (Msc1) Seq ID No 66

M15Y (BstB1) C GAA TGG TAT

TAT

CCC AAT GAC GG (Msc1) Seq ID No 67

M15X (Msc1) CC GTC ATT GGG ACT ACG TAC CAT T (BstB1) Seq ID No 68

(bottom strand)

Underline indicates codon changes at amino acid position 15.

Conservative substitutions were made in some cases to prevent introduction of new restriction sites.

EXAMPLE 6

Bench Liquefaction with M15X Variants

Eleven alpha-amylase variants with substitutions for M15 made as per Example 5 were assayed for activity, as compared to Spezyme® AA20 (commercially available from Genencor International, Inc.) in liquefaction at pH 5.5 using a bench liquefaction system. The bench scale liquefaction system consisted of a stainless steel coil (0.25 inch diameter, approximately 350 ml volume) equipped with a 7 inch long static mixing element approximately 12 inches from the anterior end and a 30 psi back pressure valve at the posterior end. The coil, except for each end, was immersed in a glycerol-water bath equipped with thermostatically controlled heating elements that maintained the bath at 105-106° C.

Starch slurry containing enzyme, maintained in suspension by stirring, was introduced into the reaction coil by a piston driven metering pump at about 70 ml/min. The starch was recovered from the end of the coil and was transferred to the secondary hold (95° C. for 90 minutes). Immediately after the secondary hold, the DE of the liquefied starch was determined, as described in Example 4. The results are shown in FIG.

16

.

EXAMPLE 7

Characterization of M197X

As can be As can be seen in

FIG. 9

, there was a wide range of amylase activity (measured in the soluble substrate assay) expressed by the M197X (A4 form) variants. The amylases were partially purified from the supernatants by precipitation with two volumes of ethanol and resuspension. They were then screened for thermal stability (

FIG. 10

) by heating at 95° C. for 5 minutes in 10 mM acetate buffer pH 5.0, in the presence of 5 mM calcium chloride; the A4 wild-type retained 28% of its activity after incubation. For M197W and M197P we were unable to recover active protein from the supernatants. Upon sequencing, the M197H variant was found to contain a second mutation, NI90K. M197L was examined in a separate experiment and was one of the lowest thermally stable variants. There appears to be a broad correlation between expression of amylase activity and thermal stability. The

licheniformis

amylase is restricted in what residues it can accommodate at position 197 in terms of retaining or enhancing thermal stability: cysteine and threonine are preferred for maximal thermal stability under these conditions whereas alanine and isoleucine are of intermediate stability. However, other substitutions at position +197 result in lowered thermal stability which may be useful for other applications. Additionally, different substitutions at +197 may have other beneficial properties, such as altered pH performance profile or altered oxidative stability. For example, the M197C variant was found to inactivate readily by air oxidation but had enhanced thermal stability. Conversely, compared to the M197L variant, both M197T and M197A retained not only high thermal stability (FIG.

10

), but also high activity (Table III), while maintaining resistance to inactivation by peroxide at pH 5 to pH 10 (FIG.

7

).

EXAMPLE 8

Stability and Performance in Detergent Formulation

The stability of the M197T (A4 form), M197T and M197A (A4 form) was measured in automatic dish care detergent (ADD) matrices. 2 ppm Savinase™ (a protease, commercially available from Novo Industries, of the type commonly used in ADD) were added to two commercially available bleach-containing ADD's: Cascade™ (Procter and Gamble, Ltd.) and Sunlight™ (Unilever) and the time course of inactivation of the amylase variants and Termamyl™ (a thermally stable alpha-amylase available from Novo Nordisk, A/S) followed at 65° C. The concentration of ADD product used in both cases was equivalent to ‘pre-soak’ conditions: 14 gm product per liter of water (7 grams per gallon hardness). As can be seen (

FIGS. 11

a

and

11

b

), both forms of the M197T variant were much more stable than Termamyl™ and M197A (A4 form), which were inactivated before the first assay could be performed. This stability benefit was seen in the presence or absence of starch as determined by the following protocol. Amylases were added to 5 ml of ADD and Savinase™, prewarmed in a test tube and, after vortexing, activities were assayed as a function of time, using the soluble substrate assay. The “+ starch” tube had spaghetti starch baked onto the sides (140° C., 60 mins.). The results are shown in

FIGS. 11

a

and

11

b.

EXAMPLE 9

Characterization of M15X Variants

All M15X variants were propagated in

Bacillus subtilis

and the expression level monitored as shown in FIG.

13

. The amylase was isolated and partially purified by a 20-70% ammonium sulfate cut. The specific activity of these variants on the soluble substrate was determined as per Example 3 (FIG.

14

). Many of the M15X amylases have specific activities greater than that of Spezyme® AA20. A benchtop heat stability assay was performed on the variants by heating the amylase at 90° C. for 5 min. in 50 mM acetate buffer pH 5 in the presence of 5 mM CaCl

2

(FIG.

15

). Most of the variants performed as well as Spezyme® AA20 in this assay. Those variants that exhibited reasonable stability in this assay (reasonable stability defined as those that retained at least about 60% of Spezyme® AA20's heat stability) were tested for specific activity on starch and for liquefaction performance at pH 5.5. The most interesting of those mutants are shown in

FIG. 16. M

15D, N and T, along with L, outperformed Spezyme® AA20 in liquefaction at pH 5.5 and have increased specific activities in both the soluble substrate and starch hydrolysis assays.

Generally, we have found that by substituting for the methionine at position 15, we can provide variants with increased low pH-liquefaction performance and/or increased specific activity.

EXAMPLE 10

Tryptophan Sensitivity to Oxidation

Chloramine-T (sodium N-chloro-p-toluenesulfonimide) is a selective oxidant, which oxidizes methionine to methionine sulfoxide at neutral or alkaline pH. At acidic pH, chloramine-T will modify both methionine and tryptophan (Schechter, Y., Burstein, Y. and Patchomik, A. (1975) Biochemistry 14 (20) 4497-4503).

FIG. 17

shows the inactivation of

B. licheniformis

alpha-amylase with chloramine-T at pH 8.0 (AA20=0.65 mg/ml, M197A=1.7 mg/ml, M197L=1.7 mg/ml). The data shows that by changing the methionine at position 197 to leucine or alanine, the inactivation of alpha-amylase can be prevented. Conversely, as shown in

FIG. 18

, at pH 4.0 inactivation of the M197A and M197L proceeds, but require more equivalents of chloramine-T (

FIG. 18

; AA20=0.22 mg/ml, M197A=4.3 mg/ml, M197L=0.53 mg/ml; 200 mM NaAcetate at pH 4.0). This suggests that a tryptophan residue is also implicated in the chloramine-T mediated inactivation event. Furthermore, tryptic mapping and subsequent amino acid sequencing indicated that the tryptophan at position 138 was oxidized by chloramine-T (data not shown). To prove this, site-directed mutants were made at tryptophan 138 as provided below:

Preparation of Alpha-Amylase Double Mutants W138 and M197

Certain variants of W138 (F, Y and A) were made as double mutants, with M197T (made as per the disclosure of Example 3). The double mutants were made following the methods described in Examples 1 and 3. Generally, single negative strands of DNA were prepared from an M13MP18 clone of the 1.72 kb coding sequence (Pst I-Sst I) of the

B. licheniformis

alpha-amylase M197T mutant. Site-directed mutagenesis was done using the primers listed below, essentially by the method of Zoller, M. et al. (1983) except T4 gene 32 protein and T4 polymerase were substituted for klenow. The primers all contained unique sites, as well as the desired mutation, in order to identify those clones with the appropriate mutation.

Tryptophan 138 to Phenylalanine

133 134 135 136 137

138

139 140 141 142 143

Seq ID No 42

CAC CTA ATT A

AA GCT

T

TC ACA CAT TTT CAT TTT

Hind III

Tryptophan 138 to Tyrosine

133 134 135 136 137

138

139 140 141 142 143

Seq ID No 43

CAC CTA ATT A

AA GCT

T

AC ACA CAT TTT CAT TTT

Hind III

Tryptophan 138 to Alanine - This primer also engineers unique sites

upstream and downstream of the 138 position.

127 128 129 130 131 132 133 134 135 136 137

138

139 140 141 142

C CGC GTA ATT

TCC GGA

GAA CAC CTA ATT AAA GCC

GCA

ACA CAT TTT CAT

BspE I

143 144 145 146 147

Seq ID No 44

TTT

CCC GGG

CGC GGC AG

Xma I

Mutants were identified by restriction analysis and W138F and W138Y confirmed by DNA sequencing. The W138A sequence revealed a nucleotide deletion between the unique BspE I and Xma I sites, however, the rest of the gene sequenced correctly. The 1.37 kb SstII/SstI fragment containing both W138X and M197T mutations was moved from M13MP18 into the expression vector pBLapr resulting in pBLapr (W138F, M197T) and pBLapr (W138Y, M197T). The fragment containing unique BspE I and Xma I sites was cloned into pBLapr (BspE I, Xma I, M197T) since it is useful for cloning cassettes containing other amino acid substitutions at position 138.

Single Mutations at Amino Acid Position 138

Following the general methods described in the prior examples, certain single variants of W138 (F, Y, L, H and C) were made.

The 1.24 kb Asp718-SstI fragment containing the M197T mutation in plasmid pBLapr (W138X, M197T) of Example 7 was replaced by the wild-type fragment with methionine at 197, resulting in pBLapr (W138F), pBLapr (W138Y) and pBLapr (BspE I, Xma I).

The mutants W138L, W138H and W138C were made by ligating synthetic cassettes into the pBLapr (BspE I, Xma I) vector using the following primers:

Tryptophan 138 to Leucine

CC GGA GAA CAC CTA ATT AAA GCC

CTA

ACA CAT TTT CAT TTT C

Seq ID No 45

Tryptophan 138 to Histidine

CC GGA GAA CAC CTA ATT AAA GCC

CAC

ACA CAT TTT CAT TTT C

Seq ID No 46

Tryptophan 138 to Cysteine

CC GGA GAA CAC CTA ATT AAA GCC

TGC

ACA CAT TTT CAT TTT C

Seq ID No 47

Reaction of the double mutants M197T/W138F and M197T/W138Y with chloramine-T was compared with wild-type (AA20=0.75 mg/ml, M197T/W138F=0.64 mg/ml, M197T/W138Y=0.60 mg/ml; 50 mM NaAcetate at pH 5.0). The results shown in

FIG. 19

show that mutagenesis of tryptophan 138 has caused the variant to be more resistant to chloramine-T.

56 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

1
GATCAAAACA TAAAAAACCG GCCTTGGCCC CGCCGGTTTT TTATTATTTT TGAGCT 56

29 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

2
TGGGACGCTG GCGCAGTACT TTGAATGGT 29

34 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

3
TGATGCAGTA CTTTGAATGG TACCTGCCCA ATGA 34

36 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

4
GATTATTTGT TGTATGCCGA TATCGACTAT GACCAT 36

26 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

5
CGGGGAAGGA GGCCTTTACG GTAGCT 26

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

6
GCGGCTATGA CTTAAGGAAA TTGC 24

23 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

7
CTACGGGGAT GCATACGGGA CGA 23

35 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

8
CTACGGGGAT TACTACGGGA CCAAGGGAGA CTCCC 35

36 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

9
CCGGTGGGGC CAAGCGGGCC TATGTTGGCC GGCAAA 36

45 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

10
CATCAGCGTC CCATTAAGAT TTGCAGCCTG CGCAGACATG TTGCT 45

36 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

11
GATTATTTGG CGTATGCCGA TATCGACTAT GACCAT 36

21 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

12
GGGAAGTTTC GAATGAAAAC G 21

38 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

13
GTCGGCATAT GCATATAATC ATAGTTGCCG TTTTCATT 38

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

14
CGAATGAAAA CGGCAACTAT GATTATTTGA TCTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

15
CGAATGAAAA CGGCAACTAT GATTATTTGT TCTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

16
CGAATGAAAA CGGCAACTAT GATTATTTGG TTTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

17
CGAATGAAAA CGGCAACTAT GATTATTTGA GCTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

18
CGAATGAAAA CGGCAACTAT GATTATTTGC CTTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

19
CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

20
CGAATGAAAA CGGCAACTAT GATTATTTGT ACTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

21
CGAATGAAAA CGGCAACTAT GATTATTTGC ACTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

22
CGAATGAAAA CGGCAACTAT GATTATTTGG GCTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

23
CGAATGAAAA CGGCAACTAT GATTATTTGC AATATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

24
CGAATGAAAA CGGCAACTAT GATTATTTGA ACTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

25
GCAATGAAAA CGGCAACTAT GATTATTTGA AATATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

26
CGAATGAAAA CGGCAACTAT GATTATTTGG ATTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

27
CGAATGAAAA CGGCAACTAT GATTATTTGG AATATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

28
CGAATGAAAA CGGCAACTAT GATTATTTGT GTATTGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

29
CGAATGAAAA CGGCAACTAT GATTATTTGT GGTATGCCGA C 41

41 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

30
CGAATGAAAA CGGCAACTAT GATTATTTGA GATATGCCGA C 41

1968 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

31
AGCTTGAAGA AGTGAAGAAG CAGAGAGGCT ATTGAATAAA TGAGTAGAAA GCGCCATATC 60
GGCGCTTTTC TTTTGGAAGA AAATATAGGG AAAATGGTAC TTGTTAAAAA TTCGGAATAT 120
TTATACAACA TCATATGTTT CACATTGAAA GGGGAGGAGA ATCATGAAAC AACAAAAACG 180
GCTTTACGCC CGATTGCTGA CGCTGTTATT TGCGCTCATC TTCTTGCTGC CTCATTCTGC 240
AGCAGCGGCG GCAAATCTTA ATGGGACGCT GATGCAGTAT TTTGAATGGT ACATGCCCAA 300
TGACGGCCAA CATTGGAAGC GTTTGCAAAA CGACTCGGCA TATTTGGCTG AACACGGTAT 360
TACTGCCGTC TGGATTCCCC CGGCATATAA GGGAACGAGC CAAGCGGATG TGGGCTACGG 420
TGCTTACGAC CTTTATGATT TAGGGGAGTT TCATCAAAAA GGGACGGTTC GGACAAAGTA 480
CGGCACAAAA GGAGAGCTGC AATCTGCGAT CAAAAGTCTT CATTCCCGCG ACATTAACGT 540
TTACGGGGAT GTGGTCATCA ACCACAAAGG CGGCGCTGAT GCGACCGAAG ATGTAACCGC 600
GGTTGAAGTC GATCCCGCTG ACCGCAACCG CGTAATTTCA GGAGAACACC TAATTAAAGC 660
CTGGACACAT TTTCATTTTC CGGGGCGCGG CAGCACATAC AGCGATTTTA AATGGCATTG 720
GTACCATTTT GACGGAACCG ATTGGGACGA GTCCCGAAAG CTGAACCGCA TCTATAAGTT 780
TCAAGGAAAG GCTTGGGATT GGGAAGTTTC CAATGAAAAC GGCAACTATG ATTATTTGAT 840
GTATGCCGAC ATCGATTATG ACCATCCTGA TGTCGCAGCA GAAATTAAGA GATGGGGCAC 900
TTGGTATGCC AATGAACTGC AATTGGACGG TTTCCGTCTT GATGCTGTCA AACACATTAA 960
ATTTTCTTTT TTGCGGGATT GGGTTAATCA TGTCAGGGAA AAAACGGGGA AGGAAATGTT 1020
TACGGTAGCT GAATATTGGC AGAATGACTT GGGCGCGCTG GAAAACTATT TGAACAAAAC 1080
AAATTTTAAT CATTCAGTGT TTGACGTGCC GCTTCATTAT CAGTTCCATG CTGCATCGAC 1140
ACAGGGAGGC GGCTATGATA TGAGGAAATT GCTGAACGGT ACGGTCGTTT CCAAGCATCC 1200
GTTGAAATCG GTTACATTTG TCGATAACCA TGATACACAG CCGGGGCAAT CGCTTGAGTC 1260
GACTGTCCAA ACATGGTTTA AGCCGCTTGC TTACGCTTTT ATTCTCACAA GGGAATCTGG 1320
ATACCCTCAG GTTTTCTACG GGGATATGTA CGGGACGAAA GGAGACTCCC AGCGCGAAAT 1380
TCCTGCCTTG AAACACAAAA TTGAACCGAT CTTAAAAGCG AGAAAACAGT ATGCGTACGG 1440
AGCACAGCAT GATTATTTCG ACCACCATGA CATTGTCGGC TGGACAAGGG AAGGCGACAG 1500
CTCGGTTGCA AATTCAGGTT TGGCGGCATT AATAACAGAC GGACCCGGTG GGGCAAAGCG 1560
AATGTATGTC GGCCGGCAAA ACGCCGGTGA GACATGGCAT GACATTACCG GAAACCGTTC 1620
GGAGCCGGTT GTCATCAATT CGGAAGGCTG GGGAGAGTTT CACGTAAACG GCGGGTCGGT 1680
TTCAATTTAT GTTCAAAGAT AGAAGAGCAG AGAGGACGGA TTTCCTGAAG GAAATCCGTT 1740
TTTTTATTTT GCCCGTCTTA TAAATTTCTT TGATTACATT TTATAATTAA TTTTAACAAA 1800
GTGTCATCAG CCCTCAGGAA GGACTTGCTG ACAGTTTGAA TCGCATAGGT AAGGCGGGGA 1860
TGAAATGGCA ACGTTATCTG ATGTAGCAAA GAAAGCAAAT GTGTCGAAAA TGACGGTATC 1920
GCGGGTGATC AATCATCCTG AGACTGTGAC GGATGAATTG AAAAAGCT 1968

483 amino acids

amino acid

single

linear

protein

not provided

32
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Arg

511 amino acids

amino acid

single

linear

protein

not provided

33
Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe
1 5 10 15
Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu
20 25 30
Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro Asn Asp Gly
35 40 45
His Trp Lys Arg Leu Gln Asn Asp Ser Ala Tyr Leu Ala Glu His Gly
50 55 60
Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala
65 70 75 80
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe His
85 90 95
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu Gln
100 105 110
Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr Gly Asp
115 120 125
Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp Val Thr
130 135 140
Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser Gly Glu
145 150 155 160
His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg Gly Ser
165 170 175
Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp
180 185 190
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Ala Ala Glu Ile
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg Asp Trp
260 265 270
Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu Asn Lys
290 295 300
Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr Gln Phe
305 310 315 320
His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Asp
385 390 395 400
Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr Phe Asp
420 425 430
His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser Val Ala
435 440 445
Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ala Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly Trp Gly
485 490 495
Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510

520 amino acids

amino acid

single

linear

protein

not provided

34
Met Arg Gly Arg Gly Asn Met Ile Gln Lys Arg Lys Arg Thr Val Ser
1 5 10 15
Phe Arg Leu Val Leu Met Cys Thr Leu Leu Phe Val Ser Leu Pro Ile
20 25 30
Thr Lys Thr Ser Ala Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp
35 40 45
Tyr Thr Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala
50 55 60
Glu His Leu Ser Asp Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala
65 70 75 80
Tyr Lys Gly Leu Ser Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu
85 90 95
Tyr Asp Leu Gly Glu Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr
100 105 110
Gly Thr Lys Ser Glu Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg
115 120 125
Asn Val Gln Val Tyr Gly Asp Val Val Leu Asn His Lys Ala Gly Ala
130 135 140
Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg
145 150 155 160
Asn Gln Glu Thr Ser Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe
165 170 175
Arg Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp
180 185 190
Tyr His Phe Asp Gly Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg
195 200 205
Ile Phe Lys Phe Arg Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser
210 215 220
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr
225 230 235 240
Asp His Pro Asp Val Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr
245 250 255
Ala Asn Glu Leu Ser Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His
260 265 270
Ile Lys Phe Ser Phe Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala
275 280 285
Thr Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala
290 295 300
Gly Lys Leu Glu Asn Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val
305 310 315 320
Phe Asp Val Pro Leu His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly
325 330 335
Gly Gly Tyr Asp Met Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg
340 345 350
His Pro Glu Lys Ala Val Thr Phe Val Glu Asn His Asp Thr Gln Pro
355 360 365
Gly Gln Ser Leu Glu Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala
370 375 380
Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr
385 390 395 400
Gly Asp Met Tyr Gly Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser
405 410 415
Leu Lys Asp Asn Ile Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala
420 425 430
Tyr Gly Pro Gln His Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp
435 440 445
Thr Arg Glu Gly Asp Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu
450 455 460
Ile Thr Asp Gly Pro Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys
465 470 475 480
Asn Ala Gly Glu Thr Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr
485 490 495
Val Lys Ile Gly Ser Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly
500 505 510
Ser Val Ser Ile Tyr Val Gln Lys
515 520

548 amino acids

amino acid

single

linear

protein

not provided

35
Val Leu Thr Phe His Arg Ile Ile Arg Lys Gly Trp Met Phe Leu Leu
1 5 10 15
Ala Phe Leu Leu Thr Ala Ser Leu Phe Cys Pro Thr Gly Arg His Ala
20 25 30
Lys Ala Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
35 40 45
Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala
50 55 60
Asn Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Ser Leu Pro Pro Ala
65 70 75 80
Tyr Lys Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
85 90 95
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
100 105 110
Gly Thr Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala
115 120 125
Gly Met Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala
130 135 140
Asp Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg
145 150 155 160
Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
165 170 175
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
180 185 190
Tyr His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg
195 200 205
Ile Tyr Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp
210 215 220
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met
225 230 235 240
Asp His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr
245 250 255
Val Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Gly Leu Lys His
260 265 270
Ile Lys Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln
275 280 285
Thr Gly Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile
290 295 300
Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Thr Met Ser Leu
305 310 315 320
Phe Asp Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly
325 330 335
Gly Ala Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp
340 345 350
Gln Pro Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Asn Pro
355 360 365
Ala Lys Arg Cys Ser His Gly Arg Pro Trp Phe Lys Pro Leu Ala Tyr
370 375 380
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
385 390 395 400
Asp Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys
405 410 415
Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
420 425 430
His Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly
435 440 445
Val Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
450 455 460
Ala Gly Arg Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys
465 470 475 480
Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
485 490 495
Ser Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val
500 505 510
Trp Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr
515 520 525
Thr Arg Pro Trp Thr Gly Glu Phe Val Arg Trp His Glu Pro Arg Leu
530 535 540
Val Ala Trp Pro
545

483 amino acids

amino acid

single

linear

protein

not provided

36
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Thr Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Arg

487 amino acids

amino acid

single

linear

protein

not provided

37
Ala Ala Ala Ala Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu
1 5 10 15
Trp Tyr Met Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp
20 25 30
Ser Ala Tyr Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro
35 40 45
Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp
50 55 60
Leu Tyr Asp Leu Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys
65 70 75 80
Tyr Gly Thr Lys Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser
85 90 95
Arg Asp Ile Asn Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly
100 105 110
Ala Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp
115 120 125
Arg Asn Arg Val Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His
130 135 140
Phe His Phe Pro Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His
145 150 155 160
Trp Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn
165 170 175
Arg Ile Tyr Lys Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn
180 185 190
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp
195 200 205
His Pro Asp Val Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala
210 215 220
Asn Glu Leu Gln Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile
225 230 235 240
Lys Phe Ser Phe Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr
245 250 255
Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly
260 265 270
Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe
275 280 285
Asp Val Pro Leu His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly
290 295 300
Gly Tyr Asp Met Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His
305 310 315 320
Pro Leu Lys Ser Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly
325 330 335
Gln Ser Leu Glu Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr
340 345 350
Ala Phe Ile Leu Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
355 360 365
Asp Met Tyr Gly Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu
370 375 380
Lys His Lys Ile Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr
385 390 395 400
Gly Ala Gln His Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr
405 410 415
Arg Glu Gly Asp Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile
420 425 430
Thr Asp Gly Pro Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn
435 440 445
Ala Gly Glu Thr Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val
450 455 460
Val Ile Asn Ser Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser
465 470 475 480
Val Ser Ile Tyr Val Gln Arg
485

32 amino acids

amino acid

single

linear

protein

not provided

38
Met Lys Gln Gln Lys Arg Leu Thr Ala Arg Leu Leu Thr Leu Leu Phe
1 5 10 15
Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu
20 25 30

33 amino acids

amino acid

single

linear

protein

not provided

39
Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu
1 5 10 15
Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys
20 25 30
Ser

35 amino acids

amino acid

single

linear

protein

not provided

40
Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu
1 5 10 15
Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Ala Ala
20 25 30
Ala Ala Asn
35

32 amino acids

amino acid

single

linear

protein

not provided

41
Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu
1 5 10 15
Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Asn Leu
20 25 30

33 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

42
CACCTAATTA AAGCTTTCAC ACATTTTCAT TTT 33

33 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

43
CACCTAATTA AAGCTTACAC ACATTTTCAT TTT 33

66 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

44
CCGCGTAATT TCCGGAGAAC ACCTAATTAA AGCCGCAACA CATTTTCATT TTCCCGGGCG 60
CGGCAG 66

42 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

45
CCGGAGAACA CCTAATTAAA GCCCTAACAC ATTTTCATTT TC 42

42 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

46
CCGGAGAACA CCTAATTAAA GCCCACACAC ATTTTCATTT TC 42

42 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

47
CCGGAGAACA CCTAATTAAA GCCTGCACAC ATTTTCATTT TC 42

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

48
GATGCAGTAT TTCGAACTGG TATA 24

26 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

49
TGCCCAATGA TGGCCAACAT TGGAAG 26

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

50
CGAATGGTAT GCTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

51
CGAATGGTAT CGCCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

52
CGAATGGTAT AATCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

53
CGAATGGTAT GATCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

54
CGAATGGTAT CACCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

55
CGAATGGTAT AAACCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

56
CGAATGGTAT CCGCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

57
CGAATGGTAT TCTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

58
CGAATGGTAC ACTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

59
CGAATGGTAT GTTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

60
CGAATGGTAT TGTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

61
CGAATGGTAT CAACCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

62
CGAATGGTAT GAACCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

63
CGAATGGTAT GGTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

64
CGAATGGTAT ATTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

65
CGAATGGTAT TTTCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

66
CGAATGGTAC TGGCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

67
CGAATGGTAT TATCCCAATG ACGG 24

24 base pairs

nucleic acid

single

linear

DNA (genomic)

not provided

68
CCGTCATTGG GACTACGTAC CATT 24

Number	Name	Date
4261868	Hora et al.	Apr 1981
4284722	Tamuri et al.	Aug 1981
4493893	Mielenz et al.	Jan 1985
4620936	Kielman et al.	Nov 1986
4634551	Burns et al.	Jan 1987
4760025	Estell	Jul 1988

Number	Date	Country
094692	Jul 1992	DK
150392	Dec 1992	DK
0 130 756	Jan 1985	EP
0285123	Oct 1988	EP
0 378 261	Jul 1990	EP
0 409 299 A2	Jan 1991	EP
0 410 498 A2	Jan 1991	EP
2 676 456	Nov 1992	FR
WO 9100353	Jan 1991	WO
WO 9116423	Oct 1991	WO
9208778	May 1992	WO
9402597	Feb 1994	WO
WO 9402597	Feb 1994	WO

	Number	Date	Country
Parent	08/016395	Feb 1993	US
Child	08/194664		US

Oxidatively stable alpha-amylase

Information

Patent Number

Date Filed

Date Issued

Inventors

Examiners

CPC

US Classifications

Field of Search

US

International Classifications

Disclaimer

Abstract

Description

Claims

RELATED APPLICATIONS

US Referenced Citations (6)

Foreign Referenced Citations (13)

Non-Patent Literature Citations (23)

Continuation in Parts (1)

Entry
Declerck, et al., “Use of Amber Suppressors to Investigate the Thermostability of Bacillus licheniformis α-Amylase” J. of Biol. Chem. 265(26):15481-15488 (1990).
Manning, et al., “Thermostable α-Amylase of Bacillus stearothermophilus” J. of Biol. Chem. 236(11):2952-2965 (Nov. 1961).
Matsui, et al., “A mutant α-amylase with enhanced activity specific for short substrates” FEBS 11596 310(3):216-218 (Oct. 1992).
Nakajima, et al., “Nucleotide Sequence of the Bacillus stearothermophilus α-Amylase Gene” J. Bacteriology 163(1):401-406 (Jul. 1985).
Ogasahara, et al., “Studies on Thermophilic α-Amylase from Bacillus stearothermophilus” J. Biochem. 67(1):65-89 (1970).
Ottesen et al., “The Subtilisins” Methods in Enzymology 19:199-215 (1970).
Takase et al., “Site-directed mutagenesis of active site residues in Bacillus subtilis a-amylase” Biochimica et Biophysica Acta 1120-281-288 (1992).
Bealin-Kelly et al, “Studies on the thermostability of the alpha-amylase of bacillus-caldovelox” Appl. Microbiol. and Biotech 36(3):332-336 (Dec. 1991).
Estell, et al., “Engineering an Enzyme by sitedirected Mutagenesis to Be Resistant to Chemical Oxidation” J. Biol. Chem. 260(11):6518-6521 (Jun. 1985).
Brosnan, et al., “Investigation of the mechanism of irreversible thermoinactivation of bacillus-stearothermophilu alpha-amylase” Eur. J. Biotech. 203(1-2):225-231 (Jan. 1992).
Joyet, P., et al., biotechnology, vol. 10, Dec. 1992, pp. 1579-1583.
Janecek, FEBS 11085, vol. 304, No. 1,1-3 (Jun. 1992.
Holm, L., et al., Protein Engineering, vol. 3, No. 3, pp. 181-191, 1990.
Suzuki, Y., et al., The Journal of Biological Chemistry, vol. 264, No. 32, pp. 18933-18938 (1989.
Vihinen, M., et al., J. Biochem, 107, pp. 267-272 (1990).
Nakajima, R., et al., Journal of Bacteriology, Jul. 1985, pp. 401-406.
Tomazic, S.J., et al., The Journal of Biological Chemistry, vol. 263, No. 7, pp. 3086-3096, 1988.
Gray, G.L., et al., Journal of Bacteriology, vol. 166, No. 2, 1986, pp. 635-643.
Jorgensen, P.L., et al., FEMS Microbiology Letters 77 (1991) pp. 271-276.
B. Svensson et al. “Mutational Analysis of Glycosylase Function” J. Biotechnol. 29: 1-37 (1993).*
M. Søgaard et al. “Site-directed Mutagenesis of Histidine 93. . .” J. Biol. Chem. 268(30)22480-22484 (Oct. 1993).*
I. Matsui et al. “An Increase in the Transglycosylation Activity” Biochim. Biophys. Acta. 1077:416-419 (1991).*
L. Holm. et al. “Random Mutagenesis Used to Probe the Structure. . .” Prot. Eng. 3(3) 181-191 (1990).