Amylase variants

The present invention relates to variants of a parent &agr;-amylase, which parent &agr;-amylase (i) has an amino acid sequence selected from the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 7, respectively; or (ii) displays at least 80% homology with one or more of these amino acid sequences; and/or displays immunological cross-reactivity with an antibody raised against an &agr;-amylase having one of these amino acid sequences; and/or is encoded by a DNA sequence which hybridizes with the same probe as a DNA sequence encoding an &agr;-amylase having one of these amino acid sequences; in which variant: (a) at least one amino acid residue of the parent &agr;-amylase has been deleted; and/or (b) at least one amino acid residue of the parent &agr;-amylase has been replaced by a different amino acid residue; and/or (c) at least one amino acid residue has been inserted relative to the parent &agr;-amylase; the variant having &agr;-amylase activity and exhibiting at least one of the following properties relative to the parent &agr;-amylase: increased thermostability; increased stability towards oxidation; and reduced Ca 2&plus; dependency; with the proviso that the amino acid sequence of the variant is not identical to any of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively.

EXPERIMENTAL SECTION The preparation, purification and sequencing of the parent &agr;-amylases having the amino acid sequences shown in SEQ ID No.1 and SEQ ID No.2 (from Bacillus strains NCIB 12512 and NCIB 12513, respectively) is described in WO 95/26397. The pI values and molecular weights of these two parent &agr;-amylases (given in WO 95/26397) are as follows: SEQ ID No. 1: pI about 8.8-9.0 (determined by isoelectric focusing on LKB Ampholine™ PAG plates); molecular weight approximately 55 kD (determined by SDS-PAGE). SEQ ID No. 2: pI about 5.8 (determined by isoelectric focusing on LKB Ampholine™ PAG plates); molecular weight approximately 55 kD (determined by SDS-PAGE). Purification of &agr;-amylase variants of the invention The construction and expression of variants according to the invention is described in Example 2, below. The purification of variants of the invention is illustrated here with reference to variants of the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, respectively: Purification of SEO ID No. 1 variants (pI approx. 9.0): The fermentation liquid containing the expressed &agr;-amylase variant is filtered, and ammonium sulfate is added to a concentration of 15% of saturation. The liquid is then applied onto a hydrophobic column (Toyopearl butyl/TOSOH). The column is washed with 20 mM dimethyl-glutaric acid buffer, pH 7.0. The &agr;-amylase is bound very tightly, and is eluted with 25% w/w 2-propanol in 20 mM dimethylglutaric acid buffer, pH 7.0. After elution, the 2-propanol is removed by evaporation and the concentrate is applied onto a cation exchanger (S-Sepharose™ FF, Pharmacia, Sweden) equilibrated with 20 mM dimethylglutaric acid buffer, pH 6.0. The amylase is eluted using a linear gradient of 0-250 mM NaCl in the same buffer. After dialysis against 10 mM borate/KCl buffer, pH 8.0, the sample is adjusted to pH 9.6 and applied to an anion exchanger (Q-Sepharose™ FF, Pharmacia) equilibrated with 10 mM borate/KCl buffer, pH 9.6. The amylase is eluted using a linear gradient of 0-250 mM NaCl. The pH is adjusted to 7.5. The &agr;-amylase is pure as judged by rSDS-PAGE. All buffers contain 2 mM CaCl 2 in order to stabilize the amylase. Purification of SEQ ID No. 2 variants (pI approx. 5.8): The fermentation liquid containing the expressed &agr;-amylase variant is filtered, and ammonium sulfate is added to a concentration of 15% of saturation. The liquid is then applied onto a hydrophobic column (Toyopearl butyl/TOSOH). The bound amylase is eluted with a linear gradient of 15%-0% w/w ammonium sulfate in 10 mM Tris buffer, pH 8.0. After dialysis of the eluate against 10 mM borate/KCl buffer, pH 8.0, the liquid is adjusted to pH 9.6 and applied onto an anion exchanger (Q-Sepharose™ FF, Pharmacia) equilibrated with the same buffer. The amylase is step-eluted using 150 mM NaCl. After elution the amylase sample is dialyzed against the same buffer, pH 8.0, in order to remove the NaCl. After dialysis, the pH is adjusted to 9.6 and the amylase is bound once more onto the anion exchanger. The amylase is eluted using a linear gradient of 0-250 mM NaCl. The pH is adjusted to 7.5. The amylase is pure as judged by rSDS-PAGE. All buffers contain 2 mM CaCl 2 in order to stabilize the amylase. Determination of &agr;-amylase activity &agr;-Amylase activity is determined by a method employing Phadebas™ tablets as substrate. Phadebas tablets (Phadebas™ Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted. For the determination of every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 MM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0. 1 mM CaCl 2 , pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The &agr;-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this &agr;-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the &agr;-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the &agr;-amylase activity. It is important that the measured 620 nm absorbance after 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given &agr;-amylase will hydrolyze a certain amount of substrate and a blue color will be produced. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure &agr;-amylase protein) of the &agr;-amylase in question under the given set of conditions. Thus testing different &agr;-amylases of interest (including a reference &agr;-amylase, in this case the parent &agr;-amylase in question) under identical conditions, the specific activity of each of the &agr;-amylases at a given temperature and at a given pH can be compared directly, and the ratio of the specific activity of each of the &agr;-amylases of interest relative to the specific activity of the reference &agr;-amylase can be determined. Mini dishwashing assay The following mini dishwashing assay was used: A suspension of starchy material was boiled and cooled to 20° C. The cooled starch suspension was applied on small, individually identified glass plates (approx. 2×2 cm) and dried at a temperature of ca. 140° C. in a drying cabinet. The individual plates were then weighed. For assay purposes, a solution of standard European-type automatic dishwashing detergent (5 g/l) having a temperature of 55° C. was prepared. The detergent was allowed a dissolution time of 1 minute, after which the &agr;-amylase in question was added to the detergent solution (contained in a beaker equipped with magnetic stirring) so as to give an enzyme concentration of 0.5 mg/l. At the same time, the weighed glass plates, held in small supporting clamps, were immersed in a substantially vertical position in the &agr;-amylase/detergent solution, which was then stirred for 15 minutes at 55° C. The glass plates were then removed from the &agr;-amylase/detergent solution, rinsed with distilled water, dried at 60° C. in a drying cabinet and re-weighed. The performance of the &agr;-amylase in question &lsqb;expressed as an index relative to a chosen reference &agr;-amylase (index 100)—in the example below (Example 1) the parent &agr;-amylase having the amino acid sequence shown in SEQ ID No. 1&rsqb; was then determined from the difference in weight of the glass plates before and after treatment, as follows: 1 Index = weight &it; &it; loss &it; &it; for &it; &it; plate &it; &it; treated &it; &it; with &it; &it; α - amylase weight &it; &it; loss &it; &it; for &it; &it; plate &it; &it; treated &it; &it; with &it; &it; reference &CenterDot; 100 The following examples further illustrate the present invention. They are not intended to be in any way limiting to the scope of the invention as claimed. 
 EXAMPLE 1 Mini dishwashing test of variants of parent &agr;-amylase having the amino acid sequence shown in SEQ ID No. 1 The above-described mini dishwashing test was performed at pH 10.5 with the parent &agr;-amylase having the amino acid sequence shown in SEQ ID No. 1 and the following variants thereof (the construction and purification of which is described below): T183*&plus;G184*; Y243F; and K269R. The test gave the following results: 26 Parent (SEQ ID No. 1) Index: 100 T183* &plus; G184* Index: 120 Y243F Index: 120 K269R Index: 131 It is apparent that the each of the tested variants T183*&plus;G184* (which exhibits, inter alia, higher thermal stability than the parent &agr;-amylase), Y243F (which exhibits lower calcium ion dependency than the parent &agr;-amylase) and K269R (which exhibits lower calcium ion dependency and higher stability at high pH than the parent &agr;-amylase) exhibits significantly improved dishwashing performance relative to the parent &agr;-amylase. 
 EXAMPLE 2 Construction of variants of the parent &agr;-amylases having the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, respectively Primers: DNA primers employed in the construction of variants as described below include the following &lsqb;all DNA primers are written in the direction from 5′ to 3′ (left to right); P denotes a 5′ phosphate&rsqb;: 27 &num;7113: GCT GCG GTG ACC TCT TTA AAA AAT AAC GGC Y296: CC ACC GCT ATT AGA TGC ATT GTA C &num;6779: CTT ACG TAT GCA GAG GTC GAT ATG GAT CAC CC &num;6778: G ATC CAT ATC GAC GTC TGC ATA CGT AAG ATA GTC &num;3811: TT A(C/G)G GGC AAG GCC TGG GAC TGG &num;7449: C CCA GGC CTT GCC C(C/G)T AAA TTT ATA TAT TTT GTT TTG &num;3810: G GTT TCG GTT CGA AGG ATT CAC TTC TAC CGC &num;7450: GCG GTA GAA GTG AAT CCT TCG AAC CGA AAC CAG B1: GGT ACT ATC GTA ACA ATG GCC GAT TGC TGA CGC TGT TAT TTG C &num;6616: P CTG TGA CTG GTG AGT ACT CAA CCA AGT C &num;8573: CTA CTT CCC AAT CCC AAG CTT TAC CTC GGA ATT TG &num;8569: CAA ATT CCC AGG TAA AGC TTG GGA TTG GGA AGT AG &num;8570: TTG AAC AAC CGT TCC ATT AAG AAG A: Construction of variants of the parent &agr;-amylase having the amino acid sequence shown in SEQ ID No. 1 Description of plasmid pTVB106: The parent &agr;-amylase having the amino acid sequence shown in SEQ ID No. 1 and variants thereof are expressed from a plasmid-borne gene, SF16, shown in FIG. 2 . The plasmid, pTVB106, contains an origin of replication obtained from plasmid pUB 110 (Gryczan et al., 1978) and the cat gene conferring resistance towards chloramphenicol. Secretion of the amylase is aided by the Termamyl™ signal sequence that is fused precisely, i.e. codon No. 1 of the mature protein, to the gene encoding the parent &agr;-amylase having the nucleotide and amino acid sequence (mature protein) shown in SEQ ID No. 4 and SEQ ID No. 1, respectively. The Termamyl promoter initiates transcription of the gene. Plasmid pTVB106 is similar to pDN1528 (see laid-open Danish patent application No. 1155/94). Some unique restriction sites are indicated on the plasmid map in FIG. 2 , including BstBI, BamHI, BstEII, EcoNI, DrdI, AflIII, DraIII, XmaI, SalI and BglII. Construction of variant M202T: The PCR overlap extension mutagenesis method is used to construct this variant (Higuchi et al., 1988). An approximately 350 bp DNA fragment of pTVB106 is amplified in a PCR reaction A using primers &num;7113 and mutagenic primer &num;6778. In a similar PCR reaction B, an approximately 300 bp DNA fragment is amplified using primers Y296 and &num;6779. The complete DNA fragment spanning the mutation site (M202) from primer &num;7113 to primer Y296 is amplified in PCR C using these primers and purified DNA fragments from reactions A and B. PCR C DNA is digested with restriction endonucleases BstEII and AflIII, and the 480 bp fragment is ligated with plasmid pTVB106 digested with the same enzymes and transformed into a low-protease and low-amylase Bacillus subtilis strain (e.g. strain SHA273 mentioned in WO 92/11357). Other M202 variants are constructed in a similar manner. Construction of variants T183*&plus;G184* and R181*&plus;G182*: The PCR overlap extension mutagenesis method is used to construct these variants (Higuchi et al., 1988). The mutagenic oligonucleotides are synthesized using a mixture (equal parts) of C and G in one position; two different mutations can therefore be constructed by this procedure. An approximately 300 bp DNA fragment of pTVB106 is amplified in a PCR reaction A using primers &num;7113 and mutagenic primer &num;7449. In a similar PCR reaction B, an approximately 400 bp DNA fragment is amplified using primers Y296 and &num;3811. The complete DNA fragment spanning the mutation site (amino acids 181-184) from primer &num;7113 to primer Y296 is amplified in PCR C using these primers and purified DNA fragments from reactions A and B. PCR C DNA is digested with restriction endonucleases BstEII and AflIII and the 480 bp fragment is ligated with plasmid pTVB106 digested with the same enzymes and transformed into a low-protease and low-amylase B. subtilis strain (e.g. strain SHA273 mentioned in WO 92/11357). Sequencing of plasmid DNA from these transformants identifies the two correct mutations: i.e. R181*&plus;G182* and T183*&plus;G184*. Construction of variant R124P: The PCR overlap extension mutagenesis method is used to construct this variant in a manner similar to the construction of variant M202T (vide supra). PCR reaction A (with primers &num;3810 and Bi) generates an approximately 500 bp fragment, and PCR reaction B (primers 7450 and Y296) generates an approximately 550 bp fragment. PCR reaction C based on the product of PCR reaction A and B and primers B1 and Y296 is digested with restriction endonucleases BstEII and AflIII, and the resulting 480 bp fragment spanning amino acid position 124 is subcloned into pTVB106 digested with the same enzymes and transformed into B. subtilis as previously described. Construction of variant R124P&plus;T183*&plus;G184*: For the construction of the variant combining the R124P and the T183*&plus;G184* mutations, two EcoNI restriction sites (one located at position 1.774 kb, i.e. between the R124P mutation and the T183* &plus;G184* mutation, and one located at position 0.146 kb) were utilized. The approximately 1630 bp EcoNI fragment of the pTVB106-like plasmid containing the T183*&plus;G184* mutation was subcloned into the vector part (approximately 3810 bp DNA fragment containing the origin of replication) of another pTVB106-like plasmid containing the R124P mutation digested with the same enzyme. Transformation into Bacillus subtilis was carried out as previously described. Construction of variants G182*&plus;G184*; R181*&plus;T183*; Y243F; K269R; and L351C&plus;M430C: These variants were constructed as follows: A specific mutagenesis vector containing a major part of the coding region for the amino acid sequence shown in SEQ ID No. 1 was prepared. The important features of this vector (which is denoted pPM103) include an origin of replication derived from the pUC plasmid, the cat gene conferring resistance towards chloramphenicol and a frameshift-mutation-containing version of the bla gene, the wild-type version of which normally confers resistance towards ampicillin (amp R phenotype). This mutated version of the bla gene results in an amp S phenotype. The plasmid pPM103 is shown in FIG. 3 , and the E. coli origin of replication, the 5′-truncated version of the SF16 amylase gene, and ori, bla, cat and selected restriction sites are indicated on the plasmid. Mutations are introduced in the gene of interest as described by Deng and Nickoloff &lsqb; Anal. Biochem. 200 (1992),pp. 81-88&rsqb;, except that plasmids with the “selection primer” (&num;6616) incorporated are selected based on the amp R phenotype of transformed E. coli cells harboring a plasmid with a repaired bla gene instead of using the selection by restriction-enzyme digestion outlined by Deng and Nickoloff. Chemicals and enzymes used for the mutagenesis were obtained from the Chameleon™ mutagenesis kit from Stratagene (catalogue number 200509). After verification of the DNA sequence in variant plasmids, the truncated gene containing the desired alteration is subcloned from the pPM103-like plasmid into pTVB106 as an approximately 1440 bp BstBI-SalI fragment and transformed into Bacillus subtilis for expression of the variant enzyme. For the construction of the pairwise deletion variant G182*&plus;G184*, the following mutagenesis primer was used: P CTC TGT ATC GAC TTC CCA GTC CCA AGC TTT TGT CCT GAA TTT ATA TAT TTT GTT TTG AAG For the construction of the pairwise deletion variant R181*&plus;T183*, the following mutagenesis primer was used: P CTC TGT ATC GAC TTC CCA GTC CCA AGC TTT GCC TCC GAA TTT ATA TAT TTT GTT TTG AAG For the construction of the substitution variant Y243F, the following mutagenesis primer was used: P ATG TGT AAG CCA ATC GCG AGT AAA GCT AAA TTT TAT ATG TTT CAC TGC ATC For the construction of the substitution variant K269R, the following mutagenesis primer was used: P GC ACC AAG GTC ATT TCG CCA GAA TTC AGC CAC TG For the construction of the pairwise substitution variant L351C&plus;M430C, the following mutagenesis primers were used simultaneously: 1) P TGT CAG AAC CAA CGC GTA TGC ACA TGG TTT AAA CCA TTG 2) P ACC ACC TGG ACC ATC GCT GCA GAT GGT GGC AAG GCC TGA ATT Construction of variant L351C&plus;M430C&plus;T183*&plus;G184*: This variant was constructed by combining the L351C&plus;M430C pairwise substitution mutation and the T183*&plus;G184* pairwise deletion mutation by subcloning an approximately 1430 bp HindIII-AflIII fragment containing L351C&plus;M430C into a pTVB106-like plasmid (with the T183*&plus;G184 mutations) digested with the same enzymes. Construction of variant Y243F&plus;T183*&plus;G184*: This variant was constructed by combining the Y243F mutation and the T183*&plus;G184* mutation by subcloning an approximately 1148 bp DrdI fragment containing T183*&plus;G184* into a pTVB106-like plasmid (with the Y243 mutation) digested with the same enzyme. Bacillus subtilis transformants were screened for &agr;-amylase activity on starch-containing agar plates and the presence of the correct mutations was checked by DNA sequencing. Construction of variant Y243F&plus;T183*&plus;G184*&plus;L351C&plus;M430C: The L351C 30 M430C pairwise substitution mutation was subcloned as an approximately 470 bp XmaI-SalI fragment into a pTVB106-like vector (containing Y243F&plus;T183*&plus;G184*) digested with the same enzymes. Construction of variant Y243F&plus;T183*&plus;G184*&plus;L351C&plus;M430C&plus;Q391E&plus;K444Q: A pPM103-like vector containing the mutations Y243F&plus;T183*&plus;G184*&plus;L351C&plus;M430C was constructed by substituting the truncated version of SF16 in pPM 103 with the approximately 1440 bp BstB1-SalI fragment of the pTVB 106-like vector containing the five mutations in question. The Q391E and K444Q mutations were introduced simultaneously into the pPM103-like vector (containing Y243F&plus;T183*&plus;G184*&plus;L351C &plus;M430C) by the use of the following two mutagenesis primers in a manner similar to the previously described mutagenesis on pPM103: P GGC AAA AGT TTG ACG TGC CTC GAG AAG AGG GTC TAT P TTG TCC CGC TTT ATT CTG GCC AAC ATA CAT CCA TTT B: Construction of variants of the parent &agr;-amylase having the amino acid sequence shown in SEQ ID No. 2 Description of plasmid PTVB112: A vector, denoted pTVB 112, to be used for the expression in B. subtilis of the &agr;-amylase having the amino acid sequence shown in SEQ ID No. 2 was constructed. This vector is very similar to pTVB106 except that the gene encoding the mature &agr;-amylase of SEQ ID No. 2 is inserted between the PstI and the HindIII sites in pTVB106. Thus, the expression of this &agr;-amylase (SEQ ID No. 2) is also directed by the amyL promoter and signal sequence. The plasmid pTVB112 is shown in FIG. 4 . Construction of variant D183*&plus;G184*: The construction of this variant was achieved using the PCR overlap extension mutagenesis method referred to earlier (vide supra). Primers &num;8573 and B1 were used in PCR reaction A, and primers &num;8569 and &num;8570 were used in PCR reaction B. The purified fragments from reaction A and reaction B and primers 1B and &num;8570 were used in PCR reaction C, resulting in an approximately 1020 bp DNA fragment. This fragment was digested with restriction endonucleases PstI and MluI, and subcloned into the expression vector and transformed into B. subtilis. Construction of further variants: By analogy with the construction (vide supra) of the plasmid pPM103 used in the production of mutants of the amino acid sequence of SEQ ID No. 1, a plasmid (denoted pTVB114; shown in FIG. 5 ) was constructed for the continued mutagenesis on variant D183*&plus;G184* (SEQ.ID No. 2). Mutations were introduced in pTVB114 (SEQ ID No. 2; D183*&plus;G184*) in a manner similar to that for pPM103 (SEQ ID No. 1). For the construction of the pairwise deletion variants R181*&plus;D183* and R181*&plus;G182*, it was chosen to alter the flanking amino acids in the variant D183&plus;G184* instead of deleting the specified amino acids in the wild type gene for SEQ ID No. 2. The following mutagenesis primer was used for the mutagenesis with pTVB114 as template: PCC CAA TCC CAA GCT TTA CCA (T/C)CG AAC TTG TAG ATA CG The presence of a mixture of two bases (T/C) at one position allows for the presence of two different deletion flanking amino acid based on one mutagenesis primer. DNA sequencing of the resulting plasmids verifies the presence of either the one or the other mutation. The mutated gene of interest is subcloned as a PstI-DraIII fragment into pTVB112 digested with the same enzymes and transformed into B. subtilis. For the construction of G182*&plus;G184* and R181*&plus;G184*, the following mutagenesis primer was used with pTVB114 as template: PCC CAA TCC CAA GCT TTA TCT C(C/G)G AAC TTG TAG ATA CG As before, the presence of a mixture of two bases (C/G) at one position allows for the presence of two different deletion flanking amino acid based on one mutagenesis primer. DNA sequencing of the resulting plasmids verifies the presence of either the one or the other mutation. The mutated gene of interest is subcloned as a PstI-DraIII fragment into pTVB112 digested with the same enzymes and transformed into B. subtilis. For the construction of D183*&plus;G 184*&plus;M202L the following mutagenesis primer was used: PGA TCC ATA TCG ACG TCT GCA TAC AGT AAA TAA TC For the construction of D183*&plus;G184*&plus;M202I the following mutagenesis primer was used: PGA TCC ATA TCG ACG TCT GCA TAA ATT AAA TAA TC 
 EXAMPLE 3 Determination of oxidation stability of M202 substitution variants of the parent &agr;-amylases having the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 A: Oxidation stability of variants of the sequence in SEQ ID No. 1 The measurements were made using solutions of the respective variants in 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl 2 , pH adjusted to the value of interest with NaOH), pH 9.0, to which hydrogen peroxide was added (at time t&equals;0) to give a final concentration of 200 mM H 2 O 2 . The solutions were then incubated at 40° C. in a water bath. After incubation for 5, 10, 15 and 20 minutes after addition of hydrogen peroxide, the residual &agr;-amylase activity was measured using the Phadebas assay described above. The residual activity in the samples was measured using 50 mM Britton-Robinson buffer, pH 7.3, at 37° C. (see Novo analytical publication AF207-1/1, available on request from Novo Nordisk A/S). The decline in activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes which was not incubated with hydrogen peroxide (100% activity). The percentage of initial activity as a function of time is shown in the table below for the parent enzyme (SEQ ID No. 1) and for the variants in question. 28 % Activity after incubation for (minutes) Variant 0 5 10 15 20 M202L 100 90 72 58 27 M202F 100 100 87 71 43 M202A 100 99 82 64 30 M202I 100 91 75 59 28 M2O2T 100 87 65 49 20 M202V 100 100 87 74 43 M202S 100 100 85 68 34 Parent 100 51 26 13 2 All the M202 substitution variants tested clearly exhibit significantly improved stability towards oxidation relative to the parent &agr;-amylase (SEQ ID No. 1). B: Oxidation stability of variants of the sequence in SEQ ID No. 2 Measurements were made as described above using the parent &agr;-amylase in question (SEQ ID No. 2), the variant M202L&plus;D183*&plus;G184* (designated L in the table below) and the variant M202I&plus;D183*&plus;G184* (designated I in the table below), respectively. In this case, incubation times (after addition of hydrogen peroxide) of 5, 10, 15 and 30 minutes were employed. As in the table above, the percentage of initial activity as a function of time is shown in the table below for the parent enzyme and for the variants in question. 29 % Activity after incubation for (minutes) Variant 0 5 10 15 30 L 100 91 85 71 43 I 100 81 61 44 18 Parent 100 56 26 14 4 The two “substitution&plus;pairwise deletion” variants tested (which both comprise an M202 substitution) clearly exhibit significantly improved stability towards oxidation relative to the parent &agr;-amylase (SEQ ID No. 2). 
 EXAMPLE 4 Determination of thermal stability of variants of the parent &agr;-amylases having the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 A: Thermal stability of pairwise deletion variants of the sequence in SEQ ID No. 1 Measurements were made using solutions of the respective variants in 50 mM Britton-Robinson buffer (vide supra), pH 9.0. The solutions were incubated at 65° C. in a water bath, and samples were withdrawn after incubation for the indicated periods of time. The residual &agr;-amylase activity of each withdrawn sample was measured using the Phadebas assay, as described above. The decline in activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes which was not incubated (100% activity). The percentage of initial activity as a function of time is shown in the table below for the parent enzyme (SEQ ID No. 1) and for the following pairwise deletion variants in question: Variant 1: R181*&plus;G182* Variant 2: R181*&plus;T183* Variant 3: G182*&plus;G184* Variant 4: T183*&plus;G184* Variant 5: T183*&plus;G184*&plus;R124P 30 % Activity after incubation for (minutes) Variant 0 5 10 15 30 45 60 1 100 81 66 49 24 14 8 2 100 80 53 39 17 8 3 3 100 64 40 28 10 4 2 4 100 64 43 34 20 8 5 5 100 78 73 66 57 47 38 Parent 100 13 2 0 0 0 0 It is apparent that all of the pairwise deletion variants tested exhibit significantly improved thermal stability relative to the parent &agr;-amylase (SEQ ID No. 1), and that the thermal stability of Variant 5, which in addition to the pairwise deletion mutation of Variant 4 comprises the substitution R124P, is markedly higher than that of the other variants. Since calorimetric results for the substitution variant R124P (comprising only the substitution R124P) reveal an approximately 7° C. thermostabilization thereof relative to the parent &agr;-amylase, it appears that the thermostabilizing effects of the mutation R124P and the pairwise deletion, respectively, reinforce each other. B: Thermal stability of pairwise deletion variants of the sequence in SEQ ID No. 2 Corresponding measurements were made for the parent enzyme (SEQ ID No. 2) and for the following pairwise deletion variants: Variant A: D183*&plus;G184* Variant B: R181*&plus;G182* Variant C: G182*&plus;G184* 31 % Activity after incubation for (minutes) Variant 0 5 10 15 30 A 100 87 71 63 30 B 100 113 85 76 58 C 100 99 76 62 34 Parent 100 72 55 44 18 Again, it is apparent that the pairwise deletion variants in question exhibit significantly improved thermal stability relative to the parent &agr;-amylase (SEQ ID No. 2). C: Thermal stability of a multi-combination variant of the sequence in SEQ ID No. 1 Corresponding comparative measurements were also made for the following variants of the amino acid sequence shown in SEQ ID No. 1: Variant 4: T183*&plus;G184* Variant 6: L351C&plus;M430C Variant 7: Y243F Variant 8: Q391E&plus;K444Q Variant 9: T183*&plus;G184*&plus;L351C&plus;M430C&plus;Y243F&plus;Q391E&plus;K444Q 32 % Activity after incubation for (minutes) Variant 0 5 10 15 30 4 100 66 41 22 7 6 100 87 73 65 43 7 100 14 2 1 0 8 100 69 46 31 14 9 100 92 93 89 82 Again, it appears that the thermostabilizing effect of multiple mutations, each which has a thermostabilizing effect, is—at least qualitatively—cumulative. 
 EXAMPLE 5 Calcium-binding affinity of &agr;-amylase variants of the invention Unfolding of amylases by exposure to heat or to denaturants such as guanidine hydrochloride is accompanied by a decrease in fluorescence. Loss of calcium ions leads to unfolding, and the affinity of a series of &agr;-amylases for calcium can be measured by fluorescence measurements before and after incubation of each &agr;-amylase (e.g. at a concentration of 10 &mgr;g/ml) in a buffer (e.g. 50 mM HEPES, pH 7) with different concentrations of calcium (e.g. in the range of 1 &mgr;M-100 mM) or of EGTA (e.g. in the range of 1-1000 &mgr;M) &lsqb;EGTA&equals;1,2-di(2-aminoethoxy)ethane-N,N,N′,N′-tetraacetic acid&rsqb; for a sufficiently long period of time (such as 22 hours at 55° C.). The measured fluorescence F is composed of contributions form the folded and unfolded forms of the enzyme. The following equation can be derived to describe the dependence of F on calcium concentration (&lsqb;Ca&rsqb;): F&equals;&lsqb;Ca&rsqb;/ ( K diss&plus;&lsqb;Ca&rsqb; )(&agr; N -&bgr; N log(&lsqb; Ca&rsqb; ))&plus; K diss /( K diss &plus;&lsqb;Ca&rsqb; )(&agr; U -&bgr; U log(&lsqb; Ca&rsqb; )) where &agr; N is the fluorescence of the native (folded) form of the enzyme, &bgr; N is the linear dependence of &agr; N on the logarithm of the calcium concentration (as observed experimentally), &agr; U is the fluorescence of the unfolded form and &bgr; U is the linear dependence of &agr; U on the logarithm of the calcium concentration. K diss is the apparent calcium-binding constant for an equilibrium process as follows: 1 In fact, unfolding proceeds extremely slowly and is irreversible. The rate of unfolding is a dependent on calcium concentration, and the dependency for a given &agr;-amylase provides a measure of the Ca-binding affinity of the enzyme. By defining a standard set of reaction conditions (e.g. 22 hours at 55° C.), a meaningful comparison of K diss for different &agr;-amylases can be made. The calcium dissociation curves for &agr;-amylases in general can be fitted to the equation above, allowing determination of the corresponding values of K diss . The following values for K diss were obtained for the parent &agr;-amylases having the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, and for the indicated &agr;-amylase variants according to the invention (the parent &agr;-amylase being indicated in parentheses): 33 Variant K diss (mol/l) D183* &plus; G184* (SEQ ID No. 2) 1.2 (±0.5) × 10 −4 L351C &plus; M430C &plus; T183* &plus; G184* 1.7 (±0.5) × 10 −3 (SEQ ID No. 1) T183* &plus; G184* (SEQ ID No. 1) 4.3 (±0.7) × 10 −3 SEQ ID No. 2 (parent) 4.2 (±1.2) × 10 −2 SEQ ID No. 1 (parent) 3.5 (±1.1) × 10 −1 It is apparent from the above that the calcium-binding affinity of the latter &agr;-amylolytic enzymes decreases in a downward direction through the above table, i.e. that the pairwise deletion variant D183*&plus;G184* (SEQ ID No. 2) binds calcium most strongly (i.e. has the lowest calcium dependency) whilst the parent &agr;-amylase of SEQ ID No. 1 binds calcium least strongly (i.e. has the highest calcium dependency). 
 REFERENCES CITED IN THE SPECIFICATION Suzuki et al., the Journal of Biological Chemistry, Vol. 264, No. 32, Issue of November 15, pp. 18933-18938 (1989). Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications. Lipman and Pearson (1985) Science 227, 1435. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989. S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869. Matthes et al., The EMBO J. 3, 1984, pp. 801-805. R. K. Saiki et al., Science 239, 1988, pp. 487-491. Morinaga et al., 1984, Biotechnology 2, pp. 646-639. Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151. Hunkapiller et al., 1984, Nature 310, pp. 105-111. R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl. Acids Res. 16, pp. 7351-7367. Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221. Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329. S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682. Boel et al., 1990, Biochemistry 29, pp. 6244-6249. Deng and Nickoloff, 1992, Anal. Biochem. 200, pp. 81-88.