The Important Role Of α-amylase Gene Dosages On The Increase Of Its Production

Mesophilicα-Amylase is one of important industrial enzymes, which has extensive applications in starch processing, brewing, desizing, organic acid and pharmaceutical industries. BF-7658 and Bacillus. sp M13 are the two main mesophilicα-amylase industrial producing strains by far in china. The production ability of the strain was not effectively improved during these twenty years and was very low compared with the most advanced technology in the world. Therefore, it is very important to enhance the secretion ability of the industrial producing strain by genetic improving the strain through modern breeding technology and lower the cost of production.The mesophilicα-amylase was purified to homogeneity from theα-amylase industrial producing strain B. amyloliquefaciens CICIM B2125 and the enzyme characterizations were studied in this project. The full length gene encoding forα-amylase was cloned and the function of the gene was validated. More over, the enzyme properties were changed by site-directed mutagenesis. An optimized eletroporation transformation method was developed for B. amyloliquefaciens CICIM B2125. The secretion ability was improved significantly by increasing theα-amylase gene copies of the industrial strain.1. Theα-amylase from B. amyloliquefaciens CICIM B2125 (BAA) was purified to homogeneity and the enzyme properties were revealed for the first time. The recover rate of the enzyme was 20.1% and fold of purification was 26.9. The molecular weight of the enzyme was 58 kDa. The optimum temperature and pH were 55℃and 6.0-9.0, respectively. The enzyme was stable at the pH range of 7.0-10.0. It kept stable at 55℃for 1 h in the presence of 10 mmoL/L Ca2+ and remained 85% residue activity. The enzyme activities were significantly activated by Ca2+, Mn2+ and Co2+. The main endo-products to soluble starch were oligosaccharides and dextrin analyzed by thinner layer chromatography.2. The full-length gene encoding for mesophilicα-amylase (amyQ) was cloned from B. amyloliquefaciens CICIM B2125. The gene consists of 220 bp of promoter region, 1544 bp of structural gene fragment and 320 bp of terminal sequence. Three bases were altered in the promoter region at the site of -49 (C→T) and -79,-80 (TA→AT), however, the structural gene sequence of amyQ was the same as the amylase gene (GenBank accession No. J01542). 3. The clonedα-amylase gene was expressed in E. coli and B. subtilis. Theα-amylase gene fragment devoid of signal peptide sequence (amyQ′) was cloned into the plasmid pET-28a. The degenerated plasmid pET28a-amyQ′was introduced into E. coli to expressα-amylase and induced with IPTG. The enzyme activity was 2.8 U/mL in the supernatant of culture medium. The full-length gene (amyQ) was cloned into plasmid pLY121 and the recombinant plasmid pLY-amyQ was transformed into B. subtilis 1A510. Theα-amylase gene was expressed and the recombinant enzyme was purified by two-phase system. The characterizations of the recombinant enzyme were almost same compared with the native enzyme.4. Role of the calcium-binding residues Asp231, Asp233 and Asp438 of Bacillus amyloliquefaciensα-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233 and Asp438 were replaced with Asn, Asn and Gly to produce muants D231N, D233N and D438G, respectively. The specific activity for D233N was decreased by 84.8%, while D231N, D438G showed a decrease of 6.3%, 3.5% to that of the wild-type enzyme respectively. No significant changes in the Km value, thermo-stability, optimum temperature and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (Kcat/Km) and thermo-stability at 60oC. The ranges of optimum temperature and optimum pH for D233N were also reduced about 10oC and 3~4 units respectively.5. The enzyme secretion ability was significantly enhanced by the recombinant strain B. amyloliquefaciens CICIM B2125. An optimized electroporation transformation method was established for B. amyloliquefaciens CICIM B2125. The transformation efficiency was satisfied for genetic manipulating the strain. The recombinant plasmid pLY-amyQ was transformed into B. amyloliquefaciens CICIM B2125. About 80% of the recombinant plasmid pLY-amyQ was kept in B. amyloliquefaciens CICIM B2125 during 100 generation growth in the medium without antibiotic. It could meet the requirement of genetic stability of recombinant strain during industrial production. The secretion ability of the recombinant strain was increased by 50% and 30% in flask and 30 ton fermentor respectively, compared with the parental strain. The process of theα-amylase production was reduced about 8-12 h.