| The acid alpha-amylases can hydrolyze alpha-1,4-linkages in starch under the acidity condition.They are widely applied in numerous areas,such as in fermentation,feed,food industry,Biological drugs manufacture.In this work Strain B-5, screened out previously as a high production strain of acid alpha-amylase, is identified as Bacillus amyloliquefaciens. According to the characteristics of morphology, physiology and biochemistry tests, as well as comparison of 16S rDNA sequence.The prime was designed based on the published conversev nucleotide sequence of alpha-amylase gene, DNA fragement of alpha-amylase gene was obtained by PCR from the genomic DNA of Bacillus amyloliquefaciens. B-5 and cloned into vector pMD-19 and sequenced. The result showed alpha-amylase gene has an ORF about 1980bp.The Amy whose signal peptide was removed was successfully expressed in E.coli BL21.The apparent molecular weight of Amy-E was about 66kDa according to SDS-PAGE.Amy-E was further purified and characterized.When Amy-E hydrolyzed soluble starch, the optimal pH value was 5.0~6.2 and the optimum of temperature was 70℃.When Amy-E hydrolyzed 1% amylose, 1% amylopectin, 1% pullulan, the result showed the optimum of substrate was amylopectin and Amy-E did not have any effect on pullulan.The kinetic parameters of Amy-E on different substrate was that: Km(amylose) was 2.58; Km(amylopectin) was 0.94; Vmax(amylose) was 11.98 Vmax (amylopectin) was 72.46.The hydrolysis products of different substrates were separated and identified by TLC.The result showed G1 and G2 were the end products of all the different substrates hydrolysis by Amy-E.
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