| ABSTRACT Acidic alpha-amylase is present application and research are many enzymes, acidic alpha-amylase is capable of hydrolyzing starch under acidic conditions, and therefore are widely applied in various fields of fermentation, animal feed, food, and biological pharmaceutical. The lab previous first screened to obtain an acidic alpha-amylase of the overproducing strain B-5, the amylase secretion of its optimum temperature is 70 ℃, the optimum pH is 5.0. Acid alpha-amylase gene was cloned and successfully expressed in E. coli BL21 (DE3), obtained recombinant AMY protein by the separation and purification, the optimum temperature is 70℃, the optimum pH is 5.0 to 6.2.According to the alpha-amylase gene have been reported, and analysis this experiment recombinant alpha-amylase gene is sequencing revealed that the present experiment recombinant alpha-amylase gene containing the A, B, C, D, E five domains, but the majority of alpha-amylase is only A, B, C three structural domains. This experiment is obtained the truncated mutant of amy-△DE and amy-△E then cloned into the expression vector pET21a, to obtain a recombinant plasmid pET21a-amy-△DE and pET21a-amy-Delta E, then transformed into E. coli BL21 (DE3). Induced by IPTG, SDS-PAGE electrophoresis, the apparent molecular weight of the AMY-△DE about 45 kDa, the apparent molecular weight of the AMY-△E about 55 kDa.AMY-△DE and AMY-△E were further purified and characterized.When AMY-△DE and AMY-△E hydrolyzed soluble starch, the optimal pH value was 5.0-6.2 and the optimum of temperature was 70℃. The kinetic parameters of the truncated alpha-amylase AMY-△DE and AMY-△E on soluble starch was that:Km was 2.422mg/ml,2.591mg/ml; Vmax was 107.08uM/min/mg,115.04uM/min/mg. The original AMY of the raw starch adsorption is higher than truncated AMY-△DE and truncated AMY-△E, the adsorption capacity of the truncated alpha-amylase AMY-△DE is lowest. |
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