[Expression of a thermostable a-amylase mutant into Escherichia coli and Pichia pastoris].
Alpha-amylase are of considerable commercial value. It can be produced by a wide variety of microorganisma. The alpha-amylase gene (amyE) from Bacillus licheniformis, which is widely used for the industrial hydrolysis of starch, was mutated (amyEM), then amplified by PCR and inserted into pBV220 and pPIC9k to obtain the recombinant vector pBV220-amyEM and pPIC9k-amyEM. These recombinant vectors were transformed into corresponding competent cell E. coli DH5alpha and P. pastoris GS115 respectively. The resulting recombinant strains, DH5alpha/pBV220-amyEM and GS115/ pPIC9k-amyEM, were then screened by measuring the enzymatic activity and SDS-PAGE. DH5alpha/pBV220-amyEM was induced by temperature and GS115/pPIC9k-amyEM by methanol. In contrast to the parent cells, the a-amylases were expressed in both the recombinant strains. In E. coli the molecular weight was approximately 55kDa; optimal temperature and pH of the recombinant a-amylase were 80 degrees C - 90 degrees C and 6.0 respectively. The recombinant amylase had high activity in pH 5.0 - 5.5 compared to wild type. In Pichia pastoris, the recombinant amylase was secreted to the medium; molecular weight was 60kDa for the putative post-translational modifications; optimal pH shifted to 5.5. The specific activities of alpha-amylase produced by E. coli and P. pastoris were 8.1U/mg and 102U/mg respectively. This result indicated that the alpha-amylase were secreted into the culture medium with high efficiency in the recombinant P. pastoris High activity in high temperature and lower pH properties impart the recombinant amylase potential applications in industry.
Du
Bing-Bing
BB
Institute of Microbiology , Chinese Academy of Sciences, Beijing 100080, China. du-bing117@sohu.com
Hao
Shuai
S
Li
Yun-Min
YM
Yue
Li-Li
LL
Jiao
Qing-Hua
QH
D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D000516:alpha-Amylases