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中文摘要：
通过分析Klebsiella variicola SHN-1普鲁兰酶的活性中心,并将K.variicola SHN-1普鲁兰酶与来自K.pneumoniae,Bacillus acidopullulyticus,B.naganoensis的普鲁兰酶氨基酸序列进行同源性比较,确定了H626D、R756E、H852D、R908E、I619N、F723Y、S850T、K851S八个突变位点,采用定点突变的方法获得了8个突变酶,经过酶学性质分析,发现H852D的最适反应pH由原来的pH 5.0降低为pH 4.7,且在pH 4.5下的稳定性得到提高。通过同源建模及结构分析发现,852位点突变为天冬氨酸后与周围的氨基酸多形成了2个氢键,因此提高了结构的稳定性,且由于活性中心p Ka值的改变,造成了普鲁兰酶最适pH发生了迁移。
英文摘要：
By analyzing the active site of Klebsiella variicola SHN-1 pullulanase and alignment of the amino acid sequences of the pullulanases from K. pneumoniae ,Bacillus acidopuUulyticus and B.naganoens/s, eight site-directed mutagenesis including H626D, R756E, H852D, R908E, I619N, F723Y, S850T,and K851S were subjected to improve the acid resistance of K. variicola pullulanase. After analysis of enzymatic properties,the optimum pH of H852D was shifted from 5.0 to 4.7, and its stability was obviously improved when stored at pH 4.5. By the analysis of homology modeling and structure of the pullulanase,the formation of two hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of stability at low pH, and the change ofpKa value in the active center ofpullulanase caused the optimum pH migration.
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