嗜麦芽窄食单胞菌S．maltophilia YHJ-1角蛋白酶基因的克隆表达及活性鉴定

Cloning and Expression of Keratinase Gene from S. maltophilia YHJ - 1 and Identification of Its Biological Activity

从长期堆积废弃羽毛的土壤中分离得到一株高效降解羽毛的菌株YHJ—1，经16S rDNA序列分析鉴定为嗜麦芽窄食单胞菌，命名为嗜麦芽窄食单胞菌S．mahophilia YHJ-1。以菌株YHJ-1基因组为模板，通过PCR方法克隆了该菌的角蛋白酶基因（GenBank登录号：FJ765514），命名为kerD。将其编码区插入到表达载体pET28a（＋）中，转化大肠杆菌（Escherichia coli）BL21（DE3），筛选获得重组菌。通过对重组菌破碎上清液酶活测定和SDS—PAG分析，证实该酶在大肠杆菌中获得了高效表达，但酶活较低，最高酶活为10．5U／mL。酶学性质研究结果表明，该酶的最适反应温度为50℃，最适反应pH值为7．8，为中性酶。实验结果为嗜麦芽窄食单胞菌角蛋白酶的基础理论和应用研究奠定了基础。

A strain YHJ - 1 highly degrading feather bacterium was isolated from the poultry farm soil. According to the sequence of 16S rDNA ,this bacterium was identied as S. mahophilia YHJ- 1. The gene kerD encoding keratinase（ GenBank Assesion No. FJ765514） from S. mahophilia YHJ- 1 was amplified by PCR and inserted into expression plasmid pET28a（ ＋ ）to construet the recombinant expression plasmid pET28 - kerD transformed into Escherichia coli BL21 and induced to express. Enzyme activity analysis to the supernatant of E. coli BL21 （ pET28 - kerD） sonicate demonsrated that the enzyme was expressed in E. coli BL21 at low keratianse activity （10.5 U/mL）. But it was higly expressed by SDS- PAGE. Enzymological characterization analysis showed that the optimal enzyme reaction 7.8. The result layed a foundation for the theory maltophilia YHJ - 1. temperature was 50℃, and the optimal pH was and application research on keratinase from S.