角蛋白酶kerC基因的克隆及序列分析

Cloning and Sequence Analysis of Keratinase kerC Gene

为克隆分析角蛋白酶ker C基因,本研究以羽毛为底物从11株实验室保存的芽孢杆菌中筛选角蛋白酶产生菌。得到了一株具有高效降解羽毛能力的枯草芽孢杆菌BS10,该菌株3 d即可降解一根完整的羽毛,以羽毛粉、天青角蛋白为底物测定其酶活力,分别达(1.88±0.10)U/mL、(1.79±0.49)U/mL。以同源克隆的方法克隆ker C基因,获得了一条全长1 149 bp的kerC基因(Gen Bank登录号:KX108888),编码383个氨基酸。利用BLAST、ProtParm、SOPMA和MEGA等生物信息学工具对其理化性质、二级结构及系统进化树等进行分析,发现其与NCBI中的角蛋白酶基因相似性达到85%;相对分子质量为39.095 kD,等电点为9.23;该基因蛋白为亲水性蛋白;系统进化树分析结果与菌株信息相吻合。羽毛降解菌的获得可促进废弃羽毛资源化利用;角蛋白酶基因的获得及其分子特征的分析,为进一步通过基因工程手段提高角蛋白酶活性提供了一定的理论依据。

In order to clone and analyze the keratinase kerC gene, a keratinase-producing strain was screened from 11 Bacilli in laboratory by using feathers as substrate. A Bacillus subtilis strain named BS10 that could effectively degrade a complete feather in 3 days was obtained, whose enzyme activity was （1.88±0.10） U/mL and （1.79±0.49） U/mL when feather and keratin azure were used as substrates. The kerC was cloned by homologous cloning, and a kerC gene （GenBank accession number： KX108888） with a total length of 149 bp was acquired, encoding 383 amino acids. BLAST, ProtParm, SOPMA, MEGA and some other bioinformatics tools were adopted to analyze its physicochemical properties, secondary structures and phylogenetic tree, the result of which showed that the similarity between kerC gene and the keratinase genes in NCBI reached as high as 85%. The relative molecular weight and isoelectric point were 39.095 kD and 9.23, respectively. It was a hydrophilic protein. In addition, the result of phylogenetic tree analysis was consistent with the strain information. The gain of a new feather-degrading bacteria could promote the utilization of waste feathers. The acquisition of kerC gene and its molecular characterization would provide a theoretical basis for further improving the activity of keratinase by means of genetic engineering.