角蛋白酶产生菌的分离鉴定及其kerC的克隆表达

以四川农业大学养鸡场堆砌废弃羽毛处土壤为样品,筛选出一株具有较强降解羽毛能力的细菌B-3菌株.经形态学、生理生化特性和16S rRNA基因序列分析,鉴定其为枯草芽孢杆菌(Bacillus subtilis),命名为枯草芽孢杆菌B-3.并成功克隆到该菌株的角蛋白酶基因kerC(GenBank No.:JN021789),在大肠杆菌BL21(Escherichia coli BL21)中获得了高效表达.该基因全长1146bp,GC含量46.5%,编码381个氨基酸,与已报道的枯草芽孢杆菌YYW-1的kerC基因(GenBank No.: EU362730)同源性达到100%.重组菌株经IPTG诱导后角蛋白酶酶活力达14.8U/mL,经His-Tag纯化和SDS-PAGE分析表明,重组角蛋白酶分子量约为60kDa(融合了硫氧还蛋白,Trx).重组角蛋白酶最适反应温度和pH值分别为65℃与7.0.

Isolation, identification of B-3 Bacillus Subtilis and cloning, expression of kerC

A feather-degrading bacterium was isolated from a sample of discarded feather in the chicken farmland of Sichuan Agricultural University. Relative experiments about its morphological, physiological and biochemical characteristics and 16SrDNA were conducted. Results showed it possessed typical characterizations of Bacillus subtilis. Therefore, it was identified as Bacillus subtilis and named Bacillus subtilis B-3. Afterwards, the nucleotide sequence of the keratinase gene was successfully amplified by PCR utilizing its genomic DNA as template. The keratinase gene was further cloned into expression vector pET32a(+) and was highly expressed using Escherichia coli BL21 (DE3) . Sequencing result revealed the kerC gene was composed of 1146 bp which encodes 381 amino acids. And the sequence alignment of kerC showed it shared a 100% similarity with the kerC gene from Bacillus subtilis strain YYW-1 submitted in GenBank (No.: EU362730). The activity of this recombinant kerC was 14.814U/mL when induced by IPTG. And this recombinant enzyme (fused with E.coli thioredoxin) was purified by His-Tag. SDS-PAGE analysis demonstrated that it had a molecular mass of 60.0 kDa. Subsequent tests validated that the optimal temperature and pH of the recombinant enzyme were 65℃ and 7.0 respectively.