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| Isolation, identification of B-3 Bacillus Subtilis and cloning, expression of kerC |
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Abstract 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.
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Received: 22 December 2011
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. [J]. CHINA ENVIRONMENTAL SCIENCECE, 2004, 24(2): 0-0. |
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