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首页> 外文期刊>Applied and Environmental Microbiology >Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1.
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Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1.

机译：地衣芽孢杆菌PWD-1角蛋白分解蛋白酶编码基因kerA的核苷酸序列和表达。

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摘要

Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.
机译：地衣芽孢杆菌PWD-1（ATCC 53757）分泌角蛋白酶，一种蛋白水解酶，对整根羽毛都有活性。通过氨基酸序列相似性和对苯甲基磺酰氟的抑制，证明了角蛋白酶是丝氨酸蛋白酶。确定了角蛋白酶结构基因kerA的编码区和侧翼区的整个核苷酸序列。来自纯化酶N端序列的固定寡核苷酸引物和第二个随机寡核苷酸引物用于称为PCR步移的程序中，该程序用于扩增和测序kerA的上游和下游区域。另一种方法是用λ噬菌体载体进行PCR筛选，以插入的PWD-1基因组DNA片段为模板，以载体臂的已知序列和酶的N端序列作为引物。 lambda文库的PCR扩增和序列分析完成了整个kerA序列，并建立了一组基因缺失。 kerA基因与来自地衣芽孢杆菌NCIMB 6816的枯草杆菌蛋白酶Carlsberg的基因具有97％的序列同一性。推定的启动子，核糖体结合位点和转录终止子在这两种细菌中也相似。推导的氨基酸序列表明两种成熟蛋白酶之间仅三个氨基酸差异。 Northern（RNA）分析表明，转录调控可控制kerA在不同生长培养基上的表达。

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