一新中温碱性蛋白酶基因的克隆及原核表达（英文）

Molecular cloning and expression of a novel mesophilicalkaline protease from Bacillus sp. L010 in Escherichia coli

【目的】从环境中分离筛选产蛋白酶、降解蛋白质的菌株,寻找使用价值较高的碱性蛋白酶。【方法】通过酪蛋白平板法分离筛选产蛋白酶菌株,经生理生化方法及16S rDNA基因序列鉴定菌株;利用简并引物及基因组步移克隆蛋白酶完整开放阅读框;蛋白酶前体蛋白及成熟肽序列在大肠杆菌(Escherichia coli)BL21(DE3)中进行重组表达;纯化活性蛋白酶后,利用化学合成多肽底物(succinyl-Ala-Ala-Pro-Phe-p-nitroanilide)检测酶活性质及其催化活力。【结果】分离到的菌株L010被鉴定命名为芽胞杆菌(Bacillus sp.)L010;蛋白酶开放阅读框包含了1149个碱基,编码382个氨基酸,氨基酸序列按其功能分为N端的30个氨基酸残基组成的信号肽,77个氨基酸残基构成的前导肽,C端275个氨基酸残基组成的成熟肽;此蛋白属于丝氨酸蛋白酶家族中枯草杆菌蛋白酶类(Subtilisins)成员,并命名为SprD;SprD的前体蛋白在大肠杆菌(Escherichia coli)BL21(DE3)中重组表达时,在前导肽辅助下自加工为活性蛋白酶;SprD呈现出较高的催化活力,其反应最适条件为温度70℃,pH 9-10。【结论】SprD在碱性(pH 7.0-10.0)、中高温(25℃-60℃)条件下的稳定性及较高的催化能力使其具有一定的研究和潜在利用价值。

[ Objective] Bacteria producing proteases were isolated and selected from the environment and the alkaline proteases with superior performance for commercial exploitation were screened. [ Methods ] The strain producing extracellular proteases was isolated by a casein plate and was identified by biochemical and morphological tests and by 16S rDNA sequence analysis. To acquire the open reading frame （ORF） of the protease, degenerate primers designing and genome walking method were used. The precursor and mature peptide of the protease were recombinant expressed in BL21 （ DE3）. After purification of the active protease, the characteristics and the catalytic ability were detected using synthetic peptide succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrates. [ Results] Strain L010 isolated was named as Bacillus sp. L010 after identification. The ORF of the protease was l149-bp long and encoded a protein of 382 amino acids comprised with a 30-residual signal peptide, a 77-residual propeptide, and a 275-residual mature protein, and the encoded protein was one of subtilisins-a member of serine proteases and designated as SprD. The precursor of SprD （pro- SprD） autoprocessed into active SprD mediated by the propeptide when pro-SprD was recombinant expressed in BL21 （DE3）. The enzyme exhibited high catalytic efficiency （Ko/Km ） towards synthesis substrates with optimal activity at 70℃ and pH 9 - 10. SprD was stable over a range of pH 7.0 to 10.0 and was thermal stable at 25℃ - 60℃. [ Conclusion] The high stability of SprD towards alkaline conditions （pH 7 - 10） and under temperature 25℃ -60℃ and the high catalytic efficiency suggested that the protease would find research value and potential applications.