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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.
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Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.

机译：嗜热芽孢杆菌属某种蛋白酶的某些特征。在大肠杆菌中表达：与天然酶的比较及其在大肠杆菌和体外的加工。

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摘要

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C. The enzyme is expressed in E. coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
机译：在芽孢杆菌固定期产生蛋白酶Ak.1。 Ak.1文化。它是一种pI为4.0的丝氨酸蛋白酶，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计分子量为36.9 kDa。该酶在60和70摄氏度下稳定，在80和90摄氏度下的半衰期分别为13小时和19分钟。以偶氮酪蛋白为底物在pH 7.5时观察到最大的蛋白水解活性，该酶还切割了内蛋白酶底物Suc-Ala-Ala-Pro-Phe-NH-Np（琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯丙氨酸对硝基苯胺）。胰岛素B链的主要切割位点被确定为Leu-15-Tyr-16，Gln-4-His-5和Glu-13-Ala-14。将该蛋白酶基因克隆到大肠杆菌中，并在75摄氏度的细胞外培养基中检测到该活性酶的表达。该酶在大肠杆菌中于37摄氏度表达为无活性的蛋白酶，并通过酶转化为成熟酶。将无细胞培养基加热到60摄氏度或更高。原蛋白酶被纯化至同质，pI为4.3，分子量为45 kDa。 NH 2-末端序列为Ala-Ser-Asn-Asp-Gly-Val-Glu-，显示出精确的信号肽切割点。加热原酶导致产生具有与天然酶相同的NH 2-末端序列的活性蛋白酶。克隆的蛋白酶的特征与天然酶的特征相同。（摘要截短为250字）

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