We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

中文翻译：

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对温度敏感的重组枯草杆菌蛋白酶蛋白酶变体，可有效降解分子生物学酶

我们使用易于出错的PCR在枯草杆菌蛋白酶编码基因中产生突变，并筛选了表达温度敏感（TS）变体的重组体。从重组基因中检测到的数十种突变中，我们发现影响天冬氨酸75的那些突变对温度稳定性的影响最大。因此，我们将分析的重点放在枯草杆菌蛋白酶C的两个变体上，即对热敏感的变体24（V24），其氨基酸变化为D75G，L234M和Q274P。和变体25（V25），只有一个氨基酸变化D75A。对于V24，在50°C下10分钟内，活性降低了2个对数倍。对于V25，在60°C（该温度会使野生型酶的活性降低约30％）时发生两个对数倍的降低。V24变体完全灭活了分子生物学研究和分子诊断中常用的酶，并通过浓度为10％或更高的丙二醇稳定了其自溶能力。枯草杆菌蛋白酶变体由菌株缺乏其天然分泌的蛋白酶表达的枯草芽孢杆菌，可以使用镍亲和层析从上清液中分离出变异体。