Abstract

Site-directed mutagenesis was made to obtain a glucose isomerase (GI) working at high temperatures and low pH values and having high affinity to the substrate and more pH and termal stability in comparison with the native enzyme. For this purpose, GI gene from Geobacillus caldoxylosilyticus TK4 was cloned to pET-28a(+) vector and H99Q, V184T and D102N mutations were performed. Biochemical features of the recombinant and mutant enzymes were identified. These mutations enabled the mutant enzymes to increase the optimum temperature and K_M and decrease the optimum pH and V_max values of the reaction. Furthermore, the mutant proteins had more thermal and pH stability than that of the recombinant GI. The mutant enzymes showed the highest activities in the presence of Co²⁺, Cu²⁺ and Mn²⁺ and were more resistant to some metals than the recombinant GI. In conclusion, the site-directed mutations led to improve the performance of the recombinant enzyme, which means that the mutant enzyme (TK4GI mutation) may be practicable in high fructose corn syrup production process.

中文翻译：

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通过突变开发嗜热重组葡萄糖异构酶的一些特性

摘要

进行定点诱变以获得葡萄糖异构酶（GI），其在高温和低pH值下工作，与底物具有高亲和力，与天然酶相比具有更高的pH和末端稳定性。为此目的，将来自caldoxylosilyticus TK4的GI基因克隆到pET-28a（+）载体中，并进行了H99Q，V184T和D102N突变。鉴定了重组和突变酶的生化特征。这些突变使突变酶能够增加最佳温度和K _M并降低最佳pH和V _max反应值。此外，突变蛋白比重组GI具有更高的热稳定性和pH稳定性。突变酶在Co 2 ⁺，Cu ²⁺和Mn ²⁺的存在下表现出最高的活性，并且比重组GI对某些金属更具抗性。总之，定点突变导致重组酶性能的提高，这意味着该突变酶（TK4GI突变）在高果糖玉米糖浆生产过程中是可行的。