通过定点突变提高耐热发酵肠杆菌来源葡萄糖异构酶的催化效率和热稳定性。
Enhanced catalytic efficiency and thermostability of glucose isomerase from Thermoanaerobacter ethanolicus via site-directed mutagenesis.
机构信息
The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, PR China.
The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, PR China.
出版信息
Enzyme Microb Technol. 2021 Dec;152:109931. doi: 10.1016/j.enzmictec.2021.109931. Epub 2021 Oct 6.
Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 ℃ and pH 6.5 with the divalent cations Co and Mg. The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased K from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process.
葡萄糖异构酶（GI）是制备高果糖玉米糖浆（HFCS）的关键酶。本研究通过定点突变获得了来源于热厌氧菌乙醇丁醛CCS D1 的突变体 TEGI-M-L38 M/V137 L（TEGI-M2）。TEGI-M2 在 85℃和 pH6.5 时具有最佳活性，二价阳离子 Co 和 Mg 存在时具有最佳活性。基于同源建模和分子对接研究了 TEGI-M 和 TEGI-M2 之间的结构差异，以阐明酶性质提高的机制。与原始酶相比，TEGI-M2 的酶活性提高了 2.0 倍，K 值从 234.2mM 降低到 85.9mM。最后，尝试将突变体 TEGI-M2 应用于 HFCS 一步法生物合成中，得到了 67.3%的 d-果糖产率，比 TEGI-M 提高了 14.3%。TEGI-M2 的这种改进的催化性能对于一步法制备 d-果糖的工业生产具有重要意义。
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