定点突变改善β-甘露聚糖酶AuMan5A的酶学性质

Improvement in the Enzymatic Properties of β-Mannanase（AuMan5A） by Site-Directed Mutagenesis

DOI：10.3969/j.issn.1673-1689.2017.08.006

中文关键词: β-甘露聚糖酶 定点突变 温度特性 比活性 催化效率

英文关键词: β-mannanase,site-directed mutagenesis,temperature characteristics,specific activity,catalytic efficiency

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中文摘要:

基于糖苷水解酶5家族（GHF5）β-甘露聚糖酶一级、三维结构的比对和分析，对宇佐美曲霉GHF5 β-甘露聚糖酶AuMan5A的关键位点氨基酸实施定点突变以获得酶学性质优良的突变酶AuMan5AG320D。采用大引物PCR技术将AuMan5A基因（Auman5A） 中编码Gly320的密码子GGT突变为Asp320的GAC，构建出突变酶基因Auman5AG320D。分别将Auman5A和Auman5AG320D在毕赤酵母GS115中进行了表达，分析了表达产物AuMan5A和AuMan5AG320D的酶学性质。结果表明：AuMan5AG320D的最适温度Topt由突变前的65 ℃提升至70 ℃，在70 ℃的半衰期t1/270由原酶的10 min延长至25 min；AuMan5AG320D的比活性由突变前的351.2 U/mg提高到1 729.1 U/mg，其催化效率（kcat/Km） 是原酶的9.3倍。将Gly320突变为Asp320不仅改善了AuMan5A的温度特性，而且显著提高了该酶的比活性和催化效率。

英文摘要:

Based on comparison and analysis of primary and three-dimensional（3-D） structures of glycoside hydrolase family 5（GHF5） β-mannanases，an amino acid at the key site of Aspergillususamii GHF5 β-mannanase（AuMan5A） was subjected to site-directed mutagenesis to obtain a mutant enzyme AuMan5AG320D with superior enzymatic properties. Using the megaprimer PCR method，the AuMan5AG320D-encoding gene，Auman5AG320D，was constructed by mutating the Gly320-encoding codon GGT of Auman5A into Asp320-encoding GAC. Then，two genes，Auman5A and Auman5AG320D，were extracellularly expressed in Pichia pastoris GS115 and the enzymatic properties of expressed products were analyzed. Results indicated that the optimal temperature of AuMan5AG320D was 70 ℃ and and its half-life at 70 ℃（t1/270） was 25 min. The specific activity of AuMan5AG320D was increased from 351.2 U/mg to 1 729.1 U/mg and its catalytic efficiency（kcat/Km） was 9.3 times higher than that of AuMna5A. Through mutating Gly320 into Asp320，this work not only improved the temperature characteristics of AuMan5A，but also significantly enhanced its specific activity and catalytic efficiency.

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