葡萄糖氧化酶在毕赤酵母中的热稳定性改良及其酶学性质表征
首发时间:2021-02-23
摘要:葡萄糖氧化酶(GOD)在毕赤酵母中的热稳定性改良及其酶学性质表征葡萄糖氧化酶在工业应用中非常广泛,但热稳定性差限制了其广泛的应用。本研究通过生物信息学分析和序列比对在野生GOD基础上设计了4个初级突变体,并进一步组合设计构建了突变体GODm,并利用毕赤酵母表达、纯化、研究酶学性质。酶学性质表明: GODm的比酶活是野生型的2.10倍,(Kcat/Km)值提高了1.45倍。GODm经55℃处理3h后仍剩余37.5%的酶活,在55℃、65℃下的t1/2较野生型提高了2.28、3.36倍。分析突变体GODm的蛋白质三级结构发现,T30V形成了新的氢键、强化了疏水作用,D70K、D315K优化了表面静电相互作用,A162T提高了电子传递链的传递效率,从而获得了一个热稳定性、催化效率同步提升的突变体。
For information in English, please click here
Improvement of thermal stability and characterization of glucose oxidase in Pichia pastoris
Abstract:Glucose oxidase(GOD) is widely used in industry, but its poor thermal stability limits its broader application. In this study, four primary mutants were designed on the basis of wild God by bioinformatics analysis and sequence alignment, and the mutant godm was further constructed by combinatorial design.The expression, purification and enzymatic properties of the mutant godm were studied by Pichia pastoris.The enzyme properties showed that the specific enzyme activity of GODm was 2.10-fold of that of wild type, and the value of (Kcat / Km) was increased by 1.45-fold. The enzyme activity of GODm remained 37.5% after treated at 55 ℃ for 3 h, and the t1/2 of GODm at 55 ℃ and 65 ℃ was 2.28-fold and 3.36-fold higher than that of wild type. By analyzing the protein tertiary structure of mutant GODm, it was found that T30V formed a new hydrogen bond and enhanced the hydrophobic interaction, D70K and D315K optimized the surface electrostatic interaction, and A162T improved the transfer efficiency of the electron transfer chain, thus a mutant with improved thermal stability and catalytic efficiency was obtained.
Keywords: glucose oxidase Pichia pastoris mutation thermostability
基金:
引用
No.****
动态公开评议
共计0人参与
勘误表
葡萄糖氧化酶在毕赤酵母中的热稳定性改良及其酶学性质表征
评论
全部评论0/1000