蜡样芽孢杆菌聚乙烯醇脱氢酶的表达及酶学特性

Expression and properties of polyvinyl alcohol dehydrogenase from Bacillus cereus

摘要:

【背景】聚乙烯醇脱氢酶(polyvinyl alcohol dehydrogenase, PVADH)能够使聚乙烯醇(polyvinyl alcohol, PVA)氧化脱氢，在PVA的生物降解过程中起到重要作用。【目的】从PVA降解菌株蜡样芽孢杆菌DG01中获取pvadh基因，实现PVADH在毕赤酵母中的异源表达并探究其对不同型号PVA的降解特异性，为PVADH在PVA实际降解中的应用提供指导。【方法】通过反转录扩增技术获得长度为1 965 bp的pvadh基因片段，构建pPIC9K-cpvadh重组表达质粒并在毕赤酵母GS115中实现异源表达，甲醇诱导表达蛋白，进行分离纯化后对其酶学性质及降解特异性进行研究。【结果】最佳发酵条件下PVADH粗酶液酶活达到54.55 U/mL。经分离纯化后表达蛋白PVADH的比酶活为173.42 U/mg，分子量为67.1 kDa，等电点为6.06，该酶最适作用温度为41 ℃，最适作用pH值为7.5，在27-32 ℃、pH 7.0-8.0条件下酶的半衰期超过4 h，1 mmol/L的Ca²⁺对酶活力有激活作用。PVADH分别作用于PVA1788、PVA1799及PVA2488，K_m值分别为1.17、1.49、1.21 mg/mL。【结论】毕赤酵母表达的PVADH产酶及纯化方便，酶学性质稳定，对醇解度小的PVA具有更好的降解能力。

关键词:聚乙烯醇;聚乙烯醇脱氢酶;异源表达;降解特异性

Abstract:

[Background] Via oxydehydrogenation of polyvinyl alcohol (PVA), polyvinyl alcohol dehydrogenase (PVADH) plays an important role in the biodegradation of PVA.[Objective] pvadh gene was extracted from the PVA-degrading Bacillus cereus sp. DG01 for the expression of PVADH in Pichia pastoris and specificity of the enzyme for degrading different PVA species was explored. The findings are expected to guide the application of PVADH in PVA degradation.[Methods] The 1 965 bp pvadh as obtained by reverse-transcription PCR amplification. pPIC9K-cpvadh plasmid was constructed for expression in P. pastoris GS115. Methanol was employed to induce the expression of the protein which was then isolated and purified. The enzymatic properties and degradation specificity were investigated. [Results] The activity of crude PVADH solution yielded under optimal fermentation conditions reached 54.55 U/mL. The purified PVADH had the specific activity of 173.42 U/mg, molecular weight of 67.1 kDa, and isoelectric point of 6.06, and the optimum temperature and pH for PVADH were 41 ℃ and pH 7.5, respectively. The half-life of PVADH was more than 4 h at 27-32 ℃ and pH 7.0-8.0, and 1 mmol/L Ca²⁺ can activate the enzyme. K_mvalues of PVADH for the three substrates PVA1799, PVA1788, and PVA2488, were 1.49 mg/mL, 1.17 mg/mL, and 1.21 mg/mL, separately. [Conclusion] Heterogeneous expression in P. pastoris is a simple method to obtain PVADH and the purification features ease of implementation. The yielded PVADH has stable enzymatic properties and high efficiency in degrading the PVA with low alcoholysis degree.

Key words:polyvinyl alcohol;polyvinyl alcohol dehydrogenase;heterologous expression;enzymatic properties