嗜碱细菌Cellulomonas bogoriensis 69B4^(T)木聚糖酶的表达纯化及性质研究

Expression,purification and characterization of xylanase from alklinphilic bacterium Cellulomonas bogoriensis 69B4^(T)

【目的】对嗜碱细菌Cellulomonas bogoriensis 69B4^(T)产碱性木聚糖酶进行研究,克隆来源于该菌株的木聚糖酶基因,并对其进行异源表达、纯化及酶学性质的表征,为后续研究碱性木聚糖酶的耐碱机制及应用奠定基础。【方法】采用单因素分析法对菌株产碱性木聚糖酶情况进行研究;通过基因组分析,锚定5个内切木聚糖酶基因,利用同源扩增的方法进行克隆,并在大肠杆菌中重组表达,利用亲和层析对重组酶进行纯化,以木聚糖为底物表征木聚糖酶的酶学性质。【结果】来源于C.bogoriensis 69B4^(T)的5种木聚糖酶Xyn370、Xyn393、Xyn425、Xyn466和Xyn486均在大肠杆菌内实现了异源表达,并经亲和层析获得纯酶组分,其最适反应温度分别为60、50、40、40、60℃,在50℃范围内保温2h,残余酶活均在90%以上;最适反应p H分别为7.0、8.0、8.0、8.0、9.0,在p H5.0–9.0时具有较好的稳定性;5种重组木聚糖酶对部分金属离子和高浓度盐表现出较好的耐受性,对榉木木聚糖的酶活性最高,均为内切型木聚糖酶。【结论】本研究表达纯化的5种重组木聚糖酶具有耐盐碱的优良特性,且对温度、某些金属离子和化学试剂耐受,为研究木聚糖酶的耐碱机制及工业应用提供了酶源。

[Objective]This study aims to investigate the production of alkaline xylanase from alkaliphilic bacterium Cellulomonas bogoriensis 69B4^(T);clone the xylanase genes derived from this strain;and perform heterologous expression,purification,and characterization of the enzymatic properties.[Methods]We used the single-factor culture method to study the alkaline xylanase production of the strain and cloned five endo-xylanase genes through the homologous amplification.We expressed recombinant xylanases in Escherichia coli and purified them using the affinity chromatography.The enzymatic properties of purified enzymes were characterized using xylan as substrate.[Results]Five xylanases,i.e.,Xyn370,Xyn393,Xyn425,Xyn466,and Xyn486,derived from C.bogoriensis 69 B4T achieved heterologous expression in E.coli and obtained pure enzyme components.Their optimal temperature was 60℃,50℃,40℃,40℃,and 60℃,and the remaining enzyme activity was more than 90%when kept at 50℃for 2 h.The optimum p H for Xyn370,Xyn393,Xyn425,Xyn466,and Xyn486 were 7.0,8.0,8.0,8.0,and 9.0,respectively.The five recombinant xylanases had good stability at a p H range of 5.0–9.0 and showed good tolerance to some metal ions and high salt concentrations,and the enzyme activity on beech xylan was the highest.All xylanases were endo-xylanases.[Conclusion]The five recombinant xylanases expressed and purified in this paper have excellent salt and alkali resistance and are resistant to temperature,certain metal ions,and chemical reagents,providing novel enzymes for studying the alkali resistance mechanism and the industrial applications of the xylanase source.