糙皮侧耳(Pleurotus ostreatus)漆酶POXA1在里氏木霉中的高效表达及酶学性质

摘要

[Objective] Expression, purification and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei. [Methods] The strong promoter and terminator of cellobiohydrolase I (cbhl) gene from T. reesei were amplified by PCR and inserted into pBluescriptIISK(+) to form vector pSKCST. The laccase gene from Pleurotus ostreatus was de novo synthesized according to T. reesei condon bias and cloned into the vector pLacdt resulting in the expression vector pSKLDT. The linearized pSKLDT was introduced into T. reesei strain Tu6 by protoplast-mediated transformation. The screened laccase expression transformants were grown in shake flasks on minimal medium and the recombinant laccase was purified and characterized. [Results] Transformants were isolated in selective screening medium plate and identified by PCR. The enzyme activity of laccase in transformant LC-7 was 237.134 U/mL which was 28. 6 — fold higher than that in P. ostreatus. The specific activity of the purified enzyme was 9852 IU/mg. Enzymatic assay revealed that the optimum temperature for its activity was 501 and pH was 3.0. The optimum substrate was ABTS and the Km and Vmax for ABTS were 7. 58 × 10-2 mmol/L and 9. 752 × 10-3 mmol/L/min. Metal ions like Cu2+ , Zn2+ , Fe3+ , Mn2+ , Ba2+ , Mg2+ and Fe2+ had different inhibitory effect on purified laccase. [Conclusions] Under the regulation of cbhl promoter and cbhl signal peptide, heterologous laccase was successfully overexpressed in T. reesei.%[目的]将优化后的糙皮侧耳(Pleurotus ostreatus)漆酶基因poxl,在里氏木霉中进行高效表达并对重组表达的漆酶进行酶学性质测定.[方法]:根据里氏木霉的密码子偏好性,对漆酶POXA1密码子进行优化并合成.以质粒pBluscriptⅡSK(+)为骨架,利用里氏木霉纤维二糖水解酶基因(cbhl)的启动子和终止子序列构建里氏木霉外源蛋白表达载体pSKLDT.PEG介导的原生质体转化方法转化里氏木霉菌株Tu6,筛选获得漆酶表达工程菌；通过木霉工程菌摇瓶发酵培养后对发酵液上清中的漆酶进行纯化,并对异源表达的漆酶酶学性质进行研究.[结果]经尿嘧啶缺陷培养基筛选获得木霉阳性转化子,通过PCR分析及漆酶酶活性筛选获得漆酶高效表达重组菌LC-7,该重组菌经摇瓶发酵144h后粗酶液酶活达237.134IU/mL,酶活较其出发菌Pleurotus ostreatus提高了28.6倍.酶学性质研究表明,纯酶的比活为9852IU/mg,最适温度为50℃,最适pH为3.0,最适底物为ABTS,该漆酶催化ABTS的Km和Vmax分别为7.58×10-2 mmol/L及9.752×10-3mmol/L/min,金属离子Cu2+、Zn2+、Fe3+、Mn2+、Ba2+、Mg2+等对漆酶有不同程度的抑制作用,但Fe2+能明显的抑制漆酶的催化活性.[结论]外源漆酶基因能在里氏木霉中实现高效分泌表达.