黑曲霉葡萄糖氧化酶在里氏木霉中的分泌表达及其对内源纤维素酶表达的影响

doi:10.13304/j.nykjdb.2016.574

中国农业科技导报 ›› 2017, Vol. 19 ›› Issue (5): 42-50.DOI: 10.13304/j.nykjdb.2016.574

黑曲霉葡萄糖氧化酶在里氏木霉中的分泌表达及其对内源纤维素酶表达的影响

吴毅兰1，2§,薛鲜丽2§,谢响明1*,苏小运2*

1.北京林业大学生物科学与技术学院, 北京 100083;

2.中国农业科学院饲料研究所, 农业部饲料生物技术重点开放实验室, 北京 100081

收稿日期:2016-09-13 出版日期:2017-05-15 发布日期:2016-11-21
通讯作者: 谢响明，教授，博士生导师，主要从事资源与环境微生物学研究。E-mail：xiangmingx@sina.com。苏小运，研究员，博士生导师，主要从事木质纤维素降解和利用的研究。E-mail：suxiaoyun@caas.cn
作者简介:§吴毅兰与薛鲜丽为本文共同第一作者。吴毅兰，硕士研究生，研究方向为资源与环境微生物学。E-mail：wyllanlan@hotmail.com。薛鲜丽，博士研究生，研究方向丝状真菌表达系统研究。
基金资助:
国家自然科学基金项目（31400067）资助。

Expression of a Glucose Oxidase from Aspergillus niger in Trichoderma reesei and its Effects on Production of Endogeneous Cellulases

WU Yilan1,2§, XUE Xianli2§, XIE Xiangming1*, SU Xiaoyun2*

1.College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083;

2.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of
Agricultural Sciences, Beijing 100081, China

Received:2016-09-13 Online:2017-05-15 Published:2016-11-21

摘要/Abstract

摘要： 目前饲料工业中所使用的纤维素酶和葡萄糖氧化酶来自不同的表达系统，其生产成本较高；而在同一种微生物宿主中同时表达这两种重要的饲料工业用酶则有可能会降低成本。根据里氏木霉中密码子的偏好性优化黑曲霉来源的葡萄糖氧化酶基因god，并利用DNA assembler方法构建pRS424-cbh1P-god-cbh1T质粒（pPGT质粒）；通过PEG介导的原生质体转化法，将其转入里氏木霉Tu-6Δtku70尿嘧啶缺陷型菌株中，经筛选得到葡萄糖氧化酶酶活较高的一株转化子（2号），SDS-PAGE分析显示重组GOD的蛋白分子量大小约为70 kDa，进一步使用质谱确证了GOD得到成功表达。摇瓶发酵表明，2号转化子在微晶纤维素诱导92 h后，GOD酶活达到4.63 U/mL。该转化子的滤纸酶活、内切葡聚糖酶活和外切纤维素酶活分别为7.88 U/mL、2.58 U/mL和0.84 U/mL，而出发菌株分别为6.79 U/mL、3.19 U/mL和0.57 U/mL，而β-葡萄糖苷酶由原来的0.66 U/mL提高到1.65 U/mL（1.5倍）。实验表明，在里氏木霉中表达GOD能显著提高β-葡萄糖苷酶的酶活，也能使总体纤维素酶活得到提高。

关键词: 葡萄糖氧化酶, 里氏木霉, 纤维素酶

Abstract: The cellulase and glucose oxidase (GOD) currently used in feed industry are from different expressing systems, which brings about the high production cost. Co-expression of these 2 important feed enzymes in one microbial host may decrease production cost. The god gene from Aspergillus niger was codon-optimized to the T. reesei codon usage bias. The expression plasmid pRS424-cbh1P-god-cbh1T (pPGT) was constructed by DNA assembler and then transformed into the T. reesei uridine auxotroph stain Tu-6Δtku70 via a PEG-mediated protoplast transformation method. By screening transformants, a transformant Tu-6Δtku70god2 was selected for its high glucose oxidase activity. The Tu-6Δtku70god-2 showed that the recombinant glucose oxidase had an apparent molecular mass of ca. 70 kDa, which was further verified by mass spectrometry. In the shaking flask experiment, Tu-6Δtku70god-2 had a GOD activity of 4.63 U/mL at 92 h after Avicel induction. The filter paper cellulase activity, endo-glucanase, exo-glucanase (or cellobiohydrolase), and β-glucosidase activities of transfomant Tu-6Δtku70god-2 were 7.88 U/mL, 2.58 U/mL and 0.84 U/mL, respectively. In comparison, those of the parent strain were 6.79 U/mL, 3.19 U/mL, and 0.57 U/mL, respectively. The β-glucosidase activity in this transformant was 1.65 U/mL, much higher than that (0.66 U/mL) of the parent strain (1.5-fold increased). It was demonstrated that expressing of a GOD could significantly improve the activity of the endogenous-glucosidase activity and the overall cellulase activity in T. reesei.

Key words: glucose oxidase, Trichoderma reesei, cellulase

吴毅兰1，2§,薛鲜丽2§,谢响明1*,苏小运2* . 黑曲霉葡萄糖氧化酶在里氏木霉中的分泌表达及其对内源纤维素酶表达的影响[J]. 中国农业科技导报, 2017, 19(5): 42-50.

WU Yilan1,2§, XUE Xianli2§, XIE Xiangming1*, SU Xiaoyun2*. Expression of a Glucose Oxidase from Aspergillus niger in Trichoderma reesei and its Effects on Production of Endogeneous Cellulases[J]. Journal of Agricultural Science and Technology, 2017, 19(5): 42-50.

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黑曲霉葡萄糖氧化酶在里氏木霉中的分泌表达及其对内源纤维素酶表达的影响

Expression of a Glucose Oxidase from Aspergillus niger in Trichoderma reesei and its Effects on Production of Endogeneous Cellulases

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