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

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