两种不同种属β-葡萄糖苷酶基因在酿酒酵母中克隆表达与酶活比较研究.docVIP

  两种不同种属β-葡萄糖苷酶基因在酿酒酵 母中克隆表达与酶活比较研究# 胡佳，唐根云，李晶博，林蒋海，肖文娟，刘泽寰** 5 10 15 20 25 30 （暨南大学生命与健康工程研究院，分子生物研究中心，广州 510632） 摘要：本研究将来源于绿色木霉(Trichoderma viride)AS3.3711、黑曲霉（Aspergillus niger）CICC 40179 的β-葡萄糖苷酶基因（bgl1）分别整合到工业酿酒酵母基因组上，构 建 了 两 株 能 分 泌 表 达 β - 葡 萄 糖 苷 酶 （ BGLI ） 的 酿 酒 酵 母 菌 株 ： AS2.489/tBGLI 和 AS2.489/aBGLI。对每种重组菌的酶活采用 pNPG 的方法进行检测。结果表明：培养 84 h 后， AS2.489/tBGLI 重组菌最高酶活达到 0.836 U/ml，AS2.489/aBGLI 重组菌最高酶活达到 1.099 U/ml；对培养 84h 的两种重组酶蛋白进行酶活比较实验，结果表明 AS2.489/tBGLI 产生的酶 比活力更大，为 1.036 U/mg。绿色木霉的 BGLI 更适合在酿酒酵母中表达，具有进一步研究 和应用的价值。 关键词：β-葡萄糖苷酶；酿酒酵母；克隆；表达；比酶活 中图分类号：Q78 Studies on the cloning and expression of two different β-glucosidase genes in Saccharomyces cerevisiae and comparisons of their activities HU Jia, TANG Genyun, LI Jingbo, LIN Jianghai, XIAO Wenjuan, LIU Zehuan (Researeh Center for Molecular Biology, Institutes of Life and Health Engineering, Jinan University, GuangZhou 510632) Abstract: In this paper, β-glucosidase genes (bgl1) from Trichoderma viride AS3.3711 and Aspergillus niger CICC 40179 were integrated into the genome of an strain of industrial Saccharomyces cerevisiae genome respectively. The two recombinant S.cerevisiaes strains can secrete β-glucosidase (BGLI) and were named AS2.489/tBGLI and AS2.489/aBGLI. Enzyme Activities of the two BGLIs were tested by pNPG method after 48 h cultivation.. The results showed that the highest enzyme activity of AS2.489/tBGLI was 0.978 U/ml, while that of AS2.489/aBGLI was 0.849 U/ml. In addition, activities of two recombinant enzymes were also compared after cultivation of 84 h. The specific activity of AS2.489/tBGLI (1.036 U/mg)）was higher than that of AS2.489/aBGLI (0.762 U/mg), which indicated that . BGLI from Trichoderma viride was expressed better in Saccharomyces cerevisiae than that from Aspergillus niger and will be of great value of further studies and industrial application . Keywords: β-glucosidase; Saccharomyces cerevisiae; cloning; expression; specific activity 35 0 引言 纤维素酶是指能水解纤维素的 β-1，4-糖苷键生成纤维二