Huang Xiao-Mei, Li Qing-Qing, Chen Xiu-Ling, Fan Jin-Xia, Xu Xiu-Hong, Sun Xu-Dong, Li Dong-Yu, Zhao Hong-Xiao
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.
College of Resources and Environment, Northeast Agricultural University, Harbin, 150030, China.
Appl Biochem Biotechnol. 2017 Jul;182(3):1158-1170. doi: 10.1007/s12010-016-2389-9. Epub 2017 Feb 2.
Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257 bp, encoding 418 amino acids with a molecular weight of 44.23 kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose. A highly efficient integrated expression vector (pYPIGH-B includes a sequence of the α-mating factor signal peptide (MF-α)) was constructed. The enzymatic activity of the supernatant of recombinant yeast YPIGH-B3 was 1.29 times higher than that of YES2-egII, demonstrating that the MF-α can significantly improve the expression of the recombinant EGII in S. cerevisiae. The recombinant endoglucanase TaEGII produced by S. cerevisiae showed maximum activity at pH 5.0 and temperature 60 °C. Under these conditions, the Km and Kcat values for Avicel and raffinose hydrolysis were 1.22 × 10 mg ml, 9.09 × 10 s and 1.06 × 10 mg ml , 9.18 × 10 s, respectively. The enzymatic activity of recombinant TaEGII was stable when incubated from 40 to 60 °C for 1 h. It was stable in a wide range of pH (4.0-7.0) and sensitive to various metal ions. Transgenic yeast strain YPIGH-B3 might be applied to cellulosic ethanol production.
由深绿木霉分泌的内切葡聚糖酶是一种纤维素酶。从深绿木霉AS3.3013中克隆了一个内切葡聚糖酶基因egII，并在酿酒酵母INVScI中进行表达。egII基因的开放阅读框由1257个碱基对组成，编码418个氨基酸，分子量为44.23 kDa，外加一个21个氨基酸的信号肽。基于序列相似性，TaEGII属于糖基水解酶家族5。egII基因在深绿木霉AS3.3013中的表达可被微晶纤维素（MCC）、麸皮、羧甲基纤维素（CMC）、稻草和玉米秸秆诱导，但受到葡萄糖的抑制。构建了一个高效整合表达载体（pYPIGH-B，包含α-交配因子信号肽（MF-α）序列）。重组酵母YPIGH-B3上清液的酶活性比YES2-egII高1.29倍，表明MF-α能显著提高重组EGII在酿酒酵母中的表达。酿酒酵母产生的重组内切葡聚糖酶TaEGII在pH 5.0和温度60℃时表现出最大活性。在这些条件下，水解微晶纤维素和棉子糖的Km和Kcat值分别为1.22×10 mg/ml、9.09×10 s和1.06×10 mg/ml、9.18×10 s。重组TaEGII的酶活性在40至60℃孵育1小时时稳定。它在广泛的pH范围（4.0-7.0）内稳定，对各种金属离子敏感。转基因酵母菌株YPIGH-B3可能应用于纤维素乙醇生产。
