Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
Appl Biochem Biotechnol. 2010 Sep;162(1):103-15. doi: 10.1007/s12010-009-8700-2. Epub 2009 Jul 10.
Endoglucanase is a major cellulolytic enzyme produced by the fungus Trichoderma viride. The 1,317 bp cDNA of endoglucanase gene egVIII was cloned from T. viride AS3.3711, encoding a 438 amino acid protein with a calculated molecular mass of 46.86 kDa and isoelectric point of 4.32. Sequence analysis suggested that EGVIII belonged to the glycosyl hydrolase family 5. The N-terminal region of EGVIII contains a signal peptide sequence of 19 amino acid residues, indicating that it is an extracellular enzyme. Transcription of the egVIII gene in T. viride AS3.3711 can be induced by carboxymethyl cellulose sodium (CMC-Na), sucrose, microcrystalline cellulose, and corn stalk, and inhibited by glucose and fructose. The alpha-mating factor signal can effectively enhance the secretion of the recombinant EGVIII in Saccharomyces cerevisiae, as demonstrated by the enzymatic activity of recombinant yeast IpYEMalpha-xegVIII in the supernatant, which was 0.86 times higher than that of the IpYES2-egVIII. Recombinant endoglucanase EGVIII showed optimal activity at a temperature of 60 degrees C and pH of 6.0. It was stable when incubated from 35 degrees C to 70 degrees C for 1 h. The enzymatic activity of recombinant EGVIII was stable at a pH 3.0 to 7.5 at 50 degrees C and reached the highest level at 0.174U when activated by 75 mM of Zn(2+). The Michaelis-Menten constant (Km) and Kcat values for CMC-Na and cellotriose hydrolysis were 3.82 mg/ml, 9.56 s(-1) and 1.75 mg/ml, 7.08 s(-1), respectively. Transgenic yeast strain IpYEMalpha-xegVIII might be useful for renewable fuels industries.
内切葡聚糖酶是木霉属真菌产生的一种主要的纤维素酶。内切葡聚糖酶基因 egVIII 的 1317bp cDNA 从 T.viride AS3.3711 中克隆得到，编码一个 438 个氨基酸的蛋白质，计算分子量为 46.86kDa，等电点为 4.32。序列分析表明，EGVIII 属于糖苷水解酶家族 5。EGVIII 的 N 端区域含有 19 个氨基酸残基的信号肽序列，表明它是一种细胞外酶。在 T.viride AS3.3711 中，egVIII 基因的转录可以被羧甲基纤维素钠（CMC-Na）、蔗糖、微晶纤维素和玉米秸秆诱导，被葡萄糖和果糖抑制。α-交配因子信号可以有效地增强重组 EGVIII 在酿酒酵母中的分泌，重组酵母 IpYEMalpha-xegVIII 上清液中的酶活性比 IpYES2-egVIII 高 0.86 倍。重组内切葡聚糖酶 EGVIII 在 60°C 温度和 pH6.0 下表现出最佳活性。在 35°C 到 70°C 孵育 1 小时时保持稳定。在 50°C 时，重组 EGVIII 的酶活性在 pH3.0 到 7.5 之间稳定，在 75mM Zn(2+)激活时达到最高水平 0.174U。CMC-Na 和纤维三糖水解的米氏常数（Km）和 Kcat 值分别为 3.82mg/ml、9.56s(-1)和 1.75mg/ml、7.08s(-1)。转基因酵母菌株 IpYEMalpha-xegVIII 可能对可再生燃料工业有用。
