Northeast Agricultural University, Harbin 150030, China, Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
J Microbiol Biotechnol. 2012 Mar;22(3):390-9. doi: 10.4014/jmb.1107.06064.
Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, pYES2Malpha-egIV, and pYES2Malpha-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants IpYES2Malpha-xegIV was higher than that of transformant IpYES2-xegIV or IpYES2Malpha-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that MFalpha signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of 35 degrees C to 65 degrees C. The optimal reaction condition for EGIV enzyme activity was at the temperature of 55 degrees C, pH of 5.0, 0.75 mM Ba²⁺, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant IpYES2Malpha-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.
内切葡聚糖酶基因 egIV 是从纤维素产生真菌绿色木霉 AS 3.3711 中通过 RT-PCR 方案克隆的。egIV cDNA 长 1297bp，包含一个 1035bp 的开放阅读框，编码一个 344 个氨基酸的蛋白质，估计分子量为 35.5kDa，等电点（pI）为 5.29。在绿色木霉 AS 3.3711 中，基因 egIV 的表达可以被蔗糖、玉米秸秆、羧甲基纤维素（CMC）或微晶纤维素诱导，但特别能被 CMC 诱导。在这些底物下，egIV 的转录被调节，但 egIV 基因的表达水平可被葡萄糖和果糖抑制。构建了三个重组载体，pYES2-xegIV、pYES2Malpha-egIV 和 pYES2Malpha-xegIV，以在酿酒酵母 H158 中表达 egIV 基因。酵母转化子 IpYES2Malpha-xegIV 的 CMCase 活性高于转化子 IpYES2-xegIV 或 IpYES2Malpha-egIV，在诱导 48h 时最高活性为 0.13U/ml，表明修饰后的 egIV 基因可提高 CMCase 活性，并且来自酿酒酵母的 MFalpha 信号肽可在酿酒酵母中更有效地调节外源基因表达。重组 EGIV 酶在 pH3.5 到 7.5 和 35 到 65°C 的温度下稳定。EGIV 酶活性的最佳反应条件为温度 55°C，pH5.0，0.75mMBa²⁺，以 CMC 为底物。在这些条件下，转化子 IpYES2Malpha-xegIV 中 EGIV 酶的最高活性为 0.18U/ml。这些特性将为利用纤维素进行工业生物乙醇生产提供技术参数。
