title: Cloning, expression, and characterization of endoglucanase gene egIV from Trichoderma viride AS 3.3711.
abstract: 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.
authors.affiliation: Northeast Agricultural University, Harbin 150030, China, Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Huang
authors.forename: Jinxia
authors.initials: J
authors.lastname: Fan
authors.forename: Qian
authors.initials: Q
authors.lastname: Yang
authors.forename: Xiuling
authors.initials: X
authors.lastname: Chen
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Liu
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Daqing
authors.initials: D
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D002480:Cellulase