Cloning and heterologous expression of a novel endoglucanase gene egVIII from Trichoderma viride in Saccharomyces cerevisiae.
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.
Huang
Xiao-Mei
XM
Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
Yang
Qian
Q
Liu
Zhi-Hua
ZH
Fan
Jin-Xia
JX
Chen
Xiu-Ling
XL
Song
Jin-Zhu
JZ
Wang
Yun
Y
D000595:Amino Acid Sequence; D002244:Carbon / Q000737:chemistry; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D021382:Protein Sorting Signals; D002244:Carbon; D002480:Cellulase