Expression and Characteristics of an Endoglucanase from Trichoderma atroviride (TaEGII) in Saccharomyces cerevisiae.
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 × 10-2mg ml-1, 9.09 × 10-2s-1and 1.06 × 10-2mg ml-1, 9.18 × 10-2s-1, 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.
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.
Xiao-Mei
XM
Huang
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.
Qing-Qing
QQ
Li
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.
Xiu-Ling
XL
Chen
College of Engineering, Northeast Agricultural University, Harbin, 150030, China. fanjxneau@neau.edu.cn.
Jin-Xia
JX
Fan
College of Resources and Environment, Northeast Agricultural University, Harbin, 150030, China. xuxiuhong@neau.edu.cn.
Xiu-Hong
XH
Xu
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.|College of Resources and Environment, Northeast Agricultural University, Harbin, 150030, China.
Xu-Dong
XD
Sun
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.|College of Resources and Environment, Northeast Agricultural University, Harbin, 150030, China.
Dong-Yu
DY
Li
College of Engineering, Northeast Agricultural University, Harbin, 150030, China.|College of Resources and Environment, Northeast Agricultural University, Harbin, 150030, China.
Hong-Xiao
HX
Zhao
D002480:Cellulase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002480:Cellulase
Endoglucanase gene; Integrated expression vector; Trichoderma atroviride; Yeast expression