Cloning And Expression Of Endoglucanase Ⅱ Gene In Saccharomyces Cerevisiae From Trichoderma Atroviride

Cellulose are abundant renewable resources in nature. In the face of energy crisis, it is the current urgent problems that cellulases can hydrolyze cellulose into small molecules to produce fuel ethanol. What is possible for mankind that cellulose which are broken down by microorganism. Fungi can secret cellulases on earth, especially Trichoderma spp..Based on the strain of Trichoderma atroviride AS3.3013 as the materials, the 1,257 bp cDNA of endoglucanaseⅡ(EGⅡ) was cloned by the method of RT-PCR. The fragment was sent to sequencing and submitted to Gen Bank with access NO.KP098496, encoding a 418 amino acid protein. The size and isoelectric point of protein was predicted the 44.21 kDa and 4.96 by particular site. Sequence analysis suggested that EGⅡ belonged to the glycosyl hydrolase family 5. The N-terminal region of EGⅡcontains a signal peptide sequence of 21 amino acid residues, indicating that it is an extracellular enzyme.In order to realize the efficient expression of cellulase, a yeast efficient recombinant expression vector YPIGH was constructed this paper. EGⅡwas connected into the vector YPIGH and free carrier pYES2, becoming the recombinant plasmid YPIGH-EGⅡ and p YES2-EGⅡwhich were electrotransformed into the Saccharomyces cerevisiae INVSc Ⅰ. The enzymatic activity of yeast transformants were demonstrated by the method of DNS, and found out the enzymatic activity of YPIGH-EGⅡwas 1.29 times higher than that of the pYES2-egⅡ. After beta-galactose induced expression and using SDS-PAGE electrophoresis analysis, the results show that the purpose protein of EGⅡwas expressed in S.cerevisiae INVScⅠ, and the size of protein was in accordance with the theoretical value. The studies that were researched by real-time RT-PCR to analyze the relative expression of T.atroviride induced by different substrates and transformants induced by beta-galactose have shown that the expression amount of microcrystalline cellulose(MCC)was highest and of transformants was highest on the fourth day. In this paper, the fermentation conditions of transformants were optimized, optimizing the best enzyme production conditions as follows: the beta-galactose content is 3%, YNB content is 0.84%, oxygen flux is 100 mL, culture temperature is 29℃. The largest producing enzyme activity was 116.10U/g.