Expression Of Novel Cellulase Gene From Ampullaria Crossean In P.pastoris

Cellulose is the most abundant renewable resource in the world. Every year, half of the hundred million ton biomaterial are produced by photosynthesis, but only a few were utilized. It has great realistic significance for solving environment pollution and energy crisis by transforming cellulose effectively to energy, chemical stuff and food. Compared to the traditional methods such as acid, alkaline hydrolysis or steam heating, enzymatic hydrolysis carries out under mild conditions with less environmental contamination and byproducts. The efficient and multi-functional cellulase should be needed to hydrolysis of the comples cellulose.Ampullaria crossean is one kind of inbreaking pest to the crop. In view of the reports of isolation and purification of multi-functional endogenous cellulase from A.crossean, and poor expression of the cellulase gene in Pichia pastoris, this paper focused on cloning and expressing novel gene from A. crossean.In this paper, the cDNA sequence from Ampullaria crossean stomach named Dy1300 was obtained by RT-PCR firstly and cloned into vector pMD 18-T. Its ORF named SXYN contains 882bp nucleotide encoding a 294 amino-acid protein. The percentage of similarity of SXYN with the reported cellulase EGX gene sequence from Ampullaria crossean was 89.8%. Amino acid sequence-based classification has revealed that the putative protein belongs to the glycosyl hydrolase family 10(GHF 10). No typical cellulose binding domain was found, and three potential O-GlcNAc sites were showed through software(YinOYang V1.2).Enzyme digestion sites EcoRI and Not I were added to the two ends of interest gene respectively, and they were inserted into pPIC9K digested by the same restrictive enzymes. Then recombinant pPIC9K-SXYN was constructed, and was transformed into Pichia pastoris cell GS115 by electroporation. The G418 hyper-resistant colonies were isolated and tested to identify the Mut+ and MutS phenotype. After inducement of Mut+ phenotype strain by methanol, the activity of CMCase was detected in supernatant, but no xylase was detected. The optimal methanol concentration for inducement was 1.5% and the initially optimal pH of the medium was 6.0. The...