Studies On The Expreesion, Optimazation And Application Of Endochitinase Gene From Trichoderma Viride In P.pastoris

Chitin, aβ-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc), was considered as one of the most abundant biopolymers in nature, with a production of 1011 tons per year after cellulose and lignin. In recent years, there is a growing interest in the derivatives obtained from the hydrolysis of chitin, especial chitooligosaccharide. It was demonstrated to be physiologically functional and led to many potential applications in food, cosmetics and medicine, etc.Chitinases (EC 3.2.1.14) are a group of complex hydrolytic enzymes that catalyze depolymerization of chitin. In recent years, endochitinases have been attracting worldwide research interests due to their great potential industrial application in the preparation of chitooligosaccharide. However, the yields of endochitinase from naturally isolated microorganisms are often low and not satisfied with the requirements for the large-scale industrial production. Therefore, it is essential to enhance the yield of the endochitinase so that it can be widely used at the large scale.In this study, the mature endochitinase cDNA from Trichoderma viride was cloned from pMD19-ECH and ligated into the plasmid pPIC9K to generate the recombinant expression vector pPIC9K-ECH42. Linearized by the restriction enzyme Bpu1102I, this constructed expression vector was transformed into the genome of Pichia pastoris GS115 by electroporation method. The transformant ZJGSU02 with the highest endochitinase activity was screened. SDS-PAGE and zymogram indicated that the molecular weight of the recombinant endochitinase was about 45 kDa.The optimal conditions for production of endochitinase by engineered Pichia pastoris ZJGSU02 were investigated in this study. With the endochitinase activity as the target, the concentration of several important factors, metahanol, oleic acid, Tween-80, PTM1 and cell, and pH and medium volume were optimized, respectively. The optimal concentration of methanol was 0.5%. The activity of recombinant endochitinase could be improved greatly when added 0.05% oleic acid, 0.4% Tween-80 and 0.6% PTM1. The optimal pH, cell concentration and medium volume were 6.0,3:1 and 25 mL/100 mL, respectively.Furthermore, culture conditions were optimized by the Plackett-Burman and Box-Behnken design. The optimal culture medium components obtained were as follows:2.45% yeast extract, 2.00% tryptone,0.50%YNB,100 mM potassium phosphate,0.50% methanol,0.17% oleic acid,0.62% Tween-8,0.4% PTM1 and 4.00×10-5% biotin. The enzyme activity under the above optimal conditions was about 88.26 U/mL. The highest activity of endochitinase was obtained at 72 h in the optimal medium with activity of 89.3 U/mLThe recombinant P. pastoris ZJGSU02 was induced under the optimal culture conditions. The optimal conditions of powder chitin-degrading and chitosan with recombinant endochitinase were also explored and they were as follows:the powder chitin concentration 4%, pH 7.0, temperature 30 C, reaction time 10 h and the reducing sugar content was about 398.4μg/mL in this condition. The chitosan concentration 4%, pH 7.0, temperature 30 C, reaction time 12 h and the reducing sugar content was about 154.4μg/mL in this condition.