Expression Of β-1,3-glucanase In Pichia Pastoris

Chaetomium globosum is not only can inhibit the growth of many plant pathogens,it is also an important microbial pesticides family. Related research is becoming moreimportant and even successed producing the agents as biocontrol pesticide. C. globosumcan secreted β-1,3-glucanase that can hydrolyze cellulose glycosidic bond, anddestroying the cell walls of plant pathogens, inhibiting their growth. Thus, obtainedβ-1,3-glucanase from C. globosum and make it highly expressed, meanwhile studies thecontrol effect on the pathogen, it is a significant great promotion to the biologicalpesticides.In the experiment, the β-1,3-glucanase gene obtained is1683bp, the length ofcDNA is1605bp. First, the primers consists of EcoR I and Not I restriction sites wasused to amplifly gene cgglu. Subsequently, the target gene cgglu and the expressionvector pPIC9K were double digested by EcoR I and Not I, T4DNA Ligase was used toconstruct the recombinant expression vector pPIC9K-cgglu after digestion. Kpn2I wasused to linearized the recombinant expression vector pPIC9K-cgglu, and after that usingthe electroporation method to transform it into Pichai pastoris GS115. And theelectroporation voltage was set at800V,1000V,1200V and1400V. Which, the1000V has the maximum conversion rate of about80transformants per μg.48transformantswere randomly selected for MD and MM plate detection, and the result showed that allthe transformants were Mut+.Five of the transformants were selected for induced expression and SDS-PAGEresult showed that four of the transformants successful to secret the recombinant proteinCGGLU. The targeted band was found between50kDa and85kDa, larger than thetheoretical value size, which is54kDa. The optimum reaction temperature for therecombinant protein CGGLU is45℃. Induced transformants for96h has the highestactivity, and the value of enzyme activity was0.186U. Three different plant pathogens,Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum, were used toverify enzyme activity between the recombinant protein CGGLU and their cell wall.Among them, the cell wall of F. oxysporum has the highest enzyme activity that was0.165U. R. solani and S. sclerotiorum cell wall enzyme activities were0.123U and0.132U. The recombinant protein CGGLU has showed ability of antibacterial, and hasa great potential for biological control studies.