嗜热毁丝霉果胶酯酶MtCE12-1的克隆表达及其酶学性质和应用研究OA北大核心CSTPCD

[Objective]To explore a novel pectin esterase enzyme,a homologous pectin esterase significantly up-regulated in tobacco biomass induction was highly expressed in Myceliophthora thermophila.The enzymatic properties of this pectin esterase were investigated,along with its role in assisting the degradation of tobacco biomass.[Method]RT-qPCR was used to analyze the expression of the pectin esterase gene Mtce12-1 in M.thermophila under the condition of tobacco biomass.Using a viral 2A peptide-mediated expression screening system,the pectin esterase gene Mtce12-1 was highly expressed in the M.thermophila wild-type strain ATCC 42462.The positive transformants overexpressing MtCE12-1-His-2A-GFP were subjected to recombinant enzyme production and protein purification,and the enzymatic properties of the pectin esterase MtCE12-1 were characterized.[Result]The transcription level of the pectin esterase gene Mtce12-1 was significantly up-regulated by about 109-110 folds under tobacco biomass conditions as compared to glucose condition.The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis,copy number determination and Western blotting indicated that the recombinant proteins were successfully expressed and secreted in M.thermophila,with expressions reaching as the 464.08 U/mL.The MtCE12-1 presented the highest activity at 75℃and pH 8.0.It had good enzymatic activity in the range of 50-80℃and pH 7.0-9.0 and demonstrated excellent thermal stability.The addition of 100-300 μg of MtCE12-1 to the degradation system resulted in an increase in cellulose degradation efficiency in tobacco bar and tobacco stem by 18.5%-30.7%and 14.6%-30.5%,respectively.[Conclusion]The use of the 2A peptide-mediated expression system in M.thermophila facilitates efficient target protein expression and purification.The alkaline pectin esterase MtCE12-1 not only shows good temperature stability but also demonstrates exceptional effectiveness in tobacco biomass degradation,providing potential high-quality enzyme resources for tobacco industry applications.