裂解多糖单加氧酶在毕赤酵母中的异源表达

Heterologous expression of lytic polysaccharide monooxygenase in Pichia pastoris

裂解多糖单加氧酶(lysate polysaccharide monooxygenase,LPMO)是一类含铜氧化酶,在还原型辅因子存在下可氧化裂解纤维素,对纤维素的降解起重要作用.优化并合成黄孢原毛平革菌(Phanerochaete chrysosporium)lpmo基因,构建pPICZαA-Pclpmo表达载体,电转毕赤酵母GS115实现了分泌表达,重组蛋白表达量为450mg/L.重组PcPLMO对微晶纤维素催化活性为14U/mL,对碱预处理的秸秆纤维活性较高,为24U/mL.PcPLMO具有较好的热稳定性,分别在80、90、100℃保温60min,残留活性为80%、62%与48%.经EndoH脱糖基化分析,PcLPMO发生了高度糖基化修饰,从而促进了酶的高温热稳定.相关结果可为LPMO协同纤维素酶降解纤维素的应用提供参考.

Lysate polysaccharide monooxygenase (LPMO) is a class of copper oxidase,which cleave glycoside bonds of cellulose in an oxidative mechanism in the presence of reductive cofactors,playing an important role in enzymatic hydrolysis of cellulose.In this study,the lpmo gene of Phanerochaete chrysosporium had been optimited and synthetized,and the recombinant expression vector pPICZ α A- Pclpmo was constructed and transformed into Pichia pastoris GS115 by electroporation.The secretory expression of the target protein was achieved at 450 mg/L.The catalytic activity of purified PcLPMO on avicel was 14 U/mL,and the highest activity for alkali pretreatment of straw fiber substrate was 24 U/mL.PcPLMO had better thermal stability,after keeping at 80 ℃,90 ℃ and 100 ℃ for 60 min,the residual activities were 80%,62% and 48%,respectively.Endo H deglycosylation analysis showed that the PcLPMO was highly glycosylated,which promoted the thermal stability.The relevant results can provide reference for the further application of LPMO synergistic cellulase to degrade cellulose.