纤维二糖水解酶N-糖基化对其在草酸青霉中的分泌和酶活影响

Probing the Role of N-glycosylation on the Catalytic Domain in the Activity and Secretion of Fungal Cellobiohydrolase

目的:纤维素酶水解天然纤维素产生易被微生物利用的葡萄糖是进行生物炼制的关键。丝状真菌分泌的纤维素酶大多数是经过糖基化修饰的,研究丝状真菌纤维二糖水解酶(Cel7A)的催化功能域N-糖基化修饰对其分泌及酶活的影响,有助于优化纤维素酶的表达。方法:利用定点突变将草酸青霉和深绿木霉Cel7A催化功能域的N-糖基化位点去除,构建突变体PoCel7A^(*)和TaCel7A^(*)。以草酸青霉为宿主构建分泌表达PoCel7A^(*)、TaCel7A和TaCel7A^(*)的重组菌,检测N-糖基化去除对Cel7A分泌和酶活力的影响。结果:PoCel7A催化功能域的N-糖基化去除对其蛋白分泌和酶活力无影响。TaCel7A催化功能域的N-糖基化去除不影响其蛋白分泌;但突变体的pNPCase、FPase和Avicelase酶活力分别下降了21.2%,15.2%和17.6%。去除Cel7A催化功能域N-糖基化,加强了细胞内UPR响应。外源蛋白TaCel7A和TaCel7A^(*)的表达也加强了胞内UPR响应。结论:不仅可以为丝状真菌Cel7A的酶工程改造提供理性设计思路,而且为进一步了解糖基化在纤维素酶降解纤维素过程中的作用及机理奠定一定基础。

Objective:Cellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere,and thus form the foundation of enzyme engineering efforts in biofuels research and industrial processes.Many of these carbohydrate-active enzymes from filamentous fungi contain both N-glycans and O-glycans,which in turn can unpredictably affect activity and secretion.Understanding the roles of glycosylation in the function of cellulase is important for further improvement of the enzyme technology for biomass conversion.Methods:The N-glycans defective mutants PoCel7A*and TaCel7A*,removal of the N-glycans on the catalytic domain of cellobiohydrolases from Penicillium oxalicum(PoCel7A)and Trichoderma atroviride(TaCel7A),were constructed using site directed mutagenesis.The extracellular protein concentration and enzymatic activity of the recombinant strains,expressing of PoCel7A*,TaCel7A or TaCel7A*,were determined to investigate whether the removal of N-glycosylation affects the activity and secretion of cellobiohydrolases.Results:The mutant PoCel7A*homologous expression in P.oxalicum,the removal of Asn137 glycosylation site at PoCel7A hardly affected the activity and secretion of Cel7 A.However,after the Asn287 glycosylation site of TaCel7A was removed(TaCel7A*)and heterologous expression in P.oxalicum,the p NPCase,FPase and Avicelase activity of mutant decreased by 21.2%,15.2%and 17.6%,respectively.Furthermore,compared to the parental strain,the expression levels of the UPR marker genes pdi1,bip1 and hac1 were significantly induced in recombinant strains,indicating increased demand for protein folding and transport capacity in recombinant strains.Conclusion:These results indicate that differential roles of N-glycan modifications in contributing to the function of Cel7A and highlight the potential of improving the activity and secretion of Cel7A by tuning proper interactions between glycans and functional residues.