Study On Cloning? Expression And Enzymatic Properties Of GH7 Family Endonuclease From Arthrobotrys Sp.CX1

Energy sustainable development is an important development strategy in China,and biodegradation of cellulose to produce biofuel ethanol is a research hotspot.Biological enzyme degradation of cellulose is currently one of the most environmentally friendly and promising methods of cellulose degradation ?Endoglucanase plays an important role in cellulose degradation,so mining high-efficiency endocellulase for green cellulose degradation Become a research hotspot.Glycoside hydrolase family 7 protein(GH7)is the most common cellulose degrading enzyme in the genome of biomass degrading fungi secreted.It is mainly found in fungi and has a strong hydrolysis effect.Therefore,GH7 family cellulase is in an important position in the conversion of industrial biomass.In the early stage of this experiment,two GH7 family proteins were identified from the differential protein transcriptome data of Arthrobotrys sp.CX1,named cxGH7 A and cxGH7 B,respectively.Analysis of protein sequences and domains revealed that although cxGH7 A and cxGH7 B originated from the same host and contained GH7 family protein catalytic domain GH7-CBH-EG,they are different in evolution.Simulated the structural discovery of cxGH7 A and cxGH7 B proteins,both proteins contain a GH7-CBH-EG domain consisting of a ?-sandwich structure formed by anti-parallel ?-sheets around multiple loop regions.But there is a slight difference between the loop area and the surface area outside the active center.cxGH7 A contains a loop area outside the active center,forming a closed tunnel-like active site;cxGH7 B lacks the loop area at the active center site,making the catalytic center an open crack area.Theoretical analysis shows that these two proteins may be functionally different.After that,the cxGH7 A and cxGH7 B proteins were recombinantly constructed and expressed,and the recombinant plasmids p PICZ?A-cxGH7 A,p PICZ?A-cxGH7 B were constructed and introduced into Pichia pastoris X-33 to obtain p PICZ?A-cxGH7 A / X-33,p PICZ?A-cxGH7 B / X-33 and perform heterologous expression.The two proteins acted on different substrates at the same time.The experimental results showed that the cxGH7 A protein showed exocellulase activity and the cxGH7 B protein showed endoglucanase activity,so the relevant enzymatic properties of the cxGH7 B protein were characterized.The experimental results are as follows:(1)cxGH7 B protein showed the highest enzyme activity at 40?,p H=6;(2)cxGH7 B protein can maintain more than 80 % of enzyme activity within 30?—65?,has wide temperature stability,and has a high stability at the optimal temperature of40?;(3)In the presence of Ni2+ and other metal ions,the enzyme activity of cxGH7 B protein is increased to about 108 %.In the presence of EDTA chelating agent,the enzyme activity is lost,so cxGH7 B protein is dependent on the presence of metal ions;(4)After the cxGH7 B acts on CMC,the reaction solution is analyzed by ion chromatography to produce glucose,disaccharide and oligosaccharide,which further shows that cxGH7 B protein has endocellulase activity.The above results indicate that the cxGH7 B protein is indeed a GH7 family endocellulase and maintains the ability to degrade cellulose at a wide range of temperatures.However,cxGH7 A protein also shows the ability to degrade cellulose,which provides a research basis for future research on the mechanism of GH7 family endocellulase.