Heterologous expression of endoglucanase from Sporocytophaga sp.CX11 and the effect of CBM6 on its enzymatic properties
Endoglucanase is one of the important components of cellulase,but most endoglucanases have poor acid-base tolerance.In order to excavate novel endoglucanase,an endoglucanase gene Sm_1350 was cloned from the genome of Sporocytophaga sp.CX11.The sequence of Sm_1350 was 2 196 bp in length and encoding 732 amino acids.The coding protein was consisted with a signal peptide,GH5 catalytic domain,6 family carbohydrate binding module(CBM6)and a C-terminal domain(CTD).The relative expression levels of Sm_1350 under different carbon source culture conditions were determined by real-time qPCR.The expression level of Sm_1350 in cellobiose and filter paper medium was higher than that in glucose medium,suggesting that it may play a role in the degradation of cellulose by the strain CX11.Sm_1350 and its truncated mutants(Sm_1350,Sm_1350-GH5-CBM6,Sm_1350-GH5 and Sm_1350-CBM6)were constructed and heterologous expressed in E.coli,respectively.The deletion of CTD domain had an effective effect on soluble expression level.When CMC was used as substrate,the enzyme activities of Sm_1350-GH5-CBM6 and Sm_1350-GH5 were(7 000.00±50.00)U/g and(2 542.73±35.03)U/g,respectively,indicating that the deletion of CBM6 weakened the hydrolysis capacity of Sm_1350-GH5 to soluble polysaccharides.The enzymatic properties showed that both Sm_1350-GH5-CBM6 and Sm_1350-GH5 had wider pH tolerant range.The deletion of CBM6 only changed the optimal temperature,and had no effect on the optimal pH and temperature stability.In addition,Sm_1350-CBM6 exhibites strong ability to bind chitin,which has potential applications in protein purification.
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