Construction And Characterization Of Cellulase From Sporocytophaga Myxococcoides

Cellulose is the most abundant renewable resource in the world.Due to the complex structure of cellulose,it is difficult to be directly utilized by microorganisms.The first discovery of processive endoglucanase was in 1998,since then,many scholars have investigated and discussed the mechanism of processive endoglucanase towards the degradation of cellulose.A putative endoglucanase from Sporocytophaga sp.CX11 was successfully expressed in E.coli BL21(DE3)and identified as a processive endoglucanase.The protein was designated as Sm Cel5 A and its enzymatic activities towards cel ulose were characterized.The recombinant Sm Cel5 A was purified by affinity chromatography.The enzyme activity of Sm Cel5 A towards sodium carboxymethyl cel ulose(CMC)was287.44±0.01 U/mg;the ratio of soluble sugar to insoluble sugar generated from filter paper was 2.32 to 11.22(5-180 min);CMC viscosity can be rapidly reduced in a short time(1 min);the smal est substrate for Sm Cel5 A is cel otriose.In order to further clarify the key amino acid sites of the Sm Cel5 A active center,two mutants,W174 A and W205 A,were constructed.Compared with Sm Cel5 A,the mutation of W174 A led to the complete loss of enzyme activity,while the activity of W205 A decreased slightly when compared with Sm Cel5 A.When hydrolyzing filter paper by W205 A,processive capacity is slightly higher than Sm Cel5 A.It is suggested that the mutation sites W174 and W205 may play key roles in the process of filter paper degradation by Sm Cel5 A.W174should be a key amino acid residue of the catalytic center,while W205 may be directly affect the sustained hydrolysis capacity of Sm Cel5 A.