Enzymatic Identification And Functional Site Study Of A Novel Cold-active Cellulase

With the aggravation of resource shortage,people begin to focus on renewable energy.As one of the most abundant cellulose resources in the world,it can produce glucose through cellulase decomposition,and provide raw materials for further processing.The cloning,expression,enzymatic properties and functional sites of the new cellulase gene were systematically studied in this experiment.The main research contents are as follows:Two cellulose-decomposing strains Microbacterium kitamiense and Zhihengliuella halotolerans were obtained from samples collected from Tibet in low temperature climate.Four new cellulase genes,Mkcel5A,MkcelB,ZhcelA and ZhcelB,were successfully cloned.The proteins encoded by these four genes were verified to have cellulase activity by Congo red staining,and further expression reveals that three of the proteins are inclusion bodies,and the other is low in expression.A cellulase gene MkCel5A,one of the four genes,was studied in depth.The results showed that the size of the open reading frame of MkCel5A gene was 1449 bp,encoding a polypeptide of 482 residues with a molecular weight of 51.38 kDa.Sequence analysis showed that it belonged to the glycosylhydrolase family 5?GH5?,and had high homology with endoglucanase.After renaturation and purification of MkCel5A protein,it was found that the optimum catalytic temperature was 25 ? and the optimum catalytic pH was 7.0.Metal ions such as Rb⁺and Co²⁺stimulated the activity of the enzyme,while Cu²⁺,Mn²⁺,SDS and EDTA reduced the activity of the enzyme.When CMC-Na was used as substrate,V_maxax was 1.51 mmol/min*mg,K_m was 42.11 mg/mL,and the standard enzyme activity of wild type enzyme was 0.6 U/mg.It is noteworthy that the optimum temperature of MkCel5A is 25 ^oC and very stable at 0 ?.The enzyme is a new type of cold-active enzyme which has never been reported.It has reference significance for the function research of cold-active enzyme and has the opportunity to be applied in cellulose processing industry,especially in low temperature processing industry.Through prediction and simulation,it was found that the structure of MkCel5A protein has a typical carbohydrate binding domain?CBD?and catalytic domain?CD?of cellulase.Structural alignment analysis showed that the two glutamic acids at 312 and 404 sites of MkCel5A catalytic domain were very conservative in GH5 cellulase family and were important active sites in the catalytic process.This experiment used site-directed mutagenesis to mutate glutamic acid at positions 312 and404 to alanine,after purification and renaturation of the mutant protein,the absolute enzyme activities of mutant E312 and E404 were 0.003 and0.005 U/mg,respectively.The hydrolysis circle of mutant E312 and E404was smaller on CMC plate.The single point mutation experiment showed that the two glutamic acids at position 312 and 404 were important active sites in the catalytic process.In this study,four new cellulase genes were identified,and MkCel5A,as a new cold-active enzyme,has potential value in industrial production and functional research of enzymes in order to discover fewer cold-active cellulases with new members.