Expression And Characterization Of Alkalitolerant Cellulase

Alkalitolerant cellulase is cellulase having high activity in alkaline environment,which has wide application in the wash,textile,feed,printing and other fields.In this paper,Cellulomonas bogoriensis as experimental strains I optimized its enzyme production conditions,obtained two endo-cellulase Cel619 and Cel800 by cloning and expression,and research their enzymatic properties.By single factor experiments to determine the optimum conditions for cellulase production from the experimental strain Cellulomonas bogoriensis: cultivation time 3 d,medium initial pH 10.5,cultivation temperature 30 ?,carbon 2.5%?w/v?corn stover,nitrogen 2.0%?w/v?of yeast extract and mixed beef extract?1: 1?,liquid volume 100 mL/500 mL,inoculation content 4.0%?v/v?.Its simple research of the crude enzyme showed optimum pH is 7.0 with a wide range of pH stability;optimum reaction temperature is 50 ? with good temperature stability in the range of 4-40 ?.I obtained the whole genome DNA of strains Cellulomonas bogoriensis by NCBI,of which there are two kinds of endo-glucanase.According to their gene sequences,primers were designed for cloning and expression.Recombinant protein Cel619 was induced and expressed,the results showed the optimal induction conditions are: induction temperature of 16 ?,IPTG concentration of 0.8 mM.I obtained Cel619 and Cel800 pure enzyme components by affinity chromatography,molecular sizes of 66.5 kDa and 88 kDa respectively,specific activity was 78.25 U/mg and 145.3 U/mg respectively.Recombinase Cel619 and Cel800 optimum pH are 5.0 and 7.0 respectively,the residual activity of the enzyme at pH of 10.0 are more than 40%,indicating that the recombinant enzyme is alkalitolerant cellulase;in the range of pH 5.0-11.0 the remaining activity of the cellulase is more than 70%,indicating that the cellulase enzyme has good pH stability.The optimum temperature was 70 ? and 60 ?respectively.Both of these two enzymes have good thermal stability,it indicated that they may be thermal cellulase.Cel619 and Cel800 showed good tolerance for certain metal ions and chemical reagents,the appropriate concentration of Na⁺/K⁺ salt ions has promoted activity,and has good tolerance for salinity.Cel619 has the highest activity for soluble substrate CMC,it has certain activity for the insoluble substrate as microcrystalline.Cel619 of K_m is 11.33 mg/mL,V_max is 166.67 ?mol/min/mg.Cel800 has broad substrate specificity,it has good degrading activity for the soluble substrate CMC,xylan and laminarin,in which the highest activity is for CMC,it also has certain activity for the insoluble substrate as microcrystalline cellulose and paper.Cel800 of K_m is 9.5 mg/mL,V_max is 304.87 ?mol/min /mg.Because of the tolerance for high temperature,salinity and alkalinity,and the metal ions and organic reagents well tolerated of recombinase Cel619 and Cel800,it has broad application prospects in textile industry,washing industry and environmental protection.