Prokaryotic Expression Of The Cellulase Of Xanthomonas Campestris PV. Campestris And Its Immobilization

Cellulose is a linear glucose polymer in which the subunits arelinked through 1,4-β-glycosidic bonds. The cellulose polymers coalesceto produce microfibrils that have a crystalline structure. An estimated rateof cellulose synthesis is approximately 4 x 107 tons per year. For a longrange solution for problems of energy, chemicals, and food, cellulose isthe most promising renewable carbon source that is available in largequantities[1]. Cellulases are complex enzymes which can digestlignocellulose. They have been widely used in textile, printing and dyeing,energy, paper, washing, food, feed, wine and other industries. Thecellulases produced by Microorganism are effective way to digest andtransform cellulose.Cellulase is a kind of glycoside hydrolase, refers to the degradationenzyme for cellulose into glucose. Full hydrolysis of cellulose requiressynergistic action of three major types of enzymatic activity:1,4-β-D-endoglucanases (EC 3.1.2.4), 1,4-β-Dcellobiohydrolases (CBH;EC 3.1.2.91) andβ-glucosidases (EC 3.2.1.21). Among them, endoglucanase(endo-β-1, 4-D-glucanase EC 3.2.1.4 ) is most important incellulase system . It can hydrolysis internally in the cellulose chain, thenlong chain cellulose molecular become small molecular cellulose.This experiment studied the cellulase of Xanthomonas campestris pv.Campestris, which is a phytopathogenic bacterium. We get the highpurity cellulose, prove its hydrolytic activity, and study its immobilizedparameters. This work is helpful for our future work. The results showthat:1 cloning and expression of the cellulase geneWe design the specific primers for Xcc cellulase according to theNCBI, using PCR technique. According to the idea for libraryconstruction, using homologous primers to reduce costs. The gene wasligated into the expression vector pET28a applying CPEC(Circularpolymerase extension cloning ) method to construct a recombinatedplasmid pET28a-0639ΔSP, and then was transformed into the E. Colirosetta ( DE 3).The protein was successfully expressed by 0.4 mmol/ LIPTG ( Isopropyl-β-D -1-thiogalactopyranoside) induction, then purifiedby the affinity Ni-NTA. Its molecular weight was about 50KDa, whichdetermined by SDS-PAGE. 2 immobilization of enzymeThe protein and resin are firmly combined with covalent mode.Theprotein immobilization efficiency is up to 70%, retained activity is 60%.Immobilized protein has higher thermal stability. The resin has negativecharge itself, the optimum pH bias to the basic direction.3 analysis of the soluble and immobilized cellulaseThe activity of recombinant enzyme was 60U·mg-1. The optimumreaction temperature and pH value for soluble enzyme and insolubleenzyme were 53℃,62℃and 5.4,5.8,respectively. The results of theirenzymatic properties using sodium carboxymethylcellulose (CMC) as thesubstrate showed that their Vmax were 411umol·ml-1·h-1 and383umol·ml-1·h-1 , while Km were 0.2500% and 0.3125%, respectively.