关键词: 野油菜黄单胞杆菌, 内切葡聚糖酶, 原核表达系统, 固定化酶

Abstract: We cloned the cellulase gene of Xanthomonas campestris pv. Campestris with deleted signal peptide, using PCR (polymerase chain reaction) technique. The gene was ligated into the expression vector pET28a applying CPEC(circular polymerase extension cloning)method to construct a recombinated plasmid pET28a-engXCAΔSP, and then was transformed into the E. Coli rosetta (DE 3).The protein was successfully expressed by 0.4 mmol·L-1 IPTG(isopropyl-βD-1-thiogalactopyranoside) induction, then purified by the affinity NiNTA. Its molecular weight was about 50 kD, which was determined by SDS-PAGE. The activity of recombinant enzyme was 60 U·mg-1. We immobilized the enzyme as well. The optimum reaction temperature and pH value for soluble enzyme and insoluble enzyme were 53℃, 62℃ and 5.4, 5.8,respectively. The results of their enzymatic properties using sodium carboxymethylcellulose (CMC) as the substrate showed that their Vmax were 411 μmol·mL-1·h-1 and 383 μmol·mL-1·h-1 ,while Km were 0.2500% and 0.3125%, respectively.

Key words: Xanthomonas campestris pv. Campestris, endo-β-1, 4-D-glucanase EC 3-2-1-4, prokaryotic expression, immobilized enzyme