Cloning, Expression And Function Of β-mannanase Gene From Bacillus Licheniformis W10

β-mannanase, a kind of endo-hydrolases belonging to hemicellulases, can catalyze the hydrolysis of theβ-1, 4-D-glycosidic linkages in mannan. Mannose-oligosaccharides are carbohydrates that consist of 2-10 glucose and mannose. Being a new elicitor, it can activate plant defense system and induce system acquired resistance. Bacillus licheniformis W10, a highly producingβ-mannanase strain, was screened in our laboratory by inspecting enzyme activity.The conserved region of about 750bp in theβ-mannanase gene was obtained from the strain W10 genome by using polymerase chain reaction (PCR). The fragment showed a strong similarity (99%) to that of B. subtilis A33 when blasted in GenBank. A pair of primers was designed to amplify the complete sequence according to the open reading frame of A33. After being cloned and sequenced, the man gene consisted of a 1017 nucleotides encoding a protein of 338 amino acids with a molecular weight of 39.5kD, which was classified into glycoside hydrolase family 26.The man gene was constructed in an expression vector pET-22b (+) with pelB signal peptide, resulted in plasmid pETW10, and expressed in E. coli BL21 (DE3) by IPTG induction. The optimal conditions forβ-mannanase production were studied. The results showed that a final enzyme activity of 19.73 U/ml was detected when the E. coli BL21 containing pETW10 was cultured in SOC medium at 37℃to the A600nm of 0.75 and then induced with 0.25mM IPTG for 8h at 28°C.After the recombinant mannanase was purified from E. coli by affinity chromatography of nickel, its qualities were studied in this work. The enzyme activity was stable in the range of pH 6.0-7.5 and 40-60°C, and the maximum occured at pH 7.0 and 55°C,. The enzyme activity was not significantly affected by most of ions, but it was strongly inhibited by Ag+, Mn7+, EDTA, SDS, and activated by DTT.The hydrolyzed products of the mannanase were sprayed on tomato leaves and could reduce the incidence of lesions infected by Botrytis cinerea. The disease index of the treated was significantly lower than that of the control, with the control effect of 34.6% and 31.4%, which showed that the enzymatic products could induce the defense activity in tomato plants and be regarded as a potential elicitor.