A Method For Quick Screening And Cloning For Novel Bacillus β-mannanase Genes And Research Of Expression

p-Mannanase is an endo-wise glycosyl hydrolase catalyzes random hydrolysis of β-1,4-mannosidic linkages in the main chain of mannan and it is a kind of hemicellulases. P-Mannanase is produced mainly by microorganism, plants and lower animal. Mannanase has wide commercial applications in industry processes such as paper and pulp, foodstuff, feed, pharmaceutical and oil industry, p-mannanases produced by microorganisms is more promising due to low cost, high production rate, and readily controlled conditions.As an important source producing Mannanase, Bacillus plays a significant role in industrial application. A pair of degenerate primers was designed through homology analysis of the nucleotide sequence. A quick screening for novel gene coding for p-mannanase has been performed with PCR and the candidate novel genes encoding for p-mannanase can be deduced by sequence analysis. Among 16 gene fragments resulted from Bacillus, 14 fragments show high sequence homology to the genes of P-mannanase published while 2 are predicted as novel one. Three complete gene sequences were obtained by method of inverse PCR (IPCR) and named as manB48, manB49 and manB36 respectively. manB48, manB49 and manB36 have been registered in Genbank under the accession number of AY907668, AY913796 and DQ351940. The highest homology of manB48 and manB49 with other genes coding P-mannanase are 62.3% and 62.4% whereas that of DQ351940 is relative high. The result confirms the validity of the method of gene quick screening.The gene manB48 from Bacillus circulans was expressed in E. coli and P. pastoris. The recombinant protein expressed in E. coli was purified using chelating chromatograph. The optimum temperature and pH for activity of the mannanase MANB48E-H is 58°C and pH 7.6, respectively. The enzyme displays 75% of the initial activity in pH 6.8—9.0 and the specific activity of MANB48E-H is 481.55 U/mg.The gene manB49 from Bacillus circulans was positively expressed in E. coli and P. pastoris too. The recombinant protein in E. coli was purified using chelating chromatograph. The optimum temperature and pH for activity of the mannanase MANB49E-H is 60°C and pH 7.6, respectively. The enzyme is stable between pH 6.0 to 10.0 and displays 28.6% of the initial activity in pH 11.0 and the specific activity of MANB49E-H is 2276.81 U/mg, which is 4.8 times of that of MANB48E-H.The mannanase MANB36 was purified to homogeneity from the culture supernatant of Bacillus subtilis B36 and characterized. The optimum temperature and pH for activity of MANB36 is 50°C andpH 6.4, respectively. The enzyme displays 90% of the initial activity after incubating at 60 °C for 1 h. The specific activity of MANB36 is 927.84 U/mg and metal ions (except Hg2+ and Ag+), EDTA and 2-mercaptoethanol (2-ME) have no effects on enzyme activity.ManB36 was positively expressed in E. coli and P. pastoris. The recombinant protein in P. pastoris was purified. The result indicated that the optimum temperature and pH for activity of MANB36P is 42°C and pH 6.2. Activities of MANB36P in thermal and acid stability are lower than that of original enzyme.A method for quick screening novel gene encoding for P-mannanase has been established in this study. Three p-mannanase genes were obtained using this method and studied. The present work provides a novel idea for p-mannanase research.