Gene Cloning Of Man_B And Expression In E.coli And B.subtilis

Mannan can be hydrolyzed into mannooligosaccharide by mannanase; mannose could be obtained when mannanase worked with other glycoside hydrolases. Mannanase has been widely used in feed, pharmaceutical, food, paper and unglued industry.We obtained a Bacillus pumilus strain Nsic-2 from the stinky tofu brine and found that the strain could secrete an enzyme which played a key role in the mannan metabolic process. The enzyme gene named manB was obtained by degenerate PCR, gene walking and nest PCR. It has a ORF of 1104bp, and codes for a polypeptide of 367 amino acids. The deduced amino acid sequence showed highest identity with the beta-mannanase from Bacillus pumilus strain CCAM080065 (NCBI database) and belongs to glycoside hydrolase family 26.Then manB was cloned into three E. coli expression vectors (pET-28a, pET-32a and pET-42a), and transformed into E. coli BL21 (DE3). The highest enzyme activity measured was 11021.3U/ml, which was the highest reported mannanase activity from Bacillus sp.. Pure enzyme was obtained by affinity chromatography and showed a single band at about 41kDa by SDS-PAGE analysis.Furthermore, we studied the enzyme properties. It has a optimal temperature of 30℃ and was stable at 30-40℃.The highest activity was showed at pH 6.0. Compared to other mannanase, mans showed much higher stability under alkaline conditions and with highly stable at pH 6.0-9.0, which made it have a advantage when applied in the paper industry. Ca2+ and DTT could obviously enhanced the enzyme activity. The recombinant enzyme could strongly resistant to neutral protease. The Km and Vmax of it using LBG as the substrate were 35.66 mg/ml and 14.86μmol/(ml-min), respectively.We also studied the expression of mannanase in GRAS organism B. subtilis. mans was cloned into two B. subtilis expression vectors (pHT43 and pBNS2), and transformed into B.subtilis WB800N and IA751 respectively. The enzyme activities measured were 389.3U/ml and 7.11U/ml.