Cloning And Expression Of Bacillus Subtilis β-mannanase Gene

β-mannanase is an enzyme hydrolyzing the (3-1,4-D-mannopyanosyl linkages of mannan-oligosaccharides and mannan-polysaccharides such as galactomannan, glucomannan and galactoglucomannan. It belongs to the enzyme family of hemicellulase and has a broad application in textile and oil industry, pharmacy, feed and food-making, papermaking, dyeing and printing as well as in biological researchs.In this paper, an enzyme produce bacteria strain A33 of Bacillus subtilis which can hydrolysis the glucomannan of Amorphophallus was used as material.Itsβ-mannanase gene that is responsible for the hydrolyze enzyme was cloned by degenerate primer combined PCR and designated it as manA. Sequencing analysis showed the sequence was 1083bp in length, with a partial ORF from neuclotide 1 to 1083, and it coded a 360 amino acid polypeptide. The sequence was submitted to GenBank and the accession number was DQ269473. Furthermore, this gene and its deduced protein were predicted and analyzed by online bioinformatics database and softwares.Based on the sequence of Bacillus subtilis A33β-mannanase gene and multi-clone site of E.coli expression vector pRSET-A and pQE-30, several designed primers were used to amplify theβ-mannanase gene to introduce the desired restriction site by PCR. These fragment were inserted into the cloning site of vector pRSET-A with T7 promoter and pQE-30 with T5 promoter by respectively. The recombined expression vectors pRSET-A-manA and pQE-30-manA were screened and transformed into E. coli Host strain BL21(DE3) and the P-mannanase gene was expressed successful after induction with IPTG. Experiments are set to study the optimize condition of induction. It showed the induction temperature, induction time and induction concentration of IPTG had effect onβ-mannanase expression. The biological characteristics of engineered P-mannanase were analyzed. It is discovered thatβ-mannanase activity is higher in pRSET-A than in pQE-30 vector when test the enzyme in fermentation supernatant. This research result preliminary indicated that pQE-30 vector is not suitable for Bacillus subtilis A33β-mannanase gene fusion expression in the host.Since fusion expression protein contains some amino acid residues from the expression vector tag in N terminus, this probably results enzymatic activity decrease or completely lost due to the conformation changes. In this research, we constructed a non-fusion expression vector pET-32a-manA by excising the His-tag sequences of pET-32a, and expressed in strain E. coli BL21(DE3). But enzyme activity was declined in different hosts. In order to improve the stability ofβ-mannanase and achieve better enzymatic characters, we generated a substitution ofβ-mannanase gene using a PCR-based site-directed mutagenesis. Recombinant plasmid pET-32a-manA* was transformed into strain E. coli BL21(DE3). Compared to normal non-fusion expressionβ-mannanas, the biological activity ofβ-mannanase with substitution of leucine to valine in the second amino acid was higher than the others indicating that this substitution increase the enzyme stability and greatly improveβ-mannanase expression and activity.