Cloning and Site-directed Mutation of manA Gene from Bacillus subtilis

Abstract:

manA is a gene that encode β-mannanase (β-1,4-mannan mannohydrolase EC 3.2.1.78). ManA gene isolated from Bacillus subtilis strain A33 was cloned into an E.coli expression vector pET-32a and be transformed into E. coli strain BL21(DE3). An expression activity 41.58 U/ml was obtained after inducing. To get a better expression level that a site-directed mutation based on PCR was used to induce a mutant β-mannanase gene. The second code of the gene CUU was changed into GUU thus the second amino acid of β-mannanase of N-terminus changed from leucine to valine. The constructed mutated gene vector pET-32a-manA^* was transformed into strain E. coli BL21(DE3) and induced. The expression activity is increased to 138.65U/ml. It is predicted that a single change of amino acid enhancing the stability of expressed β-mannanase and greatly improve the expression level. The optimum temperature and pH of the enzyme is not changed observably.