Construction, Alteration Of Acid-resistance And Optimization Of β-mannanase Production By Recombinant Bacillus Subtilis

β-l,4-D-mannanase (EC3.2.1.78) is a type of endo-hydrolase which can hydrolyzes theβ-l,4-D-mannopyranosy linkages within the backbone of various mannan-based polysaccharides. The strain Bacillus subtilis B10-02 producingβ-mannanase was screened, then the gene encoding matureβ-mannanase from B10-02 was amplified and expressed in B. subtilis 168. Recombinant strain pMA5-manA2a/B. subtilis 168 was constructed which can highly express theβ-mannanase. Then the enzyme was purified successfully by Ni affinity chromatography and enzymatic properties were studied. In order to improve the pH stability of theβ-mannanase, theβ-mannanase gene was mutated by site-directed mutagenesis using overlap extension PCR technique. Recombinant strain pMA5-gmuGb54/B. subtilis 168 was obtained. Then fermentation conditions of recombinant strain pMA5-gmuGb54/B. subtilis 168 were studied. The main contents and results were as follows.(1) A strain B10-02 producingβ-mannanase was screened, and the activity of theβ-mannanase is 17.5 U/ml. Then the B10-02 was identified as B. subtilis based on the results of 16s rRNA identification and physiological biochemical identification.(2)Then the plasmids pMA5-manA1(excluding the signal peptide) and pMA5-manA2 (containing the signal peptide) were constructed and transformed into B. subtilis 168, respectively, then the recombinant strains pMA5-manA1/B. subtilis 168 and pMA5-manA2/B. subtilis 168 were obtained, the enzyme activity of strain pMA5-manA2/B. subtilis 168 (236.53 U/mL) was 9.53-fold higher than that of pMA5-manA1/B. subtilis 168 (24.83 U/mL). Theβ-mannanase gene manA2a was cloned with His-Tag added downstream of the gene manA2, then the recombinant strain pMA5-manA2a/B. subtilis 168 was obtained.(3) The enzyme MANA2a was successfully purified by Ni affinity chromatography. And enzymatic properties were studied. The results showed that the optimum activity of theβ-mannanase was obtained at pH 6.5 and 65 oC.(4) In order to improve the pH stability ofβ-mannanase, theβ-mannanase gene was mutated by site-directed mutagenesis using overlap extension PCR technique. Recombinant strain pMA5-gmuGb54/B. subtilis 168 was obtained, and the mutantedβ-mannanase showed a shift in optimal pH from 6.5 to 5.5.(5) Then fermentation conditions of the recombinant strain pMA5-gmuGb54/B. subtilis 168 was studied. The activity of theβ-mannanase reached 2207.82 U/mL in a 5 L fermentor which was 1.47-fold higher than that in Shake-flask culture(1501 U/mL).