Studies On Breeding Of β-mannanase-overproducting Strain And Purification Of The Enzyme

In this study,80 strains producingβ-mannanase were firstly isolated from the soil samples taken from Nanchong konjac production base in Sichuan, by means of enrichment culture, separation and purification. From the 80 strains,10 strains showing higher enzyme activity were then screened by using the Congo-red transparent circle method. Finally, after flask-shaking fermentation of the 10 strains, a strain with the highestβ-mannanase activity up to 1594 U/mL was isolated by detecting crude enzyme activity using DNS method. The stain, named MM5, was identified as Bacillus subtilis through morphological observation and physiological biochemical tests as well as 16S rDNA sequence analysis.The strain MM5 was then used as an original strain, and was induced twice consecutively through UV mutagenesis. A mutant strain LD24H4 was obtained, of which the enzyme activity was increased to 3717 U/mL. Through single factor tests and orthogonal tests, it has been determined that the optimal culture medium components (g/L) of LD24H4 forβ-mannanase production were 20.0 g konjac powder,2.5 g casein,1.0 g NaCl,0.5 g MgSO4,0.5 g KH2PO4, and pH 7.5; the optimal culture conditions were 47℃,90 mL medium (in a 250 mL flask),1% inoculation volume and 200 r/min rotary speed. Under the optimal conditions, theβ-mannanase activity was increased to 12534 U/mL after fermentation for 26 h, which was 3.37 times higher than the original activity.Theβ-mannanase from Bacillus subtilis LD24H4 was purified by ammonium sulfate precipitation, DEAE-sepharose FF ion exchange chromatography and Sephadex G-75 gel filtration, and the purified enzyme presented as a single protein band on SDS-PAGE. The calculation results indicated that the molecular weight of theβ-mannanase was 32 kDa, the final yield was 2.9% and purification fold was 3.47.The enzymatic properties ofβ-mannanase produced by strain LD24H4 have been studied preliminarily. The results showed that the optimal reactive temperature was 50℃with enzyme activity keeping stable at 40℃; the optimal pH was 7.0, keeping most enzyme activity in pH 5.4 to 7.4; Co2+ and Cu2+ stimulated the enzyme activity, while Mn2+,K+, Mg2+, Na+, Li+, NH4+, Fe3+, Hg+, and Ca2+ inhibited it at different levels, and Zn2+ and Ba2+ had little inhibition effect on it. With konjac powder as substrate, its Km value was 0.05 mg/mL and its Vm value was 50000μmol/(min·mL), while with locust bean gum as substrate, its Km value was 0.4 mg/mL, and its Vm value was 50000μmol/(min·mL) respectively.