Study On Production And Characteristics Of β-Mannanase From Bacillus Megaterium

The main content of this study included the flask fermentation and fermentor fermentation technics ofβ-Mannanase produced by Bacillus megaterium,its enzymatic characteristics and the type and ability of enzymolysis.Through the optimization of the flask fermentation technics,theβ-Mannanase activity reached 200,000 U/ml after Bacillus megaterium was cultivated in 500 ml flask containing 50 ml LB（OXOID,10μg/ml tetracycline was added） at 37℃on a rotary shaker at an agitation rate of 200 r/min for 24 hours.The initial pH,the amount of inoculation and the amount and addition time of inducement were 7.0,2%,0.75%and 1 hour after inoculation respectively.The LB medium of industrial production grade was used when scale-up cultivation in 50 L automated fermentor.The initial culture volume,temperature,fermentor pressure, pH,agitation rate were 35 L,37℃,0.04 MPa,6.8～7.0 and 120 r/min respectively.1 hour after inoculation,0.75%inducement was added.When the bacteria were in the logarithmic phase,the dissolved oxygen was kept at 30%～35%and 2 L A was added at some slow flow speed.The enzyme activity reached 220,000 U/ml after 20 hours in this condition.This fermentation technics also worked well in the 250 L fermentor.The broth was concentrated by the micro-filtration and ultrafiltration equipment.After concentration, the activity reached 1,450,000 U/ml,which reached the requirement of industrial enzyme preparation.β-Mannanase was purified through（NH₄）₂SO₄ precipitation,dialysis and centrifugal ultrafiltration.The preliminary studies indicated the molecular weight of theβ-Mannanase was about 39 KD;its optimal pH was 7.0 and the enzyme activity was stable between pH 6.0 and pH 10.0 relatively.So it belongs to the neutral or alkaline Mannanase family.Its optimal reaction temperature was 50℃,and the enzyme activity was stable below 50℃. Theβ-Mannanase was hardly influenced by mental ions.It can be enhanced by Ca²⁺ slightly,but can be inhibited by Mn²⁺ and Fe²⁺ distinctly.Theβ-Mannanase was proved to be an inducible endo-β-Mannanase by the experiment.After 6 hours,10%konjak powder could be hydrolyzed into oligosaccharides which were consisted of four monoses on average when 4000 units’ enzyme was added into one gram konjak powder.