Studies On The Optimization, Purification And Characterization Of β-mannanase From Bacillus Subtilis

P-mannanase is an enzymes capable of hydrolyzing hemicellulose including mannan linkaged ofβ-1,4-D-manno-pyanosyl. In recent years, with the deepening of research,β-mannanase is concerned in many fields and widely used in feed industry:foodstuff manufacture, paper industry, biological degumming, oil fracturing and molecular biology.The main research includes optimizing the culture conditions of the fermentation, purifying theβ-manannase produced by Bacillus subtilis DY-14 and finding out the main enzymatic properties by a preliminary study of the purified enzyme. The results showed that:(1) Bacillus subtilis DY-14 was selected as the target strain forβ-mannanase production after comparison of Congo red staining in selecting culture plate and P-mannanase activity in fermented liquid. DY-14 shows the highest activity around the available series strain from microbe laboratory of Northeast Forestry University.(2) Single factor and orthogonal array design tests were performed to obtain the optimum fermentation conditions for Bacillus subtilis DY-14, The results showed that DY-14 grew best when cultured at the inoculum volume of 1% in 10mL medium per 100mL flask and with the medium composed of 3% konjac powder as source,1.5% peptone as nitrogen source, pH 8.0. After 24 hours culturing at 37℃, the enzyme could show the highestβ-mannanase activity as 3216 u/mL that 3.29 times before the experiment.(3) Pureβ-mannanase from Bacillus subtilis DY-14 was obtained after purifying with DEAE-Sepharose FF anion-exchange chromatography and Sephadex G-75 gel filtration. The purification fold was 15.6 and recovery of total laccase activity was 20.98%.(4) Studied on enzymatic properties of purifiedβ-mannanase from Bacillus subtilis DY-14, the results showed that:the optimized pH is 6.0 and more than 80% activity can be maintained with pH6.0-7.5 below 37℃(2h). The optimum temperature is 55℃while the activity could be in a rapid loss above 60℃and almost be zero at 70 C.β-mannanase is activated by Various cations especially Mn2+, while inhibited by the same concentration of Ag2+...