Study On Alkaline β-mannanase And Phytase Applying In Feed

β-mannanase is widely existed in nature.β-mannanase from microorganisms which is known asβ-1.4-D-mannan mannanohydrolase (EC.3.2.1.78 )for short is investigated relatively late. The nucleic acid sequence and enzyme character of Alkalineβ-mannanase which is from Alkaliphilic Bacillus sp. N 16-5 was investigated by Ma Y et.al. In this research, the alkalineβ-mannanase gene from alkaliphilic Bacillus sp. N16-5 was subcloned into plasmid pDG148-Stu yielding hybrid pDG-man. The Escherichia coli JM109 harboring pDG-man was functionally expressed the alkalineβ-mannanase with the enzyme activity of 4.5 U/ml after induced with 0.8 mmol/L IPTG. Subsequently, E. coli DE3-RIL and B. subtilis WB700 were used as host cells. E. coli DE3-RIL harboring pDG-man expressed nearly a double enzyme of that E. coli JM109 did. B. subtilis WB600 harboring pDG-man expressed 19.2U/mL of the enzyme.To express Alkalineβ-mannanase highly in B. subtilis,we investigated expression using zymotic medium. 29.32 U/mL enzyme activity can be detected in medium after fermentation. By optimizing the zymotic medium and fermentation conditions, recombination B. subtilis can express as high as 110.02 U/mL of Alkalineβ-mannanase。Cycle of fermentation needs 5 days(4 days is needed in induced expression ) , then enzyme activity maintained at the highest level, less than 5 % enzyme activity was lost in next 2 days. Glucose and bean cake powder are important in enzyme fermentation. Research showed that the best concentration of glucose is 20 g/L and the best concentration of bean cake powder is 80g/L. Another important factor of fermentation is charge of flask, results showed that when 50mL zymotic medium is charged in 500 mL flask, the highest enzyme activity can be got by fermentation which tell us that high concentration of oxygen is needed in Alkalineβ-mannanase fermentation.About phytase, in research, phytase from Neurospora crassa was subcloned from recombination plasmid pPIC9K-phy, then recombination plasmid pBL-phy and pET-phy was constructed.After transformed into E. coli JM109, no enzyme activity was detected, no band can be found in SDS-PAGE.