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Study on Alkaline β-mannanase and Phytase Applying in Feed

Author: LuZhiQun
Tutor: WangZhengXiang
School: Jiangnan University
Course: Microbiology
Keywords: E. coli Bacillus subtilis Neurospora crassa Alkaline beta - mannanase Phytase Heterologous expression
CLC: S816
Type: Master's thesis
Year: 2006
Downloads: 138
Quote: 2
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Abstract


The beta-mannase widely exist in nature. Microbial beta-mannanase beta-1.4-D-mannan (β-1.4-D-mannan mannanohydrolase, EC.3.2.1.78) referred to a study relatively late enzymes. Ma Y, etc. [1] reported the alkalophilic Bacillus the alkaline mannanase enzyme N 16-5 (the Bacillus sp.N16-5) nucleic acid sequence and enzymatic properties. In the present study, the gene and the E. coli - B. subtilis shuttle inducible plasmid pDG148-STU constructed recombinant plasmid PDG-Man, and then the recombinant plasmids were introduced into E. coli (Escherichia coli) and Bacillus subtilis (Bacillus subtilis ) in E. coli JM109 by 0.8 mmol / L IPTG induced enzyme activity up to 4.5 U / mL. Under the same conditions, the expression of recombinant E. coli DE3-RIL (PDG-man) alkaline beta-mannanase enzyme levels close to E. coli JM109 (PDG-MAN) 2 times. Secretory expression of the gene in Bacillus subtilis. After culture in LB medium induced expression of 19.2 U / mL alkaline beta-mannanase. Alkaline beta-mannanase high expression in order to continue the test the Bacillus fermentation medium for enzyme production, the initial induction of the enzyme activity reached 29.32 U / mL, through the optimization of fermentation conditions, the recombinant Bacillus The Bacillus enzyme production levels have increased significantly, up to 110.02 U / mL. Experiments show that induction of expression of the recombinant Bacillus subtilis period of 5 days (4 days for the induction of enzyme production phase), and thereafter the amount of the enzyme is no longer increased, the enzyme activity remained stable, constant at a maximum level, not decrease after 2 days more than 5%. Glucose and soybean meal plus the amount of the enzyme activity in the enzyme production process, experimental results show that the best glucose plus the amount of 20 g / L, the best soybean meal plus 80 g / L. In addition the the shake flask fluid volume recombinant Bacillus Bacillus alkaline beta-mannanase impact test results show the highest level of enzyme activity in 500 mL shake flask volume of 50 mL. Recombinant Bacillus subtilis Fermentation demand for oxygen. Phytase, in the present study was subcloned phytase gene of Neurospora crassa (Neurospora crassa) (from recombinant the plasmid pPIC9K-phy), respectively recombinant plasmid pET-28a and PBL-WZX, constitute pBL -phy and PET-phy functional expression in E. coli without SDS-PAGE not be expressed bands.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > General Animal Science > Feed
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