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The Molecular Cloning and Overexpression of Bacillus Sp. Mx β-mannanase Gene in Escherichia Coli

Author: MaZuo
Tutor: LinMin
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: β-mannanase Bacillus sp. overexpression purification enzyme activity
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 60
Quote: 0
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Abstract


Hemicellulose is a widely used substance which accounts for from 1/4 to 1/3 of plants dry weight. Degradation and utilization of hemicellulose is of great significance in resolving energy crisis, food shortage and environmental pollution. Thus, finding a hemicellulosic hydrolase with high activity and tolerance to adversity seems crucial.β-mannanase is a important kind of hemicellulose hydrolases, which is ubiquitous in animal feed,food, medicine and widely used in paper making and textile industry. In this study, a Bacillus sp. strain with highβ-mannanase activity was isolated from an extreme environment in Xinjiang.Further efforts of expression optimization and enzyme identification were made in the E.coli.Three strains with highβ-mannanase activity were isolated from an extremely dry environment by comparing the size of hydrolyzed circle produced by each strain, which were further identified as belonging to Bacillus sp., Citrobacter sp. and Paenibacillus sp. respectively using 16S rDNA sequence analysis and colonial morphology methods. The Bacillus sp. strain with the biggest hydrolyzed circle, named Bacillus sp. MX, was selected for further research.Theβ-mannanase gene was cloned from Bacillus sp. MX chromosome and inserted into pET-28a, further transduction of this recombinant plasmid into an E.coli BL21 strain resulted in a transformant, named BL21 (pET-man).The target protein with a His-tag was expressed efficiently when BL21 (pET-man) was induced by IPTG at 37℃for 3 hours. SDS-PAGE analysis showed that most of theβ-mannanase expressed in the cytoplasm exists in the form of inclusion bodies.Different temperatures、IPTG concentrations and inducing time were tested for a higher yield of solubleβ-mannanase. The optimum condition found in this study was 0.5mM IPTG at 30℃for 5 hours. Purified fusion protein was obtained using BBST NTA Resin.The molecular mass of the purified protein determined by SDS-PAGE approximates to 43 kDa . The enzyme was identified to be active within the range of 25-95℃and pH3.0-9.6 using the dinitrosalicylic acid (DNS) method, with the optimum temperature at 55℃, pH at 5.0 (specific activity reached 4572 U/mg). Purifiedβ-mannanase remained 84%, or 33% and 14% of its initial activity, respectively, when expose in a pH 4.0 or 80℃or90℃condition for 10min, suggesting good acid resistance and thermo-stability.The gene ofβ-mannanase was cloned and inserted into the shuttle plasmid pPIC9K to develop a recombinant plasmid named pPIC9K-man. The plasmid pPIC9K-man was then digested with endonuclease BglII and integrated into pichia pastoris GS115 chromosome by PEG transformation. The fusionβ-mannanase expressed by the eukaryotic recombinant showed a highest specific activity of 345U/mg, which is lower than that expressed in the E.coli. Presumably, it’s because that the expression of the procaryotic man gene was reduced due to codon bias in the eukaryote system, or due to the reduction in copy number when man was integrated in the GS115 chromosome. Further efforts will be needed on codon optimization and improvement of the enzymic activity.

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