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| Screening of Cellulolytic Microorganisms from the Reticulitermes chinensis and Identification of Cellulase Gene and Its Prokaryotic Expression |
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Abstract Abstract Termites (Isoptera) often eat wood rich in cellulose, and there are much bacteria producing cellulase in vivo. The aim of this study was to screen a strain of producing highly active cellulase from termites to amplify the cellulase gene of this strain and express it in Escherichia coli, which had a potential basis for the the construction of cellulase gene recombinant bacteria. Collecting termites samples from Foping County of Shaanxi province, this study screened 4 strains of producing cellulase from termites (Reticulitermes chinensis) in vivo by cellulase screening medium and measurements of cellulase activity; then optimized the fermentation conditions were conducted by single factor experiment on the strain which producing the highest active cellulase, and studied the enzymatic properties of cellulase. Finally, according to the data of NCBI primers which amplified the cellulase genes were designed and prokaryotic expression was completed. The results showed that the 4 cellulase producing strains screening from termites were Enterobacter asburiae, Bacillus cereus, Bacillus subtilis and Bacillus amyloliquefaciens, respectively. The cellulase activity of the Bacillus subtilis was the highest, and the optimum fermentation temperature and pH of the strain was 42 ℃ and 6.5. The optimum temperature and pH of enzymatic reaction was 70 ℃ and 4.5, under this condition, the cellulase activity reached 2.36 U/mL. Under the condition of 50 ℃ and pH 6.5, the thermal stability and pH stability of the cellulase produced by the strain were optimum. Using crude enzyme solution to ferment crude fiber, it was found that the degradation rate of crude enzyme solution to four kinds of feed, corn (Zea mays) stalk , wheat (Triticum aestivum) straw, alfalfa (Medicago polymorpha) hay and silage corn stalk was 12.21%, 1.30%, 3.24% and 17.42%, respectively. Gene cloning results showed that the Bacillus cereus strain contained two cellulase genes BaG and Cbs. Under prokaryotic expression of the cellulase gene, the proBaG didn't show any cellulase activity, while the proCbs showed the optimum conditions of 60 ℃, pH 5.0, cellulase activity reached 4.14 U/mL. Under the condition of 50 ℃ and pH 5.0, the thermal stability and pH stability of the cellulase were optimum. Finally, the fusion expression of BaG and Cbs gene showed a new protein of about 35 kD while BaG and Cbs gene expressed the protein with a molecular weight of 27.4 kD and 55.2 kD, respectively. In the optimum conditions of 70 ℃ and pH 4.5, the cellulase activity of the fusion product expression reached 4.57 U/mL. Under the condition of 50 ℃ and pH 4.5, the thermal stability and pH stability of the cellulase were optimum. This study indicated that the BaG gene without cellulase activity could interact with the Cbs gene and may produce a new protein with higher cellulase activity and smaller molecular weight. This study should have important theoretical value for the construction of recombinant bacteria which could degradate lignocellulose.
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Received: 29 November 2017
Published: 20 July 2018
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| Fund:No. CARS-39-12 |
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