Overexpression Of An Extracellular Hybrid β-1, 3-1, 4-glucanase By Recombinant E. Coli

β-1,3-1,4-glucanase is a very important industrial enzyme,whose major application is to eliminate the negative effect ofβ-glucan on brewing and feed industry.In the domestic market so far,the production ofβ-1,3-1,4-glucanase by screened natural strains can not meet the growing industrial need for this enzyme due to low enzyme activity,poor stability and less investigation on this enzyme.Progress in molecular biology and genetic engineering allows recombinant proteins such as enzymes to be produced in bacteria,i.e.,E.coli and other microorganisms in large-scale.Thus this study mainly focused on the construction and use of recombinant E.coli strains to enhance the extracellular production and quality of the enzyme.For this purpose, different cultivation technologies were used to improve the extracellular production ofβ-1,3-1,4-glucanase by recombinant E.coli for sustainable industrial application.The main research work includes:1.Fed-batch cultivation of recombinant E.coli JM109-pLF3 for the extracellular production ofβ-1,3-1,4-glucanaseThe fed-batch fermentation was carried out in a 7 L conventional bioreactor based on the optimized medium as well as optimized cultivation conditions.The results indicated that feeding two-fold concentrated nitrogen source of the optimized medium during the cultivation period of 12-32 h could significantly enhance the biomass and enzyme activity.The highest enzyme activity and biomass reached up to 1680 U/mL and 7.67 g/L,which was 2.31 and 2.44 folds higher than those obtained in the batch fermentation at the same cultivation conditions,respectively.2.Construction of recombinant E.coli for high level secreted expression ofβ-1,3-1,4-glucanaseThe thermo-stable hybrid bgl gene encodingβ-1,3-1,4-glucanase and kil-Km secretion cassette were inserted into the plasmid pET-22b(+),yielding pET-22-bgl-(kil-Km),a newβ-1,3-1,4-glucanase expression plasmid.The recombinant plasmid was then transformed into E.coli JM109 and BL21(DE3), respectively.The IPTG induction could not enhance the enzyme production.The ability of pET-22-bgl-(kil-Km) to express extracellular target protein with high level was proved by SDS-PAGE.3.Optimization of medium components by Response Surface Methodology for high level enzyme productionStarted with the TB medium,through one-factor-at-a-time experiments,lactose and casein peptone were found to be the most suitable carbon and nitrogen sources,for enzyme production by recombinant E.coli BL21(DE3)(pET-22-bgl-(kil-Km), respectively.The statistical analyses of the effect of the carbon and nitrogen source as well as C:N ratio in the medium on enzyme production,by employing RSM and the Box-Behnken design indicated that the optimized medium should contain(g/L): casein 33.39,lactose 8.03,glycerol 9.15,NaCl 10.0,KH₂PO₄ 2.31 and K₂HPO₄ 12.54. By shake flask cultivation of the recombinant cells on the optimized medium at a temperature of 37℃and a rotation speed of 150 rpm,the enzyme activity of 1242.94 U/mL was achieved after 32.5 h of incubation,which was approximately 3.3 times higher than that obtained on the TB medium at the same conditions.4.Purification of the recombinant enzyme and characterization of enzyme properties Affinity chromatography was applied to purify the recombinant protein with 6×his tags specific covalent with Ni²⁺ column.The recovery rate was 86.44%.The molecular weight of the target enzyme with 6×his tag was about 26 kDa,in consistent with SDS-PAGE result.The enzyme activity and specific enzyme activity reached 1706.2 U/mL and 11261.6 U/mg with a separation factor of 7.69 after purification, respectively.The optimum temperature and pH for enzyme activity was 50℃and 6.0 -7.0.The enzyme was more stable under base conditions.