Cloning, Modification And Expression Of β-1,3-1,4-Glucanase Gene From Bacillus Licheniformis

β-1,3-1,4-glucanase (EC3.2.1.73), as an important industrial enzyme, has been widely used in thebrewing and animal feed industry. Nowadays the domestic researches aboutβ-1,3-1,4-glucanase is inthe beginning, the expression level and properties of natural enzyme are not sufficient for industrialpurpose, but the development of molecular biology and gene engineering provided a feasible way toresolve the problem. At present the researches aboutβ-1,3-1,4-glucanase in the country or abroadfocus on constructing gene engineering strains and improving the expression level and properties. Thepurpose of this research is to improve the expression level and properties ofβ-1,3-1,4-glucanase withthe methods of molecular biology and gene engineering. And it would be an effective approach foraccelerating the application ofβ-1,3-1,4-glucanase in industry. The studies and results aboutβ-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in this research are asfollows:1. The effectes of medium and fermentation conditions on the activity ofβ-1,3-1,4-glucanase produced by B. licheniformis EGW039Through optimization of carbon and nitrogen nutrition of the medium, it was founded that 4%complex carbon nutritions such as oat, bran are better than single nutrition such as glucose, sucrose,and 1% complex nitrogen nutrition is also better than inorganic and organic nitrogen. The naturalstrain yieldedβ-1,3-1,4-glucanase maximumly under the optimized medium and ferment conditions at150 rpm, pH5.0-6.0, 48 h.2. The cloning and expression ofβ-1,3-1,4-glucanase in Escherichia.coliThe gene encodingβ-1,3-1,4-glucanase is amplified by PCR with the template of genomic DNAof B. licheniformis EGW039, it is 648 bp, then cloned into expression vector pET28a(+), therecombinant vector is transformed into E. coli host strain BL21(DE3). The MW of recombinantβ-1,3-1,4-glucanase is about 28 KDa with 6xHis tag at the N terminal. The expression level ofrecombinantβ-1,3-1,4-glucanase is approximately 60.9% of the total protein, 12.5% of the solubleprotein in crude cell lysate supernatant. The recombinantβ-1,3-1,4-glucanase activity in cell lysatesupernatant is 1,286 and 986 U ml^-1 with l%(w/v) barleyβ-glucan and 1%(w/v) lichenan respectively.Accordingly, the specific activities are 2,479 and 1,906 U mg^-1 with these two substrates. With Ni²⁺chromatography purification the recombinant protein is concentrated by 8.74 times. The optimalacidity and temperature of this recombinant enzyme are pH 5.6 and 40℃, respectively.3. The gene modification and expression ofβ-1,3-1,4-glucanase in Pichiapastoris GSll5The gene encodingβ-1,3-1,4-glucanase is obtained by PCR with the template of genomic DNAof B. licheniformis EGW039, and modified with codon optimization. In which a total of 102nucleotides are changed, the G+C ratio is simultaneously decreased from 43.6% to 41.6%. Themodified and optimal gene(M-eg1314) is ligated into the expression vector pPIC9K, the recombinantvector pPIC9K-M-eg1314 after being linearized with Bglâ…¡is transformed into P. pastoris GSll5with electronic pulse, then the recombinant yeast is obtained through culturing on nutrition deficient medium. Theβ-1,3-1,4-glucanase is highly expressed secretly in P. pastoris GS 115. At shaking flasklevel, theβ-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml^-1 with 1%(w/v)barleyβ-glucan and1%(w/v)lichenan as substrate respectively, it is increased by about 15 times than unoptimal gene(4Uml^-1). At 7L laboratory fermentor level, the secreted protein concentration is approximately 250 mgL^-1, theβ-1,3-1,4-glucanase activity are 333.7 and 256.7 U ml^-1 with 1%(w/v)barleyβ-glucan and1%(w/v)lichenan as substrate, respectively, the specific activity are 1,334.8 and 1,026.8 U mg^-1 withthese two substrates, the most high expression level is improved about 50 times compared withoriginal host.The MW of recombinant enzyme is about 34 KDa with glycosylation, it is approximately 53.8%of the total secreted protein; The optimum acidity and temperature of this recombinant enzyme are pH6.0 and 45℃respectively. And the recombinant enzyme activity was activated by FeSO₄, COCl₂ andMnSO₄, but inhibited by CuSO4, EDTA,β-Mercaptoethanol and CaCl₂. The recombinantβ-1,3-1,4-glucanase has high substrate selectivity, and hydrolysate could promote the growth ofprobiotics such as lactobacillus. SDS-PAGE analysis suggested that modified recombinant proteinwas secreted successfully with high level in P. pastoris, and its thermostability was increasedobservably.