【作者】 刘征；

【导师】 卢英华；

【作者基本信息】 厦门大学 ， 生物化工， 2008， 硕士

【摘要】 β-葡聚糖酶是重要的工业用酶,可有效消除谷物β-葡聚糖在酿造和饲料工业中产生的负面影响。国内对β-1,3-1,4-葡聚糖酶的研究还处于初级阶段,天然菌株的产酶量及酶的性能满足不了实际应用。然而,分子生物学、基因工程方法的应用为其提供了契机。现在对β-1,3-1,4-葡聚糖酶的研究主要集中在构建基因工程菌株来提高酶的产量和性能方面。本研究应用工业生物技术进行了一系列的探索,旨在找到一种可持续发展的,可用于产业化、规模化的生产β-1,3-1,4-葡聚糖酶的方法。主要研究内容包括:1.发酵罐流加培养重组大肠杆菌JMl09-pLF3生产β-1,3-1,4-葡聚糖酶在原有优化培养基基础上,优化了发酵工艺,在7 L发酵罐上进行了分批补料发酵产酶研究。结果表明,在12～32 h之间向发酵罐内恒速流加双倍浓缩的培养基氮源,对细胞生长和产酶有极大的促进作用。最大酶活增长到1680 U/mL,比间歇发酵提高了231.40%;最大细胞密度为7.67 g/L,是间歇发酵3.44倍。2.高效分泌表达β-1,3-1,4-葡聚糖酶重组大肠杆菌的构建将热稳定性较好的杂合β-1,3-1,4-葡聚糖酶表达基因（bgl）插入质粒pET-22b（+）,并插入kil-Km分泌盒,得到了新的β-葡聚糖酶表达质粒pET-22-bgl-（kil-Km）。将重组质粒转化大肠杆菌E coli JM109和BL21（DE3）。通过SDS-PAGE证明了含有质粒pET-22-bgl-（kil-Km）的重组菌株具有目的蛋白的高效分泌表达的功能。3.响应面法优化重组E.coli BL21（DE3）-pET-22-bgl-（kil-Km）产酶培养基在TB培养基基础上,通过单因素实验选择乳糖为最佳碳源,酪蛋白胨为最佳氮源。在单因素优化基础上采用Box-Behnken设计法和RSM响应面分析法考察主要影响因素乳糖浓度、酪蛋白胨浓度和碳氮比三因素的的交互作用。确定了重组E.coli BL21（DE3）-pET-22-bgl-（kil-Km）产酶的优化培养基配方为（g/L）:酪蛋白胨33.39,乳糖8.03,甘油9.15,NaCl 10.0,KH₂PO₄ 2.31,K₂HPO₄ 12.54。利用优化培养基在37℃、150 rpm条件下,摇瓶培养32.5 h,酶活达到1242.94 U/mL,比初始培养基提高了3.3倍。4.重组酶的分离纯化和酶学性质表征利用Ni²⁺与6×His标签特异性结合的性质利用亲和层析纯化重组酶,该方法将目的蛋白纯化了7.69倍,纯化后重组酶的活力为1706.2 U/mL,比活11261.6U/mg,酶活回收率达到86.44%。通过SDS-PAGE分析,可知带有6个His标签的β-葡聚糖酶分子量为26 kDa左右。重组β-1,3-1,4-葡聚糖酶的酶学性质为:最适反应温度和耐热温度均为50℃,最适反应pH在6.0～7.0之间,碱性条件下稳定性较高。更多还原

【Abstract】 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.更多还原