【作者】 李永仙；

【导师】 顾国贤；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 硕士

【摘要】 β-1,3-1,4-葡聚糖酶能够有效地降解植物和微生物细胞壁来源的β-葡聚糖,通过添加外源β-葡聚糖酶可有效解决啤酒酿造中麦汁过滤困难,啤酒的非生物稳定性降低等问题,在饲料工业中也可提高禽畜对麦类饲料养分的吸收效率。目前啤酒工业及饲料加工业中使用的β-葡聚糖酶制剂,主要是利用真菌或芽孢杆菌经传统通风发酵而生产。近年来,将外源β-葡聚糖酶基因通过合适的质粒载体克隆到大肠杆菌中进行表达得到了广泛的研究和应用。本研究室自行构建了重组大肠杆菌E.coli BL21（DE3）-pET28a（+）-bgl,该菌克隆了源自一株淀粉液化芽胞杆菌（Bacillus amyloliquefaciens）的高产β-1,3-1,4-葡聚糖酶基因,在乳糖或IPTG的诱导下可实现重组葡聚糖酶的表达。本文针对影响重组菌表达效果的因素（如菌体的生长状态、诱导剂的最优作用条件、培养基的组成等）对诱导表达条件进行了优化,并初步进行了7升规模的放大发酵实验。所得的主要结论如下:1.在25 mL起始pH为7.0的LB培养基中,接入10%（以0.35的OD值为分值）处于对数中期的二级种子液,37℃、200 r/min振荡培养至菌液OD值为1.0左右时加入诱导剂,此时β-葡聚糖酶相对酶活最高。2.采用正交实验的方法优化了在LB培养基中诱导剂的作用条件,得到的最佳诱导参数为:IPTG浓度0.0336 mmol/L,乳糖浓度10 mmol/L,诱导温度24℃,诱导时间6 h。发酵清液酶活达到336.33 U/mL,较优化前提高44.3%,该酶活是原始菌的4.85倍。3.利用Plackett-Burman design实验设计方法对初始培养基中各组分进行了主效应分析,其中的甘油、酵母粉、氯化钠为主效应因子。采用Box-Benhken实验设计,以及MATLAB7.0的响应面回归（RSREG）和NESS数据处理软件对实验结果进行分析,得到了三个主效应因子的最佳浓度及最佳培养基组成（g/L）:甘油18.9,酵母粉45.6,NaCl5.3,胰蛋白胨5.0,KH₂PO₄2.8,K₂HPO₄.3H₂O 4.8。最优浓度下酶活预测最大值为2053.1U/mL,菌种在优化后的培养基中发酵12 h,酶活最高达到了2311.81 U/mL,超过预测值,与优化前相比,酶活提高了23.04%。4.7升规模放大发酵13 h,发酵清液中的酶活最高为4189.58 U/mL,与三角瓶发酵最优结果相比提高了81.23%,该酶活是原始菌在最优条件下的酶活（853.33 U/mL）的4.91倍。以上结果不仅为重组菌的进一步放大发酵提供了可靠的参考依据,也预示了该重组菌有着较好的工业化应用前景。更多还原

【Abstract】 β-1,3-1,4-glucanase can hydrolyzeβ-glucan in the plant and microorganism cell wall efficiently.Lower wort’s filtration speed,worse beer non-biological stability in beer brewing and lower nutrition absorptivity in feed industry could be resolved effectively by adding exogenousβ-glucanase.Now,β-glucanase used in the beer industry and feed industry is mainly produced by fungi and Bacillus with aerobic fermentation.Cloning and expression of exogenousβ-glucanase gene in Escherichia coli with different plasmids have been studied widely recent years.The strain used in this paper is a recombinant Escherichia coli pET28a（+）-bgl,which cloned aβ-glucanase gene from a strain of Bacillus amyloliquefaciens prodeucingβ-1,3-1,4-glucanase efficiently.The recombinant starin could expressβ-glucanase by lactose and IPTG inducement.This paper studied the recombinant’s performance according to the factors affecting expression,exp,growth characteristic of recombinant strain, inducing condition and composition of culture medium.The amplificatory fermentation in 7 litres fermenter was also junior studied.The conclusions are as follows:1.Seed liquid(OD₆₀₀=0.35) was inoculated into 25 mL LB medium with initial pH 7.0 in 10%volume.It was added the inducement when it was incubated in 37℃and 200 r/min to OD₆₀₀=1.The activity of recombinantβ-glucanase would be highest.2.The optimal inducing condition in LB medium were ensured by orthogonal experiment. 0.0336 mmol/L IPTG and 10 mmol/mL lactose was added as inductor.Inducing for 6 h in 24℃,the highestβ-glucanase activity was 336.33 U/mL in supernatant liquor,which was higher than non-optimization 44.3%.The activity was 4.85 fold higher than original strain in same conditions.3.Main effect analysis for initial medium using Plackett-Burman design was performed.The main effect factors were glycerol,yeast extract and NaCl.Using Box-Benhken design and Response Surface Regression（RSREG） of MATLAB 7.0 and NESS for datas of experiment, the optimal composition of culture medium was as follows:18.9 g/L glycerol,45.6 g/L yeast extract,5.3 g/L NaCl,5.0 g/L peptone,2.8 g/L KH₂PO₄ and 4.8 g/L K₂HPO₄.3H₂O.The recombinant strain fermented in the optimal medium for 12 h,the total activity could reach 2311.81 U/mL,which was higher than the forecast 2053.1 U/ml.4.Amplificatory fermentation in 7 litres fermenter for 13 h,β-glucanase activity was 4189.58 U/mL in supernatant,which was higher than shaking flask 81.23%and 4.91 fold higher than original strain in optimal condition.This paper showed the recombinant strain had great applied value in industry and layed the foundation for amplificatory fermentation.更多还原