基因重组酒香酵母菌株高产β-葡萄糖苷酶的高密度液态发酵模型及酶学性质研究
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摘要
β-葡萄糖苷酶(EC3.2.1.21),又称β-D-葡萄糖苷水解酶,是组成纤维素酶系必不可少的成分。本研究对产β-葡萄糖苷酶菌株B.anomalus PSY-001分别进行紫外诱变和超声波诱变,从诱变菌株中筛选出高产β-葡萄糖苷酶的两亲株U1和V1,进行三次种内基因重组,以p-葡萄糖苷酶活力为指标,筛选出高产β-葡萄糖苷酶的新融合菌株F3-25,对F3-25的发酵条件进行优化,并建立该菌株的高密度液态发酵模型,采用AKTA purifier-900蛋白分离纯化工作站对该菌株生产的β-葡萄糖苷酶进行分离纯化并进行酶性质分析,为工业化生产提供了可靠的依据。
主要研究内容和结果如下：
1,以B.anomalus PSY-001为出发菌株,分别进行紫外诱变和超声波诱变,从诱变菌株中筛选出高产β-葡萄糖苷酶的菌株进行三次种内基因重组。通过初筛培养基筛选后,再以β-葡萄糖苷酶活力为指标,筛选出高产β-葡萄糖苷酶的新融合菌株F3-25。融合菌株F3-25产β-葡萄糖苷酶的酶活比原始菌株提高了近8倍。
2,运用Design-Expert软件对试验数据进行分析,得到融合菌株F3-25的最佳培养基组成：当麸皮浓度为53.15g/L,酵母粉浓度为3.03g/L,KCl浓度为0.204g/L,CaCl2浓度为0.611g/L时,β-葡萄糖苷酶的酶活最高,为4.794U/mL。
通过单因素试验和正交试验确定了融合菌株F3-25的最佳摇瓶发酵条件：发酵温度为30℃、250mL锥形瓶装液量为20mL、摇床转数为150r/min、接种量为3%、初始pH值为5。
3,对F3-25的补料分批发酵条件进行了优化。运用Orign8.0软件拟合得到融合菌株F3-25在该条件下的生长动力学模型为：
4,利用AKTA purifier-900蛋白分离纯化工作站,采用葡聚糖凝胶G-100层析,使β-葡萄糖苷酶的纯化率达到了96.2%,经SDS-PAGE电泳检测为电泳纯,得到两条带,测定其两个亚基的分子量分别为50.3KDa和67.8KDa,运用电泳分析软件BandScan4.3对电泳结果进行分析,得到其亚基的含量分别为40.5%和57.3%。
融合菌株F3-25发酵产生的β-葡萄糖苷酶,最适反应温度为60℃,在40℃～50℃温度条件下,具有较好的热稳定性；最适pH值为5.0,在pH为5.0～6.0范围内,具有较好的稳定性；Fe3+、Mn2+、Ca2+对β-葡萄糖苷酶酶活有明显的促进作用,而Mg2+、Al3+、Cu2+、Zn2+对β-葡萄糖苷酶酶活有抑制作用。
P-glucosidase(EC3.2.1.21), aslo known as p-D-glucosidase glucohydrolase, which is an essential ingredient of the composition of cellulase. In this study, the two parental strains U1and V1, which were carried out three times genome shuffling, were obtained using P-glucosidase enzyme activity as indicator from B.anomalus PSY-001with UV and ultrasonic as mutagent. By using the screening medium, and P-glucosidase enzyme activity as indicator, a fusion strain F3-25high-yield beta-glucosidase was attained. Optimize the fermentation conditions of the F3-25, and develop the strains of high-density liquid fermentation model. The purification of P-glucosidase produced by F3-25were investigated with AKTA purifier-900protein separation and purification workstation. The properties of β-glucosidase was aslo discussed. This paper provides a reliable basis for the industrial production.
Main contents and results of the study are as follows:
1, In this study, the strain producing β-glucosidase B.anomalus PSY-001was UV mutagenesis and ultrasonic mutagenesis, screened from the mutant strains yielding beta-glucosidase from the two parental strains U1and V1, which was carried out three times genome shuffling. By the screening of the screening medium, and then to the P-glucosidase enzyme activity as an indicator, filter out the high-yield P-glucosidase fusion strain F3-25. The strain F3-25produced P-glucosidase activity is nearly eight times larger than the original strain.
2, The optimum culture medium of fusion strain F3-25was optimized by Design Expert software, which were composed of:the bran concentration of53.15g/L, the concentration of yeast extract of3.03g/L, KC1of0.204g/L, CaCl2of0.611g/L.
Fusion strains of the F3-25flask fermentation conditions were determined by single factor and orthogonal experiments:the fermentation temperature of30℃,250mL conical bottled liquid volume of20mL, the shaker is150r/min inoculation3%, the initial pH of5.
3, The fed-batch fermentation conditions of F3-25were optimized. The growth kinetics model of fusion strain F3-25was obtained by Orign8.0software. The model is
4, The purification of P-glucosidase produced by F3-25were investigated with AKTA purifier-900protein separation and purification workstation using Sephadex G-100 gel filtration chromatography. β-glucosidase purification rate was reached96.2%. Electrophoretic purity of β-glucosidase was detected by SDS-PAGE electrophoresis. Two electrophoresis band were attained, which molecular mass were estimated to be50.3KDa and67.8KD respectively. The content of two subunits were40.5%and57.3%respectively anlalyzed by Bandscan.
The optimum temperature of F3-25was60℃. The optimal pH was5.0. Theβ-glucosidase has good stability within the range of40℃-50℃and the pH5.0-6.0. Fe3+、Mn2+and Ca2+can promote the activities of β-galactosidase while Mg2+、Al3+、Cu2+、 Zn2+can inhibit the activities of β-galactosidase.
主要研究内容和结果如下：
1,以B.anomalus PSY-001为出发菌株,分别进行紫外诱变和超声波诱变,从诱变菌株中筛选出高产β-葡萄糖苷酶的菌株进行三次种内基因重组。通过初筛培养基筛选后,再以β-葡萄糖苷酶活力为指标,筛选出高产β-葡萄糖苷酶的新融合菌株F3-25。融合菌株F3-25产β-葡萄糖苷酶的酶活比原始菌株提高了近8倍。
2,运用Design-Expert软件对试验数据进行分析,得到融合菌株F3-25的最佳培养基组成：当麸皮浓度为53.15g/L,酵母粉浓度为3.03g/L,KCl浓度为0.204g/L,CaCl2浓度为0.611g/L时,β-葡萄糖苷酶的酶活最高,为4.794U/mL。
通过单因素试验和正交试验确定了融合菌株F3-25的最佳摇瓶发酵条件：发酵温度为30℃、250mL锥形瓶装液量为20mL、摇床转数为150r/min、接种量为3%、初始pH值为5。
3,对F3-25的补料分批发酵条件进行了优化。运用Orign8.0软件拟合得到融合菌株F3-25在该条件下的生长动力学模型为：
4,利用AKTA purifier-900蛋白分离纯化工作站,采用葡聚糖凝胶G-100层析,使β-葡萄糖苷酶的纯化率达到了96.2%,经SDS-PAGE电泳检测为电泳纯,得到两条带,测定其两个亚基的分子量分别为50.3KDa和67.8KDa,运用电泳分析软件BandScan4.3对电泳结果进行分析,得到其亚基的含量分别为40.5%和57.3%。
融合菌株F3-25发酵产生的β-葡萄糖苷酶,最适反应温度为60℃,在40℃～50℃温度条件下,具有较好的热稳定性；最适pH值为5.0,在pH为5.0～6.0范围内,具有较好的稳定性；Fe3+、Mn2+、Ca2+对β-葡萄糖苷酶酶活有明显的促进作用,而Mg2+、Al3+、Cu2+、Zn2+对β-葡萄糖苷酶酶活有抑制作用。
P-glucosidase(EC3.2.1.21), aslo known as p-D-glucosidase glucohydrolase, which is an essential ingredient of the composition of cellulase. In this study, the two parental strains U1and V1, which were carried out three times genome shuffling, were obtained using P-glucosidase enzyme activity as indicator from B.anomalus PSY-001with UV and ultrasonic as mutagent. By using the screening medium, and P-glucosidase enzyme activity as indicator, a fusion strain F3-25high-yield beta-glucosidase was attained. Optimize the fermentation conditions of the F3-25, and develop the strains of high-density liquid fermentation model. The purification of P-glucosidase produced by F3-25were investigated with AKTA purifier-900protein separation and purification workstation. The properties of β-glucosidase was aslo discussed. This paper provides a reliable basis for the industrial production.
Main contents and results of the study are as follows:
1, In this study, the strain producing β-glucosidase B.anomalus PSY-001was UV mutagenesis and ultrasonic mutagenesis, screened from the mutant strains yielding beta-glucosidase from the two parental strains U1and V1, which was carried out three times genome shuffling. By the screening of the screening medium, and then to the P-glucosidase enzyme activity as an indicator, filter out the high-yield P-glucosidase fusion strain F3-25. The strain F3-25produced P-glucosidase activity is nearly eight times larger than the original strain.
2, The optimum culture medium of fusion strain F3-25was optimized by Design Expert software, which were composed of:the bran concentration of53.15g/L, the concentration of yeast extract of3.03g/L, KC1of0.204g/L, CaCl2of0.611g/L.
Fusion strains of the F3-25flask fermentation conditions were determined by single factor and orthogonal experiments:the fermentation temperature of30℃,250mL conical bottled liquid volume of20mL, the shaker is150r/min inoculation3%, the initial pH of5.
3, The fed-batch fermentation conditions of F3-25were optimized. The growth kinetics model of fusion strain F3-25was obtained by Orign8.0software. The model is
4, The purification of P-glucosidase produced by F3-25were investigated with AKTA purifier-900protein separation and purification workstation using Sephadex G-100 gel filtration chromatography. β-glucosidase purification rate was reached96.2%. Electrophoretic purity of β-glucosidase was detected by SDS-PAGE electrophoresis. Two electrophoresis band were attained, which molecular mass were estimated to be50.3KDa and67.8KD respectively. The content of two subunits were40.5%and57.3%respectively anlalyzed by Bandscan.
The optimum temperature of F3-25was60℃. The optimal pH was5.0. Theβ-glucosidase has good stability within the range of40℃-50℃and the pH5.0-6.0. Fe3+、Mn2+and Ca2+can promote the activities of β-galactosidase while Mg2+、Al3+、Cu2+、 Zn2+can inhibit the activities of β-galactosidase.
引文
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