芽孢杆菌产β-甘露聚糖酶的纯化及其稳定性研究
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摘要
β-甘露聚糖酶是一种重要的半纤维素酶,应用广泛,多采用微生物发酵制备。本文首先对发酵培养基进行简化与优化,并通过分离纯化制得高纯酶;进而运用圆二色、二级结构预测等方法,对酶的结构及稳定性进行研究,通过添加稳定化试剂并优化操作参数,提高了喷雾干燥产品的稳定性。主要工作如下:
对发酵培养基氮源、碳源及无机盐进行筛选优化,确定了简化发酵培养基组成;最终发酵液的单位酶活成本较原始发酵培养基降低63%,大大提高了生产收益,且色素及杂质的含量也明显低于原始培养基,有利于产品的后续精制。
β-甘露聚糖酶粗酶液经盐析、超滤、离子交换层析、凝胶层析及羟基磷灰石层析制得电泳纯酶蛋白。电泳分析和凝胶排阻色谱分析估算所得β-甘露聚糖酶分子量一致,约为39kDa。
基于确定的氨基酸序列,运用四种算法-APSSP2、SSPRO4、PSIPRED、PHD对β-甘露聚糖酶的二级结构及部分性质进行了预测和计算,得到疏水性标度值及滴定曲线,pI=5.505,分子量38005.4。
利用远紫外CD对比分析了β-甘露聚糖酶的二级结构和酶活随温度和pH的变化规律,初步探讨了二级结构变化对酶活力的影响,以优选出的CDSSTR算法计算出β-甘露聚糖酶二级结构含量为:α-螺旋42%,β-折叠13%,转角17%,无规卷曲28%,其三级结构类型为:α+β型。
考察了四种添加剂(NaCl、淀粉、蔗糖、糊精)在不同浓度下对喷雾干燥制备β-甘露聚糖酶稳定性的影响,结果表明:添加蔗糖和糊精可大幅提高酶的热稳定性,加入50 mg/ml糊精,酶活提高50%,效果最佳;通过响应面法优化,确定了喷雾干燥三项主要操作参数的最优值:加料流率4.3mL/min,干燥空气流率31.3L/h,空气进口温度177℃,此时酶活较优化前提高21.9%,达3715.9U/g。
β-Mannanase is an important hemicellulase, which is widely used and usually produced by microbial fermentation method. In this thesis, firstly, the fermentation medium was simplified and optimized, and the purified enzyme was obtained by separating and purifying. Then, the circular dichroism and the prediction of protein secondary structure were employed in investigating the structures and stabilization ofβ-mannanase. Finally, the stability of spray-drying product was improved by adding stabilizer and optimizing operating parameters. The main results were shown as following:
The nitrogen sources, carbon sources and inorganic salts of the fermentation medium were screened and optimized, the simplified fermentation medium was determined. The unit enzyme activity cost of final fermentative solution deceased about 63% comparing with that before optimization, which suggested that the benefit was largely increased. Meanwhile, the pigment and impurity contents of the fermentative solution were obviously lower than that before optimization, which is beneficial to the subsequent enzyme refining.
Theβ-mannanase of electrophoretic purity was prepared from crude enzyme by ammonium sulfate precipitation, UF, DEAE-52 sepharose, SephadexG-100, and hydroxyapatite chromatography. The molecular weight ofβ-mannanase estimated through GEC is consistent with that through SDS-PAGE, which is about 39kDa.
Four sequence analysis methods-APSSP2, SSPRO4, PSIPRED and PHD were used to predict the secondary structure and some properties ofβ-mannanase based on its amino acid sequence. The results indicated the pI ofβ-mannanase was 5.505 and the molecular weight was 38005.4. The hydrophobicity curve and titration curve were also predicted.
Through investigating changes in secondary structure and enzyme activity ofβ-mannanase at different temperature and pH by far UV circular dichroism, the effects of secondary structure on enzyme stabilization were preliminarily studied. The secondary structure relative contents ofβ-mannanase were calculated by the optimal CDSSTR method asα-helix 42%,β-sheet 13%, turns 17%, and random coil 28%. And the tertiary structure class ofβ-mannanase was also determined asα+β.
The effects of four additives (NaCl, sucrose, amylum, and dextrin) on the stability ofβ-mannanase during spray drying were discussed. The experimental results of the above additives with various concentrations were shown that the addition of sucrose and dextrin might largely enhance the thermal stability of enzyme during spray drying, and the maximum activity of beta-mannanase was increased about 50 % when dextrin was 50 mg/ml. Furthermore, through RSM, the optimal conditions of spray drying were calculated as follows: feed flow rate of 4.3 mL/min, heated air flow rate of 31.3 L/h, and inlet temperature of 177 oC. Under these conditions,β-mannanase activity of 3715.9 U/g was experimentally obtained, which increased about 21.9 % comparing with that before optimization.
对发酵培养基氮源、碳源及无机盐进行筛选优化,确定了简化发酵培养基组成;最终发酵液的单位酶活成本较原始发酵培养基降低63%,大大提高了生产收益,且色素及杂质的含量也明显低于原始培养基,有利于产品的后续精制。
β-甘露聚糖酶粗酶液经盐析、超滤、离子交换层析、凝胶层析及羟基磷灰石层析制得电泳纯酶蛋白。电泳分析和凝胶排阻色谱分析估算所得β-甘露聚糖酶分子量一致,约为39kDa。
基于确定的氨基酸序列,运用四种算法-APSSP2、SSPRO4、PSIPRED、PHD对β-甘露聚糖酶的二级结构及部分性质进行了预测和计算,得到疏水性标度值及滴定曲线,pI=5.505,分子量38005.4。
利用远紫外CD对比分析了β-甘露聚糖酶的二级结构和酶活随温度和pH的变化规律,初步探讨了二级结构变化对酶活力的影响,以优选出的CDSSTR算法计算出β-甘露聚糖酶二级结构含量为:α-螺旋42%,β-折叠13%,转角17%,无规卷曲28%,其三级结构类型为:α+β型。
考察了四种添加剂(NaCl、淀粉、蔗糖、糊精)在不同浓度下对喷雾干燥制备β-甘露聚糖酶稳定性的影响,结果表明:添加蔗糖和糊精可大幅提高酶的热稳定性,加入50 mg/ml糊精,酶活提高50%,效果最佳;通过响应面法优化,确定了喷雾干燥三项主要操作参数的最优值:加料流率4.3mL/min,干燥空气流率31.3L/h,空气进口温度177℃,此时酶活较优化前提高21.9%,达3715.9U/g。
β-Mannanase is an important hemicellulase, which is widely used and usually produced by microbial fermentation method. In this thesis, firstly, the fermentation medium was simplified and optimized, and the purified enzyme was obtained by separating and purifying. Then, the circular dichroism and the prediction of protein secondary structure were employed in investigating the structures and stabilization ofβ-mannanase. Finally, the stability of spray-drying product was improved by adding stabilizer and optimizing operating parameters. The main results were shown as following:
The nitrogen sources, carbon sources and inorganic salts of the fermentation medium were screened and optimized, the simplified fermentation medium was determined. The unit enzyme activity cost of final fermentative solution deceased about 63% comparing with that before optimization, which suggested that the benefit was largely increased. Meanwhile, the pigment and impurity contents of the fermentative solution were obviously lower than that before optimization, which is beneficial to the subsequent enzyme refining.
Theβ-mannanase of electrophoretic purity was prepared from crude enzyme by ammonium sulfate precipitation, UF, DEAE-52 sepharose, SephadexG-100, and hydroxyapatite chromatography. The molecular weight ofβ-mannanase estimated through GEC is consistent with that through SDS-PAGE, which is about 39kDa.
Four sequence analysis methods-APSSP2, SSPRO4, PSIPRED and PHD were used to predict the secondary structure and some properties ofβ-mannanase based on its amino acid sequence. The results indicated the pI ofβ-mannanase was 5.505 and the molecular weight was 38005.4. The hydrophobicity curve and titration curve were also predicted.
Through investigating changes in secondary structure and enzyme activity ofβ-mannanase at different temperature and pH by far UV circular dichroism, the effects of secondary structure on enzyme stabilization were preliminarily studied. The secondary structure relative contents ofβ-mannanase were calculated by the optimal CDSSTR method asα-helix 42%,β-sheet 13%, turns 17%, and random coil 28%. And the tertiary structure class ofβ-mannanase was also determined asα+β.
The effects of four additives (NaCl, sucrose, amylum, and dextrin) on the stability ofβ-mannanase during spray drying were discussed. The experimental results of the above additives with various concentrations were shown that the addition of sucrose and dextrin might largely enhance the thermal stability of enzyme during spray drying, and the maximum activity of beta-mannanase was increased about 50 % when dextrin was 50 mg/ml. Furthermore, through RSM, the optimal conditions of spray drying were calculated as follows: feed flow rate of 4.3 mL/min, heated air flow rate of 31.3 L/h, and inlet temperature of 177 oC. Under these conditions,β-mannanase activity of 3715.9 U/g was experimentally obtained, which increased about 21.9 % comparing with that before optimization.
引文
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