β-葡萄糖苷酶的固定化及应用研究
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摘要
本实验对多孔陶瓷球、介孔TiO_2（M-TiO_2）和巯丙基官能化介孔TiO_2（SH-M-TiO_2）这三种载体固定化β-葡萄糖苷酶的技术进行研究，初步考察了陶瓷球固定化酶填充床反应器的酶解工艺和模型。得出的主要结论如下：
1、以多孔陶瓷球为载体，采用吸附交联—包封的方法固定化β-葡萄糖苷酶，I-a型陶瓷球固定化表观酶活最大约为1.75U/g，从单因素实验还可以看出，戊二醛交联和胶粘剂包封的步骤在陶瓷球固定化酶的操作过程中起关键作用。固定化酶在酸碱、热、储存稳定性方面较游离酶有明显改善；且重复测定酶活30批后，残余酶活仍在80%以上。在重复分批酶解实验中，前三批底物的转化率在90%以上，随后转化率逐渐下降，第九批的转化率降到50%左右，反应10批后固定化酶的残余酶活下降到68.5%。
2、采用连续水解的模式，将陶瓷球固定化酶装填成一单级填充床反应器，反应器的最佳工作温度和pH分别是60℃、pH4.8。底物进料流速低时，转化率高，但是体积生产效率低下；如流速为3.53ml/min时，转化率可达97.5%，而体积生产效率仅为6.29g/(l·h)，当流速增大到11.2ml/min时，转化率仅为82.1%，而体积生产效率高达15.62g/(l·h)。进料速度5.47ml/min时，反应器连续水解十天，转化率保持在90%以上，平均转化率为90.7%。建立了描述填充床的一维稳态轴向扩散模型，以STATISTICA软件对保留时间和转化率关系进行拟合，拟合因子R为0.95，模型计算结果与实验结果的平均偏差为9.15%。
3、由陶瓷球固定化酶二级串联反应器的实验可知，流速为7.67ml/min时，反应器转化率达91.4%，体积生产效率为12.69g/(l·h)，对比单级反应器转化率为91.7%时的体积生产效率为8.98g/(l·h)，二级串联反应器的体积生产效率比单级反应器提高了41%。
4、M-TiO_2吸附法固定β-葡萄糖苷酶（BG-M-TiO_2）的最大表观酶活回收率为64.14%，对应的表观酶活为4.68U/g；当起始给酶量为0.907U/ml时，BG-M-TiO_2的表观酶活力接近最大值，约为7.6U/g。M-TiO_2固载酶后N2吸附量略有下降，比表面积（SBET）从62.04m~2/g降低到47.72m~2/g，孔容（Vp）从0.37cm~3/g降低到0.29cm~3/g。BG-M-TiO_2重复测定酶活6批后，残余酶活降到50%以下。
5、分别以3-氨丙基三乙氧基硅烷（-NH2改性）和3-巯丙基三甲氧基硅烷（-SH改性）两种偶联剂，对M-TiO_2进行后嫁接法官能化改性，BG-SH-M-TiO_2在酶活、酶活回收率和重复分批测定酶活稳定性中的结果均优于BG-NH2-M-TiO_2。-SH改性后的M-TiO_2SBET从62.04m~2/g降低到53.50m~2/g，孔径（DBJH）从20.3nm下降到18.2nm，Vp从0.37cm~3/g降低到0.31cm~3/g；SH-M-TiO_2能谱图新增了Si和S元素的特征吸收峰，质量分数分别是1.78%和1.81%，巯丙基占的总质量分数约为3.6%；SH-M-TiO_2的热重曲线在355℃出现了来自巯丙基官能团的失重峰，质量损失约为2%。
6、BG-SH-M-TiO_2固定化酶的表观酶活回收率最高为92.84%，对应的表观酶活为10.99U/g，当起始给酶量为18U/ml时，BG-SH-M-TiO_2的表观酶活接近最大值为21.3U/g。BG-SH-M-TiO_2重复测定酶活30批后，残余酶活仍在50%左右。在重复分批酶解实验中，前八批对纤维二糖的转化率都在90%以上，反应10批后，BG-SH-M-TiO_2的残余酶活为反应前的80.5%。
There were three carriers, porous ceramic balls, mesoporous TiO_2(M-TiO_2),and mercapto-modified mesoporous TiO_2(SH-M-TiO_2), which were studied on theimmobilization of the enzyme β-glucosidase (BG). In additional, the hydrolysisprocess and model of packed-bed reactor, which was loaded with β-glucosidaseimmobilized onto the porous ceramic balls, were investigated. The main conclusionsare as follows:
1. The β-glucosidase was immobilized onto the porous ceramic ball of type I-a bythe crossing-linking and enveloping operation. The maximum apparent activity ofimmobilized β-glucosidase was about1.75U/g. It was known that two operationsplayed key roles to the activity of the immobilized enzyme. One was crossing-linkedwith the glutaraldehyde, the other was enveloped with a goil-based adhesive. Theimmobilized β-glucosidase showed better stabilities on pH, thermal and storage thanthe free β-glucosidase. After reacted for30cycles on β-glucosidase assay, theretained activity of the immobilized β-glucosidase was over80%. In the batchhydrolysis experiment, the conversion was over90%in the former three batches andthen declined gradually. The conversion was only50%for the ninth batch. Theretained activity of the immobilized β-glucosidase was68.5%after10batches ofreaction.
2. A single packed-bed reactor was constructed for the hydrolysis of cellubiose ina continuous mode, while the reactor was loaded with the immobilized β-glucosidaseonto the porous ceramic ball of type I-a. The optimum operation pH and temperatureof the reactor were4.8and60℃, respectively. As the flow rate was low, theconversion was high but the production efficiency on volume was low. For example,as the flow rate was3.53ml/min, the conversion attained97.5%, but the productionefficiency on volume was only6.29g/(l·h). As the flow rate raised up to11.2ml/min,the conversion was only82.1%, but the production efficiency on volume attained15.62g/(l·h). Furthermore, as the flow rate was stable at5.47ml/min, the conversionmaintained over90%with an average value of90.7%. In addition, a model ofone-dimensional steady-state axial dispersion was set up to describe the performanceof the single packed-bed reactor. The fitting factor R of the model was0.95. Theaverage deviation was9.15%between the calculated results and the experimentalresults.
3. When the two packed-bed reactors were linked in a serial mode and the flow rate was7.67ml/min, the conversion was91.4%, and the production efficiency onvolume was12.69g/(l·h). Compared with the data on single packed-bed reactor,which were the conversion of91.7%and the production efficiency on volume of8.98g/(l·h),the production efficiency on volume of the double serial reactors increased by41%.
4. When β-glucosidase was immobilized onto M-TiO_2(BG-M-TiO_2) with theabsorption action, the maximum apparent activity recovery of BG-M-TiO_2was64.14%, meanwhile the apparent activity was4.68U/g. As the initial enzymeconcentration was0.907U/ml, the apparent activity of BG-M-TiO_2was closed to themaximum value of7.6U/g. After the immobilization of BG, M-TiO_2got less on theabsorption volume to N2, the specific surface area (SBET) of M-TiO_2reduced from62.04m~2/g to47.72m~2/g, and the pore volume (Vp) of M-TiO_2reduced from0.37cm~3/g to0.29cm~3/g. After reacted for6cycles on BG assay, the retained activity ofBG-M-TiO_2was below50%.
5. NH2-M-TiO_2and SH-M-TiO_2were obtained after the M-TiO_2were respectivelymodified by3-aminopropyltriethoxysilane and3-mercaptopropyltrimethoxysilane.BG-SH-M-TiO_2showed better results than BG-NH2-M-TiO_2on the enzyme activity,activity recovery and operation stability. After M-TiO_2was functionalized with3-mercaptopropyltrimethoxysilane, SBETreduced from62.04m~2/g to53.50m~2/g,pore diameter (DBJH) changed from20.3nm to18.2nm, and Vpreduced from0.37cm~3/g to0.31cm~3/g. The elements Si and S were present on the energy dispersivespectrum of SH-M-TiO_2, whose mass fractions were1.78%and1.81%, respectively.Therefore the total mass fraction of mercapto group was about3.6%. Furthermore,there was a peak for the mercapto group loss at355℃on the thermogravimetryfigure of SH-M-TiO_2.
6. The maximum apparent activity recovery of β-glucosidase immobilized ontoSH-M-TiO_2(BG-SH-M-TiO_2) was92.84%, while the apparent activity was10.99U/g.As the initial enzyme concentration was about18U/ml, the apparent activity of BG-SH-M-TiO_2was closed to the maximum value of21.3U/g. After reacted for30cycles of BG assay, the BG-SH-M-TiO_2remained the activity of about50%. In thebatch hydrolysis experiment, the conversions were stable over90%in the former8batches. The retained activity of BG-SH-M-TiO_2was80.5%after10batches ofreaction.
1、以多孔陶瓷球为载体，采用吸附交联—包封的方法固定化β-葡萄糖苷酶，I-a型陶瓷球固定化表观酶活最大约为1.75U/g，从单因素实验还可以看出，戊二醛交联和胶粘剂包封的步骤在陶瓷球固定化酶的操作过程中起关键作用。固定化酶在酸碱、热、储存稳定性方面较游离酶有明显改善；且重复测定酶活30批后，残余酶活仍在80%以上。在重复分批酶解实验中，前三批底物的转化率在90%以上，随后转化率逐渐下降，第九批的转化率降到50%左右，反应10批后固定化酶的残余酶活下降到68.5%。
2、采用连续水解的模式，将陶瓷球固定化酶装填成一单级填充床反应器，反应器的最佳工作温度和pH分别是60℃、pH4.8。底物进料流速低时，转化率高，但是体积生产效率低下；如流速为3.53ml/min时，转化率可达97.5%，而体积生产效率仅为6.29g/(l·h)，当流速增大到11.2ml/min时，转化率仅为82.1%，而体积生产效率高达15.62g/(l·h)。进料速度5.47ml/min时，反应器连续水解十天，转化率保持在90%以上，平均转化率为90.7%。建立了描述填充床的一维稳态轴向扩散模型，以STATISTICA软件对保留时间和转化率关系进行拟合，拟合因子R为0.95，模型计算结果与实验结果的平均偏差为9.15%。
3、由陶瓷球固定化酶二级串联反应器的实验可知，流速为7.67ml/min时，反应器转化率达91.4%，体积生产效率为12.69g/(l·h)，对比单级反应器转化率为91.7%时的体积生产效率为8.98g/(l·h)，二级串联反应器的体积生产效率比单级反应器提高了41%。
4、M-TiO_2吸附法固定β-葡萄糖苷酶（BG-M-TiO_2）的最大表观酶活回收率为64.14%，对应的表观酶活为4.68U/g；当起始给酶量为0.907U/ml时，BG-M-TiO_2的表观酶活力接近最大值，约为7.6U/g。M-TiO_2固载酶后N2吸附量略有下降，比表面积（SBET）从62.04m~2/g降低到47.72m~2/g，孔容（Vp）从0.37cm~3/g降低到0.29cm~3/g。BG-M-TiO_2重复测定酶活6批后，残余酶活降到50%以下。
5、分别以3-氨丙基三乙氧基硅烷（-NH2改性）和3-巯丙基三甲氧基硅烷（-SH改性）两种偶联剂，对M-TiO_2进行后嫁接法官能化改性，BG-SH-M-TiO_2在酶活、酶活回收率和重复分批测定酶活稳定性中的结果均优于BG-NH2-M-TiO_2。-SH改性后的M-TiO_2SBET从62.04m~2/g降低到53.50m~2/g，孔径（DBJH）从20.3nm下降到18.2nm，Vp从0.37cm~3/g降低到0.31cm~3/g；SH-M-TiO_2能谱图新增了Si和S元素的特征吸收峰，质量分数分别是1.78%和1.81%，巯丙基占的总质量分数约为3.6%；SH-M-TiO_2的热重曲线在355℃出现了来自巯丙基官能团的失重峰，质量损失约为2%。
6、BG-SH-M-TiO_2固定化酶的表观酶活回收率最高为92.84%，对应的表观酶活为10.99U/g，当起始给酶量为18U/ml时，BG-SH-M-TiO_2的表观酶活接近最大值为21.3U/g。BG-SH-M-TiO_2重复测定酶活30批后，残余酶活仍在50%左右。在重复分批酶解实验中，前八批对纤维二糖的转化率都在90%以上，反应10批后，BG-SH-M-TiO_2的残余酶活为反应前的80.5%。
There were three carriers, porous ceramic balls, mesoporous TiO_2(M-TiO_2),and mercapto-modified mesoporous TiO_2(SH-M-TiO_2), which were studied on theimmobilization of the enzyme β-glucosidase (BG). In additional, the hydrolysisprocess and model of packed-bed reactor, which was loaded with β-glucosidaseimmobilized onto the porous ceramic balls, were investigated. The main conclusionsare as follows:
1. The β-glucosidase was immobilized onto the porous ceramic ball of type I-a bythe crossing-linking and enveloping operation. The maximum apparent activity ofimmobilized β-glucosidase was about1.75U/g. It was known that two operationsplayed key roles to the activity of the immobilized enzyme. One was crossing-linkedwith the glutaraldehyde, the other was enveloped with a goil-based adhesive. Theimmobilized β-glucosidase showed better stabilities on pH, thermal and storage thanthe free β-glucosidase. After reacted for30cycles on β-glucosidase assay, theretained activity of the immobilized β-glucosidase was over80%. In the batchhydrolysis experiment, the conversion was over90%in the former three batches andthen declined gradually. The conversion was only50%for the ninth batch. Theretained activity of the immobilized β-glucosidase was68.5%after10batches ofreaction.
2. A single packed-bed reactor was constructed for the hydrolysis of cellubiose ina continuous mode, while the reactor was loaded with the immobilized β-glucosidaseonto the porous ceramic ball of type I-a. The optimum operation pH and temperatureof the reactor were4.8and60℃, respectively. As the flow rate was low, theconversion was high but the production efficiency on volume was low. For example,as the flow rate was3.53ml/min, the conversion attained97.5%, but the productionefficiency on volume was only6.29g/(l·h). As the flow rate raised up to11.2ml/min,the conversion was only82.1%, but the production efficiency on volume attained15.62g/(l·h). Furthermore, as the flow rate was stable at5.47ml/min, the conversionmaintained over90%with an average value of90.7%. In addition, a model ofone-dimensional steady-state axial dispersion was set up to describe the performanceof the single packed-bed reactor. The fitting factor R of the model was0.95. Theaverage deviation was9.15%between the calculated results and the experimentalresults.
3. When the two packed-bed reactors were linked in a serial mode and the flow rate was7.67ml/min, the conversion was91.4%, and the production efficiency onvolume was12.69g/(l·h). Compared with the data on single packed-bed reactor,which were the conversion of91.7%and the production efficiency on volume of8.98g/(l·h),the production efficiency on volume of the double serial reactors increased by41%.
4. When β-glucosidase was immobilized onto M-TiO_2(BG-M-TiO_2) with theabsorption action, the maximum apparent activity recovery of BG-M-TiO_2was64.14%, meanwhile the apparent activity was4.68U/g. As the initial enzymeconcentration was0.907U/ml, the apparent activity of BG-M-TiO_2was closed to themaximum value of7.6U/g. After the immobilization of BG, M-TiO_2got less on theabsorption volume to N2, the specific surface area (SBET) of M-TiO_2reduced from62.04m~2/g to47.72m~2/g, and the pore volume (Vp) of M-TiO_2reduced from0.37cm~3/g to0.29cm~3/g. After reacted for6cycles on BG assay, the retained activity ofBG-M-TiO_2was below50%.
5. NH2-M-TiO_2and SH-M-TiO_2were obtained after the M-TiO_2were respectivelymodified by3-aminopropyltriethoxysilane and3-mercaptopropyltrimethoxysilane.BG-SH-M-TiO_2showed better results than BG-NH2-M-TiO_2on the enzyme activity,activity recovery and operation stability. After M-TiO_2was functionalized with3-mercaptopropyltrimethoxysilane, SBETreduced from62.04m~2/g to53.50m~2/g,pore diameter (DBJH) changed from20.3nm to18.2nm, and Vpreduced from0.37cm~3/g to0.31cm~3/g. The elements Si and S were present on the energy dispersivespectrum of SH-M-TiO_2, whose mass fractions were1.78%and1.81%, respectively.Therefore the total mass fraction of mercapto group was about3.6%. Furthermore,there was a peak for the mercapto group loss at355℃on the thermogravimetryfigure of SH-M-TiO_2.
6. The maximum apparent activity recovery of β-glucosidase immobilized ontoSH-M-TiO_2(BG-SH-M-TiO_2) was92.84%, while the apparent activity was10.99U/g.As the initial enzyme concentration was about18U/ml, the apparent activity of BG-SH-M-TiO_2was closed to the maximum value of21.3U/g. After reacted for30cycles of BG assay, the BG-SH-M-TiO_2remained the activity of about50%. In thebatch hydrolysis experiment, the conversions were stable over90%in the former8batches. The retained activity of BG-SH-M-TiO_2was80.5%after10batches ofreaction.
引文
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