改进溶胶-凝胶法和吸附法固定化醇脱氢酶的研究
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摘要
传统溶胶-凝胶(sol-gel)法进行酶的固定化具有酶活损失大和传质阻力大的
缺点。本文以聚乙二醇（PEG）为多功能试剂对传统sol-gel过程进行改进，以提
高固定化甲醇脱氢酶(ADH)的活性。以PEG为模板剂修饰凝胶，使凝胶的孔径
由 7.8nm增大到 9.3nm，比表面积也由 463 m2﹒g-1增加到 534m2﹒g-1，底物在
凝胶孔中的有效扩散系数提高到原来的 1.4 倍。用修饰后的凝胶固定化的ADH
的活性比传统凝胶固定化的ADH的活性高。此外，利用活化PEG(SC-PEG)为修
饰剂对ADH进行修饰，以提高ADH在固定化后的活性，ADH的修饰度约 90％。
对修饰后的ADH进行固定化，ADH的活性比未经修饰的ADH的活性有明显提
高。由甲醛转化为甲醇的酶促反应比较两条提高酶活途径下的四种固定化的
ADH的活性：未修饰凝胶固定化修饰ADH（固定化ADHⅢ）活性> 修饰凝胶固
定化修饰ADH（固定化ADHⅣ）活性> 修饰凝胶固定化未修饰ADH（固定化
ADHⅡ）活性> 传统固定化方法下的ADH（固定化ADHⅠ）活性。而且固定化
ADHⅡ、ADHⅢ、ADHⅣ的稳定性均高于固定化ADHⅠ的稳定性。可见凝胶
的修饰以及ADH的修饰均可提高固定化ADH的活性和稳定性，但是ADH的修饰
较凝胶的修饰对提高ADH的活性效果明显。
另外，为了比较吸附法与传统 sol-gel 包埋法固定化 ADH 的优劣，对两种
方法固定化的 ADH 的活性进行了比较。采用大孔硅胶对 ADH 进行吸附，得到
吸附动力学曲线和吸附等温线，ADH 在硅胶上的吸附等温线可以用 Langmuir
方程拟合。并且分析了硅胶孔径对 ADH 吸附量的影响，得到大孔径的硅胶对
ADH 有着更大吸附量的结论。由甲醛转化为甲醇的酶促反应动力学实验测得的
反应初速度及拟合得到的米氏常数表明吸附法固定化 ADH 表现出比 sol-gel 法
固定化 ADH 高的催化活性。
Enzyme immobilization by traditional sol-gel approach suffers from the
drawbacks of relatively high enzyme activity loss and high mass transfer resistance,
solutions to this problem have been tentatively exploited herein, PEGs were
employed as multifunctional additives to enhance immobilized enzyme activities: 1.
Tetraethylorthosilicate (TEOS) derived gel is modified in the presence of
polyethylene glycol (PEG, molecular weight 600), the effects of PEG addition to the
precursor solution on the microstructure of the gel are studied, the size of pores is
increased in the resultant gels on the bridge-linkage between PEG and precursor,
thus diffusion rates is comparably increased to1.4 times. 2. PEG Succinimidyl
Carbonate (SC-PEG, molecular weight 5,000) is used to modify alcohol
dehydrogenase (ADH) before being immobilized in the gel in order to avoid the
damage of enzyme in sol-gel process and also improve the properties of gels such as
hydrophilicity. The activities of diverse immobilized ADH have been investigated by
the enzymatic reaction of converting formaldehyde into methanol: unmodified gel
immobilized modified ADH（Immobilized ADHⅢ）>modified gel immobilized
modified ADH（Immobilized ADHⅣ）>modified gel immobilized unmodified ADH
（ Immobilized ADH Ⅱ ） > unmodified gel immobilized unmodified ADH
（ImmobilizedADHⅠ）.And the stabilities of immobilizedADHⅡ、ADHⅢ、ADH
Ⅳ are higher than that of immobilized ADHⅠ. This result indicated that enzyme
activities after the modification of gel and the modification of ADH respectively
significantly increased compared to the native immobilized ADH, but modification
on ADH is more effective.
In addition, in order to compare the enzyme immobilization differences
between adsorption immobilization process and sol-gel encapsulation process. ADH
is respectively immobilized onto macroporous silica carrier by adsorption and into
microporous silica gel by sol-gel encapsulation. Through the study of adsorption
kinetics and adsorption equilibrium, the kinetics curve and adsorption isotherm are
obtained. The adsorption isotherm is fitted by Langmuir equation. The effect of pore
size on the adsorption capacity is examined. The sol-gel encapsulated ADH is
prepared with silica matrix. The catalytic activities of the above-mentioned
immobilized ADHs towards the conversion of formaldehyde into methanol are
compared. The initial rates of enzymatic reaction catalyzed by adsorption
immobilized ADH is higher than that catalyzed by sol-gel encapsulated ADH,
whereas the Michaelis constants of the former are less than that of the latter. These
results indicate that the catalytic activity of adsorption immobilized ADH is higher
than that of sol-gel encapsulated ADH.
缺点。本文以聚乙二醇（PEG）为多功能试剂对传统sol-gel过程进行改进，以提
高固定化甲醇脱氢酶(ADH)的活性。以PEG为模板剂修饰凝胶，使凝胶的孔径
由 7.8nm增大到 9.3nm，比表面积也由 463 m2﹒g-1增加到 534m2﹒g-1，底物在
凝胶孔中的有效扩散系数提高到原来的 1.4 倍。用修饰后的凝胶固定化的ADH
的活性比传统凝胶固定化的ADH的活性高。此外，利用活化PEG(SC-PEG)为修
饰剂对ADH进行修饰，以提高ADH在固定化后的活性，ADH的修饰度约 90％。
对修饰后的ADH进行固定化，ADH的活性比未经修饰的ADH的活性有明显提
高。由甲醛转化为甲醇的酶促反应比较两条提高酶活途径下的四种固定化的
ADH的活性：未修饰凝胶固定化修饰ADH（固定化ADHⅢ）活性> 修饰凝胶固
定化修饰ADH（固定化ADHⅣ）活性> 修饰凝胶固定化未修饰ADH（固定化
ADHⅡ）活性> 传统固定化方法下的ADH（固定化ADHⅠ）活性。而且固定化
ADHⅡ、ADHⅢ、ADHⅣ的稳定性均高于固定化ADHⅠ的稳定性。可见凝胶
的修饰以及ADH的修饰均可提高固定化ADH的活性和稳定性，但是ADH的修饰
较凝胶的修饰对提高ADH的活性效果明显。
另外，为了比较吸附法与传统 sol-gel 包埋法固定化 ADH 的优劣，对两种
方法固定化的 ADH 的活性进行了比较。采用大孔硅胶对 ADH 进行吸附，得到
吸附动力学曲线和吸附等温线，ADH 在硅胶上的吸附等温线可以用 Langmuir
方程拟合。并且分析了硅胶孔径对 ADH 吸附量的影响，得到大孔径的硅胶对
ADH 有着更大吸附量的结论。由甲醛转化为甲醇的酶促反应动力学实验测得的
反应初速度及拟合得到的米氏常数表明吸附法固定化 ADH 表现出比 sol-gel 法
固定化 ADH 高的催化活性。
Enzyme immobilization by traditional sol-gel approach suffers from the
drawbacks of relatively high enzyme activity loss and high mass transfer resistance,
solutions to this problem have been tentatively exploited herein, PEGs were
employed as multifunctional additives to enhance immobilized enzyme activities: 1.
Tetraethylorthosilicate (TEOS) derived gel is modified in the presence of
polyethylene glycol (PEG, molecular weight 600), the effects of PEG addition to the
precursor solution on the microstructure of the gel are studied, the size of pores is
increased in the resultant gels on the bridge-linkage between PEG and precursor,
thus diffusion rates is comparably increased to1.4 times. 2. PEG Succinimidyl
Carbonate (SC-PEG, molecular weight 5,000) is used to modify alcohol
dehydrogenase (ADH) before being immobilized in the gel in order to avoid the
damage of enzyme in sol-gel process and also improve the properties of gels such as
hydrophilicity. The activities of diverse immobilized ADH have been investigated by
the enzymatic reaction of converting formaldehyde into methanol: unmodified gel
immobilized modified ADH（Immobilized ADHⅢ）>modified gel immobilized
modified ADH（Immobilized ADHⅣ）>modified gel immobilized unmodified ADH
（ Immobilized ADH Ⅱ ） > unmodified gel immobilized unmodified ADH
（ImmobilizedADHⅠ）.And the stabilities of immobilizedADHⅡ、ADHⅢ、ADH
Ⅳ are higher than that of immobilized ADHⅠ. This result indicated that enzyme
activities after the modification of gel and the modification of ADH respectively
significantly increased compared to the native immobilized ADH, but modification
on ADH is more effective.
In addition, in order to compare the enzyme immobilization differences
between adsorption immobilization process and sol-gel encapsulation process. ADH
is respectively immobilized onto macroporous silica carrier by adsorption and into
microporous silica gel by sol-gel encapsulation. Through the study of adsorption
kinetics and adsorption equilibrium, the kinetics curve and adsorption isotherm are
obtained. The adsorption isotherm is fitted by Langmuir equation. The effect of pore
size on the adsorption capacity is examined. The sol-gel encapsulated ADH is
prepared with silica matrix. The catalytic activities of the above-mentioned
immobilized ADHs towards the conversion of formaldehyde into methanol are
compared. The initial rates of enzymatic reaction catalyzed by adsorption
immobilized ADH is higher than that catalyzed by sol-gel encapsulated ADH,
whereas the Michaelis constants of the former are less than that of the latter. These
results indicate that the catalytic activity of adsorption immobilized ADH is higher
than that of sol-gel encapsulated ADH.
引文
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