固定化产酸酶的微环境pH变化规律、调控机制及两阶段催化策略的研究

Immobilized enzymes offer considerable advantages such as facility of removal and reuse, increased shelf life and thermal stability. Unfortunately, most immobilized enzyme catalysts are porous and the internal diffusional resistance in particles remarkably affects the catalytic efficiency. For these enzymes catalyzing a proton-producing or consuming reaction, the internal diffusional resistance would result in a severe microenvironmental pH change in the immobilized enzymes, sometimes leading to a low operational stability. It is undoubtedly important that the effect of microenvironmental pH changes on the activity and stability of immobilized enzymes being investigated. In this proposal, the change of microenvironmental pH is discussed in the catalysis of immobilized enzymes by introducing a variety of regulation methods. A model describing the relationship between microenvironmental pH change and the activity and stability of the immobilized enzymes will be constructed. It was found in our previous work that the initial rate of the immobilisates catalyzed reaction is usually very high, which might result in a low stability, largely owing to the sharp intraparticle pH gradient. While in the late period of the catalysis, the biocatalyst shows a very low activity due to the low substrate concentration and high product accumulation. Accordingly, a novel strategy of "two-stage regulated catalysis" is developed to realize both high performance and operational stability of the biocatalyst. In the optimized biotransformation, the reaction rate of the first stage would be down regulated to preserve the enzymatic activity, and some methods would be used in the second stage to shorten the catalyzing duration. Furthermore, two or three enzymes with high potential application in industry (such as cephalosporin C acylase, acetyl esterase and nitrilase) would be used as the research objective to develop their catalyzing process. The results to be obtained in this work are critical for better understanding the relationship between microenvironmental pH change, efficiency and stability of the immobilized enzymes in proton-producing reactions. A guideline for selection of proper regulation protocols for an optimum catalysis of immobilized enzymes will be developed, and surely the output of this work will impel a more extensive and successful application of the immobilized enzymes in industry.

固定化酶具有稳定性好、可重复使用、易与产物分离等优点，但通常存在着颗粒内部的传质阻力问题，尤其是对催化过程中pH发生变化的酶（主要是产酸酶），颗粒内部的传质阻力会导致微环境pH发生剧烈变化，对固定化酶的催化效率和稳定性都可能会造成不利影响，甚至限制了一些酶的工业应用。本课题针对固定化产酸酶在催化过程中微环境pH剧烈变化的问题，研究不同调控策略下的固定化酶微环境pH变化规律，并结合酶催化特性和稳定性数据建立相关的数学模型。在前期研究中发现，由于受微环境pH变化和底物/产物浓度影响，固定化产酸酶的催化过程普遍存在着反应前期稳定性差和后期催化效率低的问题。对此，本课题提出"两阶段催化"调控策略：通过一定手段适当减缓初期反应速度以提高酶的稳定性，加快后期反应速度以缩短催化总反应时间。在理论研究的基础上，本课题还将对2~3种有重要应用前景的产酸酶的固定化酶催化模式进行研究，以推动其在工业上的应用。

固定化酶具有稳定性好、可重复使用、易与产物分离等优点，但多孔的固定化酶通常存在着颗粒内部的传质阻力问题，尤其是对催化过程中pH发生变化的产酸酶，颗粒内部的传质阻力会导致微环境pH严重偏离主体相溶液的pH，对固定化产酸酶的催化效率和稳定性都可能会造成不利影响。本课题针对固定化产酸酶在催化过程中微环境pH剧烈变化的问题，研究了改变温度、添加产物/抑制剂、加入缓冲体系等不同调控策略下的固定化酶微环境pH变化规律，发现酶催化速率与固定化酶微环境pH及酶稳定性之间存在重要关联，也即是可以通过控制催化前期的反应速度来提高酶的催化稳定性。研究发现，由于受微环境pH变化和底物/产物浓度的影响，固定化产酸酶的催化过程普遍存在着反应前期稳定性差和后期催化效率低的问题。对此，本课题提出基于变温操作的“两阶段催化”调控策略：在反应初期采用较低温度催化，适当减缓反应速度以提高酶的稳定性，在反应后期由于存在产物对酶的保护作用，可以采用较高催化温度加快反应速度以缩短催化总反应时间。通过理论分析，采用缓冲体系可以调控产酸酶微环境pH并进而提高酶的稳定性，设计和开发了利用廉价且不影响产物分离的碳酸氢铵来调控固定化产酸酶微环境pH的方法，并取得良好的效果。相比水体系中的催化，在碳酸氢铵体系中酶的催化稳定性提高了3倍。在此基础上，成功开发了固定化产酸酶在填充床反应器（PFR）中的连续催化方式。头孢菌素C酰化酶和乙酰酯酶都可以实现连续稳定地催化，单位酶催化的产物量比搅拌釜反应器的批次催化有明显提高。固定化头孢菌素C酰化酶在填充床反应器中催化，单位酶的催化能力相比于搅拌釜反应器（STR）中水体系催化提升高了383%。为进一步发挥连续催化工艺的优势，本课题还开发了连续搅拌釜反应器（CSTR）与填充床反应器联用的连续催化工艺，可以同时实现酶的高效催化和底物的高转化率，这个新工艺的提出也将有力地推动更多产酸酶的工业应用。