Abstract We report herein new enzyme-polymer conjugates synthesized by grafting polymerization onto Candida rugosa lipase (CRL) with zwitterionic carboxyl betaine methacrylate (CBMA), hydrophobic tert-butyl methacrylate (TBMA) and their equimolar mixture, which respectively created CRL-pCBMA, CRL-pTBMA, and CRL-p(T50-C50). All the enzyme conjugates presented improved catalytic activity, thermostability and pH tolerance, and the kinetic studies indicated that the polymer-grafting resulted in significantly higher enzymatic reaction efficiency and enzyme-substrate affinity. CRL-pTBMA increased the catalytic activity by 2.26-fold and half-life by 43.1-fold at 50 °C as compared with the free enzyme due to the interfacial activation effect of TBMA. However, despite the lower activity of the copolymer conjugate (1.56 times of free enzyme), CRL-p(T50-C50), its half-life was 2.2-fold higher than CRL-pTBMA. This was due to the stabilization effect of the zwitterionic monomer component (CBMA) in the copolymer. This is evident from the similar stability of CRL-p(T50-C50) with CRL-pCBMA, but the latter showed lower activity than the former due to the lack of the interfacial activation effect of TBMA. Thus, a zwitterionic/hydrophobic balance is of vital importance to enhance the enzymatic performance. Spectroscopic characterizations revealed the changes in the microenvironment and secondary structures of the enzyme in CRL-polymer conjugates. Therefore, modification with zwitterionic-hydrophobic copolymer was found more effective in improving the catalytic performance of lipase.

中文翻译：

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通过将疏水性和两性离子单体的共聚物接枝到酶上来激活和稳定脂肪酶

摘要 我们在此报告了新的酶-聚合物偶联物，该偶联物通过将聚合反应接枝到假丝酵母脂肪酶 (CRL) 上与两性离子羧基甜菜碱甲基丙烯酸酯 (CBMA)、疏水性甲基丙烯酸叔丁酯 (TBMA) 及其等摩尔混合物，分别产生 CRL-pCBMA、CRL -pTBMA 和 CRL-p(T50-C50)。所有酶偶联物都表现出改善的催化活性、热稳定性和 pH 耐受性，动力学研究表明聚合物接枝导致酶促反应效率和酶-底物亲和力显着提高。由于 TBMA 的界面活化作用，与游离酶相比，CRL-pTBMA 在 50°C 下将催化活性提高了 2.26 倍，半衰期提高了 43.1 倍。然而，尽管共聚物缀合物的活性较低（游离酶的 1.56 倍），CRL-p(T50-C50)，其半衰期比 CRL-pTBMA 高 2.2 倍。这是由于共聚物中两性离子单体组分 (CBMA) 的稳定作用。从CRL-p(T50-C50)与CRL-pCBMA相似的稳定性可以看出这一点，但由于缺乏TBMA的界面活化作用，后者的活性低于前者。因此，两性离子/疏水平衡对于提高酶促性能至关重要。光谱表征揭示了 CRL-聚合物偶联物中酶的微环境和二级结构的变化。因此，发现用两性离子-疏水共聚物进行改性可以更有效地提高脂肪酶的催化性能。这是由于共聚物中两性离子单体组分 (CBMA) 的稳定作用。从CRL-p(T50-C50)与CRL-pCBMA相似的稳定性可以看出这一点，但由于缺乏TBMA的界面活化作用，后者的活性低于前者。因此，两性离子/疏水平衡对于提高酶促性能至关重要。光谱表征揭示了 CRL-聚合物偶联物中酶的微环境和二级结构的变化。因此，发现用两性离子-疏水共聚物进行改性可以更有效地提高脂肪酶的催化性能。这是由于共聚物中两性离子单体组分 (CBMA) 的稳定作用。从CRL-p(T50-C50)与CRL-pCBMA相似的稳定性可以看出这一点，但由于缺乏TBMA的界面活化作用，后者的活性低于前者。因此，两性离子/疏水平衡对于提高酶促性能至关重要。光谱表征揭示了 CRL-聚合物偶联物中酶的微环境和二级结构的变化。因此，发现用两性离子-疏水共聚物进行改性可以更有效地提高脂肪酶的催化性能。但由于缺乏 TBMA 的界面活化作用，后者的活性低于前者。因此，两性离子/疏水平衡对于提高酶促性能至关重要。光谱表征揭示了 CRL-聚合物偶联物中酶的微环境和二级结构的变化。因此，发现用两性离子-疏水共聚物进行改性可以更有效地提高脂肪酶的催化性能。但由于缺乏 TBMA 的界面活化作用，后者的活性低于前者。因此，两性离子/疏水平衡对于提高酶促性能至关重要。光谱表征揭示了 CRL-聚合物偶联物中酶的微环境和二级结构的变化。因此，发现用两性离子-疏水共聚物进行改性可以更有效地提高脂肪酶的催化性能。