MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels,Biomacromolecules

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MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels
Biomacromolecules ( IF 5.5 ) Pub Date : 2020-11-18 , DOI: 10.1021/acs.biomac.0c01262
Maximilian Nöth _{1,

2} , Larissa Hussmann _{2,

3} , Thomke Belthle _{2,

3} , Islam El-Awaad _{1,

2,

4} , Mehdi D Davari ₁ , Felix Jakob _{1,

2} , Andrij Pich _{2,

3,

5} , Ulrich Schwaneberg _{1,

2}

Affiliation

Microgels are an emerging class of “ideal” enzyme carriers because of their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly(N-vinylcaprolactam) (PVCL) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as a comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. P450 monooxygenases are usually challenging to immobilize, and often, high activity losses occur after the immobilization (in the case of P450 BM3 from Bacillus megaterium up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels was achieved without the loss of catalytic activity at the pH optimum of P450 BM3 (pH 8; ∼9.4 nmol 7-hydroxy-3-carboxy coumarin ethyl ester/min for free and immobilized P450 BM3); the resulting P450-microgel systems were termed P450 MicroGelzymes (P450 μ-Gelzymes). In addition, P450 μ-Gelzymes offer the possibility of reversible ionic strength-triggered release and re-entrapment of the biocatalyst in processes (e.g., for catalyst reuse). Finally, a characterization of the potential of P450 μ-Gelzymes to provide resistance against cosolvents (acetonitrile, dimethyl sulfoxide, and 2-propanol) was performed to evaluate the biocatalytic application potential.

中文翻译：

MicroGelzymes：细胞色素 P450 BM3 在微凝胶中的非 pH 依赖性固定

微凝胶是一类新兴的“理想”酶载体，因为它们具有化学和工艺稳定性、生物相容性和高酶载量能力。在这项工作中，我们合成了一种新型的永久带正电荷的聚（N-乙烯基己内酰胺）（PVCL）微凝胶，其以 1-乙烯基-3-甲基咪唑（通过 N-乙烯基咪唑部分甲基化对氮进行季铵化）作为共聚单体（PVCL/VimQ） ) 通过沉淀聚合。PVCL/VimQ 微凝胶的特征在于它们的尺寸、电荷、溶胀度和在水溶液中的温度响应性。P450 单加氧酶通常难以固定化，并且通常在固定化后会发生高活性损失（对于来自巨大芽孢杆菌的 P450 BM3高达 100% 的活动损失）。在 P450 BM3 的最佳 pH 值（pH 8；~9.4 nmol 7-羟基-3-羧基香豆素乙酯/分钟免费和固定化的 P450 BM3)；得到的 P450-微凝胶系统被称为 P450 MicroGelzymes (P450 μ-Gelzymes)。此外，P450 μ-Gelzymes 提供了可逆离子强度触发释放和重新捕获过程中生物催化剂的可能性（例如，用于催化剂再利用）。最后，对 P450 μ-Gelzymes 对共溶剂（乙腈、二甲亚砜和 2-丙醇）的耐受性进行了表征，以评估生物催化的应用潜力。

更新日期：2020-12-14

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