Understanding Design Rules for Optimizing the Interface between Immobilized Enzymes and Random Copolymer Brushes.
Understanding Design Rules for Optimizing the Interface between Immobilized Enzymes and Random Copolymer Brushes.
复制标题
了解优化固定化酶和无规共聚物刷之间界面的设计规则。
DOI：
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发表时间：
2021
影响因子：
9.5
通讯作者：
Joel L. Kaar
中科院分区：
文献类型：
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作者：
Héctor Sánchez;James S. Weltz;D. K. Schwartz;Joel L. Kaar
生物技术领域的一个长期目标是开发和理解固定到材料上的酶稳定性的设计规则。虽然固定化作为一种稳定酶的策略有时是成功的，但稳定酶的合成材料的设计在很大程度上仍然是凭经验的。我们试图通过研究固定化脂肪酶在由聚（乙二醇）甲基丙烯酸酯（PEGMA）和磺基甜菜碱甲基丙烯酸酯（SBMA）组成的无规共聚物刷表面上稳定的机制基础来克服这一挑战，它们代表了固定化酶的新型异质载体。使用几种相关但结构不同的脂肪酶，包括枯草芽孢杆菌脂肪酶 A (LipA)、米赫根毛霉脂肪酶、皱褶假丝酵母脂肪酶和南极假丝酵母脂肪酶 B (CALB)，我们发现每种脂肪酶在高温下的稳定性强烈依赖于刷层中 PEGMA 的比例。这种依赖性可以通过开发和应用一种新算法来量化蛋白质表面疏水性来解释，其中涉及使用无监督聚类分析来识别疏水原子簇。脂肪酶的表征表明，最佳刷组成与单位酶表面积的溶剂化自由能相关，范围从 LipA 的 -17.1 kJ/mol·nm2 到 CALB 的 -11.8 kJ/mol·nm2。此外，使用该算法，我们发现由脂肪族残基组成的疏水斑块比由芳香族残基组成的斑块具有更高的自由能。通过为合理调整酶和材料之间的界面提供基础，这种理解将改变材料的使用，以在极端条件下可靠地加固酶。
A long-standing goal in the field of biotechnology is to develop and understand design rules for the stabilization of enzymes upon immobilization to materials. While immobilization has sometimes been successful as a strategy to stabilize enzymes, the design of synthetic materials that stabilize enzymes remains largely empirical. We sought to overcome this challenge by investigating the mechanistic basis for the stabilization of immobilized lipases on random copolymer brush surfaces comprised of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA), which represent novel heterogeneous supports for immobilized enzymes. Using several related but structurally diverse lipases, including Bacillus subtilis lipase A (LipA), Rhizomucor miehei lipase, Candida rugosa lipase, and Candida antarctica lipase B (CALB), we showed that the stability of each lipase at elevated temperatures was strongly dependent on the fraction of PEGMA in the brush layer. This dependence was explained by developing and applying a new algorithm to quantify protein surface hydrophobicity, which involved using unsupervised cluster analysis to identify clusters of hydrophobic atoms. Characterization of the lipases showed that the optimal brush composition correlated with the free energy of solvation per enzyme surface area, which ranged from -17.1 kJ/mol·nm2 for LipA to -11.8 kJ/mol·nm2 for CALB. Additionally, using this algorithm, we found that hydrophobic patches consisting of aliphatic residues had a higher free energy than patches consisting of aromatic residues. By providing the basis for rationally tuning the interface between enzymes and materials, this understanding will transform the use of materials to reliably ruggedize enzymes under extreme conditions.