Easy stabilization of interfacially activated lipases using heterofunctional divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment
Easy stabilization of interfacially activated lipases using heterofunctional divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment
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DOI：
10.1016/j.procbio.2016.04.002
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发表时间：
2016-07-01
影响因子：
4.4
通讯作者：
Fernandez-Lafuente, Roberto
中科院分区：
文献类型：
--
作者：
de Albuquerque, Tiago L.;Rueda, Nazzoly;Fernandez-Lafuente, Roberto
辛烷基琼脂糖是一种支持，允许一步固定化，稳定和纯化的脂肪酶。然而，在极端条件下，酶可能会从载体中释放出来。本文介绍了一种新的异功能载体——二乙烯基砜活化的辛基琼脂糖珠，该载体已被证明可用于生产非常稳定和活跃的生物催化剂，用于从念珠菌（Candida rugosa， CRL）、米黑根霉（Rhizomucor miehei， RML）和热酵母菌（Thermomyces luginosus， TLL）中提取脂肪酶，能够在任何反应条件下工作而不会有酶解吸的风险。由于不同的原因，三种酶在乙氧基辛酯载体上的固定化失败。在pH为5的条件下固定化可以保持辛酯琼脂糖的良好性能。在pH 8下进一步孵化，允许每个酶分子建立至少一个共价酶支持键（防止酶解吸的风险），避免在pH 10下产生失活，最终结果是所有三种新的生物催化剂都比辛烷基-乙氧基对应物更活跃，更稳定（例如，使用CRL 20）。酶支持反应的结束是通过用不同的亲核试剂（阳离子、阴离子、疏水等）阻断乙烯基砜基团来实现的。这不仅决定了最终酶的稳定性，还决定了不同固定化制剂的活性、选择性甚至特异性。(C) 2016 Elsevier Ltd.版权所有。
Octyl-agarose is a support that permits the one step immobilization, stabilization and purification of lipases. However, the enzyme may be released from the support under drastic conditions. This paper describes a new heterofunctional support, octyl agarose beads activated with divinyl sulfone, that has proved to be useful to produce very stable and active biocatalysts of lipases from Candida rugosa (CRL), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), able to work under any reaction conditions without risking enzyme desorption. The three enzymes failed in immobilization on glyoxyl-octyl supports for different reasons. The immobilization at pH 5 permitted to keep the good properties of octyl agarose. Further incubation at pH 8 permitted to establish at least one covalent enzyme-support bond per enzyme molecule (preventing the risk of enzyme desorption), avoiding the inactivation produced at pH 10, and the final result is that all three new biocatalysts are more active than the octyl-glyoxyl counterparts and much more stable (e.g., 20 using CRL). The end of the enzyme-support reaction was achieved via blocking the vinylsulfone groups with different nucleophiles (cationic, anionic, hydrophobic, etc). This not only determined the final enzyme stability, but also the activity, selectivity and even specificity of the different immobilized preparations. (C) 2016 Elsevier Ltd. All rights reserved.