SpyTag/SpyCatcher-mediated SpyRing cyclization improves the operational stability and renaturation efficiency of immobilized enzymes after catalytic inactivation

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry Pub Date : 2025-05-27 DOI:10.1016/j.procbio.2025.05.017

Yao Ma , Jincheng Miao , Qiwei Chen , Hongxu Sun , Yanhong Chang , Hui Luo

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引用次数: 0

Abstract

L-Phenylserine aldolase (LPA), a promising industrial multi-subunit enzyme, catalyzes the condensation of aldehydes with glycine to form β-hydroxy amino acids. This study investigated the effect of SpyTag/SpyCatcher-mediated SpyRing cyclization on operational stability and renaturation after catalytic inactivation. Two immobilized forms of LPA were prepared on a glyoxyl-agarose support (Gx): the native Gx-LPA and its SpyRing-cyclized variant, Gx-SRLPA. The immobilized cyclized enzyme, Gx-SRLPA, exhibited superior operational stability compared to Gx-LPA. A renaturation strategy involving partial unfolding with low concentrations of guanidine hydrochloride (GuHCl) followed by renaturation, proved effective. Gx-SRLPA recovered up to 87.9 % of its activity at 1 M GuHCl and pH 9, exceeding Gx-LPA’s 52.9 % recovery at 1 M GuHCl and pH 7. The presence of dithiothreitol in the unfolding solution and weak alkaline conditions further enhanced Gx-SRLPA’s renaturation. This study highlights the stabilizing effect of the SpyRing structure on enzymes and provides valuable insights into the renaturation of immobilized multi-subunit proteins.

查看原文本刊更多论文

SpyTag/ spycatcher介导的SpyRing环化提高了固定化酶在催化失活后的操作稳定性和还原效率

l -苯基丝氨酸醛缩酶（L-Phenylserine aldolase， LPA）是一种很有发展前景的工业多亚基酶，可催化醛与甘氨酸缩合生成β-羟基氨基酸。本研究考察了SpyTag/ spycatcher介导的SpyRing环化对催化失活后操作稳定性和复性的影响。在乙氧基琼脂糖载体（Gx）上制备了两种固定化形式的LPA：天然Gx-LPA及其spyring环化变体Gx- srlpa。与Gx-LPA相比，固定化酶Gx-SRLPA具有更好的操作稳定性。采用低浓度盐酸胍（GuHCl）进行部分展开的复复性策略，证明是有效的。Gx-SRLPA在1 M GuHCl和pH为9条件下的活性回收率高达87.9% %，超过了Gx-LPA在1 M GuHCl和pH为7条件下的52.9% %的回收率。展开液和弱碱性条件下二硫苏糖醇的存在进一步增强了Gx-SRLPA的复性。这项研究突出了SpyRing结构对酶的稳定作用，并为固定化多亚基蛋白的再生提供了有价值的见解。

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来源期刊

Process Biochemistry 生物-工程：化工

CiteScore

8.30

自引率

4.50%

发文量

374

审稿时长

53 days

期刊介绍： Process Biochemistry is an application-orientated research journal devoted to reporting advances with originality and novelty, in the science and technology of the processes involving bioactive molecules and living organisms. These processes concern the production of useful metabolites or materials, or the removal of toxic compounds using tools and methods of current biology and engineering. Its main areas of interest include novel bioprocesses and enabling technologies (such as nanobiotechnology, tissue engineering, directed evolution, metabolic engineering, systems biology, and synthetic biology) applicable in food (nutraceutical), healthcare (medical, pharmaceutical, cosmetic), energy (biofuels), environmental, and biorefinery industries and their underlying biological and engineering principles.