Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylCA

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Alana M.M. Rangaswamy , Francis M. Roy , Jeffrey W. Keillor
{"title":"Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylCA","authors":"Alana M.M. Rangaswamy ,&nbsp;Francis M. Roy ,&nbsp;Jeffrey W. Keillor","doi":"10.1016/j.ab.2024.115598","DOIUrl":null,"url":null,"abstract":"<div><p>The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate <em>N</em>-acetyl-GABA-pNP ester (<em>k</em><sub>cat</sub> = 0.37 s<sup>−1</sup>, <em>K</em><sub>M</sub> = 256 μM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.</p></div>","PeriodicalId":2,"journal":{"name":"ACS Applied Bio Materials","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Bio Materials","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0003269724001428","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MATERIALS SCIENCE, BIOMATERIALS","Score":null,"Total":0}
引用次数: 0

Abstract

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate N-acetyl-GABA-pNP ester (kcat = 0.37 s−1, KM = 256 μM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.

Abstract Image

用于快速定量尼龙水解酶 NylCA 的酰基转移活性的小分子底物。
尼龙等聚酰胺的广泛使用导致了尼龙废料的积累,由于酰胺键的内在稳定性,尼龙废料特别不易分解。需要采用新的方法,通过解聚来真正回收利用这些废料。有人提出,能够水解聚酰胺的酶作为生物催化剂可能适用于这一应用。NylC 是一种能介导水解氨基己酸低聚物的酶,在一定程度上也能介导水解大块聚合物。然而,目前表征这种酶活性的检测方法需要较长的反应时间和/或依赖二次反应来量化水解作用。在此,我们设计了结构优化的小分子致色酯,可作为底物类似物连续监测 NylC 乙酰转移酶的活性。这种检测方法可在室温下几分钟内完成,底物 N-acetyl-GABA-pNP 酯(kcat = 0.37 s-1,KM = 256 μM)在复杂的生物介质中显示出对 NylC 的选择性。我们还证明,对这种底物类似物的活性与酰胺水解相关,而酰胺水解是这种酶的主要活性。此外,我们对底物类似物的筛选有助于深入了解 NylC 的底物特异性,这与生物催化应用息息相关。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信