High-throughput screening and identification of lignin peroxidase based on spore surface display of Bacillus subtilis.

IF 3.3 2区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY Journal of the Science of Food and Agriculture Pub Date : 2024-11-04 DOI:10.1002/jsfa.13988

Na Shi, Shouzhi Li, Lu He, Yong Feng, Muhammad Saeed, Yi Ma, Zhong Ni, Daochen Zhu, Huayou Chen

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Abstract

Background: Lignin peroxidase is closely related to agriculture and food as it improves the quality of feedstuffs, facilitates the degradation of lignin in agricultural wastes, and degrades azo dyes that have similar complex structures to lignin. However, the current status of homologous or heterologous expression of lignin peroxidase is unsatisfactory and needs to be modified with the help of immobilization and directed evolution to maximize its potential. Directed evolution technology is an effective strategy for designing and improving enzyme characteristics, and Bacillus subtilis spore surface display technology is an efficient method for preparing immobilized enzymes.

Results: A colorimetric dye decolorization assay using Congo red as a substrate was developed and optimized for high-throughput screening of spore surface display in a 96-well plate. After two rounds of screening, a superior mutant strain was selected from 2700 mutants. Its highest catalytic activity was 196.36%. The amino acid substitution sites were identified as N120D and I242T.

Conclusion: The mechanism for the enhanced catalytic activity was explained using protein modeling and functional analysis software. This study provides insights into the rational design of lignin peroxidase and its application in food and agriculture. © 2024 Society of Chemical Industry.

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基于枯草芽孢杆菌孢子表面显示的木质素过氧化物酶的高通量筛选和鉴定。

背景：木质素过氧化物酶与农业和食品密切相关，因为它能改善饲料的质量，促进农业废弃物中木质素的降解，并能降解与木质素具有相似复杂结构的偶氮染料。然而，木质素过氧化物酶的同源或异源表达现状并不令人满意，需要借助固定化和定向进化技术对其进行改造，以最大限度地发挥其潜力。定向进化技术是设计和改进酶特性的有效策略，枯草芽孢杆菌孢子表面展示技术是制备固定化酶的有效方法：结果：开发并优化了一种以刚果红为底物的比色染料脱色测定法，用于在 96 孔板中对孢子表面展示酶进行高通量筛选。经过两轮筛选，从 2700 个突变株中筛选出了一个优良突变株。其最高催化活性为 196.36%。氨基酸替换位点被确定为 N120D 和 I242T：结论：利用蛋白质建模和功能分析软件解释了催化活性增强的机理。本研究为木质素过氧化物酶的合理设计及其在食品和农业中的应用提供了启示。© 2024 化学工业协会。

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来源期刊

Journal of the Science of Food and Agriculture 工程技术-农业综合

CiteScore

8.10

自引率

4.90%

发文量

634

审稿时长

3.1 months

期刊介绍： The Journal of the Science of Food and Agriculture publishes peer-reviewed original research, reviews, mini-reviews, perspectives and spotlights in these areas, with particular emphasis on interdisciplinary studies at the agriculture/ food interface. Published for SCI by John Wiley & Sons Ltd. SCI (Society of Chemical Industry) is a unique international forum where science meets business on independent, impartial ground. Anyone can join and current Members include consumers, business people, environmentalists, industrialists, farmers, and researchers. The Society offers a chance to share information between sectors as diverse as food and agriculture, pharmaceuticals, biotechnology, materials, chemicals, environmental science and safety. As well as organising educational events, SCI awards a number of prestigious honours and scholarships each year, publishes peer-reviewed journals, and provides Members with news from their sectors in the respected magazine, Chemistry & Industry . Originally established in London in 1881 and in New York in 1894, SCI is a registered charity with Members in over 70 countries.