Identification of a novel D-amino acid oxidase and its application in deracemization of D, L-phosphinothricin.

IF 3.6 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Bioprocess and Biosystems Engineering Pub Date : 2025-11-01 Epub Date: 2025-08-09 DOI:10.1007/s00449-025-03219-0

Li-Qun Jin, Meng-Dan Liu, Zi-Yu Guan, Yi-Xin Li, Ya-Ping Xue, Zhi-Qiang Liu, Yu-Guo Zheng

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引用次数: 0

Abstract

DAAO is applied as a potential catalyst in the biosynthesis of L-PPT. However, its low solubility expression constrains its broader industrial application. Herein, a novel DAAO derived from Cladophialophora carrionii (CcDAAO) was identified, which demonstrated superior catalytic performance toward D-Ala (specific activity: 106.38 ± 1.21 U/mg, K_m: 1.56 ± 0.06 mM), along with remarkable thermostability and broad substrate spectrum. Under optimal culture conditions, the soluble expression level of CcDAAO was enhanced through a co-expression strategy with molecular chaperones, and the enzyme activity increased by 36.3% compared with the initial level. Subsequently, CcDAAO was constructed as a fusion protein (CGD) with catalase from Geobacillus sp. CHB1 (GbCAT) and applied in a D-amino acid aminotransferase (DAAT)-mediated cascade system. In a 2 L reaction system, this cascade system achieved complete conversion (> 99%) of 1 M D,L-PPT within 8 h, exhibiting a yield of 11.26 g/L/h for PPO, which represents a significant improvement over existing reports. This study presents a promising practical approach for the industrial production of optically pure L-PPT.

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一种新型D-氨基酸氧化酶的鉴定及其在D， l -膦酸三甲氨酸脱羧中的应用。

DAAO作为一种潜在的催化剂被应用于L-PPT的生物合成。但其溶解度较低，限制了其广泛的工业应用。本文从腐肉Cladophialophora carrionii （CcDAAO）中分离得到一种新的DAAO，该DAAO对D-Ala具有优异的催化性能（比活性：106.38±1.21 U/mg, Km: 1.56±0.06 mM），并且具有良好的热稳定性和广泛的底物光谱。在最佳培养条件下，通过与分子伴侣共表达策略提高CcDAAO的可溶性表达水平，酶活性较初始水平提高36.3%。随后，CcDAAO与Geobacillus sp. CHB1 （GbCAT）过氧化氢酶构建融合蛋白（CGD），并应用于d -氨基酸氨基转移酶（DAAT）介导的级联系统。在2 L的反应体系中，该级联体系在8 h内实现了1 M D，L- ppt的完全转化（> 99%），PPO的产率为11.26 g/L/h，与现有报道相比有了显著提高。本研究为光纯L-PPT的工业化生产提供了一种很有前途的实用方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioprocess and Biosystems Engineering 工程技术-工程：化工

CiteScore

7.90

自引率

2.60%

发文量

147

审稿时长

2.6 months

期刊介绍： Bioprocess and Biosystems Engineering provides an international peer-reviewed forum to facilitate the discussion between engineering and biological science to find efficient solutions in the development and improvement of bioprocesses. The aim of the journal is to focus more attention on the multidisciplinary approaches for integrative bioprocess design. Of special interest are the rational manipulation of biosystems through metabolic engineering techniques to provide new biocatalysts as well as the model based design of bioprocesses (up-stream processing, bioreactor operation and downstream processing) that will lead to new and sustainable production processes. Contributions are targeted at new approaches for rational and evolutive design of cellular systems by taking into account the environment and constraints of technical production processes, integration of recombinant technology and process design, as well as new hybrid intersections such as bioinformatics and process systems engineering. Manuscripts concerning the design, simulation, experimental validation, control, and economic as well as ecological evaluation of novel processes using biosystems or parts thereof (e.g., enzymes, microorganisms, mammalian cells, plant cells, or tissue), their related products, or technical devices are also encouraged. The Editors will consider papers for publication based on novelty, their impact on biotechnological production and their contribution to the advancement of bioprocess and biosystems engineering science. Submission of papers dealing with routine aspects of bioprocess engineering (e.g., routine application of established methodologies, and description of established equipment) are discouraged.