Identification, characterization, substrate binding mode prediction, and modification of a novel amidohydrolase from <i>Microbulbifer thermotolerans</i>.

Nana XU; Mingzhu YAN; Hao WANG; Xiao LIANG; Weidong LIU; Huimin QIN; Jian GAO

Return

Identification, characterization, substrate binding mode prediction, and modification of a novel amidohydrolase from Microbulbifer thermotolerans.

Author: Nana XU ¹ ; Mingzhu YAN ¹ ; Hao WANG ² ; Xiao LIANG ² ; Weidong LIU ² ; Huimin QIN ¹ ; Jian GAO ²
Author Information

1. School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China.
2. State Key Laboratory of Engineering Biology for Low-carbon Manufacturing, National Engineering Research Center of Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Publication Type:Journal Article
Keywords: amidohydrolase; enzymatic properties; expression and purification; ochratoxin A; protein engineering
MeSH: Amidohydrolases/chemistry*; Ochratoxins/metabolism*; Substrate Specificity; Escherichia coli/metabolism*; Recombinant Proteins/metabolism*; Actinomycetales/genetics*
From: Chinese Journal of Biotechnology 2025;41(9):3567-3578
CountryChina
Language:Chinese
Abstract: Ochratoxin A (OTA) is ubiquitous in the food and feed fields. It has strong hepatotoxicity and nephrotoxicity, seriously threatening the health of humans and animals. Enzymatic degradation of mycotoxins is considered to be a promising method to control mycotoxin contaminations. In this study, a new ochratoxin A amidohydrolase from Microbulbifer thermotolerans (MiADH) was obtained. After heterologous expression in Escherichia coli and purification, the recombinant protein was studied regarding the hydrolysis activity, hydrolysis products, enzymatic properties, and substrate binding mode. MiADH can degrade OTA into ochratoxin α (OTα) and phenylalanine, demonstrating a detoxifying ability. It demonstrated the best performance at 70 ℃ and pH 8.0, and Cu²⁺ had the strongest inhibitory effect on the activity of MiADH. MiADH with good thermal stability exhibited huge potential for industrial application. Rational design guided by three-dimensional structural models and substrate docking analysis revealed the important amino acids affecting substrate binding and obtained multiple mutants with improved activity. Among these mutants, V324A had the highest activity, which was 4.2-fold that of the wild type. The identification of MiADH enriches the ochratoxin A degradation enzyme library and provides a new candidate enzyme for the biological detoxification of ochratoxin A in the food and feed industry.