Fa Zhang, Muhammad Naeem, Bo Yu, Feixia Liu, Jiansong Ju
{"title":"Improving the enzymatic activity and stability of N-carbamoyl hydrolase using deep learning approach.","authors":"Fa Zhang, Muhammad Naeem, Bo Yu, Feixia Liu, Jiansong Ju","doi":"10.1186/s12934-024-02439-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Optically active D-amino acids are widely used as intermediates in the synthesis of antibiotics, insecticides, and peptide hormones. Currently, the two-enzyme cascade reaction is the most efficient way to produce D-amino acids using enzymes DHdt and DCase, but DCase is susceptible to heat inactivation. Here, to enhance the enzymatic activity and thermal stability of DCase, a rational design software \"Feitian\" was developed based on k<sub>cat</sub> prediction using the deep learning approach.</p><p><strong>Results: </strong>According to empirical design and prediction of \"Feitian\" software, six single-point mutants with high k<sub>cat</sub> value were selected and successfully constructed by site-directed mutagenesis. Out of six, three mutants (Q4C, T212S, and A302C) showed higher enzymatic activity than the wild-type. Furthermore, the combined triple-point mutant DCase-M3 (Q4C/T212S/A302C) exhibited a 4.25-fold increase in activity (29.77 ± 4.52 U) and a 2.25-fold increase in thermal stability as compared to the wild-type, respectively. Through the whole-cell reaction, the high titer of D-HPG (2.57 ± 0.43 mM) was produced by the mutant Q4C/T212S/A302C, which was about 2.04-fold of the wild-type. Molecular dynamics simulation results showed that DCase-M3 significantly enhances the rigidity of the catalytic site and thus increases the activity of DCase-M3.</p><p><strong>Conclusions: </strong>In this study, an efficient rational design software \"Feitian\" was successfully developed with a prediction accuracy of about 50% in enzymatic activity. A triple-point mutant DCase-M3 (Q4C/T212S/A302C) with enhanced enzymatic activity and thermostability was successfully obtained, which could be applied to the development of a fully enzymatic process for the industrial production of D-HPG.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11151596/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microbial Cell Factories","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s12934-024-02439-5","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Optically active D-amino acids are widely used as intermediates in the synthesis of antibiotics, insecticides, and peptide hormones. Currently, the two-enzyme cascade reaction is the most efficient way to produce D-amino acids using enzymes DHdt and DCase, but DCase is susceptible to heat inactivation. Here, to enhance the enzymatic activity and thermal stability of DCase, a rational design software "Feitian" was developed based on kcat prediction using the deep learning approach.
Results: According to empirical design and prediction of "Feitian" software, six single-point mutants with high kcat value were selected and successfully constructed by site-directed mutagenesis. Out of six, three mutants (Q4C, T212S, and A302C) showed higher enzymatic activity than the wild-type. Furthermore, the combined triple-point mutant DCase-M3 (Q4C/T212S/A302C) exhibited a 4.25-fold increase in activity (29.77 ± 4.52 U) and a 2.25-fold increase in thermal stability as compared to the wild-type, respectively. Through the whole-cell reaction, the high titer of D-HPG (2.57 ± 0.43 mM) was produced by the mutant Q4C/T212S/A302C, which was about 2.04-fold of the wild-type. Molecular dynamics simulation results showed that DCase-M3 significantly enhances the rigidity of the catalytic site and thus increases the activity of DCase-M3.
Conclusions: In this study, an efficient rational design software "Feitian" was successfully developed with a prediction accuracy of about 50% in enzymatic activity. A triple-point mutant DCase-M3 (Q4C/T212S/A302C) with enhanced enzymatic activity and thermostability was successfully obtained, which could be applied to the development of a fully enzymatic process for the industrial production of D-HPG.
期刊介绍:
Microbial Cell Factories is an open access peer-reviewed journal that covers any topic related to the development, use and investigation of microbial cells as producers of recombinant proteins and natural products, or as catalyzers of biological transformations of industrial interest. Microbial Cell Factories is the world leading, primary research journal fully focusing on Applied Microbiology.
The journal is divided into the following editorial sections:
-Metabolic engineering
-Synthetic biology
-Whole-cell biocatalysis
-Microbial regulations
-Recombinant protein production/bioprocessing
-Production of natural compounds
-Systems biology of cell factories
-Microbial production processes
-Cell-free systems