Improving the enzyme property of ornithine transcarbamylase from Lactobacillus brevis through site-directed mutation
Introduction
Ethyl carbamate (EC) occurs naturally in most fermented foods and beverages, such as bread, wine, cheese, soy sauce, and vinegar (Li et al., 2017; Park et al., 2007; Wu et al., 2012), and it has been proven to have carcinogenic and mutagenic effects on laboratory animals (Lachenmeier et al., 2010). Accordingly, the determination and production mechanism of EC has attracted attention (Bortoletto & Alcarde, 2015).
Urea, citrulline, cyanic acid, and carbamoyl phosphate were reported to participate in the formation of EC; urea and citrulline were the major precursors (Fang et al., 2013). The generation of citrulline is attributed to the metabolism of arginine by lactic acid bacteria via the arginine deiminase pathway (ADI pathway) (Liu et al., 1994; Arena et al., 1999). There are three enzymes: arginine deiminase (ADI), ornithine transcarbamylase (OTC), and carbamate kinase (CK), that participate in the pathway (Arena et al., 1999; Liu & Pilone, 1998) (Fig. 1). Regarding the safety issues of fermented beverages, the metabolism of arginine is not desirable for citrulline formation, but this process is coupled with ATP and ammonia formation, both of which are crucial for cellular growth and sensory quality through energy provision and pH regulation, respectively (Wibowo et al., 1985). Ornithine transcarbamylases (OTC) participate in the degradation of arginine by catalyzing the formation of ornithine and carbamoyl phosphate from citrulline.
Various methods have been adopted to eliminate EC formation, while enzymatic decomposition methods have been widely employed because of their safety and environmentally friendly characteristics. Research seldom focuses on OTC which can degrade citrulline and instead has focused on two other types of enzymes that are most commonly utilized namely urease, which can degrade urea and is now commercially available (Fidaleo et al., 2006; Yang et al., 2010), and urethanase, which can directly catalyze EC degradation (Chun et al., 1991; Mohapatra & Bapuji, 1997; Zhao and Kobashi, 1994; Zhou et al., 2013).
According to previous research, OTC has a negative relationship with EC concentration (Fang et al., 2013, 2015). In this study, we focused on increasing OTC enzyme activity or stability based on site-directed mutagenesis. Compared to the traditional methods for enhancing protein thermostability or activity, targeted mutagenesis guided by sequence or structure has emerged as a popular protein-engineering route, which could potentially reduce efforts in protein manufacturing and formulation (Asial et al., 2013). Discovery Studio is a software that can perform rational design based on protein 3D structure by calculating the interaction energy. After alanine scanning and saturation mutagenesis, H140, Q143, and D236 sites were mutated in order to increase enzyme activity or thermostability.
Section snippets
Strains and reagents
The wild-type LB-OTC cDNA gene, including the BamHI and SalI restriction sites, was stored in our laboratory. The recombinant plasmid pET28a-LB-OTC was used as a template for site-directed mutagenesis. Primer synthesis and DNA sequencing were completed using TsingKe Biological Technology (Nanjing, China). High-fidelity PrimeSTAR® Max DNA polymerase and Dpn1 were purchased from Takara Biotechnology (Kyoto, Japan). The QuickPure Plasmid Mini purification kit, PCR clean-up kit, Ni-NTA-Sefinose™
Selection of sites for site-directed mutation
Based on sequence alignment, LB-OTC displayed high identity with another reported OTC enzyme, which shares the highest sequence identity of 97% with OTC from Lactobacillus hilgardii. The catalytic domain ranged from residues 46 to 255 according to CD-Search (Search for Conserved Domains Service) of NCBI (Blanca et al., 2009). Three conserved residues: histidine (His140), glutamine (Gln143), and aspartic acid (Asp236) played a key role in catalytic action (Fig. 1A). The 3D-structure of LB-OTC
Conclusion
Based on the amino acid sequence derived from Lactobacillus brevis OTC, a mutation library was constructed using Discovery Studio software. It was found that the mutation at the H140, Q143, and D236 sites may have certain effects on the catalytic properties or thermal stability of the enzyme. The results showed that the catalytic activity of Q143W and H140A mutants was 2–3 times higher than that of the wild type. The optimal temperature decreased from 55 °C to 35 °C, and the optimal pH changed
Author contributions
In this work, Ruosi Fang designed the whole experiment and acquired funding; Jason Chandra Syahputra and Osasu Airhunmwunde did all the experiment and raw data collection; Yuan Wu was in charge of data curation; Changjiang Lv designed the mutation library with the discovery studio software; Jun Huang conceptualization the whole idea; Gongnian Xiao played a role in original writing of this paper; Qihe Chen reviewed the paper work and helped revised it.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper
Acknowledgements
This work was supported by grants from Zhejiang Natural Science Foundation (Nos. LGN19C200004, LQ18C200003).
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