Purification and characterization of Stenotrophomonas maltophilia-derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan
In the present study, we purified α-amino acid ester hydrolase (AEH) from cell-free extracts of the Stenotrophomonas maltophilia strain HS1. The approximately 70-kDa AEH from S. maltophilia HS1 (SmAEH) was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-filtration experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experiments, SmAEH had wide substrate specificity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30 °C. SmAEH also showed the greatest stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50 °C. In subsequent activity assays in the presence of various metal ions, Ag+ strongly inhibited enzyme activity. Finally, SmAEH activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that the protein is a serine protease.
Keywordsα-Amino acid ester hydrolase Stenotrophomonas maltophilia Antihypertensive dipeptide Ile-Trp
Compliance with ethical standards
Conflict of interest
The authors declare that there is no conflict of interests.
- Felsenstein J (1989) PHYLIP-phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
- Kato K (1980) Kinetics of acyl transfer by α-amino acid ester hydrolase from Xanthomonas citri. Agric Biol Chem 44:1083–1088Google Scholar
- Kato K, Kawahara K, Takahashi T, Igarasi S (1980) Enzymatic synthesis of amoxicillin by the cell-bound α-amino acid ester hydrolase of Xanthomonas citri. Agric Biol Chem 44:821–825Google Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- Malathi S, Chakraborty R (1991) Production of alkaline protease by a new Aspergillus flavus isolate under solid-substrate fermentation conditions for use as a depilation agent. Appl Environ Microbiol 57:712–716Google Scholar
- Mine Y, Li-Chan E, Jiang B (2011) Bioactive proteins and peptides as functional foods and nutraceuticals, vol 29. Wiley, New YorkGoogle Scholar
- Sheldon RA, van Rantwijk F, van Langen LM, Wegman MA, Cao L, Janssen MHA (2001) Biocatalysts and biocatalysis in the synthesis of ß-Lactam antibiotics. In: Bruggink A (ed) Synthesis of β-Lactam antibiotics. Springer, Dordrecht, Netherlands, pp 102–148Google Scholar
- Yokozeki K, Suzuki S (2010) Method for producing dipeptides. US Patent 7,754,466. Ajinomoto Co Inc, JapanGoogle Scholar