Purification and characterization of Stenotrophomonas maltophilia-derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan

Abstract

In the present study, we purified α-amino acid ester hydrolase (AEH) from cell-free extracts of the strain HS1. The approximately 70-kDa AEH from 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.

Characterization and Thermal Denaturation Kinetic Analysis of Recombinant l-Amino Acid Ester Hydrolase from Stenotrophomonas maltophilia

“…S. maltophilia HS1 was grown at 30 °C as described previously . Chromosomal DNA was obtained using phenol/chloroform extraction and used as the template for polymerase chain reaction (PCR).…”

“…S. maltophilia HS1 was grown at 30 °C as described previously. 18 Chromosomal DNA was obtained using phenol/chloroform extraction and used as the template for polymerase chain reaction (PCR). The pET21a plasmid (Novagen) was used as an expression vector, and E. coli XL10-gold (Agilent Technologies) and E. coli Rosetta-gami B(DE3) (Novagen) were used for cloning and recombinant protein expression, respectively.…”

“…By contrast, however, reports on recombinant AEHs are limited because the attempts made to clone an aeh gene have been unsuccessful. , To date, recombinant AEHs from Xanthomonas citri , A. turbidans , and X. campestris pv campestris have been characterized, but there remains great interest in further expanding the number of characterized species isoforms in this class of enzymes to enable improvement in their properties by combinatorial or data-driven protein engineering techniques. The cloning of aeh genes and the overproduction of their products would be particularly worthwhile because their expression level in their natural hosts is low, varying from 0.5% to 3% of total cellular protein. , …”

“…The cloning of aeh genes and the overproduction of their products would be particularly worthwhile because their expression level in their natural hosts is low, varying from 0.5% to 3% of total cellular protein. 17,18 Previously, we screened microorganisms exhibiting AEH activity that can synthesize dipeptides such as Ile−Trp from Ile−OMe and Trp and isolated several bacteria with this property. Among the isolates, Stenotrophomonas maltophilia HS1 exhibited higher AEH activity than the other isolates.…”

“…Among the isolates, Stenotrophomonas maltophilia HS1 exhibited higher AEH activity than the other isolates. The fermentation conditions for AEH production were investigated, 18 but AEH production was insufficient to produce a material for further investigation, such as conducting detailed kinetic studies with highly purified protein or assessing its application to dipeptide synthesis. In this study, we thus aimed to clone the aeh gene from S. maltophilia HS1 and to characterize its recombinant AEH protein, which could potentially be useful for the synthesis of the dipeptide Ile− Trp, an important antihypertensive agent.…”