Purification and characterization of a cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3

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Abstract

Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn2+ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp.

Introduction

Optically pure vicinal diol compounds, which are highly versatile chiral synthons and often used in the synthesis of biologically active molecules, are becoming increasingly important to the chemical and pharmaceutical industries [1]. One of the practical processes to obtain such building blocks is enantioselective hydrolysis of the corresponding epoxides catalyzed by epoxide hydrolases (EHs, EC 3.3.2.3).1 Because this enzymatic process is usually conducted at mild conditions with a high transformation rate and enantioselectivity with a great variety of EH sources, the application of EHs in chemical synthesis has become a focus of the organic chemical industry.

EHs can catalyze the conversion of epoxides to the corresponding diols by addition of a water molecule. They are found ubiquitously in mammals, insects, plants and microorganisms [2]. Recently, microbial EHs have been received considerable attention as they can be produced on an almost unlimited scale [3]. A number of microbial EHs have been purified and characterized to determine their applicability to enantioselective transformation [4], [5], [6], [7], [8], [9].

One of the potential applications of EHs in biotransformation is producing l(+)- or d(−)-tartaric acid. These enantiomeric tartaric acids are well-known organic chiral auxiliaries and building blocks [10], [11] that have a broad application in the pharmaceutical and food industries. At present, l(+)-tartaric acid is produced as a byproduct of the wine industry from lees and argols or by biotransformation with immobilized microorganisms on an industrial scale [12]. However, d(−)-tartaric acid rarely exists in natural resources [13] and the cost of production by chemical resolution is high. If some EHs able to produce d(−)-tartaric acid were available and could be applied in the manufacturing process, it would make the production of d(−)-tartaric acid much simpler and more economical.

The EHs that catalyze cis-epoxysuccinic acid hydrolysis to form d(−)-tartaric acid as well as l(+)-tartaric acid were also named cis-epoxysuccinic acid hydrolases (Fig. 1). At present most of the cis-epoxysuccinic acid hydrolases produced by microorganisms can only produce l(+)-tartaric acid [12] and only three microorganisms, Pseudomonas putida, Alcaligenes sp. and Bordetella sp., were reported to possess enzymes with the ability to catalyze the hydrolysis of cis-epoxysuccinic acid to form d(−)-tartaric acid [14], [15], [16]. Currently there have been few studies on the characteristics of these d(−)-tartaric acid-transforming EHs. Although the cis-epoxysuccinic acid hydrolase from Alcaligenes sp. was overexpressed in Escherichia coli for d(−)-tartaric acid production by Asai et al. [14], and it was found that the enzyme is a heterodimer with a molecular mass of 80 kDa, the detailed physiological properties of this enzyme were not determined.

In previous work, a Bordetella sp. strain 1–3 was isolated in our laboratory and found to have the ability to produce d(−)-tartaric acid from cis-epoxysuccinic acid [15]. In this paper, the cis-epoxysuccinic acid hydrolase from that strain was purified to homogeneity and the properties of the enzyme were studied.

Section snippets

Organism and culture conditions

A Bordetella sp. strain 1–3 producing the cis-epoxysuccinic acid hydrolase was isolated from soil, which was grown on the basal medium slant at 30 °C for 36 h and then stored at 4 °C. The basal medium contained 10 g/L cis-epoxysuccinic acid, 10 g/L yeast extract, 0.5 g/L KH2PO4, 2 g/L K2HPO4·3H2O, 0.5 g/L MgSO4·7H2O, and 10 ml/L trace element solution at pH 7.0. The trace element solution consisted of 1.5 g/L MgSO4·7H2O, 1.0 g/L NaCl, 0.5 g/L FeSO4·7H2O, 0.5 g/L ZnSO4·7H2O, 0.5 g/L MnSO4·H2O, 0.05 g/L CuSO4·5H

Enzyme purification

When strain 1–3 was cultured in the 10-L bioreactor with cis-epoxisuccinic acid as inducer, the cis-epoxysuccinic acid hydrolase was induced and the induction level reached 0.28 U/mg. Total 34,000 U of this hydrolase was obtained after incubation for 36 h and the enzyme was purified 177-fold to homogeneity. The purity of the enzyme after each purification step was examined by SDS–PAGE and the results are summarized in Fig. 2 and Table 1. In the first step of purification, ammonium sulfate

Discussion

The EH from Bordetella sp. strain 1–3 has been shown to be a highly enantioselective enzyme (even at unpurified state [15]) for the production of d(−)-tartaric acid with cis-epoxysuccinic acid as the substrate. So far, only a few enzymes involved in microbial biotransformation of cis-epoxysuccinic acid to d(−)-tartaric acid have been characterized. In this study, this EH was purified and its main characteristics were investigated.

EHs are a group of enzymes obtained from different sources. The

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