Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1

Abstract

Objective: This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1.

Methods: Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.

Results: The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T₅₀¹⁵) of 61 °C and the half-life at 50 °C (t_{1/2, 50 °C}) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu²⁺, Ag⁺, and anionic surfactants. Its kinetic parameters (K_m, k_cat, and k_cat/K_m) were 18.7 mmol/L, 222.3 s^-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.

Conclusions: The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.

Keywords: 3-Dihydrobutanedioic acid; Characterization; Cloning; Oxiranedicarboxylate hydrolase (ORCH); L(+)-2.

Fig. 1

Purification and analysis of ORCH from Labrys sp. WH-1 (a) Purification of the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. Samples were taken at different stages of ORCH purification (Table 1) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lanes: M, protein molecular weight standards; 1, crude enzyme; 2, 40%–60% saturated (NH₄)₂SO₄ precipitate; 3, pooled fractions after diethylaminoethyl (DEAE)-Sepharose chromatography; 4, pooled fractions after phenyl-Sepharose chromatography; 5, pooled fractions after Sephadex G100 chromatography. (b) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of the ORCH from Labrys sp. WH-1. (c) Size exclusion chromatography profile. The peak at 10.05 min corresponds to a molecular mass of 30 kDa. Inset, the standard curve (see Section 2.1). (d) Capillary isoelectric focusing profile of ORCH from Labrys sp. WH-1 and standard pI markers

Fig. 2

Effects of temperature, pH, and various reagents on the activity of ORCH from Labrys sp. WH-1 (a) The optimum temperature for enzyme activity and the thermal stability of purified oxiranedicarboxylate hydrolase (ORCH). The optimum temperature was determined by varying the temperature at pH 8.0. Thermal stability was determined after preincubation of purified ORCH at different temperatures for 30 min at pH 8.0. (b) The temperature at which the activity of the enzyme decreased by 50% in 15 min (T ₅₀ ¹⁵) was determined as follows: purified ORCH was heated for 15 min at different temperatures in thin-walled tubes, cooled immediately on ice, and the residual activity was measured. (c) The half-life at 50 °C (t _{1/2, 50 °C}) was determined by incubating purified ORCH in a water bath at 50 °C for various time, cooling immediately in an ice-water bath, and measuring the residual activity. (d) The optimum pH for enzyme activity and the pH stability of purified ORCH. The optimum pH was determined by assay at various pH values at 37 °C. pH stability was investigated by preincubation of ORCH in different buffers for 30 min at 25 °C. (e) Effects of metal ions (1 mmol/L) and chemicals (0.1% (1 g/L), except for 20 mmol/L ethylenediaminetetraacetic acid (EDTA)-Na₂) on the activity of purified ORCH. The activity of the ORCH from Labrys sp. WH-1 in standard reaction conditions (37 °C, pH 8.0) was (231±10.3) U/mg, and the residual activity was taken as 100%. Each value is expressed as mean±standard deviation (SD), n=3. SDS: sodium dodecyl sulfate-polyacrylamide; DTT: dithiothreitol

Fig. 3

Amino acid sequence alignments for ORCHs from different species The sequences of oxiranedicarboxylate hydrolases (ORCHs) from Labrys sp. WH-1 (this study), Klebsiella sp. BK-58 (GenBank accession No. KF977193) and Rhodococcus sp. ML-0004 (GenBank accession No. DQ471957) were aligned with ClustalW2. Identical amino acids are marked with an asterisk, conserved substitution residues with a colon, semiconserved substitution residues with a period. The predicted secondary structure elements of ORCHs by PredictProtein are shown. α-Helices are shaded black and β-sheets are shaded gray. The indispensable catalytic amino acids for Klebsiella and Rhodococcus are shown in the box