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 (pl) 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.

概要

目 的

克隆双头菌 WH-1 环氧乙烷二酸水解酶(ORCH) 的基因并研究其酶学性质。

创新点

首次获得双头菌 ORCH 的基因, 且该酶催化效率 高, 热稳定性好。

方 法

柱层析纯化 ORCH 后, 进行酶学性质研究; 通过 蛋白末端测序和PCR 获得其基因序列; 通过二级 结构预测和多序列比对进行 ORCH 序列分析。

结 论

来源于双头菌 WH-1 的 ORCH 是迄今报道的催化 效率和热稳定性最好的 ORCH, 为 l(+)-2,3-二羟 基丁二酸的生产提供了新的催化剂。