Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1.
- Author:
Wen-Na BAO
1
;
Zi-Sheng LUO
1
;
Shi-Wang LIU
2
;
Yuan-Feng WU
2
;
Pei-Lian WEI
2
;
Gong-Nian XIAO
2
;
Yong LIU
2
Author Information
- Publication Type:Journal Article
- Keywords: 3-Dihydrobutanedioic acid; Characterization; Cloning; Oxiranedicarboxylate hydrolase (ORCH); L(+)-2
- MeSH: Alphaproteobacteria/enzymology*; Cloning, Molecular; Dicarboxylic Acids/metabolism*; Enzyme Stability; Epoxide Hydrolases/metabolism*
- From: Journal of Zhejiang University. Science. B 2019;20(12):995-1002
- CountryChina
- Language:English
-
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 (t1/2, 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 (Km, kcat, and kcat/Km) 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.
