Project description: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.
Project description:Labrys filiformis n. sp., the second species of the rare genus Labrys , was recovered from natural forests of Gilan province and is described based upon morphological and molecular characters. The new species is characterized by its smooth cuticle under light microscopy, lateral field with two incisures forming a single plain band, lip region continuous with body contour, dorso-ventrally flattened and forming four poorly prominent lobes, having a dorso-ventrally narrower protuberant labial plate laterally extended to the amphidial margins, oral area (oral plate) dorso-ventrally elongated and embedded in the labial plate with six small labial sensilla surrounding the slightly prominent oral aperture, amphidial apertures as longitudinally lemniscatic slits bordered by the labial plate extensions which are overlapped at the middle length of amphids, stylet delicate, 6 to 7?µm long, elongate weakly developed fusiform median bulb with weak valve, wide excretory pore with long and heavily sclerotized duct, offset spermatheca filled with small spheroid sperm cells, 106 to 127?µm long elongate-conoid tail with filiform distal region and finely rounded tip. Molecular phylogenetic analyses were performed using a near-full length fragment of the 18S rDNA and the D2-D3 expansion segments of the 28S rDNA using Bayesian inference and maximum likelihood methods. In the inferred phylogenetic tree with 18S rDNA, the new species has a close affinity with several isolates of the type species, Labrys chinensis . The reconstructed phylogenetic tree using partial 28S rDNA, revealed the new species is nested inside the putative monophyletic group of several populations of L. chinensis .