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Cloning, heterologous expression, and characterization of a novel thioesterase from natural sample.

ABSTRACT: A novel thioesterse gene was successfully cloned and sequenced directly from natural sample of Domas Hot Spring, West Java, Indonesia. Homological analysis of the sequence showed that the gene appeared high homology to thioesterase genes with the highest to a putative thioesterase gene from uncultured Acidilobus sp. JCHS at 66% identity. However, phylogenetic analysis showed that the protein was separated from the branch with other known thioesterases. The size of the gene is around 500 base pairs, lied into 2 kb DNA fragment from a random PCR amplicon. The gene was overexpressed in Escherichia coli, a dominant band appeared at 17 kDa in SDS-PAGE with expression level at around 32% of total proteins. The activity of the purified protein using acetyl-CoA as substrate showed that the protein exhibited thioesterase activity. Furthermore, the enzyme also showed esterase activity on p-nitrophenyl ester as substrate. Detail characterization of esterolytic activity showed that the enzyme preferred p-nitrophenyl decanoate as substrate. The optimum activity of the enzyme was at 80 °C and pH 8. Activity of the enzyme was maintained after incubation at 80 °C up to 24 h. In addition, the enzyme was favorable on polar organic solvents. All the data obtained suggested that the enzyme is a novel alkaline thermostable thioesterase.

SUBMITTER: Suharti

PROVIDER: S-EPMC8024609 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Cloning, heterologous expression, and characterization of a novel thioesterase from natural sample.

Suharti Mahardika Gita G Raissa Dewi Laksmi L Yohandini Heni H Widhiastuty Made Puspasari MP Sakti Raden Aditya Wibawa RAW Wahyudi Setyanto Tri ST Akhmaloka

Heliyon 20210324 3

A novel thioesterse gene was successfully cloned and sequenced directly from natural sample of Domas Hot Spring, West Java, Indonesia. Homological analysis of the sequence showed that the gene appeared high homology to thioesterase genes with the highest to a putative thioesterase gene from uncultured <i>Acidilobus sp. JCHS</i> at 66% identity. However, phylogenetic analysis showed that the protein was separated from the branch with other known thioesterases. The size of the gene is around 500 b ...[more]

PMID: 33851045

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Project description:Lysoplasmalogen (LyPls)-specific phospholipase D (LyPls-PLD) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1-(1-alkenyl)-sn-glycero-3-phosphate (lysoplasmenic acid). Little is known about LyPls-PLD and metabolic pathways of plasmalogen (Pls). Reportedly, Pls levels in human serum/plasma correlate with several diseases such as Alzheimer's disease and arteriosclerosis as well as a variety of biological processes including apoptosis and cell signaling. We identified a LyPls-PLD from Thermocrispum sp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/Escherichia coli with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions (pH 8.0 and 50 °C), steady-state kinetic analysis for LyPlsCho yielded Km and kcat values of 13.2 μm and 70.6 s-1, respectively. The ORF of LyPls-PLD gene consisted of 1005 bp coding a 334-amino-acid (aa) protein. The deduced aa sequence of LyPls-PLD showed high similarity to those of glycerophosphodiester phosphodiesterases (GDPDs); however, the substrate specificity differed completely from those of GDPDs and general phospholipase Ds (PLDs). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls-PLD were highly conserved to GDPDs. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls-PLD may participate in the substrate recognition. These findings will help to elucidate differences among LyPls-PLD, PLD, and GDPD with regard to function, substrate recognition mechanism, and biochemical roles.Data accessibilityThermocrispum sp. strain RD004668 and its 16S rDNA sequence were deposited in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE BP-01628 and in the DDBJ database under the accession number AB873024. The nucleotide sequences of the 16S rDNA of strain RD004668 and the LyPls-PLD gene were deposited in the DDBJ database under the accession numbers AB873024 and AB874601, respectively.EnzymeEC number EC 3.1.4.4.

Dataset Information

Cloning, heterologous expression, and characterization of a novel thioesterase from natural sample.

Publications

Cloning, heterologous expression, and characterization of a novel thioesterase from natural sample.

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