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.

A strategy to enhance and modify fatty acid synthesis in Corynebacterium glutamicum and Escherichia coli: overexpression of acyl-CoA thioesterases

“…In vitro validation might not be consistent with in vivo assays because enzyme activity might be affected by substrate availability and presentation in vivo [ 33 ]. The substrate specificity was influenced by the geometry and hydrophobicity of the catalytic pocket [ 34 ]. Furthermore, TesA from other organisms exhibited lysophospholipase A, protease, and arylesterase activities, in addition to TE activity [ 28 ].…”

Characterization of thermostable serine hydroxymethyltransferase for β-hydroxy amino acids synthesis

“…Cell pellet (100 mg) was resuspended in 1 mL of 50 mM phosphate buffer pH 7.5 (250 mM NaCl, 1 mg/mL lysozyme) and shaken at 150 rpm 25 °C, 2 h. This was followed by heating at 70 °C for 10 min and then a centrifugation process (12,000 x g , 4 °C, 5 min). The purification was undertaken based on a previous method (Suharti et al 2021 ; Ma’ruf et al 2021 ), while the dialysis process was carried out to discard imidazole from the enzyme solution.…”

“…SDS-PAGE analysis was performed according to a previous method (Suharti et al 2021 ). For native PAGE and zymography analysis, the electrophoresis gels were treated without the addition of SDS and the boiling step (Syihab et al 2015 ).…”

“…In addition, thermostable enzymes are commonly resistant to organic solvents, proteases, chaotropic agents, and detergents (Angelaccio 2013 ). Several studies have reported extensive exploration of the thermophilic microorganisms and their enzyme application through cultivation or metagenomic cloning methods (Widhiastuty et al 2009 ; Nurhasanah et al 2015 ; Suharti et al 2014 , 2021 ). Certain thermophilic microorganisms were isolated from the thermogenic phase of the composting process and showed methanol tolerance (Syihab et al 2015 ).…”

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

“…Of those, esterases of high temperature ecosystems have received considerable attention due to their thermostability and the many biotechnological applications these biocatalysts have (Zarafeta et al 2016 ). However, the use of enzymes in industrial processes requires certain specific characteristics (Suharti et al 2021 ). Industrial processes using high temperatures require thermostability, as well as overall tolerance to protein destabilizing agents, such as organic solvents, metal ions, surfactants and others (Zarafeta et al 2016 ).…”