Abstract
The soil metagenome of Apharwat (latitude 34.209° and longitude 74.368°) was explored for the presence of esterase encoding genes using a cultivation-independent approach, metagenomics. Among the various protocols tested, the method developed by Wechter was found to be the best for metagenome isolation from the soil under investigation. The purity of the isolated metagenomic DNA was not suitable for gene cloning. To improve the yield and purity of isolated metagenomic DNA, isothermal amplification of the isolated metagenomic DNA using phi (φ) polymerase in a strand displacement technique was performed. The amplified DNA was comparatively pure and the yield increased 50-fold. A metagenomic library was constructed in Escherichia coli (DH5α) using pUC19 as a vector with an average insert size ranging between 2 and 5 kb. Out of 10,000 clones generated, one clone carrying a ~1,870-bp insert hydrolysed tributyrin, indicating esterase activity. Sequence analysis revealed that the insert harboured three open reading frames (ORFs), of which ORF 3 encoded the esterase. Open reading frame 3 comprises 1,178 bp and encodes a putative 392 amino acid protein whose size correlates with most of the bacterial esterases. The esterase isolated in the present study is suggested to be a 4-methyl-3-oxoadipyl-CoA thioesterase (Accession No. JN717164.1), as it shows 60 % sequence similarity to the thioesterase gene of Pseudomonas reinekei (Accession No. ACZ63623.1) by BLAST, ClustalX and ClustalW analysis.
Highlights
Thioesterases (EC 3.1.2.23) are a large enzyme family that catalyse the hydrolysis of the thioester bond between a carbonyl group and a sulphur atom to liberate free fatty acids and thiols [coenzyme A (CoA) and acyl carrier protein]
A metagenomic library was constructed in Escherichia coli (DH5a) using pUC19 as a vector with an average insert size ranging between 2 and 5 kb
The esterase isolated in the present study is suggested to be a 4-methyl-3-oxoadipyl-CoA thioesterase (Accession No JN717164.1), as it shows 60 % sequence similarity to the thioesterase gene of Pseudomonas reinekei (Accession No ACZ63623.1) by BLAST, ClustalX and ClustalW analysis
Summary
Thioesterases (EC 3.1.2.23) are a large enzyme family that catalyse the hydrolysis of the thioester bond between a carbonyl group and a sulphur atom to liberate free fatty acids and thiols [coenzyme A (CoA) and acyl carrier protein]. Two thioesterases (type I and type II) play equal roles. Type I thioesterases are responsible for removing the final product from the biosynthetic complex and type II thioesterases are reported to perform housekeeping functions, such as removing aberrant units from carrier domains. Thioesterases are classified into 23 families, which are further characterised on the basis of sequence and threedimensional structure similarity in the newly compiled ThYme database (Jing et al 2011). Thioesterases have roles in the processing of secondary metabolites such as antibiotics, immunosuppressants, antitumour agents and other bioactive compounds (Ansari et al 2004). A recent major application of thioesterases is the overproduction of biodiesel from fatty acids using microbial thioesterases (Steen et al 2010)
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