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. 2013 Feb;3(1):19-27.
doi: 10.1007/s13205-012-0065-5. Epub 2012 Jun 15.

Isolation of a thioesterase gene from the metagenome of a mountain peak, Apharwat, in the northwestern Himalayas

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Isolation of a thioesterase gene from the metagenome of a mountain peak, Apharwat, in the northwestern Himalayas

Avneet Kour Sudan et al. 3 Biotech. 2013 Feb.

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.

Keywords: Metagenomics; Multiple displacement amplification (MDA); Phi polymerase (φ); Thioesterase; α/β Hydrolase fold superfamily.

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Figures

Fig. 1
Fig. 1
Construction of metagenomic library from Apharwat soil. a Standardisation of isolation of metagenomic DNA from Apharwat soil. 1 λ DNA (250 ng/μl). 2 Metagenomic DNA from Zhou’s method (100 ng/g of soil). 3 Metagenomic DNA from Pang’s method (250 ng/g of soil). 4 Metagenomic DNA from Wechter’s method (75 ng/g of soil). b (i). Multiple displacement amplification applied on Apharwat soil DNA with varying concentrations of enzymes. Lane 1 Apharwat DNA with 0.2 U/μl of enzyme. Lane 2 Apharwat DNA with 0.35 U/μl of enzyme. Lane 3 Apharwat DNA with 0.5 U/μl of enzyme. Lane 4 Apharwat DNA loaded 10 ng/μl (not visible) as control b. ii Clone Aph4 showing esterase activity on tributyrin plate. c Insert confirmation of the positive clone. Lane 1 1-Kb Marker. Lane 2 pUC19 without insert. Lane38 transformants digested with HindIII and EcoR1. Arrow depicting Aph4 insert
Fig. 2
Fig. 2
Position of ORFs located on the Aph4 sequence, drawn on the basis of ORF finder results. (1) ORF 1 395 bp, (2) ORF 2 1,286 bp, (3) ORF 3 1,178 bp
Fig. 3
Fig. 3
a Alignment between Aph4 and other known thioesterases using ClustalX.1 ACZ63623.1 thioesterase (P. reinekei).2 GAA16450.1 thioesterase (P. aeruginosa).3 Aph4. The arrow depicts the catalytic triad, Ser137 Asp257 His350. b Phylogenetic tree of Aph4 and known thioesterases. The protein sequence of Aph4 and three thioesterases were aligned and the phylogenetic tree was generated using CLC Sequence Viewer software
Fig. 4
Fig. 4
a ClustalW of Aph4 and known lipases having G-X-S-X-G conserved sequence. Lipase P. Fluorescens (gi.AAA25882.1), lipase P. sp.7323 (gi.CAJ76166.1), lipase Prochlorococcus phage P-SSM-2 (gi.Yp_214431.1), lipase Thermomyces lanuginosus (gi.CAB58509.1), thioesterase component of yersiniabactin synthetase gene (B6VKU7), lysosomal thioesterase (Q6GNY7) and palmitoyl protein thioesterase (P45478). b Phylogenetic tree of Aph4, known lipases and known thioesterase that have same GXSXG motif generated by using Mega5 software

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