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

Isolation and in silico characterization of novel esterase gene with β-lactamase fold isolated from metagenome of north western Himalayas

“…Three clones showing esterase activity namely Aph2, Aph4 and Aph7 were isolated and preserved. An esterase-producing clone Aph4 has already been characterised (Sudan and Vakhlu 2012 ). In the present study, characterization of another esterase-producing clone Aph2 is being reported (Fig.…”

“…In the present study, DNA isolated using Wechter protocol (2002) contained impurities that hampered the manipulation of DNA. To reduce the interfering impurities and to increase the quantity of DNA, whole metagenome was diluted 50-fold and then multiple displacement amplification (MDA) was performed using phi (φ)29 DNA polymerase as per the method standardised previously (Sudan and Vakhlu 2012 ). Subsequent cloning and transformation generated 10,000 clones (Sudan and Vakhlu 2012 ).…”

Phylogenetic and functional diversity of soil prokaryotic communities in temperate deciduous forests with different tree species

“…Functional screening recovered 41 bacterial clones with potential lipolytic activity (Table 3.2). Insert size for these clones ranged from 1,350 -7,250 bp, and demonstrates the range of lipase-encoding genes, which falls in this range (Sudan and Vakhlu, 2013). Interestingly, most positive clones were detected in libraries from the summer (32 clones) plasmid libraries, compared to spring (5 clones) and autumn (4 clones) libraries.…”

“…Further experiments to determine the substrate affinity of proteins encoded by Lip3 and Lip49 are required to confirm whether the proteins belong in their respective families. The limitations encountered are generally unrelated to the lack of the corresponding genes in the soil environment, as function-based screening is not an indicator of abundance (Sudan and Vakhlu, 2013). Several, interdependent factors such as cloning host/vector compatibility, protein expression quality and quality of source environmental DNA can introduce heavy bias in the process (Gaida et al, 2015;Uchiyama and Miyazaki, 2009).…”

“…In addition to the closest related protein, a representative of each family of esterases was included, based on the classification scheme outlined by Arpigny and Jaegger (1999).3.4.2.2 Further screening for lignocellulolytic functionActivity-based screening conducted for laccase, xylanase and cellulases did not yield positive clones. The limitations encountered are generally unrelated to the lack of the corresponding genes in the soil environment, as function-based screening is not an indicator of abundance(Sudan and Vakhlu, 2013). Several, interdependent factors such as cloning host/vector compatibility, protein expression quality and quality of source environmental DNA can introduce heavy bias in the process(Gaida et al, 2015;Uchiyama and Miyazaki, 2009).…”