Identification and Characterization of a Novel Thermostable gh-57 Gene from Metagenomic Fosmid Library of the Juan De Fuca Ridge Hydrothemal Vent

Abstract

A novel glycoside hydrolases family 57 gene (gh-57) was found from a metagenomic fosmid library constructed from a black smoker chimney sample 4143-1 from the Mothra hydrothermal vent at the Juan de Fuca Ridge. Sequence and homology analysis using BLAST revealed that it had high similarity to gh-57 family. Conserved domain research revealed that the novel gh-57 contained a Glyco-hydro-57 domain and five conserved regions, including two putative catalytic residues Glu¹⁵⁴ and Asp²⁶³. The three-dimensional features of the protein and its homologue from Pyrococcus horikoshii OT3 known as α-amylase were generated by homology modeling. The gh-57 gene was cloned, expressed, and purified in Escherichia coli using pQE system. Enzyme activity revealed that the recombinant protein could hydrolyze soluble starch and demonstrated amylase activity. It showed an optimal pH of 7.5, an optimal temperature of 90 °C, and its thermostability at 90 °C could remain over 50% enzyme activity for 4 h. The enzyme activity could be increased by DTT and Mg²⁺ while an inhibitory effect was observed with EDTA, ATP, and Ca²⁺. These results showed that the gh-57 gene was a novel thermostable amylase from oceanic microorganisms.

Two potentially novel amylolytic enzyme specificities in the prokaryotic glycoside hydrolase α-amylase family GH57

“…no. F4ZW71; Wang et al, 2011) and the potential maltogenic amylase from P. furiosus (UniProt accession no. Q8U2G5; Comfort et al, 2008).…”

“…A threedimensional model for the non-specified amylase from an uncultured bacterium (UniProt accession no. F4ZW71; Wang et al, 2011) was created with the Phyre-2 server (Kelley & Sternberg, 2009) using the experimentally determined branching enzyme structure from Thermococcus kodakaraensis (PDB code 3N92; Santos et al, 2011) and the model of the maltogenic amylase from P. furiosus (UniProt accession no. Q8U2G5; Comfort et al, 2008) was then obtained from the SwissModel server (Arnold et al, 2006) using the modelled structure of the non-specified amylase from an uncultured bacterium as template.…”

Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57

“…In fact, five defined enzyme specificities have been classified within the family GH57 (Janecek 2010): a-amylase (EC 3.2.1.1; hydrolysis of a-1,4-glucosidic linkages in starch and related a-glucans), amylopullulanase (EC 3.2.1.1/41; hydrolysis of both a-1,4 and a-1,6 linkages in starch, pullulan and other related a-glucans), branching enzyme (EC 2.4.1.18; formation of a-1,6-branching points in glycogen and amylopectin), 4-a-glucanotransferase (EC 2.4.1.25; disproportionation of a-1,4-glucosidic linkages in a-glucans), and a-galactosidase (EC 3.2.1.22; release of galactose from melibiose and raffinose). However, based on evolutionary comparison (Zona et al 2004;Janecek 2005) and preliminary biochemical studies (Comfort et al 2008;Wang et al 2011) one may expect that additional specificities will be confirmed in the future.…”

Deciphering the role of Paenibacillus strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès

“…Furthermore, hydrolysis of polymers is of great interest for industries as it can be used in a wide range of applications, from paper industries to biofuel production [7]. For these reasons and because of the development of metagenomic approaches, many studies focused on the screening for new polymer-degrading enzymes [8-10]. Therefore, function-based screening of a DNA library allowed the discovery of various enzymes such as amylases, cellulases and lipases.…”