Identification And Characterization Of A Novel Thermostable Glycoside Hydrolase Family 57 Gene

Marine which covers about 70% of Earth's total surface area is rich in microbial resources, and is the natural treasure house of a variety of active substances and genes for enzymes with important application of industrial. Due to the ultra and peculiar environment, more than 95% of marine microorganisms under the existing experimental conditions can not be cultured, which became the bottleneck of development and utilization of marine microbial resources. Study on these microbial resources using metagenomic techniques has become a new hot spot for develope the marine microorganisms'resource.Our research is based on the sequencing data of about 3000 clones from metagenomic fosmid library of Juan de Fuca Ridge hydrothermal vent which is supplied by State Key Laboratory of Biocontrol, Sun Yat-sen University. Through the BLAST program aligment with the known sequence information, we sorted out 127 thermostable gene fragments from the metagenomic library. After prospect for industrial applications, the glycoside hydrolase family 57 (GH-57) (metagenomic library code CL2343 Contigl) with potential industrial applications was selected for activity and function analysis.In this study, technologies were composed of four steps:First of all, the gh-57 gene fragment was screened in Fosmid metagenomic library and then, was sequenced at both sides to obtain the full length of the gene. Secondly, the gh-57 gene sequence and homology were analyzed using BLAST, and the conservation and strcture of the predicted protein was compared and modeled. Thirdly, the gh-57 gene was cloned, expressed and purified in Escherichia coli using pQE system. At last, the recombinant GH-57 protein activity was assayed.The obtained DNA sequence of gh-57 was 1632 bp long encoding a predicted protein of 544 AA long with a molecular weight of 62.2 kDa and isoelectric point of 5.84. The gene sequence has been submitted to GenBank with the accession number of HQ324240. Homology analysis using BLAST revealed that the novel gh-57 showed the maximum nucleic acid sequence identity of 68% to the gh-57 from Geobacter sp. M21 (GenBank accession number CP001661) and Geobacter bemidjiensis Bern (CP001124). The deduced protein showed the maximum amino acid sequence identity of 62% to the GH-57 from Moorella thermoacetica (ABC20110), followed by 60% to the GH-57 from Desulfovibrio fructosovorans JJ (EFL49755). Conserved domain research revealed that the novel gh-57 contained a 304 AA long (from Ile 7 to Glu 310) Glyco-hydro-57 domain (Pfam 030065) in its N-terminal region, which was highly conserved and showed similarity to proteins in GH-57 family. Also, a DUF3536 domain (Pfam 12055) appeared in its C-terminal and it was found in bacteria and archaea with unknown function. Multiple sequence alignments of GH-57 protein members indicated that the novel GH-57 contained five conserved domain regions, two catalytic residues and six highly conserved amino acid residues, is a very typical GH-57 family. Protein structure simulation results also show that the GH-57 protein has a very similar three-dimensional structure with the GH-57 family PH0193 which have been reported with a-amylase activity.After expression and purification, enzyme activity revealed that the recombinant protein could hydrolyze solubale starch and demonstrated amylase activity. It showed an optimal pH of 7.5, an optimal temperature of 90℃, and its thermostability at 90℃could remain 73% enzyme activity for 4 h. What is more, the relative activity could still reach 60.0% at 100℃. In addition, the enzyme activity could be increased by DTT and Mg2+ while an inhibitory effect was observed with EDTA, ATP and Ca2+. The enzyme has a certain dependence of DTT. In a concentration of 30 mM DTT, the relative activity was 5.37 times higher than without DTT, and 0.1 mM EDTA could completely inhibited the enzyme activity. This result was similar with the characteristics of a reported GH-57 protein from the extremthermophilic bacteria Thermotoga maritima MSB8, but was significantly different with the reported mesople a-amylase and other GH-57 family proteins which had a-amylase function.