title: 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.
authors: Research Center for Molecular Biology, Institutes of Life and Health Engineering, National Engineering and Research Center for Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, People's Republic of China.
authors: Hui
authors: H
authors: Wang
authors: Yingxue
authors: Y
authors: Gong
authors: Wei
authors: W
authors: Xie
authors: Wenjuan
authors: W
authors: Xiao
authors: Junmei
authors: J
authors: Wang
authors: Yangyang
authors: Y
authors: Zheng
authors: Jia
authors: J
authors: Hu
authors: Zehuan
authors: Z
authors: Liu
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000681:Amylases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015698:Genomic Library; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D054892:Metagenome; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D000681:Amylases; D008274:Magnesium; D002118:Calcium