Recombinant expression and characterization of an organic-solvent-tolerant α-amylase from Exiguobacterium sp. DAU5.
The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545 bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57 kDa. The enzyme exhibited maximal activity at 40 °C in pH 8.5 glycine-NaOH buffer, and the activity was strongly inhibited by Zn(2+), Cu(2+), and Fe(2+). The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.
Department of Biotechnology, Dong-A University, Busan, South Korea.
Jie
J
Chang
Yong-Suk
YS
Lee
Shu-Jun
SJ
Fang
In-Hye
IH
Park
Yong-Lark
YL
Choi
D056449:Bacillales / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D012997:Solvents / Q000494:pharmacology*; D013213:Starch / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D008670:Metals; D009930:Organic Chemicals; D011994:Recombinant Proteins; D012997:Solvents; D013213:Starch; D000516:alpha-Amylases