Characterization of maltotriose production by hydrolyzing of soluble starch with α-amylase from Microbulbifer thermotolerans DAU221.
A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli, using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 °C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 °C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu(2+), Fe(3+), Hg(2+), Zn(2+), and EDTA, but exhibited extreme stability toward hexane. The K m and V max values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.
Department of Biotechnology, Dong-A University, Busan, 604-714, Republic of Korea.
Yong-Suk
YS
Lee
Dong-Ju
DJ
Park
Yong-Lark
YL
Choi
D020816:Amino Acid Motifs; D002413:Cations, Divalent / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism*; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008670:Metals; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014312:Trisaccharides; C008317:maltotriose; D013213:Starch; D004492:Edetic Acid; D000516:alpha-Amylases