Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme.
Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.
Grzybowska
Beata
B
Department of Food Chemistry and Technology, Gdansk University of Technology, ul. Gabriela Narutowicza 11/12, 80-952 Gdansk, Poland.
Szweda
Piotr
P
Synowiecki
Józef
J
D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015202:Protein Engineering / Q000379:methods*; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases