Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E.
The bifunctional activities of alpha-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E. An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system. The optimum pH of the recombinant amylopullulanase was 6.0. The most stable pH for the alpha-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the alpha-amylase activity was 90 degrees C, while its most stable temperature was 80 degrees C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75 degrees C, respectively. Pullulan was found to be the best substrate for the enzyme. The enzyme was activated and stabilized by the presence of Ca2+, whereas EDTA, N-bromosuccinimide, and alpha-cyclodextrin inhibited its bifunctional activities. A malto-2-4-oligosaccharide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen. A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase. Kinetic analysis of the enzyme indicated that the Km values of alpha-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the Vmax values were 39 and 98 micromol/(min x mg of protein), respectively.
Lin
Fu-Pang
FP
Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung, Republic of China. fpl5505@mail.ntou.edu.tw
Leu
Kuen-Lin
KL
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C082507:amylopullulanase