Abstract
The bifunctional activities of α-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 α-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the α-amylase activity was 90°C, while its most stable temperature was 80°C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75°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 α-cyclodextrin inhibited its bifunctional activities. A malto-2–4-oligosac-charide 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 K m values of α-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the V max values were 39 and 98 µmol/(min · mg of protein), respectively.
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Lin, FP., Leu, KL. Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E. Appl Biochem Biotechnol 97, 33–44 (2002). https://doi.org/10.1385/ABAB:97:1:33
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DOI: https://doi.org/10.1385/ABAB:97:1:33