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Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A

By Badal C. Saha, Raphael Lamed, Chan-Yong Lee, Saroj P. Mathupala and J. Gregory Zeikus

Abstract

A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60°C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75°C and pH 5.0, respectively. Both the enzyme activities were stable up to 70°C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70°C and 45 min at 75°C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by β- and γ-cyclodextrins but not by α-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in α1,6 linkages and produced multiple saccharides from cleavage of α-1,4 linkages in starch. The Kms for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively

Topics: Enzymology and Protein Engineering
Year: 1990
OAI identifier: oai:pubmedcentral.nih.gov:184316
Provided by: PubMed Central
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