title: Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A.
abstract: A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60 degrees 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 degrees C and pH 5.0, respectively. Both the enzyme activities were stable up to 70 degrees C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70 degrees C and 45 min at 75 degrees C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by beta- and gamma-cyclodextrins but not by alpha-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 alpha1,6 linkages and produced multiple saccharides from cleavage of alpha-1,4 linkages in starch. The K(m)s for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.
authors.affiliation: Michigan Biotechnology Institute, Lansing, Michigan 48909, and Departments of Biochemistry and Microbiology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: B C
authors.initials: BC
authors.lastname: Saha
authors.forename: R
authors.initials: R
authors.lastname: Lamed
authors.forename: C Y
authors.initials: CY
authors.lastname: Lee
authors.forename: S P
authors.initials: SP
authors.lastname: Mathupala
authors.forename: J G
authors.initials: JG
authors.lastname: Zeikus