Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A

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.