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. 1990 Apr;56(4):881–886. doi: 10.1128/aem.56.4.881-886.1990

Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A

Badal C Saha 1, Raphael Lamed 1, Chan-Yong Lee 1, Saroj P Mathupala 1, J Gregory Zeikus 1,*
PMCID: PMC184316  PMID: 16348174

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.

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Selected References

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