Abstract
Thermostable extracellular pullulanase, produced byBacillus stearothermophilus G-82 was purified to homogeneity from supernatants of continuous culture by ultrafiltration, ammonium sulphate precipitation, chromatography on Sephadex G-100, and DEAE cellulose. A mol wt of 53,000 was determined by gel filtration and 56,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point (pI) was 4.2. The pullulanase contained predominantly acidic amino acids. The enzyme was optimally active at a temperature of 60°C and pH 7.0. It preserved 100% of its activity after 10 min treatment at 60°C. The thermostability was considerably increased in the presence of pullulan. Ca2+ did not increase activity or thermostability. Enzyme activity was fully inhibited byN-bromosuccinimide and partially by phenylmethylsulfonyl fluoride.Bacillus stearothermophilus G-82 pullulanase was able to hydrolyze α1-6 as well as α1-4 glucosidic bonds in pullulan, amylopectin, amylose, glycogen, and dextrin. The enzyme showed highest affinity to pullulan (Km = 0.14).
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Kambourova, M.S., Emanuilova, E.I. Purification and general biochemical properties of thermostable pullulanase fromBacillus stearothermophilus G-82. Appl Biochem Biotechnol 33, 193–203 (1992). https://doi.org/10.1007/BF02921835
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DOI: https://doi.org/10.1007/BF02921835