Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183
Extracellular pullulanase (pullulan 6-glucanohydrolase, EC 188.8.131.52) was purified from cell free culture supernatants of Thermoanaerobium Tok6-B1 by ammonium sulphate precipitation, affinity precipitation, gel exclusion and ion exchange chromatography. A final purification factor of over 1600 was achieved. A molecular weight of 120 kD was determined by steric exclusion HPLC. Enzyme activity was specifically directed towards the α 1–6 glucosidic linkages of pullulan resulting in 100% conversion to maltotriose and also possessed activity towards α 1–4 linkages of starch, amylopectin and amylose producing maltooligosaccharides (DP2-DP4) as products. Maltotetraose was slowly hydrolysed to maltose. Values of K m (% w/v) were 7.3×10-3 for pullulan, 2.7×10-3 for amylopectin and 4.7×10-3 for Lintner's starch. Pullulanase activity was resistant to 6 M urea and was thermostable at temperatures up to 80°C (t 1/2 in the order of hours). Above 80°C thermal denaturation was significant (t 1/2=17 min at 85°C; 5 min at 90°C) but became less so in the presence of substrate (pullulan or starch). Thermostability was greatest at the pH activity optimum (pH 5.5) and was promoted by Ca2+ ions.