Characterization of Pullulanase Type II from Bacillus cereus H1.5
- 1 ,
- 2 , Afganistan
Published On: 31 December 2009
Copyright: © 2020 Hii Siew Ling, Tau Chuan Ling, Rosfarizan Mohamad and Arbakariya B. Ariff. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Problem statement: Pullulanase is one of the important enzymes in starch industry. Search for the pullulanase with distinct features, possibly from easily grown bacterium, is of interest for industrial applications Approach: The extracellular pullulanase produced by Bacillus cereus HI.5 was purified by chromatographic method of DEAE-Sepharose, followed by Superdex gel filtration. The enzyme was characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The enzyme showed optimal activity at 55°C and pH 6.0. The thermostability and the thermoactivity of the enzyme were increased considerably in the presence of Ca2+. In the present of 2 mM Ca2+, the enzyme had half-life duration of more than 2 h at 50°C. Almost all metal ions had a strong inhibitory effect, except Ca2+ and Mn2+. The Ca2+ had a very strong stimulating effect on the enzyme, increasing its activity by 170%. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, where as N-bromosuccinimide and Schardinger dextrins were inhibitors, suggesting that tryptophan and thiol residues may be important for the activity. The apparent Km and Vmax value for pullulan was 1.1 mg mL-1 and 0.275 μmol min-1, respectively. A relative substrate specificity for hydrolysis of pullulan, amylopectin and soluble starch by this pullulanase was 100%, 28.5% and 20.4%, respectively. Conclusion: The enzyme was able to attack specifically the α-1,6 linkages in pullulan to generate maltotriose as the major end product, as well as the α-1,4 linkages in amylopectin and soluble starch leading to the formation of a mixture of maltose and glucose and therefore be classified as a type II pullulanase or an amylopullulanase.
- Bacillus cereus
- substrate specificity