Abstract
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.
Highlights
Pullulan is a linear glucan of maltotriosyl units (Glcα1-4Glcα1-4Glc) linked through α-1,6 glucosidic linkages
They are: (i) pullulan hydrolase type I which hydrolyzed α-1,4 glycosidic linkage of pullulan to form panose; (ii) pullulan hydrolase type II which acts on pullulan to form isopanose; (iii) pullulan hydrolase type III which attacks α-1,4 as well as α-1,6 glycosidic linkages in pullulan forming a mixture of maltotriose, panose and maltose; (iv) pullulanase type I
Among pullulanase type II, amylopullulanases are interesting in starch processing industry due to the specific debranching capacity of hydrolyzing either α-1,6 and α-1,4 glycosidic linkages
Summary
Pullulan is a linear glucan of maltotriosyl units (Glcα1-4Glcα1-4Glc) linked through α-1,6 glucosidic linkages. Assay of pullulanase activity: Enzyme activity was determined by measuring the enzymatic release of reducing sugar from pullulan [16]. In this assay, 0.5 mL of enzyme sample was added into 0.5 mL of 1% (w/v). The reaction mixtures containing the enzyme sample and pullulan as a substrate were incubated at 55°C for 30 min in 0.02 M sodium phosphate buffer, pH 6.9. End product analysis: The pullulanase sample (10 mL) was added into different substrates (pullulan, soluble starch and amylopectin) at a concentration of 1% (w/v) in 0.02 M sodium phosphate buffer, at pH 6.9. Standard sugar mixtures containing pure glucose, maltose, maltotriose, up to maltoheptaose (DP7) were used for the generation of calibration curves; DP denotes degree of polymerization
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