The branching reaction was performed at 50°C in 5 mM PBS buffer (pH 7.0) using 0.125% amylose V (Avebe, The Netherlands) as substrate, and a final concentration of enzyme ranging from 0.03 to 0.13 mg/ml was used depending on the total activity of enzyme. Samples were taken at time intervals to monitor the reaction progress. The reaction was stopped by boiling samples at 100°C for 10 min. Branched samples were treated with Pseudomonas sp. isoamylase (0.4 U/ml) and Klebsiella planticola pulullanase M1 (1.4 U/ml) (Megazyme, Ireland) at 40°C for 24 h with constant mild shaking. Reducing ends were quantified by the 2,2′‐bicinchoninic acid (BCA) method using glucose as the standard.30 The hydrolytic and branching activity units were calculated based on reducing end profiles of samples before debranching and net increase in reducing end profiles, respectively. One unit of activity is defined as 1 μmol reducing ends released or transferred per minute under aforementioned reaction conditions.
Pseudomonas sp isoamylase
Manufactured by Megazyme
Sourced in Ireland
Pseudomonas sp. isoamylase is an enzyme used for the hydrolysis of starch. It catalyzes the cleavage of α-1,6-glycosidic linkages in amylopectin and glycogen.
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2 protocols using pseudomonas sp isoamylase
1
Starch Branching Enzyme Kinetics
2
Branching Enzyme Activity Assay
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The branching reaction was performed at 50 o C in 5 mM PBS buffer (pH7.0) using 0.125% amylose V (Avebe, The Netherlands) as substrate, and a final concentration of enzyme ranging from 0.03 mg/ml to 0.13 mg/ml was used depending on the total activity of enzyme. Samples were taken at time intervals to monitor the reaction progress. The reaction was stopped by boiling samples at 100 o C for 10 min. Branched samples were treated with Pseudomonas sp isoamylase (0.4 U/ml) and Klebsiella planticola pulullanase M1 (1.4 U/ml) (Megazyme, Ireland) at 40 o C for 24 h with constant mild shaking. Reducing ends were quantified by the 2,2'-bicinchoninic acid (BCA) method using glucose as standard 30 . The activity unit was calculated based on reducing end profiles of samples before and after debranching. One unit of activity is defined as 1 μmol reducing ends released or transferred per minute under aforementioned reaction conditions.
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