Abstract
A procedure for separation of α- and β-amylases was developed which results in their complete resolution in less than 20 min. A cyclodextrin stationary phase column was equilibrated in 10 mM phosphoric acid at pH 7.0. Purified barley malt α- or β-amylases, mixtures of both, or crude malt extracts were injected. β-Amylase did not bind to the column and was rapidly eluted with water or buffer. α-Amylases specifically bind to the immobilized dextrin. Optimal elution of α-amylase was achieved with a 10-ml gradient from 0 mg/ml β-cyclodextrin (cycloheptaamylose) in buffer to 12 mg/ml β-cyclodextrin in 15% aqueous methanol, followed by flushing with 20 ml of 12 mg/ml β-cyclodextrin in 15% aqueous methanol. Elution buffer containing β-cyclodextrin at pH values from 6.0 to 7.0 was not as effective in eluting α-amylase as was cyclodextrin in aqueous methanol. Inclusion of methanol in the gradient resulted in enhanced recoveries of α-amylase. α-Amylase did not bind to the column at pH values higher than 7.0. This procedure should be useful for rapid separation of plant α-andd β-amylases, separation of pullulanases or debranching enzymes from other carbohydrates, and purification of α-amylases.
Original language | English (US) |
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Pages (from-to) | 361-367 |
Number of pages | 7 |
Journal | Journal of Chromatography A |
Volume | 469 |
Issue number | C |
DOIs | |
State | Published - 1989 |
Externally published | Yes |
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Organic Chemistry