Rapid high-performance liquid chromatographic separation of barley malt α-amylase on cyclobond columns

Cynthia A. Henson, Julie M. Stone

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)361-367
Number of pages7
JournalJournal of Chromatography A
Volume469
Issue numberC
DOIs
StatePublished - 1989

Fingerprint

Hordeum
Amylases
Cyclodextrins
Liquids
Methanol
Buffers
Enhanced recovery
Complex Mixtures
Purification
Carbohydrates
Water
Enzymes

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Cite this

Rapid high-performance liquid chromatographic separation of barley malt α-amylase on cyclobond columns. / Henson, Cynthia A.; Stone, Julie M.

In: Journal of Chromatography A, Vol. 469, No. C, 1989, p. 361-367.

Research output: Contribution to journalArticle

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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.",
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