Affinity chromatography of cereal α-amylase

✍ Scribed by M.P. Silvanovich; R.D. Hill

• Publisher: Elsevier Science
• Year: 1976
• Tongue: English
• Weight: 219 KB
• Volume: 73
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(76)90191-3

✦ Synopsis

An affinity chromatography technique for purification of oc-amylase from triticale (X Triticosecale Wittmack) is described. Triticale ol-amylase is retained on a column of cyclohepta-amylose epoxy Sepharose 6B. Contaminating proteins pass through in the sodium acetate elution buffer whereas the cy-amylase is selectively eluted with elution buffer containing cyclohepta-amylose.

The chromatographic step gives a yield in excess of 90% with a purification of up to 180-fold over crude extracts.

The majority of chromatographic research on cereal a-amylases has been performed with barley and wheat. MacGregor et al. ( 1) separated barley a-amylase into two components using carboxymethyl cellulose chromatography.

Kruger and Tkachuk (2) isolated and separated four a-amylases from hard red spring wheat using acetone fractionation, glycogen precipitation, and ion-exchange chromatography. Recently Tkachuk (3) described a method for affinity chromatography of wheat a-amylase.

We present a simple, rapid, highly efficient method for the purification of cr-amylase from a wheat-rye hybrid, triticale (Triticosecafe Wittmack). The method consists of glycogen precipitation followed by selective adsorption of the enzyme of insoluble agarose substituted with the substrate cyclohepta-amylose.