Screening for α-amylase in cereals. Improved gel-diffusion assay using a dye-labelled starch substrate

Abstract

Radial diffusion into a gel slab containing dye-labelled starch as substrate provides a specific, reliable and rapid a-amylase estimation of potential use in screening of a large number of cereal samples, including single grains or half-grains in early stages of a breeding program. The method has also been used to monitor a-amylase activity in column chromatography eluates.

On the localisation and transport of α-amylase during germination and early seedling growth of Hordeum vulgare

“…Fractions were monitored at 280 nm for protein, a-amylase activity was measured by either the Phadebas a-amylase test and expressed as the absorbance at 620 nm (see 2.1) or the blue-starch gel diffusion assay of HEJGAARD and GmaoNs in which a-amylase activity is expressed in K(a) units (8). Selected fractions were subjected to crossed immunoelectrophoresis using anti-barley green-malt antibodies (see 2.6) or to isoenzyme analysis (see 2.4).…”

Some methods for isolation and assays of enzymes occurring in cereals and legumes

“…Substrates such as blue starch (stained with Remazol brilliant blue), Cibacron blue F3G-A, amylose azur B, etc. (7,85) or black starch (crosslinked with bifunctional reagent in the presence of black drawing ink) (86) are useful for the assay of a-amylase activity; and pullulan dye conjugate, for the assay of limit dextrinase activity. Limit dextrinase is an enzyme of interest to the starch industry for which no satisfactory activity determination has been available (87).…”

“…The subsequent extraction of the selected enzyme is based on the nature of the enzyme and the purpose of isolation. The most common extraction media are distilled water (5,6), saline solutions (2,(7)(8)(9)(10)(11)(12)(13), thioglycerol solutions (10), and various buffers such as acetate (14), veronal (15), imidazole (3), Tris (16), and phosphate (5). Reducing agents or enzyme stabilizers such as calcium chloride, 2-mercaptoethanol (14), dithiothreitol, or polyvinylpolypropilidone (16) are added to the extraction media, if necessary.…”

Development of Detection Methods for Cellulolytic Activity ofAuricularia auricula-judae

“…For this aim, we selected a method based on a dye coupled to a polysaccharide. When the dye or dye-polymer complex is hydrolyzed, pale or colorless haloes are produced [11, 12]. Cellulose was presently used as the carbon substrate as it is typically used for the detection of fungi that secrete cellulolytic enzymes.…”