New chromogenic substrates for the assay of alpha-amylase and (1→4)-β-D-glucanase.
McCleary, B. V. (1980). Carbohydrate Research, 86(1), 97-104.
New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.
Comparison of endolytic hydrolases that depolymerise 1,4-β-D-mannan, 1,5-α-L-arabinan and 1,4-β-D-galactan.
McCleary, B. V. (1991). “Enzymes in Biomass Conversion”, (M. E. Himmel and G. F. Leatham, Eds.), ACS Symposium Series 460, Chapter 34, pp. 437-449. American Chemical Society, Washington.
Hydrolysis of mannan-type polysaccharides by β-mannanase is dependent on substitution on and within the main-chain as well as the source of the β-mannanase employed. Characterisation of reaction products can be used to define the sub-site binding requirements of the enzymes as well as the fine-structures of the polysaccharides. Action of endo-arabinanase and endo-galactanase on arabinans and arabinogalactans is described. Specific assays for endo-arabinanase and arabinan (in fruit-juice concentrates) are reported.
Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.
McCleary, B. V. (1991). Chemistry in Australia, September, 398-401.
Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.
Optimising the response.
Acamovic, T. & McCleary, B. V. (1996). Feed Mix, 4, 14-19.
A fine balance exists between enzyme activity and the adverse effects associated with feed processing. Accurate estimation of enzyme activity in the feed is a pre-requisite to optimising the response.
Measurement of α-Amylase in Cereal, Food and Fermentation Products.
McCleary, B. V. & Sturgeon, R. (2002). Cereal Foods World, 47, 299-310.
In General, the development of methods for measuring α-amylase is pioneered in the clinical chemistry field and then translated to other industries, such as the cereals and fermentation industries. In many instances, this transfer of technology has been difficult or impossible to achieve due to the presence of interfering enzymes or sugars and to differences in the properties of the enzymes being analysed. This article describes many of the commonly used methods for measuring α-amylase in the cereals, food, and fermentation industries and discusses some of the advantages and limitations of each.
A major QTL controlling seed dormancy and pre-harvest sprouting/grain α-amylase in two-rowed barley (Hordeum vulgare L.).
Li, C. D., Tarr, A., Lance, R. C. M., Harasymow, S., Uhlmann, J., Westcot, S., Young, K. J., Grime, C. R., Cakir, M., Broughton, S. & Appels, R. (2003). Australian Journal of Agricultural Research, 54(12), 1303-1313.
Barley seed dormancy is controlled by multiple genes that have a strong interaction with the environment. Lack of adequate dormancy results in pre-harvest sprouting in the field under wet weather conditions. On the other hand, too much dormancy has a detrimental effect in the malting house. There is only a very 'narrow window' of dormancy for malting barley. Harrington barley, which has been a dominant malting variety in the international market and widely used in Australia barley breeding programs, is highly susceptible to pre-harvest sprouting. A doubled haploid (DH) population derived from a cross of Chebec/Harrington was used to search for molecular markers linked with seed dormancy and pre-harvest sprouting. One major quantitative trait locus (QTL) was identified to control pre-harvest sprouting measured by α-amylase activity in barley grains, and could explain >70% of the phenotypic variation. This QTL was located on chromosome 5HL and flanked by restriction fragment length polymorphism (RFLP) marker CDO506 and simple sequence repeat (SSR) marker GMS1. The SSR marker (GMS1) linked with this QTL was further validated in a Stirling/Harrington DH population. A minor QTL on chromosome 2H accounted for 8% of phenotypic variation. Two QTLs for seed dormancy were located on chromosomes 2H and 5HL. The major QTL for dormancy coincided with the QTL for pre-harvest sprouting at chromosome 5HL and explained 61% of phenotypic variation. Since the presence of the Harrington allele at this locus favoured not only pre-harvest sprouting, but also increased malting extract, diastatic power, α-amylase, and free amino acid nitrogen, development of high malting quality varieties with pre-harvest sprouting tolerance would appear to be difficult.
Associations between caryopsis dormancy, α-amylase activity, and pre-harvest sprouting in barley.
Lin, R., Horsley, R. D. & Schwarz, P. B. (2008). Journal of Cereal Science, 48(2), 446-456.
Pre-harvest sprouting (PHS) is a concern for barley (Hordeum vulgare L.) producers, grain processors, and researchers worldwide. Pre-harvest sprouting has been mainly attributed to low dormancy, which is determined by genotype, stage of plant maturation, and environmental conditions during caryopsis development. Fourteen barley genotypes were sown in field experiments at two sites in North Dakota in 2004 and 2005. Spikes were harvested at four different stages: ≈500 g kg-1 moisture content, physiological maturity, harvest maturity, and post-harvest maturity. Results indicated that barley genotypes were released from dormancy at different rates. The 14 barley genotypes were divided into three classes based on their dormancy loss rate during caryopsis development. C93-3230-24 was highly dormant, and ‘Stander’ and ‘Legacy’ were highly susceptible to PHS due to lack of dormancy from as early as 20 d after heading date. All other genotypes fell into the third group that had intermediate dormancy loss rate. No significant correlation was detected between barley α-amylase activity and germination percentage. A moderate association between malt α-amylase activity and caryopsis dormancy suggested that cultivars with increased malt α-amylase activity tend to have low dormancy and may be more prone to PHS.