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, 58, 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.
Measurement of α-amylase in cereal, food and fermentation products.
McCleary, B. V. & Sturgeon, R. (2002). Cereal Foods World, 47(7), 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.
Comparison of short-wavelength infrared (SWIR) hyperspectral imaging system with an FT-NIR spectrophotometer for predicting alpha-amylase activities in individual Canadian Western Red Spring (CWRS) wheat kernels.
Xing, J., Symons, S., Hatcher, D. & Shahin, M. (2011). Biosystems Engineering, 108(4), 303-310.
Alpha-amylase activity in individual Canadian Western Red Spring (CWRS) wheat kernels was predicted using spectral information across the wavelength region 1235–2450 nm. Reflectance spectra were collected from an SWIR (short-wavelength infrared) hyperspectral imaging system and absorbance spectra were recorded from an Fourier transform near-infrared (FT-NIR) spectrometer on the same kernels. The partial least squares (PLS) regression technique was used to model the alpha-amylase enzyme activity levels to the spectral information. The prediction accuracy varied with the pre-processing methods applied to the regressor and regressand. The highest coefficient of determination (r2) value obtained from the SWIR hyperspectral imaging system was 0.88 and 0.82 from the FT-NIR instrument. The imaging approach was more successful because it also had the advantage of being able to localise the region where spectra were extracted from.
The objective measurement of alpha-amylase in wheat kernels using spectral imaging.
Symons, S., Xing, J., Shahin, M. & Hatcher, D. (2010, September). World Automation Congress (WAC), 257-262.
When wheat kernels are wetted in the head prior to harvest, the germination processes are initiated. The symptoms range from no obvious visible signs of enzyme activation to gross kernel disfiguration. Alpha-amylase, a starch degrading enzyme is the most prevalent of the activated enzymes in the early stages of germination and may cause significant end-product quality loss. Current analytical techniques do not provide a rapid system for estimating individual kernel sprout damage. We have developed an objective approach using near-infrared spectra (1100-2400 nm) from a hyperspectral camera to predict α-amylase levels of individual kernels in two classes of Canadian wheat. Multivariate modeling gave, an R2 of up to 0.69 for predicting individual kernel α-amylase levels. Using the hyperspectral data, a multispectral model predicted α-amylase activity levels of greater than 1 SKU unit/g with a better than 90% accuracy. At this level, there is no visible sign of kernel sprouting.
Using a Short Wavelength Infrared (SWIR) hyperspectral imaging system to predict alpha amylase activity in individual Canadian western wheat kernels.
Xing, J., Van Hung, P., Symons, S., Shahin, M. & Hatcher, D. (2009). Sensing and Instrumentation for Food Quality and Safety, 3(4), 211-218.
Sprout damage (pre-harvest germination) in wheat results in highly deleterious effects on end-product quality. Alpha-amylase, the pre-dominant enzyme in the early stage of sprouting has the most damaging effect. This paper introduces a new method using a SWIR hyperspectral imaging system (1000–2500 nm) to predict the α-amylase activity of individual wheat kernels. Two classes of Canadian wheat, Canada Western Red Spring (CWRS) and Canada Western Amber Durum (CWAD), with samples of differing degrees of sprout damage were investigated. Individual kernels were first imaged with the hyperspectral imaging system and then the α-amylase activity of each kernel was determined analytically. Individual kernel α-amylase activity prediction was significant (R2 0.54 and 0.73) for CWAD and CWRS, respectively using Partial Least Square regression on the hyperspectral data. A classification method is proposed to separate CWRS kernels with high α-amylase activity level from those with low α-amylase activity giving an accuracy of above 80%. This work shows that hyper/multi-spectral imaging techniques can be used for rapidly predicting the α-amylase activity of individual kernels, detecting sprouting at early stage.
A novel α-amylase gene is transiently upregulated during low temperature exposure in apple fruit.
Wegrzyn, T., Reilly, K., Cipriani, G., Murphy, P., Newcomb, R., Gardner, R. & MacRae, E. (2000). European Journal of Biochemistry, 267(5), 1313-1322.
An α-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5′ and 3′ RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an α-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5°C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant α-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent α-amylases lack the standard signal peptide structures found in all other plant α-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic α-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous α-amylase in response to low temperature, and confirms the presence of a new family of α-amylases in plants.