New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.
Measurement of cereal α-Amylase: A new assay procedure.
McCleary, B. V. & Sheehan, H. (1987). Journal of Cereal Science, 6(3), 237-251.
A new procedure for the assay of cereal α-amylase has been developed. The substrate is a defined maltosaccharide with an α-linked nitrophenyl group at the reducing end of the chain, and a chemical blocking group at the non-reducing end. The substrate is completely resistant to attack by β-amylase, glucoamylase and α-glucosidase and thus forms the basis of a highly specific assay for α-amylase. The reaction mixture is composed of the substrate plus excess quantities of α-glucosidase and glucoamylase. Nitrophenyl-maltosaccharides released on action of α-amylase are instantaneously cleaved to glucose plus free p-nitrophenol by the glucoamylase and α-glucosidase, such that the rate of release of p-nitrophenol directly correlates with α-amylase activity. The assay procedure shows an excellent correlation with the Farrand, the Falling Number and the Phadebas α-amylase assay procedures.
A new procedure for the measurement of fungal and bacterial α-amylase.
Sheehan, H. & McCleary, B. V. (1988). Biotechnology Techniques, 2(4), 289-292.
A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end) p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.
An improved enzymic method for the measurement of starch damage in wheat flour.
Gibson, T. S., Al Qalla, H. & McCleary, B. V. (1992). Journal of Cereal Science, 15(1), 15-27.
An improved enzymic method for the determination of starch damage in wheat flour has been developed and characterized. The proposed method is simple and reliable, and enables up to 20 samples to be measured in duplicate in 2 h. A single assay takes approximately 40 min. The assay protocol is in two phases. In the first, the flour sample is incubated with purified fungal alpha-amylase to liberate damaged starch granules as soluble oligosaccharides. After centrifugation, the oligosaccharides in the supernatant are hydrolysed by amyloglucosidase to glucose in phase 2. The glucose is then quantified with a glucose oxidase/peroxidase reagent. The proposed method therefore avoids potential errors associated with existing standard assays, which employ unpurified amylase preparations and non-specific reducing group methods to quantify the hydrolytic products. Despite the use of purified assay components, the proposed starch damage method did not exhibit an absolute end-point to the action of alpha-amylase in phase 1. This was due to a low rate of hydrolysis of undamaged granules, and is a feature of enzymic methods for starch damage determination. Other amylolytic enzymes, including beta-amylase, isoamylase and pullulanase, and combinations of these enzymes, were evaluated as alternatives to alpha-amylase in the proposed method. These enzymes, when used alone, gave no benefits over the use of alpha-amylase. When used in combination with alpha-amylase, there was a synergistic action on undamaged granules. A test kit based on the assay format described in this paper is the subject of an international interlaboratory evaluation.
Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the ceralpha assay: Collaborative study.
McCleary, B. V., McNally, M., Monaghan, D. & Mugford, D. C. (2002). Journal of AOAC International, 85(5), 1096-1102.
This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 µmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.
Internal structure of amylopectin from the pericarp tissue of developing wheat kernels.
Kalinga, D. N. & Bertoft, E. (2015). Starch‐Stärke, 67(11-12), 1070-1076.
Pericarp starch is a transient starch that forms in the developing wheat kernel and decomposes concurrently with the appearance of the endosperm. The detailed structure of the amylopectin component of pericarp starch granules was investigated and compared with amylopectin from mature wheat endosperm. Clusters of chains from the macromolecule were isolated by hydrolysis with α-amylase from Bacillus amyloliquefaciens and were then transformed to limit dextrins using phosphorylase and β-amylase. The pericarp clusters consisted of 14.2 chains on average and had a 16.1% degree of branching, which was similar to the characteristics of clusters from large endosperm starch granules, as was the composition of the branched building blocks. However, the pericarp clusters exhibited a somewhat lower number of the largest building blocks. Other structural attributes, such as the average number of building blocks in the clusters and the length of the interblock segments, were also similar to those of endosperm starch. It is concluded that the cluster structure of wheat pericarp amylopectin essentially matches that of its endosperm counterpart.
Understanding the fine structure of intermediate materials of maize starches.
Han, W., Zhang, B., Li, J., Zhao, S., Niu, M., Jia, C. & Xiong, S. (2017). Food Chemistry, 233, 450-456.
Here we concern the molecular fine structure of intermediate material (IM) fraction in regular maize starch (RMS) and Starpro 40 maize starch (S40). IM had a branching degree and a molar mass (Mw) somewhere between amylopectin (AP) and amylose (AM). Compared with AP, IM had more extra-long (Fr I) and long (Fr II) chains and fb3-chains (degree of polymerization (DP) > 36), with a higher average chain length (CL). Also, IM contained less A-chains but more B-chains (both BS-chains with DP 3-25 and BL-chains with DP ≥ 26), accompanied by longer B- and BL-chains, total internal chains (TICL) and average internal chains (ICL), and a similar average external chain length (ECL). Furthermore, relative to RMS-IM, the IM of S40 (with higher apparent amylose content than RMS) showed increases in relatively-long chains, e.g., Fr II, fb3-chains and BL-chains, but reductions in Mw, relatively-short chains (those with DP 6-12, etc.).