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Chapter 1: Theory of endo-1, 4-Beta-D-Xylanase Assay Procedure
Chapter 2: Buffers & Reagents
Chapter 3: Assay Procedure
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|Content:||200 Tablets or 1,000 Tablets|
|Stability:||> 2 years under recommended storage conditions|
|Substrate For (Enzyme):||α-amylase|
|Reproducibility (%):||~ 5%|
|Method recognition:||AACC Method 22-05.01 and RACI Standard Method|
High purity dyed and crosslinked Amylazyme tablets for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.
For the assay of cereal and microbial α-amylase. Containing AZCL-Amylose. Recommended substrate for the assay of α-amylase in weather-damaged cereal grains, honey samples and food products containing low levels of this activity.
Please note the video above shows the protocol for assay of endo-xylanase using xylazyme tablets. The procedure for the assay of α-amylase using Amylazyme Tablets is equivalent to this.
Browse more of our enzyme tablet tests.
Validation of Methods
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
Gibberellins in developing wheat grains and their relationship to late maturity α-amylase (LMA).
Mares, D., Derkx, A., Cheong, J., Zaharia, I., Asenstorfer, R. & Mrva, K. (2022). Planta, 255(6), 1-18.
Late-maturity α-amylase (LMA) in wheat (Triticum aestivum L.) involves the synthesis of α-amylase by the aleurone tissue during grain development. Previous research identified a putative ent-copalyl diphosphate synthase gene, coding for an enzyme that controls the first step in gibberellin biosynthesis, that underlies the major genetic locus involved in variation in LMA phenotype. The reported results for gene transcript analysis, preliminary gibberellin analysis and the effects of DELLA mutants on LMA phenotype appeared to be consistent with involvement of gibberellin but did not provide definitive proof of a causal link. Conversely, several observations do not appear to be consistent with this hypothesis. In this current study, LMA phenotype, gibberellin profiles and ABA content were recorded for experiments involving susceptible and resistant genotypes, gibberellin biosynthesis inhibitors, genetic lines containing different LMA quantitative trait loci and treatment of distal halves of developing grains with exogenous gibberellin. The results suggested that gibberellin may not be a prerequisite for LMA expression and further that the mechanism involved in triggering α-amylase synthesis did not correspond to the model proposed for germination and gibberellin challenged aleurone of ripe grain. The results provide new insight into LMA and highlight the need to investigate alternate pathways for the induction of α-amylase gene transcription, the function of novel 1-β-OH gibberellins and other functions of DELLA proteins in developing grains.Hide Abstract
Endogenous alpha-amylase alters the pasting properties of starch during starch separation by proteases.
Zhang, H., Wu, F., Xu, D., Ali, B., Qu, J. & Xu, X. (2021). Journal of Cereal Science, 101, 103311.
The role of amylase is often neglected in the separation of starch by protease despite the fact that it likely impacts the properties of starch. This study aimed to explore the effects of endogenous alpha-amylase on the pasting properties of starch during isolation using protease. A strategy is developed according to prevent the influence of alpha-amylase on starch pasting properties. Decreased pasting viscosity was observed in the presence of endogenous alpha-amylase while an increase in reducing sugar during incubation decrease viscosity due to starch degradation. The addition of acarbose effectively inhibits starch degradation due to endogenous alpha-amylase. However, acarbose does not appear to affect protease activity. Thus, a combination of protease and acarbose can effectively prevent interference from endogenous alpha-amylase without affecting the gelatinization properties of starch.Hide Abstract
Partial purification of components in rye water extractables which improve the quality of oat bread.
Pauly, A. & Delcour, J. A. (2018). Journal of Cereal Science, 79, 141-147.
Unlike wheat bread, the dough of which has a visco-elastic network and high gas-holding capacity, oat bread generally has a low volume and a dense structure. We showed earlier that including rye water-extractable components in an oat bread batter recipe increases loaf volume by ca. 30% (Pauly and Delcour, submitted as back-to-back publication). We here report on efforts to identify the active factor(s). Anion exchange chromatography allowed enriching the active factor(s). This and the fact that only a limited volume increase was observed when oat batter was supplemented with boiled rye extract indicate that proteins are likely the most important components responsible for the volume increase. While the most active factor(s) had a pI below 4.5, components with pI values between 4.5 and 8.5 also contributed to oat loaf volume. Alkaline rye components (pI > 8.5) or rye arabinoxylan had no impact. Rye water-extractable components smaller than 6–8 kDa also had a positive impact on loaf volume.Hide Abstract
Impact of water-extractable components from different cereals on the quality of oat bread.
Pauly, A. & Delcour, J. A. (2018). Journal of Cereal Science, 79, 134-140.
Loaf volume and crumb structure of oat bread are not comparable to those of bread from wheat flour. Hydrocolloids, surfactants and/or enzymes are often included in oat batter recipes for quality enhancement reasons. In this study, we examined the impact of water-extractable components from barley, oat, rye and wheat flour on oat bread quality. We speculated that such water extracts contain components which also would enhance the quality of oat bread. As expected, extract protein, non-starch polysaccharide, lipid and enzyme levels varied widely amongst the different cereal flours used. The extracts also varied in foaming properties and extract viscosities. Rye flour contained the highest level of water-extractable components. Inclusion of rye aqueous extract resulted in the largest loaf volume increase and in softer crumb than noted for control oat bread. Rheofermentometer analyses showed that the moment of gas cell opening was delayed when rye extract was added, indicating improved batter gas cell stabilization, while collapse during baking was not affected. The oat bread improving effect of the rye extract is likely due to a combination of the impact of different of its constituents such as enzymes and surface active components.Hide Abstract
Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in wheat.
Simsek, S., Burgess, K., Whitney, K. L., Gu, Y. & Qian, S. Y. (2012). Food Control, 26(2), 287-292.
Deoxynivalenol (DON) is a mycotoxin which can be produced in cereal grains infected by Fusarium Head Blight (FHB). Alteration of DON by the plant often involves conjugation of the respective mycotoxin to certain functional groups or molecules. Conjugation of DON with glucose results in Deoxynivalenol-3-β-D-glucopyranoside (D3G), which has been found to be the main DON metabolite in wheat. The objective of this research was to identify the fate of D3G and DON during wheat processing using LC–MS–MS and GC, respectively. There was an approximate reduction of 61.8% in the detected DON level of the flour compared to the whole wheat. DON levels detected during the fermentation stage were significantly higher (P < 0.05) than the mixed dough. D3G detected in the flour was 23.7% lower than detected in the whole wheat. There were no significant differences (P < 0.05) in the D3G detected in the dough samples. However, the baked bread had significantly (P < 0.05) less D3G detected than the dough. Experiments were conducted to determine the effect of enzyme hydrolysis on DON detection in whole wheat. There were significant differences (P < 0.05) between the wheat treated with α-amylase, cellulase, protease, and xylanase. DON detection levels were significantly (P < 0.05) higher after treatment with protease (16%) and xylanase (39%) compared to the wheat composite. These results suggest that DON may be bound or embedded to the cell wall matrix or protein component of the wheat kernel due to the rise in detection of DON after these enzyme treatments.Hide Abstract
Leman, P., Goesaert, H. & Delcour, J. A. (2009). Food Hydrocolloids, 23(1), 153-164.
Some amylases can delay bread staling and/or starch (amylopectin) retrogradation, but the molecular basis of this effect remains little understood. In order to increase our insight in these aspects of amylase functionality, several amylases were added in a pure wheat-starch-containing model system and subjected to a heating step corresponding to that in the baking phase in bread making. Next, the effects of the limited amylolytic degradation on the rapid visco analyser (RVA) rheological properties of starch were studied and the accompanying changes in the amylopectin molecular properties (such as chain length distribution) investigated. The different amylases clearly affected the molecular structure of amylopectin to a different extent, which could be related to their mode of action and the enzyme activity levels added. Bacillus subtilis and Aspergillus oryzae α-amylases had only a limited impact on the side chain distribution of the amylopectin molecules, presumably due to their preferential hydrolysis of internal chain segments and the low enzyme activity added in the RVA. In contrast, porcine pancreatic α-amylase and Bacillus stearothermophilus maltogenic α-amylase, both with higher degree of multiple attack and used at higher enzyme activity levels, had a marked influence on the amylopectin molecular structure. More in particular, under the test conditions, the maltogenic α-amylase reduced the average chain length of the outer chains by 50%. Presumably, this will affect amylopectin retrogradation to a large extent. The results contribute to a better understanding of amylase functionality in starchy foods.Hide Abstract