Play Training Video

00:03   Introduction
00:50   Principle
01:45     Product Description
02:23    Reagent Preparation
03:40    Enzyme Extraction & Dilution "Powdered Samples"
05:53    Enzyme Extraction & Dilution "Liquid Samples"
07:17     Assay Procedure
10:50    Calculations

Content: 10 g
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 2 years under recommended storage conditions
Substrate For (Enzyme): Protease
Assay Format: Spectrophotometer, Petri-dish (Qualitative)
Detection Method: Absorbance
Wavelength (nm): 440
Reproducibility (%): ~ 7%

High purity dyed, soluble Azocasein (Sulphanilamide Dyed) for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.

A highly sensitive, soluble substrate for the assay of endo-protease activity. This substrate has a 5-fold greater sensitivity than similar products supplied by other companies.

Please note the video above shows the protocol for assay of endo-cellulase using Azo-CM cellulose. The procedure for the assay of protease using Azocasein is equivalent to this.

Display our full product list of soluble chromogenic enzyme substrates.

Certificate of Analysis
Safety Data Sheet
FAQs Application Note Assay Protocol
Megazyme publication

A novel enzymatic method discriminating wheat pre-harvest sprouting from Late Maturity alpha-amylase. 

Mangan, D., Draga, A., Ivory, R., Cornaggia, C., Blundell, M., Howitt, C., McCleary, B. V. & Ral, J. P. (2022). Journal of Cereal Science, 105, 103480.

The primary quality assessor of wheat grain is the Hagberg Falling Number (FN) method. This is a viscometric test surrogate for α-amylase activity. Despite being used for over sixty years, FN has been increasingly scrutinised due to its low throughput, poor reproducibility and inability to differentiate between the causes of low FN including Pre-harvest Sprouting (PHS) and Late Maturity α-Amylase (LMA). Our study describes initial efforts to analyse a specific wheat flour set tailored for the identification of enzymatic candidates that would allow discrimination between PHS and LMA affected grains. Using the sensitive enzyme-coupled assay substrate R-AMGR3, results suggest that α-glucosidase (exo-α-glucosidase) is a potential enzyme marker candidate to specifically detect sprouted but not LMA-affected grain.

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Characteristics of Koji Using Liquid Starter for Soy Sauce Production.

Choi, J., Kwon, T., Park, Y. & Kim, A. Y. (2023). Fermentation, 9(11), 979.

Soy sauce is a widely consumed seasoning derived from soybeans and wheat. This study explored the application of innovative techniques to enhance the traditional soy sauce preparation process. Fungi were isolated from a commercial koji starter, and the Aspergillus oryzae strain BJ-1 was identified. Additionally, an examination of the methods to optimize the medium composition for liquid starters revealed the impact of varying the medium composition on mycelial growth and enzyme activity. Specifically, compositions containing >10% defatted soybean meal and wheat in a 55:45 ratio resulted in elevated mycelial growth and enzymatic activity, making them promising candidates for koji production. The effect of different inoculation ratios of liquid starter on the characteristics of koji was also investigated, and a 10% inoculum was found to be preferable because of its advantageous characteristics of enzyme activities and pH for soy sauce production. This study contributes to the enhancement of the efficiency and safety of soy sauce production through innovative liquid culture techniques.

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Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases.

Kellock, M., Rahikainen, J., Borisova, A. S., Voutilainen, S., Koivula, A., Kruus, K. & Marjamaa, K. (2022). Biotechnology for Biofuels and Bioproducts, 15(1), 1-18.

Background: Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared. Results: Lignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22–39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified. Conclusions: High thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions.

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Two Heat Resistant Endopeptidases from Pseudomonas Species with Destabilizing Potential during Milk Storage.

Volk, V., Glück, C., Leptihn, S., Ewert, J., Stressler, T., & Fischer, L. (2018). Journal of Agricultural and Food Chemistry, 67(3), 905-915.

In the current study, the extracellular endopeptidases from Pseudomonas lundensis and Pseudomonas proteolytica were investigated. The amino acid sequence identity between both endopeptidases is 68%. Both endopeptidases were purified to homogeneity and partially characterized. They were classified as metallopeptidases with a maximum activity at pH 10.0 (P. lundensis) or 8.5 (P. proteolytica) at 35°C. Both remained active in skim milk with 39.7 ± 2.4% and 24.5 ± 3.3%, respectively, of the initial enzyme activity after UHT processing (138°C for 20 s), indicating the relevance for milk destabilization. The transition points in buffer were determined at 50°C (P. lundensis) and 43°C (P. proteolytica) using circular dichroism spectroscopy. The loss of the secondary structure at different temperatures was correlated with residual peptidase activities after heat treatment. The ability to destabilize UHT milk was proven by supplementation of skim milk with endopeptidase and storage for 4 weeks.

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Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima.

Theron, L. W., Bely, M. & Divol, B. (2017). Journal of the Science of Food and Agriculture., 97(11), 3584-3593.

BACKGROUND: MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10°C to 50°C. Optimal conditions for protease activity were found at 40°C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+. Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions. CONCLUSION: : Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking.

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Batch-to-batch variation and storage stability of the commercial peptidase preparation Flavourzyme in respect of key enzyme activities and its influence on process reproducibility.

Merz, M., Appel, D., Berends, P., Rabe, S., Blank, I., Stressler, T. & Fischer, L. (2016). European Food Research and Technology, 242(7), 1005-1012.

The synergy of endopeptidases and exopeptidases is the key for an efficient hydrolysis of proteins. Flavourzyme is sold as a commercial peptidase preparation from Aspergillus oryzae that exhibits various endo- and exopeptidase activities and, therefore, generates protein hydrolysates with high degrees of hydrolysis. The manufacturer (Novozymes) standardizes the enzyme preparation for one peptidase activity, determined with the marker substrate H-Leu-pNA. However, seven peptidases of Flavourzyme were recently identified and purified, and the significant contribution of six of them to wheat gluten hydrolysis was demonstrated. The knowledge about the batch-to-batch variation and storage stability of the Flavourzyme preparation regarding the other peptidase activities are still unclear, and this is important information for the usage of the enzyme preparation to gain reproducible protein hydrolysis processes. In the present study, we tested 12 Flavourzyme batches for the activity of the seven peptidases. The impact of the storage time on the peptidase activities and the magnitude of the batch-to-batch variation were investigated. In contrast to the activity determined with H-Leu-pNA as a substrate, the variations of the other peptidase activities were noticeable. The variation of the endopeptidase activity was most distinct and the activity decreased during the storage time of the preparation. The variation of the Flavourzyme composition also affected the reproducibility of a casein batch hydrolysis process, which should be taken into account for any future research and industrial application.

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Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species.

Dammannagoda, L. K., Pavasovic, A., Hurwood, D. A. & Mather, P. B. (2015). Aquaculture Research, 46(3), 626-636.

The current study evaluated the effect of soluble dietary cellulose on growth, survival and digestive enzyme activity in three endemic, Australian freshwater crayfish species (redclaw: Cherax quadricarinatus, marron: C. tenuimanus, yabby: C. destructor). Separate individual feeding trials were conducted for late-stage juveniles from each species in an automated recirculating freshwater, culture system. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch, over a 12-week period. Redclaw fed with RD showed significantly higher (P < 0.05) specific growth rates (SGR) compared with animals fed the TD, while SGR of marron and yabby fed the two diets were not significantly different. Expressed cellulase activity levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD (P < 0.05). Amylase and protease activity in all three species were significantly higher in the animals fed with RD (P < 0.05). These results indicate that test animals of all three species appear to utilize starch more efficiently than soluble dietary cellulose in their diet. The inclusion of 20% soluble cellulose in diets did not appear, however, to have a significant negative effect on growth rates.

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Isolation and characterization of a Bacillus licheniformis strain capable of degrading zearalenone.

Yi, P. J., Pai, C. K., & Liu, J. R. (2011). World Journal of Microbiology and Biotechnology, 27(5), 1035-1043.

The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.

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Protease-induced solubilisation of carbohydrates from brewers' spent grain.

Faulds, C. B., Collins, S., Robertson, J. A., Treimo, J., Eijsink, V. G. H., Hinz, S. W. A. Schols, H. A., Buchert, J. & Waldron, K. W. (2009). Journal of Cereal Science, 50(3), 332-336.

The impact of microbial proteases on the release of carbohydrates from BSG was studied. The proteases were able to release the non-cellulosic glucose, a portion of feruloylated arabinoxylan and over 50% of the protein from brewers' spent grain (BSG) after 24 h hydrolysis. The non-cellulosic glucose was derived from residual starch-derived products persisting in BSG after mashing. The proteases did not cleave the hydroxycinnamate ester linkages present on the arabinoxylan backbone, and thus do not behave as feruloyl esterases. However, the material solubilised from spent grain by the proteases contained up to 198 µg bound ferulic acid/g extract, which represented 8.6% of the total ferulic acid present in BSG. These results suggest that a portion of water-extractable feruloylated arabinoxylan and starch is trapped within the BSG matrix by a proteinaceous barrier.

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Effect of pH on the solubilization of brewers’ spent grain by microbial carbohydrases and proteases.

Faulds, C. B., Robertson, J. A. & Waldron, K. W. (2008). Journal of Agricultural and Food Chemistry, 56(16), 7038-7043.

The potential for enzymatic solubilization of brewers’ spent grain by carbohydrases and proteases was examined over a broad pH range (pH 3.2−11.2). Enzymes from Trichoderma (Depol 686) were most efficient at a lower pH, while enzymes from the Humicola preparation (Depol 740) were the best performer over the whole range. Profiling of key glycoside hydrolase, esterase and protease activities across the pH range demonstrated that solubilization of spent grain by the Trichoderm enzymes corresponded to the range of maximum activities. This was not the case with the Humicola enzymes, where maximum solubilization of the substrate occurred at pH 9.1, at which pH the determined activities were low. Protease activity in Depol 740 was associated with a high solubilization, but inhibition of proteolytic activity resulted in only a 5% decrease in spent grain solubilization. These results suggest that while enzymes can be used to exploit agro-industrials byproduct, the use of high pH increases the extent of hydrolysis and an unidentified factor produced by Humicola improves the enzyme-catalyzed solubilization of lignocellulosic material.

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Safety Data Sheet
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