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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

Product code: S-AZCAS

10 g

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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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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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Suppressing expression of a soluble acid invertase (BoINV2) in broccoli (Brassica oleracea) delays postharvest floret senescence and downregulates cysteine protease (BoCP5) transcription.

Eason, J. R., Ryan, D. J., Watson, L. M., Pinkney, T., Hedderley, D., Christey, M. C., Braun, R. H. & Coupe, S. A. (2007). Physiologia Plantarum, 130(1), 46-57.

We report on the production and selection of transgenic Brassica oleracea var. Italica lines with a downregulated soluble acid invertase (BoINV2). Explants of broccoli (cv. Triathlon) were transformed with an antisense construct of BoINV2 under the control of an Asparagus officinalis-derived harvest-induced promoter using Agrobacterium tumefaciens-mediated transformation. BoINV2 is upregulated in wild-type broccoli floret tissue after harvest. Transgenic broccoli lines showed reduced BoINV2 mRNA accumulation immediately after harvest compared with wild-type. Downregulation of BoINV2 had no significant impact on the expression of a second broccoli acid invertase gene (BoINV1), but plants with downregulated BoINV2 also had lower expression of a senescence-associated cysteine protease (BoCP5) compared with wild-type. The total soluble sugar levels in floret tissue of antisense BoINV2 lines were greater than wild-type tissue after harvest (up to 1.5 times higher). Soluble protein content of wild-type tissue decreased from 48 h after harvest with an increase in protease activity. In comparison, two antisense BoINV2 lines retained at-harvest levels of soluble protein until 72 and 96 h after harvest and had lower postharvest endoprotease activity compared with wild-type. Antisense BoINV2 lines also had a slower rate of floret sepal chlorosis after harvest compared with wild-type.

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Harvested broccoli (Brassica oleracea) responds to high carbon dioxide and low oxygen atmosphere by inducing stress-response genes.

Eason, J. R., Ryan, D., Page, B., Watson, L. & Coupe, S. A. (2007). Postharvest Biology and Technology, 43(3), 358-365.

Broccoli (Brassica oleracea L.) tissue held in a controlled atmosphere (CA; 10% carbon dioxide and 5% oxygen) senesces more slowly than tissue held in air. CA-treated broccoli tissues lose less water and soluble sugars, have lower protease activity, and have no significant loss of color (hue angle, chlorophyll content) for 96 h after harvest (20°C, dark) compared to tissue held in air that starts to senesce and yellow after 48 h. The current study examined differential gene expression in broccoli tissues in response to postharvest CA treatment. This genetic analysis was undertaken to identify CA-responsive genes that may act as signaling elements and repress postharvest senescence processes. CA-responsive genes with up- and down-regulated expression (compared to air controls) were isolated after a 6 h CA treatment by differential display-polymerase chain reaction. The candidate CA-responsive genes included a number of novel genes without previously assigned functions, and genes of known function previously found to be regulated by stress (e.g. dehydration, salt stress, low temperature, and sugar starvation).

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Hydrolysis of raw hide using proteolytic enzyme extracted from papaya latex.

Pitpreecha, S. & Damrongsakkul, S. (2006). Korean Journal of Chemical Engineering, 23(6), 972-976.

Crude proteolytic enzyme was extracted from papaya latex using two solvents, water and phosphate buffer pH 6. The yield of extracted enzyme using water as a solvent was similar to that using phosphate buffer. Following the solvent extraction, the extracted enzyme was precipitated in 45 wt% saturated ammonium sulfate solution. The yield and activity of precipitated enzyme considerably decreased. Crude proteolytic enzyme extracted using water as an extracting liquid was, therefore, selected to use in gelatin production from raw hide hydrolysis, comparing to the use of commercial papain. The effects of hydrolysis conditions on gelatin recovery and properties of obtained gelatin were investigated. The optimum conditions for the activities of both crude extracted enzyme and commercial papain were at 75°C and pH 7. At this condition, the highest percentages of gelatin recovery were obtained from raw hide hydrolysis reactions. The gelatin recovery and gel strength of gelatin obtained from crude extracted enzyme and commercial papain hydrolysis were similar. This proved that crude extracted enzyme from papaya latex could be effectively used in gelatin production, instead of the use of commercial papain, with a comparatively low cost.

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Suppression of the cysteine protease, aleurain, delays floret senescence in Brassica oleracea.

Eason, J. R., Ryan, D. J., Watson, L. M., Hedderley, D., Christey, M. C., Braun, R. H. & Coupe, S. A. (2005). Plant Molecular Biology, 57(5), 645-657.

An aleurain-like protein, BoCP5, is up-regulated during harvest-induced senescence in broccoli floret and leaf tissue. BoCP5 is most closely related to an Arabidopsis protein (91%, AAF43041) and has 71% identity to barley aleurain (P05167). The mRNA for this gene accumulates within 6 h after harvest in broccoli florets, and its expression is reduced in tissue that has been held in senescence-delaying treatments (e.g. water, sucrose feeding, controlled atmosphere). The gene is also expressed in leaves during aging-related and harvest-induced senescence. Analysis of protein bands that cross-react with antibodies raised to the bacterial BoCP5 fusion protein, revealed prominent immunoreactive bands at ca. 26, 28, 31, and 38 kD in floret tissue. The 31 kD band was absent in protein extracts from leaf tissue. Agrobacterium-mediated transformation was used to produce transgenic broccoli plants with down-regulated BoCP5. A reduction in the postharvest expression of BoCP5 in floret tissue was achieved for four transgenic lines in the current study. In three of these lines postharvest floret senescence (yellowing) was delayed, and florets contained significantly greater chlorophyll levels during postharvest storage at 20°C than wild-type plants. Line 4 showed the greatest down-regulation of BoCP5, and in this line postharvest protease activity remained at pre-harvest levels, and the yield of soluble proteins extracted from florets after harvest was significantly greater than that of wild-type tissue.

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Identification of dehydration‐responsive cysteine proteases during post‐harvest senescence of broccoli florets.

Coupe, S. A., Sinclair, B. K., Watson, L. M., Heyes, J. A. & Eason, J. R. (2003). Journal of Experimental Botany, 54(384), 1045-1056.

Harvest‐induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo‐protease activity (measured with azo‐casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20°C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO2, 5% O2) conditions. Four protease‐active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73–89% at the amino acid level) to dehydration‐responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest‐induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO2, 5% O2). The putative cysteine protease mRNAs are expressed before measurable increases in endo‐protease activity, loss of protein, chlorophyll or tissue chlorosis.

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Programmed cell death during flower senescence: isolation and characterization of cysteine proteinases from Sandersonia aurantiaca.

Eason, J. R., Ryan, D. J., Pinkney, T. T. & O'Donoghue, E. M. (2002). Functional Plant Biology, 29(9), 1055-1064.

Cysteine protease inhibitors delayed the senescence of Sandersonia aurantiaca Hook. flowers. Tepal fading and wilting occurred later in the 2,2´ -dipyridyl-treated flowers, and these flowers had a greater soluble protein content and less active endoproteases compared with control flowers that were held in water. Biochemical analysis revealed the presence of several protease-active bands in the soluble protein fraction Sandersonia tepals. Activity of the polypeptides increased as flower senescence progressed. Western analysis with an antibody raised against the castor bean cysteine proteinase identified homologous proteins in Sandersonia flowers (ca46, 41 and 31 kDa). Three cDNAs encoding cysteine proteinases were isolated from Sandersonia tepals (PRT5, PRT15 and PRT22). Expression of all three increased in tepals as senescence progressed. mRNAs for PRT5 were detected only in senescing flower tissue, whereas PRT15 and PRT22 were expressed in leaf, stem and root tissue. PRT5 has significant homology to C-terminus KDEL proteins, which have a role in the degradation of plant cell contents during programmed cell death. PRT15 is most similar to cysteine proteinases with a long C-terminal extension, whereas PRT22 is homologous to stress-induced cysteine proteinases.

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Safety Data Sheet
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