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Azo-Barley Glucan

Play Training Video

00:02   Principle of the Assay Procedure
00:34    Substrate & Kit Description
01:02    Dissolution of Azo-CM-Cellulose
03:10    Precipitant Solution
04:59    Preparation of Buffer Solution
05:10    Assay Procedure
08:49    Calculation

 
Product code: S-ABG100
€220.00

100 mL (1% w/v)

Prices exclude VAT

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Content: 100 mL (1% w/v)
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Physical Form: Liquid
Stability: > 1 year under recommended storage conditions
Substrate For (Enzyme): endo-Cellulase, β-Glucanase/Lichenase
Assay Format: Spectrophotometer, Petri-dish (Qualitative)
Detection Method: Absorbance
Wavelength (nm): 590
Reproducibility (%): ~ 7%

High purity dyed, soluble Azo-Barley Glucan for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.

Highly purified, low viscosity barley 1,3:1,4-β-D-glucan dyed with Remazol Brilliant Blue R dye. Recommended substrate for the measurement of β-glucanase in malt flour.

Please note the video above shows the protocol for assay of endo-cellulase using Azo-CM cellulose. The procedure for the assays of endo-cellulase and β-glucanase/lichenase using Azo-Barley Glucan is equivalent to this.

See other soluble substrates for the measurement of enzyme activity.

Documents
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol
Publications
Megazyme publication
Novel approaches to the automated assay of β-glucanase and lichenase activity.

Mangan, D., Liadova, A., Ivory, R. & McCleary, B. V. (2016). Carbohydrate Research, 435, 162-172.

We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 3.2.1.73) in crude enzyme extracts. Two assay formats based on a) a direct cleavage or b) an enzyme coupled substrate were initially investigated. The ‘direct cleavage’ substrate, namely 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-31-cellotriosyl-β-glucopyranoside (MBG4), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt β-glucanase extract, a bacterial lichenase (Bacillus sp.) and a non-specific endo-1,3(4)-β-glucanase from Clostridium thermocellum (EC 3.2.1.6). Standard curves were derived that allow the assay absorbance response to be directly converted to β-glucanase/lichenase activity on barley β-glucan. The specificity of MBG4 was confirmed by analysing the action of competing glycosyl hydrolases that are typically found in malt on the substrate. Manual and automated assay formats were developed for the analysis of a) β-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated.

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Megazyme publication
Assay of malt β-glucanase using azo-barley glucan: an improved precipitant.

McCleary, B. V. & Shameer, I. (1987). Journal of the Institute of Brewing, 93(2), 87-90.

A procedure recently described for the assay of malt β-glucanase, which employs a dye-labelled and chemically-modified barley β-glucan substrate, has been improved by changing the precipitant solution used to terminate the reaction. The new precipitant solution contains 0•4% (w/v) zinc acetate and 4% (w/v) sodium acetate dissolved in 80% (v/v) aqueous methyl cellosolve. With this precipitant the procedure can be directly applied to the assay of cellulase activity, and with minor modification, to the assay of lichenase activity.

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Megazyme publication

Measurement of malt beta-glucanase.

McCleary, B. V. (1986). Proceedings of the 19th Convention of the Institute of Brewing (Aust. and N.Z. section), 181-187.

A Procedure has been developed for the assay of malt β-glucanase [a(1→3)(1→4)-β-D-glucanase] which employs as substrate, barley β-glucan dyed with Remazolbrilliant Blue and chemically modified with carboxymethyl groups to increase solubility. The described assay procedure together with a modified extraction format allows analysis of up to ten malt samples in less than 80 min. Also, the procedure is specific for enzymes active on barley β-glucan, is accurate and reliable, and can be readily applied to the analysis of β-glucanase in malt, green malt and wort.

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Megazyme publication
A soluble chromogenic substrate for the assay of (1→3)(1→4)-β-D-glucanase (lichenase).

McCleary, B. V. (1986). Carbohydrate Polymers, 6(4), 307-318.

A simple procedure for the assay of (1→3)(1→4)-β-D-glucanase (lichenase) has been developed. This assay employs as substrate barley (1→3)(1→4)-β-D-glucan dyed with Remazolbrilliant Blue R and chemically modified with carboxymethyl groups to increase solubility. Preparation of this substrate required the development of an improved procedure for the extraction and purification of barley β-glucan. Assays based on the use of the described chromogenic substrate at pH 6•5 are sensitive and specific for enzymes active on barley β-glucan.

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Megazyme publication

Problems caused by barley beta-glucans in the brewing industry.

McCleary, B. V. (1986). Chemistry in Australia, 53, 306-308.

Brewing, the oldest application of bio-technology is now a mix of trade art and modern science. This article describes new applications of enzyme chemistry to trouble-shooting in beer production.

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Publication

Production of a halotolerant endo-1, 4-β-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme.

Djelid, H., Flahaut, S., Vander Wauven, C., Oudjama, Y., Hiligsmann, S., Cornu, B., Cherfia, R., Gares, M. & Kacem Chaouche, N. (2022). Archives of Microbiology, 204(11), 1-15.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-β-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC–MS analysis showed 40% identity with an endo-1,3-1,4-β-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley β-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-β-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65°C. It was fairly thermotolerant, retaining 76.9% of the activity at 70°C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a Vmax of 625 U/min/mL and a high affinity with barley β-glucan resulting a Km of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-β-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.

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Publication

Enhanced acidic resistance ability and catalytic properties of Bacillus 1, 3-1, 4-β-glucanases by sequence alignment and surface charge engineering.

Li, Z., Niu, C., Yang, X., Zheng, F., Liu, C., Wang, J. & Li, Q. (2021). International Journal of Biological Macromolecules, 192, 426-434.

High stability at acidic environment is required for 1,3-1,4-β-glucanase to function in biofuel, brewing and animal feed industries. In this study, a mesophilic β-glucanase from Bacillus terquilensis CGX 5-1 was rationally engineered through sequence alignment and surface charge engineering to improve its acidic resistance ability. Nineteen singly-site variants were constructed and Q1E, I133L and V134A variants showed better acidic stability without the compromise of catalytic property and thermostability. Furthermore, four multi-site variants were constructed and one double-site variant Q1E/I133L with better stability at acidic environment and higher catalytic property was obtained. The fluorescence spectroscopy and structural analysis showed that more surface negative charge, decreased exposure degree of residue No.1, shifted side chain direction of residue No.133 and the lower total and folding free energy might be the reason for the improvement of acidic stability of Q1E/I133L variant. The obtained Q1E/I133L variant has potential applications in industries.

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Publication

Innovative microscale workflow from fungi cultures to Cell Wall‐Degrading Enzyme screening.

Raulo, R., Heuson, E., Siah, A., Phalip, V. & Froidevaux, R. (2019). Microbial Biotechnology, 12(6), 1286-1292.

This study aimed at developing a complete miniaturized high‐throughput screening workflow for the evaluation of the Cell Wall‐Degrading Enzyme (CWDE) activities produced by any fungal strain directly cultivated on raw feedstock in a submerged manner. In this study, wheat straw was selected as model substrate as it represents an important carbon source but yet poorly valorised to yield high added value products. Fungi were grown in a microbioreactor in a high‐throughput (HT) way to replace the fastidious shaking flask cultivations. Both approaches were compared in order to validate our new methodology. The range of CWDE activities produced from the cultures was assayed using AZO‐died and pNP‐linked substrates in an SBS plate format using a Biomek FXp pipetting platform. As highlighted in this study, it was shown that the CWDE activities gathered from the microbioreactor cultivations were similar or higher to those obtained from shake flasks cultures, with a lower standard deviation on the measured values, making this new method much faster than the traditional one and suitable for HT CWDE production thanks to its pipetting platform compatibility. Also, the results showed that the enzymatic activities measured were the same when doing the assay manually or using the automated method.

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Publication
Production of a thermostable 1,3-1,4-β-glucanase mutant in Bacillus subtilis WB600 at a high fermentation capacity and its potential application in the brewing industry.

Niu, C., Liu, C., Li, Y., Zheng, F., Wang, J. & Li, Q. (2017). International Journal of Biological Macromolecules, 107, 28-34.

1,3-1,4-β-glucanase was an important biotechnological aid in the brewing industry. In a previous research, a Bacillus BglTO mutant (BglTO) with high tolerance towards high temperature and low-pH conditions was constructed and expressed in Escherichia coli. However, E. coli was not a suitable host for enzyme production in food industry. Therefore, the present work aimed to achieve the high-level expression of BglTO in Bacillus subtilis WB600 and to test its effect in Congress mashing. The β-glucanase mutant was successfully expressed in B. subtilis WB600 and favorable plasmid segregation and structural stability were observed. The maximal extracellular activity of β-glucanase in recombinant B. subtilis WB600 reached 4840.4 U mL−1 after cultivation condition optimization, which was 1.94-fold higher than that before optimization. The fermentation capacity of recombinant B. subtilis reached 242.02 U mL−1 h−1, which was the highest among all reported β-glucanases. The addition of BglTO in Congress mashing significantly reduced the filtration time and viscosity of mash by 29.7% and 12.3%, respectively, which was superior to two commercial enzymes. These favorable properties indicated that B. subtilis WB600 was a suitable host for production of BglTO, which was promising for application in the brewing industry.

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Publication
Comparison of releasing bound phenolic acids from wheat bran by fermentation of three Aspergillus species.

Yin, Z., Wu, W., Sun, C., Lei, Z., Chen, H., Liu, H., Chen, W., Ma, J., Min, T., Zhang, M. & Wu, H. (2017). International Journal of Food Science & Technology, In Press.

Wheat bran was fermented at 28 °C for 7 days under 70% humidity by Aspergillus niger, Aspergillus oryzae and Aspergillus awamori. Total phenolic content (TPC) of the unfermented sample was 1531.5 µg g-1 wheat bran. After the fermentation of Aspergillus awamori, Aspergillus oryzae and Aspergillus niger, TPC reached 5362.1, 7462.6 and 10 707.5 µg g-1, respectively. The antioxidant activity in the extractions of fermented wheat bran also increased significantly compared with the unfermented sample (P < 0.05). Aspergillus niger showed the greatest capacity to release bound ferulic acid (416.6 µg g-1). Aspergillus oryzae and Aspergillus awamori had the advantages of releasing more chlorogenic acid (84.0 µg g-1) and syringic acid (142.3 µg g-1), respectively. The destructive effect of Aspergillus niger on wheat bran structure was the strongest, followed by that of Aspergillus oryzae. This effect of Aspergillus niger may be due to its higher cellulase, xylanase, arabinofuranosidase and β-xylosidase activities. Besides, Aspergillus oryzae possessed higher β-glucosidase activity, and Aspergillus awamori had higher α-amylase and feruloyl esterase activities. Aspergillus niger may be the best to release bound phenolic acids in the three Aspergillus species. These will provide the helpful information for understanding mechanism of the fermentation by Aspergillus species releasing bound phenolic in wheat bran.

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Safety Data Sheet
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