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|Storage Temperature:||Below -10oC|
|Stability:||> 9 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-Arabinanase|
|Assay Format:||Spectrophotometer, Petri-dish (Qualitative)|
|Reproducibility (%):||~ 7%|
High purity dyed, soluble Red Debranched Arabinan (Sugar Beet) for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.
Debranched arabinan dyed with Procion Red dye. Substrate for the assay of endo-1,5-α-L-arabinanase.
Please note the video above shows the protocol for assay of endo-cellulase using Azo-CM cellulose. The procedure for the assay of endo-arabinanase using Red Debranched Arabinan (Sugar Beet) is equivalent to this.
See our complete list of soluble chromogenic substrates.
Inácio, J. M. & De Sá-Nogueira, I. (2008). Journal of Bacteriology, 190(12), 4272-4280.
The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-α-1,5-L-arabinanase (EC 188.8.131.52) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-α-1,5-L-arabinan as the preferred substrate, the enzyme exhibited an apparent Km of 2.0 mg ml-1 and Vmax of 0.25 mmol min-1·mg-1·at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a σA--dependent and a σH-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.Hide Abstract
Canakci, S., Kacagan, M., Inan, K., Belduz, A. O. & Saha, B. C. (2008). Applied Microbiology and Biotechnology, 81(1), 61-68.
The gene, AbfAC26Sari, encoding an α-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant α-L-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-L-arabinofuranoside as a substrate gave a V max and K m, values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu2+ and Hg2+. The recombinant arabinofuranosidase released L-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.Hide Abstract
Leal, T. F. & Sá‐Nogueira, I. (2004). FEMS Microbiology Letters, 241(1), 41-48.
Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-α-L-arabinan.Hide Abstract
Purification and functional characterization of a novel α-L-arabinofuranosidase from Bifidobacterium longum B667.
Margolles, A. & De los Reyes-Gavilan, C. G. (2003). Applied and Environmental Microbiology, 69(9), 5096-5103.
The gene encoding a novel α-L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70°C), and it had an optimum pH of 6.0 and an optimum temperature of 45°C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55°C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu2+, Hg2+, and Zn2+ markedly reduced enzymatic activity. The protein exhibited a high level of activity with p-nitrophenyl α-L-arabinofuranoside, with apparent Km and Vmax values of 0.295 mM and 417 U/mg, respectively. AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides.Hide Abstract
Skjøt, M., Pauly, M., Bush, M. S., Borkhardt, B., McCann, M. C. & Ulvskov, P. (2002). Plant Physiology, 129(1), 95-102.
Pectin is a class of complex cell wall polysaccharides with multiple roles during cell development. Assigning specific functions to particular polysaccharides is in its infancy, in part, because of the limited number of mutants and transformants available with modified pectic polymers in their walls. Pectins are also important polymers with diverse applications in the food and pharmaceutical industries, which would benefit from technology for producing pectins with specific functional properties. In this report, we describe the generation of potato (Solanum tuberosum L. cv Posmo) tuber transformants producing pectic rhamnogalacturonan I (RGI) with a low level of arabinosylation. This was achieved by the expression of a Golgi membrane-anchored endo-α-1,5-arabinanase. Sugar composition analysis of RGI isolated from transformed and wild-type tubers showed that the arabinose content was decreased by approximately 70% in transformed cell walls compared with wild type. The modification of the RGI was confirmed by immunolabeling with an antibody recognizing α-1,5-arabinan. This is the first time, to our knowledge, that the biosynthesis of a plant cell wall polysaccharide has been manipulated through the action of a glycosyl hydrolase targeted to the Golgi compartment.Hide Abstract
Saha, B. C. & Bothast, R. J. (1998). Current Microbiology, 37(5), 337-340.
A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-L-arabinofuranosidase (α-L-AFase) when grown in liquid culture on sugar beet arabinan, wheat arabinoxylan, L-arabinose, L-arabitol, xylose, xylitol, oat spelt xylan, corn fiber, or arabinogalactan. L-Arabinose was most effective for production of both whole-broth and extracellular α-L-AFase activity, followed by L-arabitol. Oat spelt xylan, sugar beet arabinan, xylose, xylitol, and wheat arabinoxylan were intermediate in their ability to support α-L-AFase production. Lower amounts of enzyme activity were detected in corn fiber- and arabinogalactan-grown cultures.Hide Abstract
Purification and characterization of a novel thermostable α-L-arabinofuranosidase from a color-variant strain of Aureobasidium pullulans.
Saha, B. C. & Bothast, R. J. (1998). Applied and Environmental Microbiology, 64(1), 216-220.
A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-L-arabinofuranosidase (α-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular α-L-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75°C, displayed optimal activity at 75°C and pH 4.0 to 4.5, and had a specific activity of 21.48 µmol min-1·mg-1·of protein against p-nitrophenyl-α-L-arabinofuranoside (pNPαAF). The purified α-L-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The Km values of the enzyme for the hydrolysis of pNPαAF, arabinan, and debranched arabinan at 75°C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The α-L-AFase activity was not inhibited at all by L-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).Hide Abstract