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|Storage Temperature:||Below -10oC|
|Stability:||> 9 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-Arabinanase|
|Assay Format:||Spectrophotometer (Semi-quantitative), Petri-dish (Qualitative)|
High purity dyed and crosslinked insoluble AZCL-Arabinan (Debranched) for identification of enzyme activities in research, microbiological enzyme assays and in vitro diagnostic analysis.
Substrate for the assay of endo-1,5-α-L-arabinanase.
Pitt, J. I., Lange, L., Lacey, A. E., Vuong, D., Midgley, D. J., Greenfield, P., Bradbury, M. I., Lacey, E., Busk, P. K., Pilgaard, B., Chooi, Y. H. & Piggott, A. M. (2017). PloS One, 12(4), e0170254.
Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.Hide Abstract
Huang, Y., Yi, Z., Jin, Y., Huang, M., He, K., Liu, D., Luo, H., Zhao, D., He, H., Fang, Y. & Zhao, H. (2017). Frontiers in Microbiology, 8, 1747.
Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.Hide Abstract
Aalbers, F., Turkenburg, J. P., Davies, G. J., Dijkhuizen, L. & van Bueren, A. L. (2015). Journal of Molecular Biology, 427(24), 3935-3946.
Glycoside hydrolases are clustered into families based on amino acid sequence similarities, and belonging to a particular family can infer biological activity of an enzyme. Family GH115 contains α-glucuronidases where several members have been shown to hydrolyze terminal α-1,2-linked glucuronic acid and 4-O-methylated glucuronic acid from the plant cell wall polysaccharide glucuronoxylan. Other GH115 enzymes show no activity on glucuronoxylan, and therefore, it has been proposed that family GH115 may be a poly-specific family. In this study, we reveal that a putative periplasmic GH115 from the human gut symbiont Bacteroides thetaiotaomicron, BtGH115A, hydrolyzes terminal 4-O-methyl-glucuronic acid residues from decorated arabinogalactan isolated from acacia tree. The three-dimensional structure of BtGH115A reveals that BtGH115A has the same domain architecture as the other structurally characterized member of this family, BoAgu115A; however the position of the C-terminal module is altered with respect to each individual enzyme. Phylogenetic analysis of GH115 amino sequences divides the family into distinct clades that may distinguish different substrate specificities. Finally, we show that BtGH115A α-glucuronidase activity is necessary for the sequential digestion of branched galactans from acacia gum by a galactan-β-1,3-galactosidase from family GH43; however, while B. thetaiotaomicron grows on larch wood arabinogalactan, the bacterium is not able to metabolize acacia gum arabinogalactan, suggesting that BtGH115A is involved in degradation of arabinogalactan fragments liberated by other microbial species in the gastrointestinal tract.Hide Abstract
Nafisi, M., Stranne, M., Zhang, L., van Kan, J. & Sakuragi, Y. (2014). Molecular Plant-Microbe Interactions, 27(8), 781-792.
The plant cell wall is one of the first physical interfaces encountered by plant pathogens and consists of polysaccharides, of which arabinan is an important constituent. During infection, the necrotrophic plant pathogen Botrytis cinerea secretes a cocktail of plant cell-wall-degrading enzymes, including endo-arabinanase activity, which carries out the breakdown of arabinan. The roles of arabinan and endo-arabinanases during microbial infection were thus far elusive. In this study, the gene Bcara1 encoding for a novel α-1,5-L-endo-arabinanase was identified and the heterologously expressed BcAra1 protein was shown to hydrolyze linear arabinan with high efficiency whereas little or no activity was observed against the other oligo- and polysaccharides tested. The Bcara1 knockout mutants displayed reduced arabinanase activity in vitro and severe retardation in secondary lesion formation during infection of Arabidopsis leaves. These results indicate that BcAra1 is a novel endo-arabinanase and plays an important role during the infection of Arabidopsis. Interestingly, the level of Bcara1 transcript was considerably lower during the infection of Nicotiana benthamiana compared with Arabidopsis and, consequently, the δBcara1 mutants showed the wild-type level of virulence on N. benthamiana leaves. These results support the conclusion that the expression of Bcara1 is host dependent and is a key determinant of the disease outcome.Hide Abstract
Yang, X., Shi, P., Huang, H., Luo, H., Wang, Y., Zhang, W. & Yao, B. (2014). Food Chemistry, 148(1), 381-387.
Two β-xylosidases of family 43 (Xyl43A and Xyl43B) and one xylanase of family 11 (Xyn11A) were identified from the genome sequence of Humicola insolens Y1, and their gene products were successfully expressed in heterologous hosts. The optimal activities of the purified Xyl43A, Xyl43B, and Xyn11A were found at pH 6.5–7.0 and 50–60°C. They were stable over a pH range of 5.0–10.0 and temperatures of 50°C and below. Xyl43A and Xyl43B had the activities of β-xylosidase, α-arabinosidase and xylanase, and showed xylose tolerance up to 79 and 292 mM, respectively. Xyn11A and Xyl43A or Xyl43B showed significant synergistic effects on the degradation of various xylans, releasing more reduced sugars (up to 1.29 folds) by simultaneous or sequential addition. This study provides several enzymes for synergistic degradation of xylan and contributes to the formulation of optimised enzyme mixtures for the efficient hydrolysis of plant biomass.Hide Abstract
Giacobbe, S., Vincent, F. & Faraco, V. (2014). New Biotechnology, 31(3), 230-236.
In this study, the α-L-arabinofuranosidase from Pleurotus ostreatus was subjected to directed evolution by expressing a library of around 7000 randomly mutated variants by error prone Polymerase Chain Reaction. High-throughput screening of the library for the most active variants was performed by assaying activity towards p-nitrophenyl α-L-arabinofuranoside, and a variant with higher activity than the wild type was selected, purified and characterised. It exhibited a Kcat of 7.3 × 103 ± 0.3 min-1, around 3-fold higher than that of the wild type (2.2 × 103 ± 0.2 min-1), and a KM (0.54 ± 0.06 mM) 30% lower than that of the wild type (0.70 ± 0.05 mM) towards this substrate. The mutant also showed improved catalytic properties towards pNP-β-D-glucopyranoside (Kcat of 50.85 ± 0.21 min−1 versus 11.0 ± 0.6 min-1) and it was shown able to hydrolyse larch arabinogalactan which is not recognised by the wild type. The mutant was also more active than the wild type towards arabinoxylan and was able to hydrolyse arabinan, which was not transformed by the wild type. The ability of rPoAbf F435Y/Y446F to hydrolyse these insoluble substrates expands its potential for application also to hemicelluloses, which in some types of pretreatment are recovered in solid fractions.Hide Abstract
Byg, I., Diaz, J., Øgendal, L. H., Harholt, J., Jørgensen, B., Rolin, C., Rolin, C., Svava, R. & Ulvskov, P. (2012). Food Chemistry, 131(4), 1207-1216.
Potato pulp is rich in dietary fibres and is an underutilised material produced in large quantities by the potato starch factories. Potato fibres are especially rich in rhamnogalacturonan I (RG I). RG I is a pectic polysaccharide with a high degree of branching and until now undegraded RG I has only been extracted in small amounts limiting the application possibilities for RG I. The present paper describes a large-scale extraction process providing large quantities of undegraded RG I readily available. The extraction process includes enzymatic starch removal using purified Termamyl, enzymatic RG I solubilisation using a highly purified polygalacturonase, and finally purification using depth filtration and ultrafiltration. The extracted RG I has a high molecular weight and a monosaccharide composition comparable to RG I extracted by analytical extraction procedures. The large amount of RG I available by the presented method allows for thorough structure–function analyses and tailoring of RG I to specific functionalities.Hide Abstract
Shi, P., Li, N., Yang, P., Wang, Y., Luo, H., Bai, Y. & Yao, B. (2010). Applied Biochemistry and Biotechnology, 162(3), 707-718.
An α-L-arabinofuranosidase gene, abf51S9, was cloned from Streptomyces sp. S9 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene consisted of 1,506 bp and encoded 501 amino acids with a calculated mass of 55.2 kDa. The deduced amino acid sequence was highly homologous with the α-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by Ni-NTA affinity chromatography and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.0 and 60∼65°C, respectively. The enzyme showed a broad pH range of stability, retaining over 75% of the maximum activity at pH 5.0 to 11.0. The specific activity, K m, and V max with p-nitrophenyl-α-L-arabinofuranoside as substrate were 60.0 U mg-1, 1.45 mM, and 221 µmol min-1 mg-1, respectively. Abf51S9 showed a mild but significant synergistic effect in combination with xylanase on the degradation of oat-spelt xylan and soluble wheat arabinoxylan substrates with a 1.19- and 1.21-fold increase in the amount of reducing sugar released, respectively. These favorable properties make Abf51S9 a good candidate in various industrial applications.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
Skjøt, M., Kauppinen, S., Kofod, L., Fuglsang, C., Pauly, M., Dalbøge, H. & Andersen, L. (2001). Molecular Genetics and Genomics, 265(5), 913-921.
Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-α-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.Hide Abstract