Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. oryzae and tobacco.
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.
Gene Cloning, Expression, and Characterization of a Family 51 α-L-Arabinofuranosidase from Streptomyces sp. S9.
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.
Development of an improved variant of GH51 α-L-arabinofuranosidase from Pleurotus ostreatus by directed evolution.
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.
Cloning, purification, and characterization of a thermostable α-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari.
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.
The endo-arabinanase BcAra1 is a novel host-specific virulence factor of the necrotic fungal phytopathogen Botrytis cinerea.
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.
Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan.
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.
Large-scale extraction of rhamnogalacturonan I from industrial potato waste.
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.