Cloning, Isolation, and Properties of a New Homologous Exoarabinase from the Penicillium canescens Fungus
Abstract
A novel exo-arabinase (GH93, exo-ABN) enzyme produced by the ascomycete Penicillium canescens has been studied. Cloning of the abn1 gene coding for exo-ABN into the recipient P. canescens strain RN3-11-7 yielded recombinant producing strains characterized by a high yield of extracellular exo- ABN production (20–30% of the total amount of extracellular protein). Chromatographic purification yielded a homogenous exo-ABN with a molecular weight of 47 kDa, as shown by SDS-PAGE. The enzyme showed high specific activity towards linear arabinan (117 U/mg) and low specific activity towards branched arabinan and arabinoxylan (4–5 U/mg) and para-nitrophenyl-α-L-arabinofuranoside (0.3 U/mg), whereas arabinogalactan and para-nitrophenyl-α-L-arabinopyranoside, the substrates that contained the pyranose form of arabinose, were not hydrolyzed. Arabinohexaose was the major product of linear arabinan hydrolysis. Exo-ABN had a pH optimum at 5.0 and a temperature optimum at 60°C. The enzyme was stable in a broad pH range (4.0–7.0) and upon heating to 50°C during 180 min. Extensive hydrolysis of linear and branched arabinans by exo- and endo-arabinase mixtures, arabinofuranosidase, and arabinofuran-arabinoxylan hydrolase has been performed. The degree of substrate conversion amounted to 67 and 83% of the maximal possible value, respectively.
Keywords
exo-arabinase arabinan arabinohexaose Penicillium canescensPreview
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References
- 1.Sakamoto, T. and Thibault, J.F., Appl. Environ. Microbiol., 2001, vol. 67, pp. 3319–3321.CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Proctor, M.R., Taylor, E.J., Nurizzo, D., Turkenburg, J.P., Lloyd, R.M., Vardakou, M., Davies, G.J., and Gilbert, H.J., Proc. Natl. Acad. Sci. U.S.A., 2005, vol. 102, pp. 2697–2702.CrossRefPubMedPubMedCentralGoogle Scholar
- 3.McKie, V.A., Black, G.W., and Willward-Sadler, S.J., Biochem. J., 1997, vol. 323, pp. 547–555.CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Wong, D.W.S., Chan, V.J., and Batt, S.B., Appl. Microbiol. Biotechnol., 2008, vol. 79, pp. 941–950.CrossRefPubMedGoogle Scholar
- 5.Kuhnel, S., Hinz, S.W.A., Pouvreau, L., Wery, J., Schools, H.A., and Gruppen, H., Biores. Technol., 2010, vol. 101, pp. 8300–8307.CrossRefGoogle Scholar
- 6.Kaji, A. and Shimokawa, K., Agric. Biol. Chem., 1984, vol. 48, pp. 67–72.Google Scholar
- 7.Whitaker, J.R., Voragen, A.G., and Wong, D.W.S., Handbook of Food Enzymology, New York; Basel: Marcel Dekker, 2003.Google Scholar
- 8.Hemicellulose and Hemicellulases, Coughlan, M.P. and Hazlewood, G.P., Eds., London and Chapel Hill: Portland Press Research Monograph, 1993.Google Scholar
- 9.Rubtsova, E.A., Bushina, E.V., Rozhkova, A.M., Korotkova, O.G., Nemashkalov, V.A., Koshelev, A.V., and Sinitsyn, A.P., Appl. Biochem. Microbiol., 2015, vol. 51, no. 5, pp. 591–599.CrossRefGoogle Scholar
- 10.Matys, V.Yu., Bubnova, T.V., Koshelev, A.V., Vel’kov, V.V., Okunev, O.N., Bravova, G.B., Shishkova, E.A., Semenova, M.V., and Sinitsyn, A.P., in Mikrobnye biokatalizatory i perspektivy razvitiya fermentnykh tekhnologii v pererabatyvayushchikh otraslyakh APK (Microbial Biocatalysts and Prospects for the Development of Enzyme Technologies in the Processing Industries of the Agro-Industrial Complex), Moscow: Pishchepromizdat, 2004.Google Scholar
- 11.Sinitsyna, O.A., Bukhtoyarov, F.E., Gusakov, A.V., Okunev, O.N., Bekkarevich, A.O., Vinetskii, Yu.P., and Sinitsyn, A.P., Biochemistry (Moscow), 2003, vol. 68, no. 11, pp. 1200–1209.CrossRefGoogle Scholar
- 12.Sinitsyn, A.P. and Rozhkova, A.M., Penicillium canescens Host as a Platform for Development of a New Recombinant Strain Producers of Carbohydrases, Microbiology Monographs, Berlin, Heidelberg: Springer-Verlag, 2015.Google Scholar
- 13.Sanger, F., Nicklen, S., and Chase, A.R., Proc. Natl. Acid. Sci. U.S.A., 1977, vol. 74, no. 12, pp. 5463–5467.CrossRefGoogle Scholar
- 14.Aleksenko, A., Makarova, N., Nikolaev, I., and Clutterbuck, A., Curr. Genet., 1995, vol. 28, pp. 474–478.CrossRefPubMedGoogle Scholar
- 15.Sinitsyn, A.P., Chernoglazov, V.M., and Gusakov, A.V., Metody izucheniya i svoistva tsellyuloliticheskikh fermentov (Methods of Study and Properties of Cellulolytic Enzymes), Itogi nauki i tekhniki, Ser. Biotekhnol., Moscow: VINITI, 1990, vol. 25, p. 148.Google Scholar