Purification and characterization of α-amylase and protease of the phytopathogenic fungus Fusarium graminearum
Main Article Content
Authors
A. Abaildayev
M.A. Aitkhozhin’s institute of molecular biоlogy and biochemistry CS MES RK, Dosmukhamedov st., 86, Almaty, 050012, Kazakhstan
V. Kuzovlev
M.A. Aitkhozhin’s institute of molecular biоlogy and biochemistry CS MES RK, Dosmukhamedov st., 86, Almaty, 050012, Kazakhstan
K. Sharipov
M.A. Aitkhozhin’s institute of molecular biоlogy and biochemistry CS MES RK, Dosmukhamedov st., 86, Almaty, 050012, Kazakhstan
A. Khakimzhanov
M.A. Aitkhozhin’s institute of molecular biоlogy and biochemistry CS MES RK, Dosmukhamedov st., 86, Almaty, 050012, Kazakhstan
Abstract
One of the modern approaches in increasing the resistance of cereal crops to fungal diseases is the study of digestive enzymes of pathogens and their protein inhibitors in grain. Recently, proteases and amylases have been considered as some of the important pathogenicity factors of fungi. To obtain these enzymes, the phytopathogenic fungus F. graminearum was grown by solid state fermentation (SSF) using wheat bran. The maximum accumulation of enzymes at the addition of 2,0×106 conidia/ml occurred on day 8th of cultivation of the fungus. Extracellular protease and α-amylase with molecular weight 25 and 29 kDa, respectively, were purified by affinity chromatography.. Using specific inhibitors, the purified protease was found to belong to the serine trypsin-like enzyme. According to isoelectric focusing data, α-amylase consisted of 4 isoenzymes with strongly acidic pI of 3.0-3.5. The pH, temperature optimum, and thermostability of the enzymes were determined. The data obtained for α-amylase and trypsin of F. graminearum are presented for the first time. The results of the study can be used in the search for specific inhibitors of amylases and proteases in grain as protective proteins for their application in estimation of wheat varieties resistance to fungal attack.
Keywords
F. graminearum, α-amylase, trypsin-like protease, purification, physicochemical properties
Article Details
References
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Lowe, R. G. T., McCorkelle, O., Bleackley, M., Collins, C., Faou, P., Mathivanan, S., & Anderson, M. Extracellular peptidases of the cereal pathogen Fusarium graminearum // Front. Plant Sci. - 2015. Vol. 6(962). - P. 1-13. Crossref
Rawlings, N. D., Morton, F. R., Kok, C. Y., Kong, J., & Barrett, A. J. MEROPS: the peptidase database // Nucleic Acids Res. - 2008. Vol. 36 (Database issue). - P. 320-325. Crossref
Dubovenko, A. G., Dunaevsky, Y. E., Belozersky, M. A., Oppert, B., Lord, J. C., & Elpidina, E. N. Trypsin-like proteins of the fungi as possible markers of pathogenicity // Fungal Biol. - 2010. Vol. 114(2-3). - P. 151-159. Crossref
Chandrasekaran, M., Thangavelu, B., Chun, S. C., & Sathiyabama, M. Proteases from phytopathogenic fungi and their importance in phytopathogenicity // J. Gen. Plant Pathol. - 2016. Vol. 82(5). – P. 233–239. Crossref
Saranraj, P., Stella, D. Fungal amylase—a review //Int J Microbiol Res. -2013. Vol. 4(2). – P. 203–211. Crossref
Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K.M., Soccol, C.R., & Pandey, A. ά-Amylases from microbial sources – an overview on recent developments // Food Technol. Biotechnol. -2006. Vol. 44(2). – P. 173–184. URL
Valaparla, V.K. Characterization and partial purification studies on α-amylase activity by Fusarium verticillioides // Int. J. Biotechnol. Biochem. - 2018. Vol. 14(3). - P. 225-240. URL
Bakri, Y., Jawhar, M., & Arabi, M. I. E. Enhanced amylase production by Fusarium solani in solid state fermentation // Pak. J. Sci. Ind. Res. B: Biol. Sci. - 2014. Vol. 57(3). - P. 123-128. Crossref
Eggert, K., Rawel, H. M., & Pawelzik, E. In vitro degradation of wheat gluten fractions by Fusarium graminearum proteases // Eur. Food Res. Technol. - 2011. Vol. 233(4). - P. 697-705. Crossref
Ortega, L. M., Kikot, G. E., Astoreca, A. L., & Alconada, T. M. Screening of Fusarium graminearum isolates for enzymes extracellular and deoxynivalenol production // J. Mycol. - 2013. Vol. 2013(358140). - P. 1-7. Crossref
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Onofre, S. B., Abatti, D., Refosco, D., Tessaro, A. A., Onofre, J. A. B., & Tessaro, A. B. Characterization of α-amylase produced by the endophytic strain of Penicillium digitatum in solid state fermentation (SSF) and submerged fermentation (SMF) // Afr. J. Biotechnol. - 2016. Vol. 15(28) - P. 1511-1519. Crossref
Fortelius, C., & Markkanen, P. Nutritional regulation of proteinase production in the fungus, Tritirachium album // J. Ind. Microbiol. - 2000. Vol. 24(6). - P. 369–373. Crossref
Farooq, M.A., Ali, S., Hassan, A.H., Tahir, H.M., Mumtaz, S., & Mumtaz, S. Biosynthesis and industrial applications of α-amylase: a review // Arch. Microbiol.- 2021. Vol. 203. - P. 1281-1292. Crossref
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