Summary
Assay methods for chitinase, β-glucanase and α-glucanase in the presence of the osmotic stabilizers used in fungal protoplast liberation were developed. Chitinase activity with an inorganic osmotic stabilizer system was in the order of NO −3 , Cl−>SO 2−4 >PO 3−4 and Na+, K+>Ca2+, Mg2+. Monovalent anion salts with monovalent cations improved chitinase activity, whereas divalent and trivalent anion and cation salts caused appreciable inhibition; phosphate salts induced very serious inhibition. These phenomena suggest that a suitable electrical state is required for optimal chitinase activity. MgSO4, KCl and NH4Cl were equally efficient as stabilizers for protoplast liberation, although they had different effects on chitinase activity. α-glucanase was inhibited more by sucrose than by mannitol and sorbitol; β-glucanase was relatively stable to both organic and inorganic osmotic stabilizers. As chitin is the major component of the fungal cell wall, chitinase is thought to be more important for protoplast liberation than are β-glucanase and α-glucanase.
Résumé
Des méthodes ont été mises au point pour mesurer les activités β-et α-glucanase en présence des stabilisateurs osmotiques utilisés pour la production de protoplastes. En présence d'un système minéral de stabilisation osmotique, l'activité chitinase est dans l'ordre NO −3 , Cl−>SO 2−4 >PO 3−4 et Na+, K+>Ca2+, Mg2+. Les anions monovalents ainsi que les cations monovalents améliorent l'activité chitinase, tandis que les anions et les cations di- et tri-valents l'inhibent de façon appréciable; les phosphates sont fortement inhibiteurs. Ces phénomènes suggèrent que l'activité chitinase optimale exige un état électrique approprié. MgSO4, KCl et NH4Cl stabilisent de façon identique la formation des protoplastes, bien que leurs effets respectifs sur l'activité chitinase soient différents. L' α-chitinase est inhibée par le saccharose davantage que par les sucres-alcools; la β-glucanase est relativement stable en présence des stabilisateurs organiques ou minéraux. Comme la chitine est le constituant majeur des parois cellulaires fungiques, on considère que la chitinase est plus importante que la β-et l' α-glucanase pour la formation des protoplastes.
Resumen
Se han desarrollado técnicas para la determinación de: quitinasa, β-glucanasa y α-glucanasa en presencia de estabilizadores osmóticos usados en la liberación de protoplastos fúngicos. La actividad quitinasa con estabilizadores inorgánicos siguió el orden: NO −3 , Cl−>SO 2−4 >PO 3−4 Na+, K+>Ca2+, Mg2+. Los aniones monovalentes junto con los cationes monovalentes mejoraron la actividad quitinasa, mientras que tanto aniones como cationes, divalentes y trivalentes causaron una inhibición apreciable. Las sales de fosfato indujeron inhibiciones muy severas. Estos fenómenos sugieren que un estado eléctrico adecuado es necesario para una actividad quitinasa óptima. MgSO4, KCl y NH4Cl fueron igualmente eficientes como estabilizadores para la liberación de protoplastos, aunque tuvieran distintos efectos en la actividad quitinasa. La α-glucanasa se inhibió más por sucrosa que por azucares-alcoholes; la β-glucanasa se mantuvo relativamente estable frente a estabilizadores osmóticos tanto orgánicos como inorgánicos. Al ser la quitina el componente mayoritario de la pared celular de los hongos, se cree que la quitinasa es más importante que la β-glucoanasa y la α-glucanasa en lo que concierne a la liberación de protoplastos.
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References
Barry, D. 1985 Factors influencing protoplast isolation. InFungal Protoplast, eds Peberdy, J.F. & Ferenczy, L. pp 45–71. New York, Basel: Marcel Dekker, Inc.
De Vries, O.M.H. &Wessels.J.G.H. 1972 Release of protoplasts fromSchizophyllum commune by a lytic enzyme preparation fromTrichoderma viride.Journal of General Microbiology 73, 13–22.
Gascon, S., Ochoa, A.G., Novaes, M. &Villanueva, J.R. 1965 Lytic action of strepzymes fromMicromonospora AS.Archives of Microbiology 51, 156–167.
Hamlyn, P.F., Bradshaw, R.E., Mellon, F.M., Santiago, C.M., Wilson, J.M. &Peberdy, J.F. 1981 Efficient protoplast isolation from fungi using commercial enzymes.Enzyme Microbial Technology 3, 321–325.
Jeffries, T.W., Eveleigh, D.E., Macmillan, J.D., Parrish, F.W. &Reese, E.T. 1977 Enzymatic hydrolysis of the walls of yeast cells and germinated fungal spores.Biochimica et Biophysica Acta 499, 10–23.
Kumar, S. &Hansen, P.M.T. 1972 New reaction mixture for spectrophotometric determination ofN-acetylhexosamines.Analytical Chemistry 44, 398–400.
Miller, G.L. 1959 Use of dinitrosalicylic acid reagent for determination of reducing sugar.Analytical Chemistry 31, 426–428.
Peberdy, J.F. (ed.) 1979Protoplasts—Applications in Microbial Genetics. Department of Botany, Nottingham: University of Nottingham, England.
Reissig, J.L., Strominger, J.L. &Leloir, L.F. 1955 A modified colorimetric method for the determination ofN-acetyl aminosugars.Journal of Biological Chemistry 217, 959–966.
Skujiņš, J.J., Potgieter, H.J. &Alexander, M. 1965 Dissolution of fungal cell walls by aStreptomycete chitinase and β-(1 → 3)-glucanse.Archives of Biochemistry and Biophysics 111, 358–364.
Wessels, J.G.H. &Sietsma, J.H. 1979 Wall structure and growth inSchizophyllum commune. InFungal Walls and Hyphal Growth, eds Burnett, J.H. & Trinci, A.P.J. pp. 27–48. Cambridge: Cambridge University Press.
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Yu, M.Y., Chang, S.T. Effects of osmotic stabilizers on the activities of mycolytic enzymes used in fungal protoplast liberation. Mircen Journal 3, 161–167 (1987). https://doi.org/10.1007/BF00933616
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DOI: https://doi.org/10.1007/BF00933616