Abstract
Penicillium velutinum van Beyma grown on water hyacinth Eichhornia crassipes (Mart.) Solms. wastes as a rich organic source under conditions of solid state fermentation was selected in a preliminary screening for producing interesting levels of pectin lyase (PL) and protease. Addition of some natural additives to the fermentation medium improved the potentiality of enzymes production. PL and protease secreted by P. velutinum under these conditions were purified to electrophoretic homogeneity using ammonium sulphate fractionation and 2-step-column chromatography. The purified PL expressed its maximal activity at 50 °C and pH value of 5.5, showed good stability in the pH range of 5.5 to 6.0 and its midpoint of thermal inactivation (Tm) was 65 °C after 45 min of exposure. Protease expressed its maximal activity at 65 °C and pH value of 7.0, showed good stability in the pH range of 6.5 to 7.5 and its Tm was 75 °C after 45 min of exposure. Ions of Ca, Na and K showed a stimulatory effect and ions of Zn and Mn showed an inhibitory effect. Moreover, Ag and Hg showed complete inhibitory effect on the two enzymes activity. Molecular mass of the purified PL and protease was found to be 42 and 63 kDa respectively.
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References
Abd-el-Naby M.A. (1988). Biochemical study on fungal cellulase. Ph.D thesis. Faculty of Science, Mansoura Univ.
Afifi A.F., Fawzi E.M., Foaad M.A. (2002). Purification and characterization of a pectin lyase produced byCurvularia inaequalis NRRL 13884 on orange peels waste, solid state culture. Ann. Microbiol., 52: 287–297.
Alana A., Alkorta I., Liama M.J., Serra J.L. (1990). Pectin lyase activity in aPenicillium italicum strain. Appl. Environ. Microbiol., 50: 3755–3759.
Albersheim P., Killias U. (1962). Studies relating to the purification and properties of pectin transelirninase. Arch. Biochem. Biophys., 97: 107–115.
Ali U F., Saad El-Dein H S. (2008). Production and partial purification of cellulase complex byAspergillus niger and A. nidulans grown on water hyacinth blend. J. Appl. Sci. Res., 4 (7): 875–891.
Ammar M.S., Louboudy S.S., Azab M.S., EI-Deeb A.A., Afifi M.M. (1994). Pecteolytic activities and identification of the fungal flora of Tut-Ankhamen Tomb (TAT) allowed to grow under solid state fermentation (SSF) condition. AI-Azhar Bull. Sci., 6: 361–374.
Bayoumi R.A., Yassin H.M., Swelim M.A., Abdel-All E.Z. (2008). Production of bacterial pectinase(s) from agro-industrial wastes under solid state fermentation conditions. J. Appl. Sci. Res., 4 (12): 1708–1721.
Chen W.C., Hsieh H.J., Tseng T.C. (1998). Purification and characterization of a pectin lyase fromPythium splendens infected cucumber fruits. Bot. Bull. Acad. Sin., 39: 181–186.
Crueger W., Crueger A. (2000). Substrates for industrial fermentation. In: Crueger W., Crueger A., Eds, Biotechnology, A Text Book of Industrial Microbiology, Panima Publishing Corporation, New Delhi, pp. 59–62.
Datta A. (1992). Purification and characterization of a novel protease from solid substrate cultures ofPhanerochaete chrysosporium. J. Biol. Chem., 267: 728–736.
Deichevalerie G. (1871) Les plantes exotiques cultivees en Egypte a l’Institute d’ Egypte, [updated English version: Taechholmia, 18 (2) 1998 Cairo University Herbarium.
Delgado L, Blanca A.T., Huitron C, Aguilar G. (1992). Pectin lyase fromAspergillus sp. CH-Y-1043. Appl. Microbiol. Biotechnol., 39: 515–519.
Deshpandel P., Nair S., Khedkar S. (2008). Water hyacinth as carbon source for the production of cellulase byTrichoderma reesei. Appl. Biochem. Biotechnol. 10: 8476–8489.
Dixon M., Webb E.C. (1979). Enzyme kinetics. In: Enzymes, 3rd edn., Academic Press, New York, San Francisco. A subsidiary of Harcourt Brace Jovanovich, Publishers. Longman Group, p. 47.
El-Gindy A.A., Ali U.F., Ibrahim Z.M., Isaac G.S. (2008). A cost-effective medium for enhanced production of extracellular a-galactosidase in solid substrate cultures ofAspergillus awamori and A.carbonarius. Aust. J. Bas. Appl. Sci., 2 (4): 880–899.
Fawzi E.M. (2003). Production and purification of beta-glucosidase and protease byFusarium proliferatum NRRL 26517 grown onFicus nitida wastes. Ann. Microbiol., 53 (4). 463–476.
Friedrich J., Cimerman A., Steiner W. (1989). Submerged production of pectolytic enzymes byAspergillus niger: effect of different aeration/agitation regimes. Appl. Microbiol. Biotechnol., 31: 490–494.
Gervais P., Molin P., Grajek W., Bensoussan M. (1988). Influence of the water activity of a solid substrate on the growth rate and sporogenesis of filamentous fungi. Biotechnol. Bioeng., 31: 457–463.
Glantz A.S. (1992). Primer of Biostatistics. McGraw Hill, Inc., USA, pp. 2–18.
Gunnarsson C.C., Petersen CM. (2007). Water hyacinths as a resource in agriculture and energy production: A literature review. Waste Management, 27, (1): 117–129.
Hamdy H.S. (2005). Purification and characterization of the pectin lyase produced byRhizopus oryzae grown on orange peels. Ann. Microbiol., 55 (3) 205–211.
Han Y.W., Anderson A.W. (1975). Semi solid fermentation of age grass straw. Appl. Microbiol., 30: 930–934.
Henrissat B., Hefferon S.E., Yoder M.D., Lietzke S.E., Jurnak F. (1995). Structural implication of structure-based sequence alignment of proteins in the pectate lyase superfamily. Plant Physiol., 107: 963–976.
Hossain M.T, Das F.,Marzan L.W., Rahman M.S., Anwar M.N. (2006). Some Properties of protease of the fungal strainAspergillus flavus. Int. J. Agricul. Biol., 2: 162–164.
Kunitz M. (1947). Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol., 30: 291–310.
Liao C.H., McCallus D.E., Fett W.F. (1994). Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase inPseudomonas viridi-flava. Mol. Plant-Microbe In., 7: 391–400.
Liao C.H., Sullivan J., Grady J., Wong L.J.C. (1997). Biochemical characterization of pectate lyases produced by fluorescentPseudomonas associated with spoilage of fresh fruits and vegetables. J. Appl. Microbiol., 83: 10–16.
Louboudy S.S., El-Gamal M.S., Ammar M.S., Ali M.O. (2001). Microbial utilization ofEichhornia crassipes for pectinases and cellulases enzyme production under solid state fermentation (SSF) conditions, Fourth Int. Sci. Conf. “Science Development and Environment”. Science Fac, Al-Azhar Univ., Cairo, Egypt, 27-29 March, p. 32.
Manachini P.L., Parini C, Fortina G. (1988). Pectic enzymes fromAureobasidium pullulans LV 10. Enzyme Microb. Technol., 10: 682–685.
Markwell M.A.K., Haas S.M., Bieber L.L, Tolbert N.E. (1978). A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87: 206–210.
Merck (1989). Merck Index, 11th edn.
Oriol E., Schettino B., Viniegra-Gonzalez G., Raimbault M. (1988a). Solid state culture ofAspergillus niger on support. J. Ferm. Technol., 66: 57–62.
Oriol E., Raimbault M., Roussos S., Viniegra-Gonzales G. (1988b). Water and water activity in the solid state fermentation of cassava starch byAspergillus niger. Appl. Microbiol. Biotechnol., 27: 498–503.
Palmer T. (1991). Extraction and Purification of Enzymes. In: Understanding Enzymes, Ellis Horwood. Ltd., England, pp. 301–317.
Palmieri G., Bianco C, Cennamo G., Giardina P., Marino G., Monti M., Sannia G. (2001). Purification, characterization and functional role of a novel extracellular protease fromPleurotus ostreatus. Appl. Environ. Microbiol., 67: 2754–2759.
Peterson E.A., Sober H.A. (1962). Column chromatography of protein: Substituted cellulases. In: Colowich S., Kaplan N., Eds, Methods in Enzymology, Vol. 5, New York, pp. 3-27.
Plummer D.T. (1978). The practice of column chromatography. In: An Introduction to Practical Biochemistry, McGraw-Hill Book Company Ltd, UK, pp. 61–66.
Reese E.T (1977). Degradation of polymeric carbohydrates by microbial enzymes. Rec. Adv. Biochem., 11: 311–367.
Rhodes W.G., Lindberg R.A., Drucker H. (1983). Purification and characterization of an extracellular protease fromNeurospora crassa. Arch. Biochem. Biophys., 223: 514–520.
Spagna G., Pifferi P.G., Gilioli E. (1995). Immobilization of a pectinlyase fromAspergillus niger for application in food technology. Enzyme Microb. Technol., 17: 729–738.
Wijesundera R.L.C., Bailey J.A., Byrde R.J.W. (1984). Production of pectin lyase byCollectotrichum lindemuthianum in culture and in infected bean(Phaseolus vulgaris) tissue. J. Gen. Microbiol., 130: 285–290.
Yang J., Shih I., Tzeng Y, Wang S. (2000). Production and purification of protease from aBacillus subtilis that can deproteinize crustacean wastes. Ezyme Microb. Technol., 26: 406–413.
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Fawzi, E.M. Purification and characterization of the pectin lyase and protease produced byPenicillium velutinum grown onEichhornia crassipes under solid state fermentation. Ann. Microbiol. 59, 755–761 (2009). https://doi.org/10.1007/BF03179220
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DOI: https://doi.org/10.1007/BF03179220