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Isolation, purification and characterization of enzyme(s) responsible for .conversion of sterigmatocystin to aflatoxin B1

Isolation, Reinigung und Charakterisierung der für die Umwandlung von Sterigmatocystin in Aflatoxin B1 verantwortlichen Enzyme

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Zusammenfassung

Zellfreie Extrakte vonAspergillus flavus ATCC 5517/A 228 waxen aktiv bei der Umwandlung von Sterigmatocystin in Aflatoxin B1. Der Extrakt wurde mit Ultrogel ACA-54 gereinigt und ergab 10 Proteinpeaks, von denen Peak VI aktiv bei der Sterigmatocystin-Umwandlung war. Dieses Protein ergab eine Bande bei der PAG-Elektrophorese; zusätzlich wurde dieses auf der DEAE-Sephadex A-50-Säule gereinigt und dabei 2 Proteinpeaks festgestellt. Nur einer dieser Gipfel zeigte enzymatische Aktivitat und ergab eine Bande bei der PAG- und SDS-Elektrophorese. Optimal für die enzymatische Aktivität waren 28 °C und ein pH-Wert von 8. Die maximale Umwandlung wurde mit 0,6 mg Enzymprotein auf 48 × 10−8 mol Sterigmatocystin gefunden. Zn2+, Co2+ und Mn2+ beschleunigten die Umwandlung, während EDTA, PHMB und PMSF die enzymatische Aktivität in Abhängigkeit von der angewandten Menge hemmten. Die Aminosäure-Analyse zeigte, daß das Enzym 22 Aminosäuren enthält, von denen drei unbekannt waren. Das Enzym hatte ein Molekulargewicht von 64 000 dalton bei der Gelfiltration bzw. 70 000 dalton bei der SDS-PAGE.

Summary

The cell-free extract prepared fromAspergillus flavus ATCC 5517/A 228 showed activity in converting sterigmatocystin to aflatoxin B1. The extract was purified on Ultrogel AcA-54 and resulted in ten protein peaks, one of which (peak VI) showed activity in sterigmatocystin conversion. The protein in this peak gave one protein band using polyacrylamide gel (PAG)-disc electrophoresis. For further purification, protein(s) in peak VI were applied on DEAE-Sephadex A-50 and two protein peaks were detected. Only one peak showed enzyme activity which showed homogeneity as one band on PAGE and sodium dodecyl sulphate (SDS)-PAGE. The optimum temperature for the enzyme activity was 28 °C and the optimum pH was 8. The maximum conversion resulted from the action of 0.6 mg enzyme protein on 48 × 10−8 mol Sterigmatocystin. Zn2+, Co2+ and Mn2+ enhanced the enzyme acitivity, while ethylenediaminetetraacetic acid, parahydroxymercuric benzoate and phenylmethylsulphonic fluoride inhibited the enzyme activity in a dose-dependent manner. Amino-acid analysis showed the presence of 22 amino acids, three of which are unknown. The enzyme has a molecular weight of 64,000 daltons (by gel filtration) and 70,000 daltons (by SDS-PAGE).

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References

  1. Scott AM (1983) Other mycotoxins. Proceedings of the International Symposium on Mycotoxins, Cairo, Egypt, 19–24th March 1983, pp 87–110

  2. Elsworthy GC, Holker JSE, McKeown JM, Robinson JB, Mulherin LJ (1970) The biosynthesis of the aflatoxin. J Chem Soc Chem Commun 1970:1069–1070

    Google Scholar 

  3. Singh R, Hsieh DPH (1976) Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts ofAspergillus parasiticus. Appl Environ Microbiol 31:743–745

    Google Scholar 

  4. Hsieh DPH, Lin MT, Yes RC (1973) Conversion of sterigmatocystin to aflatoxin B1 byAspergillus parasiticus. Biochem Biophys Res Commun 52:992–997

    Google Scholar 

  5. Jeenah MS, Dutton MF (1983) The conversion of sterigmatocystin to O-methylsterigmatocystin and aflatoxin B1 by cell-free preparation. Biochem Biophys Res Commun 116:1114–1118

    Google Scholar 

  6. Mashaly RI, El-Deeb SA (1983) Effect of some minor elements and other additives on aflatoxin production by Aspergillus. Proceedings of the International Symposium on Mycotoxines, Cairo, Egypt, 19–24th March 1983, pp 469–476

  7. Lowry ON, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  8. Davis B (1964) Disc electrophoresis. II. Method and application to human serum protein. Ann NY Acad Sci 121:404–1414

    Google Scholar 

  9. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:681–685

    Google Scholar 

  10. Association of Official Analytical Chemists (1975) Official methods of analysis. Benjamin Franklin Station, Washington DC, pp 463–481

    Google Scholar 

  11. Heathcote JG, Dutton MF, Hibbert JR (1973) Biosynthesis of aflatoxins. Chem Ind (London) 1973:1027–1030

    Google Scholar 

  12. Detroy RW, Freer SN (1974) Methionine regulation of aflatoxin biosynthesis byAspergillus parasiticus. Dev Ind Microbiol 15:124–132

    Google Scholar 

  13. Maggon KK, Cupta SK, Venkitasubramanian TB (1977) Biosynthesis of aflatoxins. Bacteriol Rev 41:822–855

    Google Scholar 

  14. Detroy RW, Hesseltin CW (1970) Secondary biosynthesis of aflatoxin B1 inAspergillus parasiticus. Can J Microbiol 16:959–963

    Google Scholar 

  15. Hsieh DPH, Mateles RI (1971) Preparation of labelled aflatoxins with high specific activities. Appl Microbiol 22:79–83

    Google Scholar 

  16. Thomas R (1965) In: Vanek Z, Hostolex Z (eds) Biogenesis of antibiotic substances. Academic Press, New York, pp 155–172

    Google Scholar 

  17. Tulpnle WB (1969) Aflatoxicosis. Indian J Med Res 17:102–114

    Google Scholar 

  18. Marsh PB, Simpson ME, Trucksess MW (1975) Effects of trace metals on the production of aflatoxins byAspergillus parasiticus. Appl Microbiol 30:52–57

    Google Scholar 

  19. Gupta SK, Maggon KK, Venkitasubramanian TB (1977) Effect of zinc on TCA cycle intermediates and enzymes in relation to aflatoxin biosynthesis. J Gen Microbiol 99:43–48

    Google Scholar 

  20. Whitaker DR (1973) Serine proteases. A symposium sponsored by the division of Agriculture and Food Chemistry at the 166th Meeting of American Chemical Society, Chicago, pp 186–201

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Authors and Affiliations

  1. Biotechnology Laboratory, University of Alexandria Research Center (UNARC), Alexandria, Egypt

    Reda I. Mashaly, Samy L. Habib, Samy A. El-Deeb, Mohamed H. Salem & Mohamed M. Safwat

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  1. Reda I. Mashaly
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  2. Samy L. Habib
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  3. Samy A. El-Deeb
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  4. Mohamed H. Salem
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  5. Mohamed M. Safwat
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Mashaly, R.I., Habib, S.L., El-Deeb, S.A. et al. Isolation, purification and characterization of enzyme(s) responsible for .conversion of sterigmatocystin to aflatoxin B1 . Z Lebensm Unters Forch 186, 118–124 (1988). https://doi.org/10.1007/BF01042704

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  • DOI: https://doi.org/10.1007/BF01042704

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