Partial purification and characterization of Aflatoxin B₁ degrading enzyme produced by Flavobacterium aurantiacum

Author

Ronald Derike Smiley

Date of Award

12-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Food Science and Technology

Major Professor

F. Ann Draughon

Committee Members

David A. Golden, Elizabeth E. Howell, Sharon L. Melton

Abstract

The ability of crude protein extracts, obtained from the enzymatic lysis of F. aurantiacum, to remove Aflatoxin B₁ (AB₁) and Aflatoxin B₂ (AB₂) was investigated. Crude protein extracts (800 µg/ ml total protein), prepared by enzymatic lysis (0.5 mg/ ml lysozyme) of a 48 h culture, removed 83% of AB₁ and 78% of AB₂ from aqueous solution after 24 h incubation. The buffer composition, used during the enzymatic lysis of F. aurantiacum affected the amount of AB₁ degraded due to the total amount of protein released by the cells. Maximum degradation (91%) was observed by crude protein extracts lysed in 50 mM Tris-HCl (pH 7.2) containing: 50 mM EDTA, 10% sucrose, 200 mM NaCl, and 0.1% (v/v) Triton X-100. As much as 78% of AB₁ was degraded, after 24 h, in aqueous solution by crude protein extracts from F. aurantiacum lysed in only 50 mM Tris-HCl (pH 7.2). Increasing the incubation did not result in increased degradation of either AB₁or AB₂ by crude protein extracts. Degradation of AB₁ and AB₂ by crude protein extracts was lower than degradation observed by live cells of F. aurantiacum. Live cells of F. aurantiacum degraded 93% of AB₁ and 90% of AB₂ from aqueous solution after 24 h incubation.

The ability of crude protein extracts from F. aurantiacum to degrade aflatoxin B₁ (AB₁) in aqueous solution was evaluated. Crude protein extracts (800 µg/ 111 ml total protein) degraded 75% of AB₁ in solution. Heat treated crude protein extracts (800 µg/ ml total protein) degraded 6% of AB₁ in aqueous solution. DNase I treated crude protein extracts degraded 80.5% of AB₁ in solution indicating that the removal of aflatoxin by F. aurantiacum is not due to non specific binding with the bacterium's genomic DNA. Proteinase K treated crude protein extracts degraded 34.5% of AB₁ providing the most conclusive evidence yet that the degradation of aflatoxin is enzymatic. Buffer pH affected the amount of AB₁ degraded after 24 h. Maximum degradation was observed at pH 7 of those pH levels tested. Minimum degradation of AB₁ by crude protein extracts was determined to be pH 5. Acidic pH tended to be more detrimental to the enzyme responsible for degradation of AB₁ than was basic pH. The results from this work are conclusive that the degradation of AB₁ by F. aurantiacum is enzymatic.

The effects of pH and total protein concentration on the degradation of aflatoxin B, (AB₁) and B2 (AB₂) by crude protein extracts from Flavobacterium aurantiacum were evaluated. Crude protein extracts (adjusted to 800 /itg/ ml) degraded 25% of AB₁ at pH 5, 50% of AB₁ at pH 6, 70% of AB₁ at pH 7, and approximately 50% of AB₁ at pH 8. Of those pH values tested, maximum degradation of AB₁ by crude protein extracts was observed at pH 7. Crude protein extracts (adjusted to 800 /xg/ ml) degraded approximately 60% of AB2 IV at pH 5, 6, and 7 and degraded approximately 75% of AB₂ at pH 8. Of those pH values tested, maximum degradation of AB₁ by crude protein extracts was observed at pH 8. The results of this work indicate the importance of pH and on the degradation of aflatoxins by crude protein extracts from F. aurantiacum.

Ammonium sulfate precipitation, size exclusion chromatography, anion exchange chromatography, and membrane filtration were used to separate proteins from crude protein extracts from f. aurantiacum. Precipitated proteins corresponding to 60% saturation with ammonium sulfate degraded an average of 72% of AB₁ during 24 h incubation at 30°C. Some AB₁ degradation activity was observed at 20, 40, 80, and 100% saturation as well. The elution of crude protein extracts through DEAE cellulose using increasing concentrations of NaCl yielded multiple peaks. Two peaks, corresponding to pooled fraction 65-150 minutes and 155-175 minutes degraded approximately 40-45% of AB₁ from aqueous solution during 24 h incubation at 30°C. Elution of crude protein extracts through Sephadex G-125 yielded multiple peaks. The peak corresponding to pooled volumes of 100.8-109.2 mL decreased AB₁ by 63%. Two other gel filtration fractions demonstrated considerable activity. The peak corresponding to pooled volumes of 27.3-42 mL decreased AB₁by 44.5% and the peak corresponding to pooled volumes of 81.9-88.2 decreased AB₁ 38%. Ultra-filtration indicated that the protein responsible for AB₁ degradation was between 50 and 20 kD.

Recommended Citation

Smiley, Ronald Derike, "Partial purification and characterization of Aflatoxin B₁ degrading enzyme produced by Flavobacterium aurantiacum. " PhD diss., University of Tennessee, 1998.
https://trace.tennessee.edu/utk_graddiss/7495