Screening Of Aspergillus Oryzae With Glyphosate-degradation Ability By UV Mutagenesis And The Characterization Of Its Intracellular Enzyme

Ultraviolet spectrophotometry, reverse phase HPLC with pre-column derivatization and HPLC with post-column derivatization was used to determine glyphosate content of in glyphosate formulations. Compared with another two glyphosate-determination methods, reverse phase HPLC with pre-column derivatization had the advantage of high precision and accuracy, with rapid and simple analysis. Therefore reverse phase HPLC with pre-column derivatization was used as the activity assay method of glyphosate-degrading enzyme produced from Aspergillus oryzae FUJX 001.Six Aspergillus strains screened in the early research were selected as test strains. Through glyphosate resistance selection, Aspergillus oryzae A-F02 was found to be the dominant strain. Using Aspergillus oryzae A-F02 as the original strain, four mutant Aspergillus strains named FUJX 001, FUJX 002, FUJX 003 and FUJX 004 was obtained after UV mutagenesis. Glyphosate degradation test showed that Aspergillus oryzae FUJX 001 had the best degradation performance.On the basis of the single factor experiment(glyphosate adding period, plus carbon source type and concentration of carbon source, nitrogen source and nitrogen source concentration, concentration of glyphosate, initial pH, liquid volume in 250 mL flask and culture temperature), the fermentation process for producing glyphosate-degrading enzyme by Aspergillus oryzae FUJX 001 was optimized by response surface method, and the suitable fermentative conditions were as follows: soluble starch content 0.3%, glyphosate content 1492.6mg/L and initial pH 7.23. Under this condition, the intracellular glyphosate-degrading enzyme activity of Aspergillus oryzae FUJX 001 is improved by 0.43U/mL than before optimization.After fermentation, intracellular substance from Aspergillus oryzae FUJX 001 was preliminarily purified by ammounium sulfate. First ammounium sulfate is added into intracellular substance to a final concentration of 30% to precipitate the others. After centrifugation, the concentration of ammounium sulfate is increased to 80% to get crude glyphosate-degrading enzyme. Comppared with the initial crude enzyme, purification times and recovery rate of glyphosate-degrading enzyme are 1.5 and 64.7%respectively.Enzymatic analysis revealed that the optimum reaction temperature, pH and substrate concentration were 37μ,7.5 and 1000mg/L respectively. The degrading enzyme was stable and showed a higher enzyme activity when temperature was between 20℃ and 40℃and pH was between 6.5 and 8.5. The enzyme only could tolerate 1 mmol/L concentration of NaCl, and the inhibition of degrading enzyme activity would be enhanced with the increase of salt concentration. Effect of different metal ions on degrading enzyme stability was various. The degrading enzyme activity could be activated by Mg2+, Ca2+, Co2+, Mn2+, Cu2+, Ni2+ and Cs+ in different extent. Co2+, Ni2+ and Cu2+ had significant activation. The degrading enzyme activity could be inhibited by Ba2+, Pb2+ and Hg2+ in different extent. Zn2+ and Fe2+ had little effect on the degrading enzyme activity. Tween 20, Tween 80, Triton X-100 and SDS had an inhibitory effect on the degrading enzyme in different extent. EDTA had little impact on the degrading enzyme. Methanol, glycerin, isoamyl alcohol, isopropyl alcohol and n-butyl alcohol could promote the degrading enzyme activity. Ethanol had little effect on the degrading enzyme.