BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 249, 719–722 (1998) ARTICLE NO. RC989111 Hydroxamate Iron Complex with Phenoloxidase Activity Acting on Lignin and Chlorolignins Carolina Parra,* Maria ˆ ngela F. Santiago,† Jaime Rodriguez,* and Nelson Dura ´n† ,1 †Instituto Quimica, Biological Chemistry Laboratory, Universidade Estadual de Campinas, C.P. 6154, Campinas, CEP 13083-970, S.P., Brazil; and *Department of Chemistry, Renewable Resources Laboratory, Universidad de Concepcio ´n, Concepcio ´n, Chile Received June 25, 1998 Until now, no reports of these compounds with these The properties of a siderophore model, acetohydrox- properties acting on wood degradation and other ligno- amic acid (AHA), of desferral were studied. The pH, cellulosic materials were reported. In this work, one ionic strength, and AHA/Fe(III) ratios for o-dianisidine model of siderophore was studied (hydroxamate type, oxidation were optimized. Phenoloxidase activity of acetohydroxamic acid-AHA). The oxidation capacity of hydroxamates/Fe(III) acting on o-dianisidine at pH 7.0 iron (II) and iron (III) complex of o-dianisidine was and pH 3.0 was observed. Under these conditions, AHA studied. The iron (III) reduction mechanism of this was able to reduce Fe(III) to Fe(II) followed by ferroz- compound and its action on lignin and chlorolignin in ine complexation. AHA/Fe(III) complex degraded lig- kraft E1 effluents were also studied. nin and chlorolignins from kraft effluent E1 65% and 85%, respectively, after 24 h.  1998 Academic Press MATERIALS AND METHODS AHA (13.0 mmol L 01, ALDRICH) stock solutions were prepared in distilled water at 1 mg/mL concentration. The optimal concentrations Chelants are widely used in agriculture cellulose of Fe(III) and Fe(II) were measured by titration of the iron solutions pulp bleaching and pharmaceutical industrial pro- (20 mmol L 01 ) and followed by spectrophotometry UV-Vis at 220 and cesses. These compounds can solve an important num- 600 nm. Dioxane-lignin was obtained from rice husk according to Pepper et al. (11). A 1:30 dilution of 5 g L 01 dioxan-lignin in 0.2 mol ber of problems associated with the presence of metals L 01 KOH was prepared. The reaction mixture contain 0.11 mL of (1,2). The use of siderophores which are natural che- 0.16 g L 01 of dioxane-lignin, 25 mL 13 mmol L 01 AHA and 16 mL 20 lants with high affinity constant for metals such as iron mmol L 01 FeCl 3 . After the reaction, 0.5 mL of KOH 5 mol L 01 was represents a good alternative for solving the potential added to 1 mL of each mixture for the fluorescence measurement. pollution and environmental impacts of the normal Controls were carried out to eliminated interference of Fe(III) and Fe(III) complexes with dioxane-lignin. The degradation of dioxane- chelant used in industrial processes (EDTA and lignin was followed by a spectrofluorimetric technique (l emission: DTPA). Several authors have studied their biodegrad- 380-540 nm and l excitation: 360 nm). Three mL of kraft E1 effluent ability and in some situations found that EDTA and were treated with 0.75 mL of 13 mmol L 01 AHA and 16 mL 20 mmol DTPA were recalcitrant to biological and chemical deg- L 01 FeCl 3 then 0.5 mL of 5 mol L 01 KOH was added and the final radation. In addition to these problems, the nitrogen volume adjusted to 9 mL for the fluorescence measurement. Controls were carried out to eliminated the interactions of formations of content (10%) contributes to the nitrogen content re- Fe(III) complex and Fe(III) with effluent. The effluent degradation leased to the effluents (1). Siderophores are specific was followed by the same procedure described above for dioxan-lignin chelants with high affinity constants for metals form- by UV-Vis spectrophotometry (220-400 nm) and by the spectrofluo- ing complex with high stability produced by microor- rimetric technique described above. Fe(II) was determined by addi- ganism for sequestering iron where the environment tion of ferrozine (1% in deionized water). The reaction mixture con- tained 0.2 mL ferrozine, 0.4 mL 50 mmol L 01 acetate buffer at pH is deficient in these metals. Catecholate siderophores 4.5 and after addition of 25 mL of 13 mmol L 01 AHA and 16 mL 20 presence was detected in several fungi with lignin deg- mmol L 01 FeCl 3 the final volume was adjusted to 1 mL. The Fe(II) radative capacity (3,4), but very few of the hydroxa- formation was followed for 3 minutes at 562 nm. Phenoloxidase- mate-type were detected (5-10). like activity was measured using o-dianisidine (e 460 Å 29.000 L mol 01 cm 01 ) as substrate. The reaction mixture was the following: 0.1 mL 1 mmol L 01 o-dianisidine, 25 mL 13 mmol L 01 AHA and 16 mL 20 mmol L 01 FeCl 3 in 1 mL final volume at 25°C. Three different pH 1 Corresponding author. Fax: /55 19 788 3023. E-mail: duran@ iqm.unicamp.br. were tested: 50 mmol L 01 citrate-phosphate buffer pH 3.0; in the absence of buffer pH 7.0, and at pH 3.0 by addition of 0.1 mol L 01 HCl. Abbreviations used: AHA, acetohydroxamic acid; EDTA, ethylenedi- aminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid. The reaction was initiated by addition of FeCl 3 and the oxidation rate 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 719