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Peroxidases Depolymerize Lignin in Organic Media but Not in Water

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Peroxidases Depolymerize Lignin in Organic Media but Not in Water Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6255-6257, September 1986 Biochemistry Peroxidases depolymerize lignin in organic media but not in water (lignin degradation/biopolymers/enzymes in nonaqueous solvents/ligninase/enzymatic degradation) JONATHAN S. DORDICK, MICHAEL A. MARLETTA, AND ALEXANDER M. KLIBANOV Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139 Communicated by Nathan 0. Kaplan,* April 7, 1986 ABSTRACT Horseradish peroxidase and milk lactoper- Both unlabeled and 14C-labeled synthetic lignins and kraft oxidase, while unable to degrade either synthetic or natural pine lignin were fractionated prior to use. In each case, a lignins in aqueous solutions, vigorously depolymerize polycon- 25-mg/ml solution of lignin in dimethylformamide was pre- iferyl alcohol, milled wood lignin, and kraft pine lignin in pared and subjected to gel filtration on a Sephadex LH-20 dioxane, dimethylformamide, or methyl formate containing column equilibrated with anhydrous dioxane. Only material 5% aqueous buffer (10 mM acetate, pH 5). Horseradish of molecular weight greater than 1500 was employed in peroxidase, solubilized in organic media by chemical modifi- subsequent experiments. cation, can also degrade lignin in native lignocellulose (wheat A suspension of a lignin in a buffered aqueous solution straw). containing peroxidase and H202 or a suspension of peroxi- dase in a 95% (vol/vol) organic solvent containing dissolved After cellulose, lignin is the most prevalent organic substance lignin and H202 was shaken on a rotary shaker under the on Earth (1). Due to lignin's challenging structural complex- conditions described in the legend to Fig. 1. (In independent ity, its potential as the renewable resource of aromatic experiments, it was determined that after 2 days of shaking chemicals, and the fact that its degradation is required for in 95% dioxane, horseradish peroxidase lost about half its subsequent enzymatic hydrolysis of wood's cellulose and enzymatic activity; in the aqueous buffer, the corresponding hemicellulose, a great deal of work has been performed on loss was 10%.) Following such incubation for a given period lignin biodegradation (2-4). A fungal enzyme, ligninase, that of time, the resultant mixture was analyzed by gel-permea- can depolymerize lignin has been isolated (5, 6). Biotechno- tion chromatography (see Results and Discussion and the logical applications of ligninase, however, are severely re- legend to Fig. 1). stricted by the scarcity of the enzyme. In order to degrade lignocellulose, horseradish peroxidase Ligninase catalyzes a one-electron oxidation oflignin in the was solubilized in 95% dioxane. To that end, 50 mg of the presence of H202 (7-9) and thus mechanistically resembles a enzyme were suspended in 10 ml of dioxane (containing 5% peroxidase (10). Peroxidases are ubiquitous and abundant in aqueous buffer, pH 5.0), and 3 mM palmitoyl chloride was it was to test whether they added. The reaction mixture was stirred at room temperature nature (11), and therefore tempting for 30 min, followed by removal of the solids by centrifuga- can act as ligninases. [Thus far, it has been found that tion. The concentration of dissolved peroxidase was about 2 horseradish and other common peroxidases can degrade only mg/ml (determined spectrophotometrically), and the specific monomeric and dimeric phenols (12-17) or water-soluble activity of the modified enzyme was approximately one-third lignosulfonates (18-21).] We report here that horseradish of the initial. peroxidase and milk lactoperoxidase, while inactive toward lignin in water, can vigorously depolymerize both synthetic and natural lignins in organic media. RESULTS AND DISCUSSION A copolymer of coniferyl alcohol (98%) and vanillyl alcohol MATERIALS AND METHODS (2%) has commonly been used as a synthetic model of lignin (22, 23). This synthetic lignin was suspended in an aqueous Horseradish peroxidase and cow's milk lactoperoxidase (EC solution (pH 5.0), followed by addition of horseradish per- 1.11.1.7) were purchased from Sigma and had specific activ- oxidase and hydrogen peroxide. The suspension was shaken ities of 200 and 100 purpurogallin units/mg of solid, respec- at 20°C for 96 hr, then 2 volumes of dimethylformamide was tively. added to solubilize the lignin, and the resultant solution was A copolymer ofconiferyl alcohol and vanillyl alcohol (98:2) subjected to gel filtration (Fig. LA). No low molecular weight was prepared according to the method of Kirk et al. (22). products were detected by UV absorbance. The lack of Kraft pine lignin (Indulin-AT) was obtained from Westvaco horseradish peroxidase-catalyzed depolymerization of syn- (Charleston Heights, SC). Milled wood lignin, 14C-labeled thetic lignin was confirmed with [ring-14C]lignin; no appre- synthetic lignin, and 14C-labeled wheat straw were gifts of ciable radioactivity was found beyond the void volume. W. G. Glasser (Virginia Polytechnic Institute and State It was possible that the failure ofperoxidase to break down University), T. K. Kirk (U.S. Department of Agriculture lignin might be due to the fact that the latter, being completely Forest Products Laboratory), and T. P. Abbott (U.S. De- insoluble in water and having a very compact structure (1, partment of Agriculture Northern Regional Research Cen- 23), was simply inaccessible to the enzyme. To overcome ter), respectively. this, we endeavored to find a solvent that would both dissolve Lipophilic Sephadex LH-20 was purchased from Sigma, lignin and sustain the catalytic activity of horseradish per- and polystyrene standards from Polysciences (Warrington, oxidase. Dioxane (containing 5% aqueous buffer, pH 5.0), PA). All chemicals and solvents used in this work were ofthe which, among several others, met both of those require- highest purity commercially available. ments, was therefore used as the reaction medium for the enzymatic lignin degradation. At the same reagent concen- trations as in water, significant peroxidase-catalyzed depoly- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *Deceased, April 15, 1986. 6255 Downloaded by guest on September 25, 2021 6256 Biochemistry: Dordick et al. Proc. Natl. Acad. Sci. USA 83 (1986) A 0.6 0.50 A < 0.3 0.25 c | b a? a 0.61 B B rn r- C14 0.31 10 20 30 40 Effluent, ml Effluent, ml FIG. 2. Gel filtration of milled wood lignin (A) and kraft pine FIG. 1. Gel filtration of synthetic lignin following its treatment lignin (B) treated with horseradish peroxidase either in water or in with horseradish peroxidase in water (A) and in dioxane (B). A dioxane. Both lignins were fractionated prior to use. Conditions were Sephadex LH-20 column (22 x 1.5 cm) was used with anhydrous the same as described in the legend to Fig. 1. (A) Enzymatic dioxane as the mobile phase (flow rate of0.6 ml/min). The calibrants, treatment was for 0 (A) or 72 hr in water (o) or dioxane (e). (B) whose elution volumes are indicated by the arrows, were ethyl Enzymatic treatment was for 0 (v) or 63 hr in water (o) or dioxane ferulate (Mr 222) (a) and polystyrenes of Mr 500 and 2000 (b and c, (U). In both cases, no lignin degradation was observed without respectively). Reaction conditions: 0.5 mg of horseradish peroxidase peroxidase (with only H202 present). per ml, 2 mM H202, 0.3 mg oflignin per ml; 200C; shaking at 250 rpm. Reaction medium was either 1 ml of aqueous buffer (10 mM acetate, well. 2 pH 5.0) that contained 1o dioxane (brought in with the lignin after not only synthetic lignin but natural lignins as Fig. fractionation) (A) or 1 ml ofdioxane containing 5% aqueous buffer (10 shows the enzymatic depolymerization of milled wood lignin mM acetate, pH 5.0) (B) and 0.15 ml of it was applied to the column (A) and kraft pine lignin (B) in 95% dioxane; again, no lignin after removal of the enzyme by centrifugation. Reaction times: 0 (o) degradation was observed in water. Furthermore, the ability and 96 hr (o) in A; 0 (o), 6 (n), and 96 hr (A) in B. to depolymerize lignin was not limited to horseradish perox- idase, since under the same conditions, milk lactoperoxidase merization of synthetic lignin occurred in 95% dioxane after was almost as effective (the reaction took place in 95% just 6 hr, and after 96 hr it was rather striking (Fig. 1B). This dioxane but not in water). lignin degradation was confirmed using [ring-14C]lignin- In addition to the solubilization of lignin in organic sol- after a 96-hr enzymatic treatment in dioxane, about one-third vents, other effects may also contribute to the observed of the total radioactivity was found in the lower molecular peroxidase-catalyzed depolymerization of lignin in dioxane weight fractions. The products of peroxidase-catalyzed deg- and dimethylformamide but not in water. Repolymerization radation of a copolymer of coniferyl and vanillyl alcohols in of the free-radical lignin fragments should be greatly dimin- 95% dioxane were subjected to both thin-layer chromatog- ished in these solvents, as compared to water, because the raphy [silica gel plates, benzene/ethanol/propionic acid rate constants of solvent quenching of similar free radicals in (88:8:2) as a solvent] and HPLC (C18 reversed-phase column, dioxane and dimethylformamide exceed that in water by 30% acetonitrile/70% water as a solvent). Both chromatog- more than two orders of magnitude (24). Also, in organic raphies revealed a complex mixture of components, of which media, in contrast to water, the enzymatically produced two were positively identified: ferulic acid and vanillin (16% hydrophobic free-radical lignin fragments will partition from and <1% of the total products, respectively, by HPLC). No the aqueous layer surrounding peroxidase (where they are appreciable depolymerization of lignin in dioxane was de- formed) into the bulk solvent, thereby becoming "diluted" tected in the absence of H202 or in its presence with and hence less able to take part in repolymerization reac- peroxidase preinactivated by boiling.
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