Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes

Abstract

Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H(2)O(2) (2 mM) for optimum activity. The apparent K(m) values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H(2)O(2) were 37, 298, 1,001, 2,367 and 1,313 microM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.

FIG. 1.

(A) Effect of soybean meal (▴) and Bacto Peptone (▪) on the production of peroxidase by A. aegerita TM A1. The concentration of one component was varied in the presence of a constant amount of the other component (5 g of Bacto Peptone per liter or 20 g of soybean meal per liter). (B) Time course of peroxidase and laccase titers in a 5-liter bioreactor with a soybean meal-Bacto Peptone medium. Peroxidase activity (•) was measured by determining the oxidation of veratryl alcohol into veratraldehyde at pH 7; laccase activity (⧫) was measured with ABTS. The dotted line indicates the time course of pH.

FIG. 2.

FPLC elution profile of peroxidases from A. aegerita TM A1 on a Mono S column. Absorption at 405 nm (thick line) and 280 nm (thin line), AaP activity (•), and the NaCl gradient (dotted line) were determined. AaP activity was measured with veratryl alcohol as the substrate.

FIG. 3.

Electrophoretic characterization of purified AaP. (A) SDS-PAGE after each purification step. From left to right the lanes contained crude extract, AaP fractions after Q Sepharose separation, AaP I after SP Sepharose separation, AaP I after Mono S separation, protein standards, AaP II after SP Sepharose separation, and AaP II after Mono S separation. (B) 2-D plot of purified AaP II after isoelectric focusing and SDS-PAGE. Spots b (basic form) and a (acidic form) were plotted and used for determination of N-terminal amino acid sequences.

FIG. 4.

Spectral characteristics of AaP (3.3 μM), including the resting enzyme (thick line), the enzyme oxidized by 15 mM H₂O₂ (dotted line) or 100 mM H₂O₂ (dashed line), and the reduced enzyme (after addition of a sodium dithionite crystal) (thin line).

FIG. 5.

N-terminal sequence alignment of two AaP II isoforms and several fungal peroxidases, including A. aegerita peroxidase (AaP II spot a and AaP II spot b after 2-D electrophoresis), CPO from C. fumago (CP), C. cinereus peroxidase (CiP), LiP H8 b from P. chrysosporium (PcLiP), MnP from A. bisporus (AbMnP), and versatile peroxidase from P. eryngii (PeVP).

FIG. 6.

Effect of pH on the oxidation of ABTS (0.3 mM) (•) and veratryl alcohol (5 mM) (▴) (A) and on the oxidation of DMP (1 mM) (▪) and benzyl alcohol (⧫) (B) by AaP II. Reactions were performed in sodium phosphate/citrate buffer in the presence of 2 mM H₂O₂ at 25°C. The data are means for three parallel experiments, and the error bars indicate standard deviations.