Efficient Heterologous Expression in Aspergillus oryzae of a Unique Dye-Decolorizing Peroxidase, DyP, of Geotrichum candidum Dec 1

Abstract

Efficient expression of the dye-decolorizing peroxidase, DyP, from Geotrichum candidum Dec 1 in Aspergillus oryzae M-2-3 was achieved by fusing mature cDNA encoding dyp with the A. oryzae ␣-amylase promoter (amyB). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in A. oryzae. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by A. oryzae exhibits a function similar to that of native DyP.The newly isolated Geotrichum candidum Dec 1 was found to decolorize 21 kinds of synthetic dyes (13), and its degradation spectrum in relation to synthetic dyes is wider than that of any other decolorizing organisms reported so far. In our previous study, an extracellular enzyme, DyP (for dye-decolorizing peroxidase), was found to be responsible for the decolorization of dyes. DyP degraded phenolic compounds, such as 2,6-dimethoxyphenol and guaiacol, while it did not degrade nonphenolic veratryl alcohol (14). Considering its substrate specificity and molecular mass, DyP was found to be a novel peroxidase distinct from other peroxidases reported previously (25,26,28,41). Furthermore, the absorption spectrum of DyP exhibited a Soret band at 406 nm corresponding to a hemoprotein, and its Na 2 S 2 O 4 -reduced form revealed a peak at 556 nm that indicates the presence of a protoheme as its prosthetic group (14). We also reported the cloning of cDNA of the dyp gene (32).So far, several microorganisms capable of decolorizing some synthetic dyes have been reported (17-20, 22, 23). In particular, the white-rot fungus Phanerochaete chrysosporium was extensively studied as a dye-decolorizing fungus (5,9,19,20,21,30) and several lignin peroxidases (LiPs) of P. chrysosporium have been reported to show decolorizing activity. However, their decolorizing spectrum toward dyes is not extensively investigated, mainly because research on their lignin-degrading reaction is focused. Furthermore, although cloning and expression of several LiPs have been reported (6,11,29), there is no report on the enhancement of the yield of these enzymes by heterologous expression.Therefore, we focused on producing a large amount of DyP having dye-decolorizing activity in Aspergillus oryzae under the control of the amyB promoter. A. oryzae is known to exhibit a high growth rate and to be a safe host (1); furthermore, it can secrete gram-per-liter quantities of heterologous protein (4). From this study and our previous data, we show that DyP is a unique peroxidase.Chemicals, enzymes, and other materials. Ten kinds of synthetic dyes kindly provided by Nippon Kayaku Co., Ltd. (Tokyo, Japan), and Bayer Japan Co., Ltd. (Tokyo, Japan), were used. Cellulase and lysing enzymes were obtained from SigmaAldrich Japan...

DyP, a Unique Dye-decolorizing Peroxidase, Represents a Novel Heme Peroxidase Family

“…Enzyme-We purified DyP by the method described in our previous report (15). The enzyme activity was defined as the amount of enzyme capable of decolorizing (decreasing absorbance at 623 nm) 1 mol of 1-amino-2-sulfonyl-4-aminomethyl-9,10-anthraquinone sodium salt at 30°C for 1 min.…”

“…DyP, a glycoprotein having one heme as a cofactor, has a molecular mass of 58 kDa and requires H 2 O 2 for all enzyme reactions, indicating that it functions as a peroxidase. DyP has several characteristics that distinguish it from all other peroxidases, including a particularly wide substrate specificity, a lack of homology to most other peroxidases, and the ability to function well under much lower pH conditions (3-3.2) compared with the other plant peroxidases (13,15). In terms of substrate specificity, DyP degrades the typical peroxidase substrates, but also degrades hydroxyl-free anthraquinone, which is not a substrate of other peroxidases (7,13,15).…”

“…So far, we isolated and characterized a novel extracellular peroxidase, DyP, from the fungus Thanatephorus cucumeris Dec 1 (13)(14)(15)(16). DyP, a glycoprotein having one heme as a cofactor, has a molecular mass of 58 kDa and requires H 2 O 2 for all enzyme reactions, indicating that it functions as a peroxidase.…”

“…Wastewater containing synthetic dyes (dye-contaminated water) is blamed for multiple environmental problems because of its recalcitrant and xenobiotic characteristic but there is little effective treatment toward the wastewater (17). In contrast, DyP is a promising enzyme for the treatment of the dye-contaminated water because it degrades those dyes effectively (13,15,18). The amino acid sequence of DyP shows no homology to any other peroxidase registered in the DNA Data Bank of Japan (DDBJ) (16), indicating it does not belong to plant peroxidase superfamily.…”

“…Previously, we performed extensive studies to describe the unique characteristics and industrial merits of DyP (7,15,18,21). Here we present the crystal structure of DyP, along with spectrophotometric analyses.…”