Characterization of a Novel Manganese Peroxidase-Lignin Peroxidase Hybrid Isozyme Produced by Bjerkandera Species Strain BOS55 in the Absence of Manganese

Abstract

A novel manganese-dependent peroxidase (MnP) isozyme produced in manganese-free cultures ofBjerkandera sp. strain BOS55 was purified and characterized. The production of the enzyme was greatly stimulated by the exogenous addition of various physiological organic acids such as glycolate, glyoxylate, and oxalate. The physical properties of the enzyme are similar to those of MnP isozymes from different white rot fungi (M r= 43,000, pI 3.88, and ε407 nm = 123 mm−1cm−1). The Bjerkandera MnP was efficient in the oxidation of Mn(II), as indicated by the kinetic constants (lowK m of 51 μm and turnover number of 59 s−1). Furthermore, the isozyme was able to oxidize various substrates in the absence of manganese, such as 2,6-dimethoxyphenol, guaiacol, ABTS, 3-hydroxyanthranilic acid, and o- andp-anisidine. An interesting characteristic of the isozyme was its ability to oxidize nonphenolic substrates, veratryl alcohol and 1,4-dimethoxybenzene, without manganese addition. The affinity for veratryl alcohol (K m = 116 μm) and its turnover number (2.8 s−1) are comparable to those of lignin peroxidase (LiP) isozymes from other white rot fungi. Manganese at concentrations greater than 0.1 mm severely inhibited the oxidation of veratryl alcohol. The results suggest that this single isozyme is a hybrid between MnP and LiP found in other white rot fungi. The N-terminal amino acid sequence showed a very high homology to those of both MnP and LiP isozymes from Trametes versicolor. A novel manganese-dependent peroxidase (MnP) isozyme produced in manganese-free cultures ofBjerkandera sp. strain BOS55 was purified and characterized. The production of the enzyme was greatly stimulated by the exogenous addition of various physiological organic acids such as glycolate, glyoxylate, and oxalate. The physical properties of the enzyme are similar to those of MnP isozymes from different white rot fungi (M r= 43,000, pI 3.88, and ε407 nm = 123 mm−1cm−1). The Bjerkandera MnP was efficient in the oxidation of Mn(II), as indicated by the kinetic constants (lowK m of 51 μm and turnover number of 59 s−1). Furthermore, the isozyme was able to oxidize various substrates in the absence of manganese, such as 2,6-dimethoxyphenol, guaiacol, ABTS, 3-hydroxyanthranilic acid, and o- andp-anisidine. An interesting characteristic of the isozyme was its ability to oxidize nonphenolic substrates, veratryl alcohol and 1,4-dimethoxybenzene, without manganese addition. The affinity for veratryl alcohol (K m = 116 μm) and its turnover number (2.8 s−1) are comparable to those of lignin peroxidase (LiP) isozymes from other white rot fungi. Manganese at concentrations greater than 0.1 mm severely inhibited the oxidation of veratryl alcohol. The results suggest that this single isozyme is a hybrid between MnP and LiP found in other white rot fungi. The N-terminal amino acid sequence showed a very high homology to those of both MnP and LiP isozymes from Trametes versicolor. Lignin is an abundant natural aromatic polymer occurring in the woody tissue of higher plants. Due to its hydrophobicity and complex random structure lacking regular hydrolyzable bonds, lignin is poorly biodegraded by most microorganisms. The best degraders of lignin are white rot fungi that produce extracellular peroxidases (1Kirk T.K. Farrell R.L. Annu. Rev. Microbiol. 1987; 41: 465-505Crossref PubMed Scopus (2059) Google Scholar). These enzymes are involved in the initial attack of the lignin polymer (2Hammel K.E. Jensen Jr., K.A. Mozuch M.D. Landucci L.L. Tien M. Pease E.A. J. Biol. Chem. 1993; 268: 12274-12281Abstract Full Text PDF PubMed Google Scholar). Manganese-dependent peroxidase (MnP) 1The abbreviations used are: MnP, manganese-dependent peroxidase; LiP, lignin peroxidase; FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography; DMP, 2,6-dimethoxyphenol. is one of the most frequently encountered peroxidases among white rot fungi (3de Jong E. Field J.A. de Bont J.A.M. FEMS Microbiol. Rev. 1994; 13: 153-188Crossref Scopus (1) Google Scholar). MnP is a glycosylated hemeprotein occurring mostly as a number of isozymes (1Kirk T.K. Farrell R.L. Annu. Rev. Microbiol. 1987; 41: 465-505Crossref PubMed Scopus (2059) Google Scholar). MnP has the same catalytic cycle as other peroxidases, involving a 2-electron oxidation of the heme by H2O2 and subsequent reduction of compound I via compound II in two 1-electron steps to the native enzyme (4Glenn J.K. Akileswaran L. Gold M.H. Arch. Biochem. Biophys. 1986; 251: 688-696Crossref PubMed Scopus (375) Google Scholar, 5Wariishi H. Akileswaran L. Gold M.H. Biochemistry. 1988; 27: 5365-5370Crossref PubMed Scopus (278) Google Scholar). A distinctive characteristic of this enzyme is that the best reducing substrate for compounds I and II is Mn(II), a metal naturally present in wood. The Mn(III) formed oxidizes other substrates (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar). Crystallographic and site-directed mutant studies confirmed the presence of a unique manganese-binding site in MnP from the best studied white rot fungus, Phanerochaete chrysosporium (7Sundaramoorthy M. Kishi K. Gold M.H. Poulus T.L. J. Biol. Chem. 1994; 269: 32759-32767Abstract Full Text PDF PubMed Google Scholar,8Sundaramoorthy M. Kishi K. Gold M.H. Poulus T.L. J. Biol. Chem. 1997; 272: 17574-17580Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Organic acids such as oxalate, glyoxylate, and lactate were shown to have an important role in the mechanism of MnP and lignin degradation (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar, 9Kuan I.C. Tien M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1242-1246Crossref PubMed Scopus (160) Google Scholar, 10Shimada M. Ma D.B. Akamatsu Y. Hattori T. FEMS Microbiol. Rev. 1994; 13: 285-296Crossref Scopus (83) Google Scholar). Mn(III) is stripped from the enzyme by organic acids, and the formed Mn(III)-organic acid complex acts as a diffusible mediator in the oxidation of lignin by MnP (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar, 9Kuan I.C. Tien M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1242-1246Crossref PubMed Scopus (160) Google Scholar). Mn(III) can also oxidize organic acids, yielding radicals (11Khindaria A. Grover T.A. Aust S.D. Arch. Biochem. Biophys. 1994; 314: 301-306Crossref PubMed Scopus (58) Google Scholar). There are two possible sources of organic acids during lignin degradation. The fungi are able to produce de novo aliphatic organic acids, mainly oxalate (12Dutton M.V. Evans C.S. Can. J. Microbiol. 1996; 42: 881-895Crossref Scopus (519) Google Scholar). Moreover, organic acids are formed from the degradation of lignin (13Hammel K.E. Mozuch M.D. Jensen K.A. Kersten P.J. Biochemistry. 1994; 33: 13349-13354Crossref PubMed Scopus (61) Google Scholar, 14Roy B.P. Archibald F. Appl. Environ. Microbiol. 1993; 59: 1855-1863Crossref PubMed Google Scholar). The ligninolytic enzymes are produced during the secondary metabolism triggered by nutrient limitation (1Kirk T.K. Farrell R.L. Annu. Rev. Microbiol. 1987; 41: 465-505Crossref PubMed Scopus (2059) Google Scholar, 15Gold M.H. Alic M. Microbiol. Rev. 1993; 57: 605-622Crossref PubMed Google Scholar). Manganese is an absolute requirement for expression of mnp genes in several white rot fungi (16Brown J.A. Alic M. Gold M.H. J. Bacteriol. 1991; 173: 4101-4106Crossref PubMed Google Scholar, 17Périé F.H. Gold M.H. Appl. Environ. Microbiol. 1991; 57: 2240-2245Crossref PubMed Google Scholar). Consistent with manganese requirement, putative metal response elements were found in the promoter regions (15Gold M.H. Alic M. Microbiol. Rev. 1993; 57: 605-622Crossref PubMed Google Scholar, 18Alic M. Akileswaran L. Gold M.H. Biochim. Biophys. Acta. 1997; 1338: 1-7Crossref PubMed Scopus (57) Google Scholar). Furthermore, enhanced MnP production is associated with increased manganese concentrations in many white rot fungi (17Périé F.H. Gold M.H. Appl. Environ. Microbiol. 1991; 57: 2240-2245Crossref PubMed Google Scholar, 19Bonnarme P. Jeffries T.W. Appl. Environ. Microbiol. 1990; 56: 210-217Crossref PubMed Google Scholar, 20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar). Bjerkandera sp. strain BOS55 is a good MnP producer (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar). Previous studies have shown that this fungus is nitrogen-unregulated, and MnP production is greatly increased by nutrient nitrogen sufficiency and excess (21Mester T. Peña M. Field J.A. Appl. Microbiol. Biotechnol. 1996; 44: 778-784Google Scholar). MnP production is also enhanced by supplementing the culture medium with simple organic acids (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar). Following the general trend of most white rot fungi, manganese is stimulatory for MnP in this fungus (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar). However,Bjerkandera sp. strain BOS55 is atypical since it produces considerable MnP in manganese-free media (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar, 22Moreira M.T. Feijoo G. Sierra-Alvarez R. Lema J. Field J.A. Appl. Environ. Microbiol. 1997; 63: 1749-1755Crossref PubMed Google Scholar). In this study, we characterized a MnP isozyme produced in the absence of manganese. Furthermore, the manganese dependence for the oxidation of various substrates was tested. Bjerkandera sp. strain BOS55 (ATCC 90940) was maintained in glucose/peptone/yeast extract slants as described earlier (23Mester T. de Jong E. Field J.A. Appl. Environ. Microbiol. 1995; 61: 1881-1887Crossref PubMed Google Scholar). Prior to the experiment, the fungus was transferred to glucose/malt extract plates (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar), which were incubated for 4–6 days at 30 °C. As inocula, 6-mm diameter agar plugs from the leading mycelial edge were used in the experiments. The standard basal medium used contained 2.2 mm nitrogen as diammonium tartrate, 56 mmglucose, 2 mg/liter thiamin, and manganese-free BIII mineral medium (pH 4.5), and the standard buffer (2,2-dimethyl succinate) was omitted (24Tien M. Kirk T.K. Methods Enzymol. 1988; 161B: 238-248Crossref Scopus (1292) Google Scholar). All glassware was previously washed with 5 mHNO3 to remove contaminating manganese. The measured concentration in the manganese-free medium was always <0.01 μm. In some experiments, the basal medium was also supplemented with manganese nutrients, providing a final concentration of 0.033 mm. Glycolic acid, glyoxylic acid, and oxalic acids (5 mm) were individually added (neutralized to pH 4.5) to the cultures at the time of incubation in order to study their stimulatory effect on MnP production. Filter-sterilized 25-ml aliquots were placed in 250-ml cylindrical flasks, which were previously autoclaved with a drop of demineralized water (22Moreira M.T. Feijoo G. Sierra-Alvarez R. Lema J. Field J.A. Appl. Environ. Microbiol. 1997; 63: 1749-1755Crossref PubMed Google Scholar). Each flask was inoculated with three agar plugs. Cultures were incubated statically under an air atmosphere at 27 °C. The collected supernatant was separated from the mycelial mat by centrifugation (20,000 × g). The extracellular fluid was washed with 10 mm sodium acetate (pH 6.0) and concentrated by ultrafiltration with a PM-10 membrane (10 kDa; Amicon, Rotterdam, The Netherlands). The concentrated supernatants were loaded in 10 mm sodium acetate onto a MonoQ anion-exchange column (Pharmacia, Uppsala, Sweden), and heme-containing proteins were monitored with a wavelength of 405 nm. Proteins were eluted with a linear gradient of sodium acetate up to 450 mm (pH 6.0) in 45 min, and 1-ml fractions were collected (1 ml/min) for 45 min. The fractions, 33–35, that contained the most MnP activity were collected and then washed and concentrated by ultrafiltration. The concentrate was eluted by creating a decreasing pH gradient in the pH range of 4.5–2.7 on a MonoP chromatofocusing column (Pharmacia) as described earlier (25Johansson T. Nyman P.O. Arch. Biochem. Biophys. 1993; 300: 49-56Crossref PubMed Scopus (69) Google Scholar), and 1-ml fractions were collected (1 ml/min) for 40 min. The MnP-containing fraction was washed with demineralized water and concentrated by ultrafiltration. The concentrate was used for enzyme characterization, kinetic, and substrate oxidation studies. The MonoP enzyme concentrate was brought through two additional FPLC steps to confirm its purity. First, gel filtration was carried out using a Superdex 75 HR 10/30 column (10 × 300 mm, volume of 24 ml; Pharmacia). The enzyme was eluted in 10 mm sodium acetate buffer (pH 6.0) with a flow rate of 0.5 ml/min for 50 min, and 0.5-ml fractions were collected. A single symmetrical peak was observed, and the enzyme-containing fractions, 22 and 23, were pooled and concentrated by ultrafiltration. The enzyme was washed with 10 mm potassium phosphate buffer (pH 6.0). Diammonium sulfate was added to the protein, reaching a final concentration of 1m, and loaded onto a 1-ml hydrophobic interaction chromatography column (phenyl-Sepharose HP, Pharmacia). Proteins were eluted in a decreasing linear gradient of 1 m diammonium sulfate in 10 mm potassium phosphate buffer (pH 6.0) in 45 min (1 ml/min). Fractions (1 ml) were also collected every minute. Again, a single symmetrical peak was observed eluting from 19 to 22 min. Protein concentration was determined according to Bradford (26Bradford M.M. Anal. Bichem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) using bovine serum albumin as a standard. The concentration of purified MnP was also measured at a wavelength of 406 nm and calculated using ε406 nm = 129 mm−1 cm−1 (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar), which gave approximately the same result. The molecular mass of the enzyme was determined by SDS-polyacrylamide gel electrophoresis (12% polyacrylamide gel) in a Phastsystem (Pharmacia) using marker proteins (low molecular mass calibration kit from Pharmacia) as standards. The isoelectric point was estimated by chromatofocusing by measuring the pH of the collected fraction containing MnP isozyme. The enzyme absorbance spectrum was determined with 86 mg/liter MnP in the presence of 50 mm malonate buffer (pH 4.5) by scanning the absorbance of the enzyme at a wavelength range of 350–700 nm using a Perkin-Elmer Lambda1 UV-visible spectrophotometer. The oxidized enzyme was scanned after addition of 0.2 mm H2O2. Kinetic constants of MnP activities for H2O2 and Mn(II) were calculated by the formation of Mn(III) malonate complex at 270 nm (ε = 11,590m−1 cm−1) (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar). The oxidation of 2,6-dimethoxyphenol to coerulignone (ε = 49,600 m−1cm−1), ABTS to ABTS⨥ (ε = 36,000 m−1 cm−1), and veratryl alcohol to veratraldehyde (ε = 9300 m−1 cm−1) was measured at wavelengths of 469, 420, and 310 nm, respectively (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar,24Tien M. Kirk T.K. Methods Enzymol. 1988; 161B: 238-248Crossref Scopus (1292) Google Scholar, 27Wolfenden B.S. Willson R.L. J. Chem. Soc. Google Scholar). MnP was incubated in 0.2 of 50 mm buffer (pH at with without to study the effect of 0.1 mm veratryl alcohol on the collected at time were 50 to activity with Mn(II) in 50 (pH were carried out in the oxidation of veratryl 1,4-dimethoxybenzene, and Bjerkandera MnP was incubated with mm compound in 50 mm buffer (pH The was with 0.1 H2O2 was added every reaching a final concentration of mm. of the was with 1 volume of oxidation was by All were carried out in were by a with an The column × mm) was with The Netherlands). The gradient 30 was and of to at and min, were on and with those of standards. In some experiments, the effect of Mn(II) on veratryl alcohol oxidation was The oxidation of 0.1 mm veratryl alcohol was during a incubation with in 50 mm sodium buffer (pH with 2 mm sodium and Mn(II) a similar was with purified isozyme from Bjerkandera sp. strain BOS55 R. S. Field J.A. Lett. PubMed Scopus Google Scholar) at the same in of veratryl alcohol of veratraldehyde was by Manganese of the culture media was measured by a mass by the of and The Netherlands). The for manganese is μm. The N-terminal amino acid sequence was carried out at the The Netherlands). All were and used without Bjerkandera sp. strain BOS55 was in manganese-free medium in the presence and absence of simple organic The FPLC studies using the MonoQ column the increased production of hemeprotein with MnP activity as a of organic acid addition to the As an the FPLC and enzyme activities measured in the collected fractions of extracellular fluid from a culture in the absence presence of 5 mm are shown in results were with oxalate and In the cultures without the organic acids, very hemeprotein and MnP activities were The addition of stimulated the production of several heme most of the MnP activity was to one peak In other studies with and other organic acids the MnP activity was between two fractions and MnP activity was present in fractions 33–35, this isozyme was and used for The is in of the MnP isozyme from Bjerkandera sp. strain activity was determined by Mn(III) malonate formation as described under and extracellular MnP activity was determined by Mn(III) malonate formation as described under and extracellular in a As the in the enzyme characterization, the molecular mass was measured by the SDS-polyacrylamide gel electrophoresis and was found to gel electrophoresis in one a The pI was as estimated by the chromatofocusing The spectrum of the enzyme showed a peak for native enzyme at nm with a of 123 mm−1 and the addition of excess in a to nm The N-terminal amino acid sequence of the protein was also determined up to amino of the native and native substrates were used to the manganese dependence of their oxidation at pH in 50 The results are in The substrate 2,6-dimethoxyphenol was oxidized in the presence and absence of manganese. The oxidation rate of without manganese was of that in the presence of manganese. trend was observed in the of other substrates such as guaiacol, ABTS, and The enzyme was also able to oxidize veratryl alcohol at pH without manganese dependence of substrate oxidation by the MnP (1 without with × activity of the enzyme was in the absence and presence of 1 mm manganese at pH in 50 mm malonate The concentration of the enzyme was without with × in a constants of the MnP isozyme from Bjerkandera sp. strain in 50 mm malonate pH in 50 mm for DMP, ABTS, and veratryl alcohol were determined in the absence of of was used since coerulignone (ε = cm−1) to two for DMP, ABTS, and veratryl alcohol were determined in the absence of alcohol for DMP, ABTS, and veratryl alcohol were determined in the absence of alcohol for DMP, ABTS, and veratryl alcohol were determined in the absence of pH in 50 mm malonate pH in 50 mm Kinetic for DMP, ABTS, and veratryl alcohol were determined in the absence of An of was used since coerulignone (ε = cm−1) to two in a The activity of the enzyme was in the absence and presence of 1 mm manganese at pH in 50 mm malonate The concentration of the enzyme was The of the substrate and of various reducing substrates of the enzyme were in of m and turnover number a physiological pH of Mn(II) was the best reducing with a turnover of 59 veratryl DMP, and ABTS were oxidized by the enzyme at this with a turnover of to in the absence of The enzyme a high affinity for Mn(II), DMP, and ABTS, the affinity for veratryl alcohol at pH was an order of The pH dependence of the oxidation of several substrates was also the pH dependence of Mn(II) and The rate of Mn(II) oxidation was observed at pH pH the Mn(II) activity was The oxidation of was the at pH and at this the rate was comparable in the presence and absence of manganese. the oxidation of ABTS and veratryl alcohol in the absence of manganese was higher at pH with pH pH the affinity of the Bjerkandera MnP for veratryl alcohol was greater than at pH that the manganese and veratryl alcohol oxidation activities were to one the enzyme was purified by two additional FPLC gel filtration and hydrophobic one symmetrical peak was observed, and the of 0.1 mm Mn(II) oxidation rate to 0.1 alcohol oxidation rate at pH at nonphenolic compounds were incubated in the absence of manganese for with the Bjerkandera MnP at pH in 50 mm The of the oxidation were by The results are in The of veratryl alcohol oxidation was with a of was also as a of with a of was oxidized to a by the isozyme. alcohol was oxidized by this of various nonphenolic compounds by the purified Bjerkandera of of oxidation = × = × are the S.D. of three 123 contained mg/liter MnP, and mm H2O2 in 50 mm buffer (pH of oxidation = × = × are the S.D. of three in a The contained mg/liter MnP, and mm H2O2 in 50 mm buffer (pH The veratryl alcohol oxidation to veratraldehyde by and LiP was in the presence of manganese LiP was by the presence of mm Mn(II) However,Bjerkandera MnP Mn(II) in the concentration range of mm inhibited the oxidation of veratryl alcohol. Mn(II) at a concentration mm) stimulated the veratryl alcohol oxidation to of manganese on the oxidation of veratryl alcohol to veratraldehyde by the purified Bjerkandera MnP in with Bjerkandera formed are as the S.D. of three mm mm mm mm contained 10 mg/liter MnP, 0.1 and mm H2O2 in 50 mm buffer with 2 mm (pH The incubation time was 10 min. The isozyme was added at veratryl as are as the S.D. of three tested. in a The contained 10 mg/liter MnP, 0.1 and mm H2O2 in 50 mm buffer with 2 mm (pH The incubation time was 10 min. The isozyme was added at veratryl as Bjerkandera sp. strain BOS55 produces MnP in high under different culture An interesting characteristic of this fungus is the ability to produce MnP in manganese-free media (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar, 22Moreira M.T. Feijoo G. Sierra-Alvarez R. Lema J. Field J.A. Appl. Environ. Microbiol. 1997; 63: 1749-1755Crossref PubMed Google Scholar). the production of MnP under manganese was for two and P. and the isozymes were characterized F. J. Biochem. 1996; PubMed Scopus Google Scholar, S. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar). In the MnP production was inhibited by of manganese. In Bjerkandera sp. strain BOS55 MnP production is stimulated by manganese The same MnP isozymes produced under manganese were also in cultures A study showed that the addition of organic acids enhanced MnP production under (20Mester T. Field J.A. FEMS Microbiol. Lett. 1997; 155: 161-168Crossref Google Scholar). In this study, glycolate, glyoxylate, and oxalate stimulated MnP production under These acids are physiological of white rot fungi (6Wariishi H. Valli K. Gold M.H. J. Biol. Chem. 1992; 267: 23688-23695Abstract Full Text PDF PubMed Google Scholar, M.V. Evans C.S. Can. J. Microbiol. 1996; 42: 881-895Crossref Scopus (519) Google B.P. Archibald F. Appl. Environ. Microbiol. 1993; 59: 1855-1863Crossref PubMed Google Scholar, P.J. Kirk T.K. J. Bacteriol. 1987; PubMed Google Scholar). The role of the organic acids in MnP production studies. The physical in of molecular isoelectric and heme absorbance of the purified MnP from those of other MnP isozymes described from other white rot fungi J.K. Gold M.H. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Gold M.H. Biochim. Biophys. Acta. 1996; PubMed Scopus Google Scholar, U. S. J. R. Lett. 1995; PubMed Scopus Google Scholar, Appl. Environ. Microbiol. 1994; PubMed Google Scholar). The turnover number of Mn(II) oxidation by MnP sp. strain BOS55 that this isozyme is efficient and comparable to other MnP isozymes described in the R. S. Field J.A. Lett. PubMed Scopus Google Scholar, F. J. Biochem. 1996; PubMed Scopus Google Scholar, Kishi K. Alic M. Gold M.H. Appl. Environ. Microbiol. 1994; PubMed Google Scholar, I.C. K.A. Tien M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, M. J. T. M. Y. Appl. Environ. Microbiol. 1996; PubMed Google Scholar). the Bjerkandera MnP to unique since it has a substrate with most MnP isozymes isozyme is able to oxidize various aromatic as as and in the absence of manganese. the oxidation rate was in the absence of manganese than in its presence at pH the oxidation of compounds was of manganese at pH was the that Bjerkandera MnP was able to oxidize veratryl alcohol and 1,4-dimethoxybenzene, which are LiP substrates, in the absence of manganese. veratryl alcohol was the oxidation of veratryl alcohol most via a mechanism than a The observed oxidation of to also a The m and for veratryl alcohol oxidation are comparable to the results for LiP isozymes from other white rot fungi M. Kirk T.K. J.A. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar, R.L. K.E. Tien M. Mozuch M.D. Kirk T.K. Scopus Google Scholar). LiP isozymes S.D. Lett. 1987; Scopus Google Scholar, R.L. 1993; Scopus Google Scholar), Mn(II) concentrations greater than 0.1 mm were found to severely the oxidation of veratryl alcohol by the Bjerkandera since both compounds as substrates of the enzyme have to for the same oxidized heme The that a concentration mm) of Mn(II) stimulated veratryl alcohol oxidation that Mn(II) to the enzyme from H2O2 by the catalytic was also observed that veratryl alcohol mm) incubated with the enzyme without Mn(II) the H2O2 of the enzyme as measured by ability These can by a single enzyme both compounds as a alcohol is a de novo secondary produced by many white rot fungi, Bjerkandera sp. strain BOS55 (3de Jong E. Field J.A. de Bont J.A.M. FEMS Microbiol. Rev. 1994; 13: 153-188Crossref Scopus (1) Google Scholar). from to in liquid cultures as as during of (23Mester T. de Jong E. Field J.A. Appl. Environ. Microbiol. 1995; 61: 1881-1887Crossref PubMed Google Scholar, T. Sierra-Alvarez R. Field J.A. Scholar). The oxidation of veratryl alcohol has to LiP as to MnP to the high of the which is oxidized by alcohol has in several in the of the oxidation of aromatic compounds with than that of veratryl alcohol Aust S.D. Grover T.A. Biochemistry. 1995; PubMed Scopus (83) Google Scholar, L. Tien M. J. Biotechnol. 1997; Scopus (57) Google Scholar). As the best reducing substrate for LiP compound veratryl alcohol can the enzyme to the native the catalytic cycle Tien M. Biochemistry. 1994; 33: PubMed Scopus Google Scholar). the presence of veratryl alcohol the of LiP by H2O2 Tien M. Appl. Environ. Microbiol. 1993; 59: PubMed Google Scholar). The that the Bjerkandera MnP isozyme oxidizes veratryl alcohol that this can have a role in the of MnP under manganese-free a MnP isozymes have shown to out aromatic were oxidized by MnP from U. S. J. R. Lett. 1995; PubMed Scopus Google manganese was for the oxidation of The MnP from was shown to oxidize in the absence of manganese F. J. Biochem. 1996; PubMed Scopus Google Scholar, S. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar). The MnP from to oxidize veratryl MnP, manganese was an requirement A. E. J. Biotechnol. 1996; Scopus Google Scholar). it is that purified MnP from P. P. oxidizes veratryl alcohol without manganese F. J. Biochem. 1996; PubMed Scopus Google Scholar, S. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar), the m of MnP was MnP under physiological All the kinetic suggest that the Bjerkandera MnP isozyme described has of both MnP and The enzyme with the most comparable substrate spectrum in the to that of the Bjerkandera MnP is MnP F. J. Biochem. 1996; PubMed Scopus Google Scholar). the N-terminal amino acid of the MnP isozymes have amino acids that in the amino acids, that the Bjerkandera MnP is a The N-terminal amino acid of MnP from other white rot fungi such as P. L. were also very to that ofBjerkandera MnP Gold M.H. Biochim. Biophys. Acta. 1996; PubMed Scopus Google Scholar, Appl. Environ. Microbiol. 1994; PubMed Google Scholar, E.A. A. Tien M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.A. Gold M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. J. L. R. 1994; PubMed Scopus Google Scholar, Appl. Microbiol. Biotechnol. 1990; 33: PubMed Scopus Google Scholar). The homology of the N-terminal amino acid sequence of Bjerkandera MnP is with those of both and from Trametes T. Nyman P.O. Arch. Biochem. Biophys. 1993; 300: PubMed Scopus (61) Google Scholar), with 1 and 2 amino acids in the the substrate of Trametes MnP isozymes has to In the MnP from Bjerkandera sp. strain BOS55 is a unique enzyme that can best described as a hybrid between manganese peroxidase and lignin The catalytic in Mn(II) and veratryl alcohol oxidation by this single isozyme are comparable to those of MnP and LiP, from other white rot fungi.