Previous Article | Next Article 
Applied and Environmental Microbiology, July 1999, p. 3071-3074, Vol. 65, No. 7
Departments of
Microbiology1 and
Chemistry,2 Montana State University,
Bozeman, Montana 59717
Received 4 January 1999/Accepted 26 April 1999
We purified a secreted fungal laccase from filtrates of
Gaeumannomyces graminis var. tritici cultures
induced with copper and xylidine. The active protein had an apparent
molecular mass of 190 kDa and yielded subunits with molecular masses of
60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of
2,6-dimethoxyphenol (Km = 2.6 × 10 Laccase
(p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) is a
copper-containing enzyme that catalyzes the oxidation of a phenolic
substrate by coupling it to the reduction of oxygen to water. Fungal
laccases display a wide substrate range, are known to catalyze the
polymerization, depolymerization, and methylation and/or demethylation
of phenolic compounds (11, 12), and may play a role in plant
pathogenicity (23, 24) or lignin degradation (8).
Many fungi produce dihydroxynaphthalene (DHN) melanin, and even though
phenol oxidase(s), including laccases, is hypothesized to catalyze the
oxidation of DHN into polymeric melanin, the in vivo mechanism of
polymerization is unknown (1, 21). While cell-free
homogenates of Cochliobolus heterostrophus catalyzed the
oxidation of DHN, purified laccases that efficiently catalyze the
polymerization of DHN in vitro have not yet been described (21).
The fungus Gaeumannomyces graminis var. tritici
is a phytopathogenic ascomycete that causes take-all, a severe root
disease of wheat and barley in temperate regions worldwide (9,
25). G. graminis var. tritici produces DHN
melanin and initiates root infection with melanized, ectotrophic
"runner" hyphae; these produce hyaline infection hyphae that
penetrate the root cortex and endodermis. Albino mutants of G. graminis are nonpathogenic, perhaps because runner hyphae must be
melanized for anchoring or for producing the invasive infection hyphae
(10). The host forms lignin-impregnated papillae, or
lignitubers, around invading hyphal tips (20); these are
physical barriers that impede hyphal tip advancement. The means by
which G. graminis var. tritici penetrates the
lignitubers is unknown at present, but the use of lignin-degrading
enzymes, such as laccase, is a plausible mechanism. Hence, G. graminis laccase(s) may be involved in melanin biosynthesis and/or
lignin degradation. Our objective in this study was to purify and
characterize the secreted laccase of G. graminis var.
tritici. This laccase is of interest because it polymerizes
DHN and oxidizes a lignin-like dye in vitro. These activities suggest
that this secreted laccase facilitates fungal infection of host plants.
Strains, media, and culture conditions.
G. graminis
var. tritici was isolated (by D. Mathre, strain DM528) from
Montana wheat (Triticum aestivum cv. Pondera) and purified
by single-ascospore isolation. The fungal strain was maintained on
Luria-Bertani medium (19) plus 1% (wt/vol) agar at 24 to 26°C. Long-term stocks were maintained on potato dextrose agar
(Difco, Detroit, Mich.) slants stored at 4°C.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification and Characterization of a Secreted
Laccase of Gaeumannomyces graminis var.
tritici
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
5 ± 7 × 106 M), catechol
(Km = 2.5 × 104 ± 1 × 105 M), pyrogallol (Km = 3.1 × 104 ± 4 × 105 M),
and guaiacol (Km = 5.1 × 104 ± 2 × 105 M). In addition,
the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a
natural fungal melanin precursor, into a high-molecular-weight melanin
and catalyzed the oxidation, or decolorization, of the dye poly B-411,
a lignin-like polymer. These findings indicate that this laccase may be
involved in melanin polymerization in this phytopathogen's hyphae
and/or in lignin depolymerization in its infected plant host.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Laccase assay.
Laccase activity was determined
spectrophotometrically by monitoring the conversion of 2 mM
2,6-dimethoxyphenol (DMOP) to 3,5,3',5'-tetramethoxydiphenoquinone in
pH 4.5 citrate-phosphate (15 mM) buffer at 468 nm (
= 49.6 mM1 cm1) (5). All
spectrophotometric assays were performed with a Gilford (Oberlin, Ohio)
model 2600 spectrophotometer. A unit of laccase activity is defined as
the amount of enzyme needed to oxidize 1 µmol of DMOP per min.
Protein concentration.
Protein concentrations were estimated
spectrophotometrically at 280 nm ( = 10%1
cm1) (17) with the absorbance corrected for
DNA contamination, determined by the
A280/A260 ratio
(17).
Laccase purification. Cultures were filtered through Whatman no. 1 filter paper, and culture filtrates were combined and concentrated to 50 ml with an Amicon (Beverly, Mass.) Stirred Cell protein concentrator with a 50,000-molecular-weight (MW) cutoff membrane. The protein was concentrated to 1 ml with a Millipore (Bedford, Mass.) Ultrafree 15 centrifuge cell with a 50,000-MW cutoff membrane. The concentrated protein solution was cooled on ice for 20 min and centrifuged at 15,000 × g for 10 min. The concentrated protein solution was decanted from the precipitate, and 100 µl of glycerol was added.
The enzyme was electrophoresed with a Bio-Rad (Hercules, Calif.) Prep Cell by using a 28-mm-inner-diameter gel tube at 2 W constant power, a running buffer of pH 6.6 His-MOPS [25 mM histidine-30 mM 3-(N-morpholino)propanesulfonic acid], and a 15-ml, 5.5% polyacrylamide nondenaturing gel. The elution buffer was pH 5.6 MES [10 mM 3-(N-morpholino)-ethanesulfonic acid], and laccase was eluted after 7 to 8 h as determined by DMOP assays. Collected fractions with laccase activity were concentrated to 0.5 ml with a Millipore Ultrafree 15 filter (50,000-MW cutoff) and quick-frozen in liquid nitrogen for storage. Samples (2 µl) of laccase in 2× loading buffer [75 mM sodium dodecyl sulfate (SDS), 20% (vol/vol) glycerol, 40 µM bromophenol blue, 125 mM tris(hydroxymethyl)aminomethane (Tris), and 5% (vol/vol)
-mercaptoethanol adjusted to pH 6.8 with
HCl] were electrophoresed on a 7.5% (wt/vol) SDS-polyacrylamide gel
by using a Bio-Rad Fast Gel System. Samples were visualized after
electrophoresis by activity staining with DMOP or protein staining with
silver stain (2) or Pierce (Rockford, Ill.) Gelcode Blue
Stain Reagent.
Laccase characterization. To detect monomer subunits, 4-µl samples of pure laccase were electrophoresed on a 7.5% (wt/vol) SDS-polyacrylamide gel by using a Bio-Rad Fast Gel System. The first sample was loaded with pH 6.8 loading buffer. The second sample was incubated in pH 8.8 loading buffer for 2 min at 23°C before loading. The third sample was incubated in pH 8.8 loading buffer for 10 min at 23°C before loading. The gel was silver stained for protein detection after electrophoresis.
Pure laccase (~20 µg) was denatured at 100°C in a solution of 0.5% (wt/vol) SDS and 1% (vol/vol)-mercaptoethanol. Denatured subunits were deglycosylated according to the manufacturer's
instructions with 2,000 U of New England Biolabs (Beverly, Mass.)
PNGase F added to the denatured protein solution and incubated at
37°C for 1 h. The laccase monomer was detected by
electrophoresis with a 7.5% (wt/vol) SDS-polyacrylamide gel.
The isoelectric point was determined by isoelectric focusing using a
Bio-Rad Fast Gel System. A pH 3 to 9 wide-range isoelectric focusing
gel with Bio-Rad wide-range ampholytes (pH 3 to 9) as standards and a
low-range (pH 4.5 to 6) isoelectric focusing gel with Bio-Rad low-range
ampholytes (pH 5.3 to 6.4) as standards were stained with Pierce
Gelcode Blue Stain Reagent.
Kinetic measurements were made at 23°C, with initial velocity
measurements performed in 3-ml glass cuvettes with 1-cm path lengths.
Reactions were initiated by the addition of laccase, and initial rates
were obtained from the linear portion of the progress curve. The
velocities of laccase-catalyzed reactions were measured at 468 nm for
DMOP, 450 nm for catechol, 450 nm for pyrogallol, 436 nm for guaiacol,
and 275 nm for L-tyrosine. Kinetic data were fitted to the
appropriate equation with the programs of Cleland (6) to
obtain the desired kinetic parameters.
Inhibitor studies were performed by using the DMOP assay with separate
pH 4.5 citrate-phosphate buffer solutions containing either 0.5 mM
sodium azide, 0.5 mM EDTA, 0.5 mM potassium cyanide, 0.5 mM hydrogen
peroxide, or 200 U of catalase/ml (all from Sigma, St. Louis, Mo.).
The pH rate profile for laccase was determined by using 2 mM DMOP in 15 mM citrate-phosphate buffer (buffering range from pH 3 to 8). The
activity was monitored spectrophotometrically at 486 nm.
Lignin degradation potential was estimated by diluting 20 µl of a
0.2% (wt/vol) poly B-411 dye solution (in H2O) to 4 ml
with pH 4.5 citrate-phosphate buffer. Laccase (400 U) was added, and decolorization was assayed after 0, 10, 20, 30, 60, and 90 min at
23°C. Decolorization was monitored by diluting 0.5 ml of the dye
solutions to 2 ml with a 10 mM sodium azide solution in 10 mM pH 4.5 citrate-phosphate buffer. The ratio of absorbance
(A593/A483) was measured
for the azide-dye solution. Decolorization assays were performed in
triplicate, and absorbances were compared with that of a control
without added laccase.
Polymerization of 1,8-DHN was measured by the addition of 15 U of
laccase to a 2-ml solution of 1 mM DHN in 5 mM pH 4.5 citrate-phosphate buffer in 50% ethanol. The polymerization solution was incubated at
23°C and monitored spectrophotometrically by scanning the solution from 320 to 520 nm at 0 to 120 min. An identical control solution without the enzyme that auto-oxidized for 16 h was also scanned. Partially solubilized polymer (in 50% ethanol) and DHN monomer allowed
to auto-oxidize overnight were dialyzed with 60,000-MW-cutoff dialysis
tubing to determine if the oxidized products were larger than 60 kDa.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
We concentrated laccase from culture filtrates of G. graminis var. tritici and purified the enzyme by
preparative gel electrophoresis (Table
1). We did not detect laccase in minimal
medium without copper, and only low levels of laccase production were
detected in the absence of xylidine (data not shown). We used pH 6.6 buffer in the Prep Cell because higher pH running buffers irreversibly denatured the protein. We purified laccase, as identified by activity staining (Fig. 1A, lane 3), to
homogeneity, as visualized by a single, silver-stained protein band on
acrylamide gels (Fig. 1A, lane 2). Because protein assay methods, such
as the Bradford method (3), produced inconsistent results,
we estimated protein concentrations spectrophotometrically.
|
|
The exact molecular mass of the active, glycosylated laccase complex is unknown, as glycosylation slows protein migration in polyacrylamide gels in an unpredictable manner (15) and the protein did not denature on SDS-polyacrylamide gels (Fig. 1A, lane 3), making the protein's migration partially dependent on the enzyme shape (27). However, purified undenatured laccase had an apparent molecular mass of approximately 190 kDa (Fig. 1A, lane 2). During long-term storage, approximately 2 months at 4°C, the enzyme degraded to a band at 70 kDa that lacked activity (Fig. 1A, lane 4). The process of subunit dissociation accelerated at pH values of 7 and higher (data not shown). After deglycosylation, the molecular mass of the protein subunits was 60 kDa (Fig. 1B, lane 3). Based on the observed molecular mass of the glycosylated monomer (70 kDa), the estimated molecular mass of a glycosylated homotrimer would be ~210 kDa and that of a homodimer would be ~140 kDa. Since the active complex migrated at ~190 kDa, the active complex was likely a homodimer (15, 28).
The wide substrate specificity of laccase sometimes makes it difficult
to distinguish from tyrosinase (EC 1.14.18.1) and peroxidase (EC
1.11.1.7). The enzyme oxidized common laccase substrates, such as DMOP
(Km = 2.6 × 105 ± 7 × 106 M), catechol (Km = 2.5 × 104 ± 1 × 105 M),
pyrogallol (Km = 3.1 × 104 ± 4 × 105 M), and guaiacol
(Km = 5.1 × 104 ± 2 × 105 M). The enzyme did not oxidize tyrosine and
was reversibly inhibited by hydrogen peroxide but not by catalase, and
thus it was not a tyrosinase or peroxidase by the classical definitions
(16, 22).
Irreversible inhibition of the enzyme by metal chelators (sodium azide, potassium cyanide, and EDTA) suggested that there was at least one metal center in the enzyme required for activity. Laccases commonly contain 4 Cu atoms in three different types of copper binding sites (a type 1, a type 2, and a type 3 binuclear site). Spectral studies did not reveal the characteristic peak near 600 nm expected for the type 1 copper site found in most laccases (data not shown). However, inductively coupled plasma spectroscopy conducted by Little Bear Laboratories (Denver, Colo.) detected copper in the purified G. graminis laccase enzyme complex. Recently, laccases that lack the type 1 copper site were reported (14). However, sequencing of five cloned putative laccase genes of G. graminis demonstrated that the consensus sequences for all three copper sites typical of laccases are encoded in all five genes (13). Therefore, the absence of a peak in the blue spectral region may be due to a low extinction coefficient for the type 1 copper site in this protein.
We determined by isoelectric focusing that the pI of the protein was 5.6 (data not shown), which was slightly higher than the pH at which the maximal activity for DMOP occurred (pH 4.5, as determined by the pH rate profile). Low-range isoelectric focusing also indicated that there were no laccase isoforms with similar molecular weights but slightly different pI's (data not shown).
The ability to decolorize poly B-411 dye by ring-opening oxidation is utilized to predict the lignocellulose degradation abilities of fungi (4). Although a direct comparison to previously tested lignin degraders cannot be made due to the difference in reaction conditions, the laccase described here did oxidize poly B-411 (Fig. 2), an activity that suggested a possible role in lignin degradation (4). The enzyme also polymerized DHN into a high-molecular-mass melanin (Fig. 3). No polymerization occurred in control reactions without added laccase; the absorbance maximum remained at 360 nm with an increase of less than 0.1 optical density unit (data not shown). While the melanin polymer was not soluble in many solvents tested, it did remain soluble in the initial 50% ethanol solution for a few hours after the polymerization reaction was initiated. This partially solubilized melanin did not dialyze through 60,000-MW-cutoff dialysis tubing, but the DHN monomer, allowed to auto-oxidize, did. Thus, the polymer synthesized by laccase had a molecular mass greater than 60 kDa.
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The pathway for DHN melanin biosynthesis in fungi is almost completely characterized (1). However, the enzyme that catalyzes the final oxidation step in the pathway, the conversion of DHN to polymerized melanin, is unknown at present (1). The laccase characterized in this paper is the first purified laccase shown to catalyze the polymerization of DHN and may polymerize DHN in vivo.
It is not known if this laccase is secreted when G. graminis var. tritici infects wheat; however, reverse transcription-PCR has shown that the mRNA for this laccase is present in infected rice and wheat plants (13). Wheat plants are known to contain enough copper to allow the laccase to be active if it is expressed (20). Melanin synthesis catalyzed by laccase is required for the pathogenicity of Cryptococcus neoformans; melanin protects this fungal pathogen from animal host oxidative immune responses (18, 26). Similarly, G. graminis var. tritici laccase may function to confer protection from plant host oxidative defense responses.
In conclusion, these studies provide evidence that the extracellular laccase produced by G. graminis var. tritici is a glycosylated homodimer that may be involved in lignin degradation and/or melanin synthesis. Future studies will focus on environmental factors that affect laccase expression and the role(s) of laccase in melanization, delignification, and/or protection from antimicrobial compounds produced by the infected plant host.
| |
ACKNOWLEDGMENTS |
|---|
The work presented in this paper was funded by the U.S. Army Research Office (grant DAAH04-96-1-0194).
We thank Jeffrey Dean and Michele McGuirl for valuable discussions and help with equipment, and we thank L. J. Cookson for the gift of poly B-411.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Microbiology Department, Montana State University, Bozeman, MT 59717. Phone: (406) 994-4690. Fax: (406) 994-4926. E-mail: jhenson{at}montana.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Bell, A., and M. Wheeler. 1986. Biosynthesis and functions of fungal melanins. Annu. Rev. Phytopathol. 24:411-451. |
| 2. | Blum, H., H. Beier, and H. Gross. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93-99. |
| 3. | Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254[Medline]. |
| 4. | Chet, I., J. Trojanowski, and A. Huttermann. 1985. Decolourisation of the dye poly B-411 and its correlation with lignin degradation by fungi. Microbios Lett. 29:37-43. |
| 5. | Claus, H., and Z. Filip. 1997. The evidence of a laccase-like enzyme activity in a Bacillus sphaericus strain. Microbiol. Res. 152:209-216. |
| 6. | Cleland, W. 1979. Kinetics. Methods Enzymol. 63:103-138[Medline]. |
| 7. | Fahraeus, G., and B. Reinhammer. 1967. Large scale production and purification of laccase from cultures of the fungus Polyporus versicolor and some properties of laccase. Acta Chem. Scand. 21:2367-2378[Medline]. |
| 8. | Hatakka, A. 1998. Lignin-modifying enzymes from select white-rot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13:125-135. |
| 9. | Huber, D., and T. McCay-Buis. 1993. A multiple component analysis of the take-all disease of cereals. Plant Dis. 77:437-447. |
| 10. | Kelly, C., A. Osbourn, and C. Caten. 1997. The genetics of Gaeumannomyces graminis with particular reference to pigment production and pathogenicity. Fungal Genet. Newsl. 44A:108. |
| 11. | Leonowicz, A., G. Szklarz, and M. Wojtas-Wasilewska. 1985. The effect of fungal laccase on fractionated lignosylphonates (Peritan Na). Phytochemistry 24:393-396. |
| 12. | Leonowicz, A., J. Trojanowski, and O. Barbara. 1979. Basidiomycetes: apparent activity of the inducible and constitutive forms of laccase with phenolic substrates. Acta Biochim. Pol. 25:369-378. |
| 13. | Litvintseva, A., and J. Henson. 1999. Personal communication. |
| 14. |
Palmier, G.,
P. Giardina,
C. Bianco,
A. Scaloni,
A. Capasso, and G. Sannia.
1997.
A novel white laccase from Pleurotus ostreatus.
J. Biol. Chem.
272:31301-31307 |
| 15. |
Perry, C.,
S. Matcham,
D. Wood, and C. Thurston.
1993.
The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus.
J. Gen. Microbiol.
139:171-178 |
| 16. | Robb, D. 1992. Tyrosinase, p. 207-240. In R. Lontie (ed.), Copper proteins and copper enzymes, vol. III. CRC Press, Boca Raton, Fla. |
| 17. | Robyt, J., and B. White. 1987. Biochemical techniques: theory and practice, p. 232-234. Waveland Press, Inc., Prospect Heights, Ill. |
| 18. |
Salas, S.,
J. Bennett,
K. Kwon-Chung,
J. Perfect, and P. Williamson.
1996.
Effect of the laccase gene, CNLAC1, on virulence of Cryptococcus neoformans.
J. Exp. Med.
184:377-386 |
| 19. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., p. A1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 20. | Soon, Y., G. Clayton, and P. Clarke. 1997. Content and uptake of phosphorus and copper by spring wheat: effect of environment, genotype, and management. J. Plant Nutr. 20:925-937. |
| 21. |
Tanaka, C.,
S. Tajima,
I. Furusawa, and M. Tsuda.
1992.
The Prg1 mutant of Cochliobolus heterostrophus lacks a  -diphenol oxidase involved in naphthalenediol melanin synthesis.
Mycol. Res.
96:959-964.
|
| 22. | Tijssen, P. 1985. Practice and theory of enzyme immunoassays, p. 173-189. In R. Burden, and P. VanKnippenberg (ed.), Laboratory techniques in biochemistry and molecular biology. Elsevier, Amsterdam, The Netherlands. |
| 23. | VanEtten, H., D. Matthews, and P. Matthews. 1989. Phytoalexin detoxification: importance for pathogenicity and practical implications. Annu. Rev. Phytopathol. 27:143-164. |
| 24. | VanEtten, H., R. Sandrock, C. Wasmann, S. Soby, K. McCluskey, and P. Wang. 1995. Detoxification of phytoanticipins and phytoalexins by phytopathogenic fungi. Can. J. Bot. 73:S518-S525. |
| 25. | Walker, J. 1981. Taxonomy of take-all fungi and related genera and species, p. 15-74. In M. Asher, and P. Shipton (ed.), Biology and control of take-all. Academic Press Inc., London, United Kingdom. |
| 26. | Wang, Y., P. Aisen, and A. Casadevall. 1995. Cryptococcus neoformans melanin and virulence: mechanism of action. Infect. Immun. 63:3131-3136[Abstract]. |
| 27. |
Weber, K., and M. Osborn.
1969.
The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.
J. Biol. Chem.
244:4406-4412 |
| 28. | Wood, D. 1980. Production, purification and properties of extracellular laccase of Agaricus bisporus. J. Gen. Microbiol. 117:327-338. |
Applied and Environmental Microbiology, July 1999, p. 3071-3074, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Enguita, F. J., Martins, L. O., Henriques, A. O., Carrondo, M. A.
(2003). Crystal Structure of a Bacterial Endospore Coat Component: A LACCASE WITH ENHANCED THERMOSTABILITY PROPERTIES. J. Biol. Chem.
278: 19416-19425
[Abstract] [Full Text] -
Endo, K., Hosono, K., Beppu, T., Ueda, K.
(2002). A novel extracytoplasmic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis. Microbiology
148: 1767-1776
[Abstract] [Full Text] -
Martins, L. O., Soares, C. M., Pereira, M. M., Teixeira, M., Costa, T., Jones, G. H., Henriques, A. O.
(2002). Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase That Occurs as a Structural Component of the Bacillus subtilis Endospore Coat. J. Biol. Chem.
277: 18849-18859
[Abstract] [Full Text] -
Litvintseva, A. P., Henson, J. M.
(2002). Cloning, Characterization, and Transcription of Three Laccase Genes from Gaeumannomyces graminis var. tritici, the Take-All Fungus. Appl. Environ. Microbiol.
68: 1305-1311
[Abstract] [Full Text] -
Solano, F., Lucas-Elío, P., Fernández, E., Sanchez-Amat, A.
(2000). Marinomonas mediterranea MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant. J. Bacteriol.
182: 3754-3760
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»