Physical and Chemical Characteristics of Glycopeptide from Wood

Chapter 8

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Physical and Chemical Characteristics of Glycopeptide from Wood Decay Fungi Akio Enoki, Hiromi Tanaka, and Shuji Itakura Department of Agricultural Chemistry, Faculty of Agriculture, K i n k i University, Nara, Japan

A low-molecular-weight (7,200~12,000) substance was isolated from cultures of brown-rot basidiomycete Tyromyces palustris. It contained about 61% protein, 24% neutral carbohydrate, and 0.12% Fe(II) by weight. The glycopeptide containing Fe(II) alone reduced O2 to • O H and catalyzed redox reactions between an electron donor, such as N A D H , and O to produce H O and to reduce H O to • OH. The glycopeptide reduced Fe(III) to Fe(II) (1.7 μ M/mg). The glycopeptide was a glycosylated peptide and had about 0.83 μM glycosylamine/mg. 2

140

2

2

2

2

© 2003 American Chemical Society Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

141


The Role of Hydroxyl Radicals in Brown-rot, White-rot, and Soft-rot Degradation of Wood The one-electron oxidation activity in intact cultures of wood-decay fungi is generally related with the rate of weight loss during wood degradation in those cultures. In brown-rot, most of the oxidation activity is caused by hydroxyl radical, while in white- and soft-rots, most of the activity is due to a combination of phenol oxidase and • OH. Most hydroxyl radicals produced in cultures of some white- and brown-rot fungi are caused in the redox reaction between 0 and one-electron donors catalized by extracellular low-molecularweight glycopeptides. Therefore, hydroxyl radicals in the redox reaction mediated by the glycopeptides are important in wood degradation by wood decay fungi. The role of the hydroxyl radical in wood degradation by wood decay fungi, and the physical and chemical characteristics of the glycopeptide are discussed. 2

Brown-rots Brown-rot fungi can degrade crystalline cellulose in wood, even though they lack exo-l,4-glucanse activity (1,2). Although these fungi can also degrade lignin, they preferentially metabolize the cellulose and hemicellulose, leaving an amorphous, brown, crumbly residue that is rich in lignin (3,4). Despite this, brown-rot fungi degrade and metabolize crystalline cellulose only when their ligninolytic systems are active. Furthermore, the cellulolytic systems of brownrot fungi are always active when these organisms are degrading lignin (4). This indicates that brown-rot fungi may possess a unique wood-componentdegrading system that is capable of fully degrading cellulose as well as modifying lignin. Hydroxyl radicals ( • OH) have been suggested as being involved in the degradation of wood by brown-rot fungi, since they can depolymerise cellulose (5) and attacks the aromatic rings in lignin, causing a variety of reactions including hydroxylation and ring-opening (6) without delignifying wood significantly (7). There is increasing experimental evidence to suggest that "OH is involved in the wood-component-degrading system of brown-rots. For example, some brown-rot fungi can degrade three 1,2diarylethane lignin-related model compounds, yielding the same products in relatively large yields as those rising from the degradation of the lignin model compounds by hydroxyl radicals (8). High concentration of hydroxyl radicals are also detected in cultures of brown-rot fungi in large yields (9). Brown-rot fungi cause rapid depolymerization of cellulose in wood before losses in total wood substance are detected. The pattern of progressive change in the mean degree of polymerization of the holocellulose in wood caused by Fenton's reagent is similar to that caused by brown-rot fungi (1,10). The rates of OHproduction in cultures of brown-rot fungi are directly proportional to the

Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

142 degradation rates of wood, and of crystalline cellulose and lignin substrate model compounds when these are added to fungal cultures (11,12). In the early stages of degradation of wood by brown-rot fungi, the S layer of wood cell wall is degraded extensively. The S layer adjacent to the cell lumen is less affected when it is attacked by fungal hyphae from the lumens (13,14). During these early stages of degradation, fungal enzymes such as cellulase and peroxidase are too large to penetrate wood cell walls (15,16). This further suggests that brown-rot fungi have a unique non enzymatic degradative system possibly involving "OH that degrades wood cell walls at some distance from hyphae in the lumen. Pretreatment of wood with • O H results in a marked increase in the amount of hydrolysis of the cellulose in the wood by endoglucanases which degrade little of the cellulose if the wood is not pretreated with • O H (5). When wood is treated with • OH, the hemicellulose is mainly lost and the contents of the lignin and cellulose increase (5). This suggests that in brownrot lacking exo-l,4-glucanase activity, endo-l,4-glucanse play an important role since brown-rot fungi preferentially metabolize the cellulose in wood. 2


3

Extracellular low-molecular-weight glycopeptides that catalyze a redox reaction between 0 and electron donors to produce the hydroxyl radicals have been isolated from wood degrading cultures of the brown-rot fungi, Gloeophyllum trabeum and Tyromyces palustris (Figure 1) (17,18). The extracelluar substance from T. palustris reduces Fe(III) to Fe(II) and strongly adsorbes Fe(II) (18). Most of the hydroxyl radical yield from cultures of T. palustris are produced in the redox reaction between 0 and certain electron donors catalyzed by the low-molecular-weight glycopeptide (19). During early stages of degradation, the glycopeptide is localized in the fungal cytoplasm and fungal cell wall, and in the extracellular slime sheath surrounding the fungal cell wall. The cell wall remains almost intact as long as the fungal hyphae remains in the lumen (20). Subsequently the glycopeptide is found throughout the wood cell wall, suggesting that it diffuses through the S layer into the S layer and the middle lamella. 2

2

3

2

White-rots White-rot fungi deplete all components of the wood cell wall during decay, but some species cause selective removal of the lignin in wood. In both the decay types, the lignin in wood cell walls being decayed is completely depleted. Some white-rot fungi can degrade the lignin in the wood preferentially to cellulose. Only in white-rot cultures with high levels of phenol oxidase activity (that is, at least one phenol-oxidizing enzyme such as LiP, MnP, or laccase) can significant degradation of wood with preferential degradation of the lignin occur (21-23). However, the phenol oxidase-activity in cultures of the white-rot fungi is not necessarily correlated with the rate of wood degradation by the



143

Electron donors (reduced form)

Electron accepter (oxidized form)

(o >

^ H

2

0

OH

2

2

L M W substance containing Fe(H)

L M W substance containing Fe(IH)

.reductioi

a

L M W substance

L M W substance (oxidized form)

L M W substance (reduced form)

Fig . 1. Proposed mechanism for generation of activated oxygen species by the extracellular one-electron oxidizing substance produced by wood decay fungi. Low-molecular-weight a


144


fungi in the cultures (23-25). In contrast, the generation of "OH in cultures of white-rot fungi is proportional to the rate of wood degradation (23,25,26). Although neither MnP or laccase are able to modify or degrade the nonphenolic lignins, both can degrade the non-phenolic portions of the polymer that are linked by j3 -O-4-alkyl aryl ether bonds, provided that some terminal phenols are present (27). Thus phenoloxidases such as LiP, MnP or laccase can degrade the lignin attacked by • O H since hydroxyl radicals attack the aromatic rings in lignin, causing aromatic hydroxylations. During early stages of wood degradation by white-rot fungi, only lowmolecular-weight agents are able to diffuse into the wood cell wall (28-30). During wood decay, white-rot fungi secrete a low-molecular-weight glycopeptide that catalyzes a redox reaction between 0 and electron donors to produce "OH; furthermore the glycopeptide reduces Fe(III) to Fe(II) and strongly binds Fe(II) (Figure 1) (23,24,31). Hydroxyl radical in combination with phenol oxidase may play a role in lignin degradation by white-rot fungi (23,25,26). Most of the hydroxyl radicals produced in wood-degrading cultures of the white-rot fungi, Phanerochaete crysosporium and Trametes versicolor are produced in the redox reaction between certain electron donors and 0 catalyzed by the low-molecular-weight glycopeptide (23,25). 2

2

Soft-rots The typical microstuctural features of soft rot decay are cavity formation in the S layers of wood cell walls (Type 1), and erosion of the S layers adjacent to cell lumens (Type 2). In both of these decay types, all the woods chemical components in an area proximal to the hypha are simultaneously degraded and eliminated, that is, simultaneous removal of all the components at one location of the cell wall results from soft-rot. This means that soft-rot fungi as well as white-rot fungi completely deplete the lignin in wood cell walls being decayed. Soft-rot and white-rot fungi generally produce the full cellulolytic enzyme complement (endo-l,4-glucanase, exo-1,4- j3 -glucanase, and 1,4- /3 glucosidase) and can hydrolyze highly-crystalline cellulose substrates. But a complete cellulase sysytem alone is insufficient for degrading the cellulose in wood cell walls because the lignin covering the cellulose prevents their contact (32). Therefore in soft-rot as well as white-rot, a ligninolytic system must be produced. The one-electron oxidation activity in cultures of soft-rot fungi is related to the rate of weight loss during wood degradation in the cultures; most of the oneelectron oxidation activity results from phenol oxidase and "OH action; although soft-rot fungi can degrade wood and the lignin in the wood only in 2

3


145 cultures with significant levels of phenol oxidase activity, the phenol oxidase activity in cultures of soft-rot fungi is not correlated with the rate of wood degradation in the cultures. (32). This suggests that in soft-rot, phenol oxidase activity is essential for lignin degradation, but that "OH may be involved since "OH does not delignify wood significantly, and works in combination with phenol oxidase, to play an important role in lignin degradation by white-rot fungi(25,26). Low-molecular-weight glycopeptides that catalyze a redox reaction to produce "OH between 0 and electron donors have been isolated from wood-degrading cultures of the soft-rot fungi Chaetomiun globosum and Xylaria polymorpha (33). Thus, hydroxyl radicals produced in the redox reaction between 0 and electron donors, mediated by extracellular low-molecular-weight gycopeptides seem to be involved in wood decay by brown-rot, white-rot and soft-rot fungi.


2

2

Glycopeptide Background A n extracellular substance that showed one-electron oxidation activity was isolated from wood-degrading cultures of the brown-rot fungus T. palusutris. The substance was partially purified by acetone- and ammonium sulfateprecipitations, and gel filtration chromatography on Sephadex G-50 and G-25, and D E A E Affi-Gel Blue gel chromatography according to the procedures previously described (31). The content of protein, neutral carbohydrates, and Fe(II) in the substance are shown in Table 1. The protein content assayed by the method of Lowry et al. (34) is 61%. Carbohydrate content, as measured by the phenol-sulfuric acid method with glucose as the standard (35) was 24%.

Table 1. Content of Protein, Neutral Carbohydrate and Fe(II) in the Glycopeptide Isolated from Wood Degrading Cultures of T. palustris, and the Molecular Weight of the Glycopeptide. Protein 61%

Neutral carbohydrate 24%

Fe(II)

Molecular weight

0.12%

12,000-7,200


146


The Fe(II) content measured with ferrozine (18) was 0.12%. The molecular weight determined by size-exclusion chromatography and gel-filtration on Sephadex G-25, was in a range of 1,000-5,000 D. But Tricine-SDS-PAGE showed that the glycopeptide consisted of at least three bands of 12.0, 10.5, and 7.2 kDa. The amino acid composition analyzed with an amino acid analyzer is shown in Table 2. The glycopeptide has an abundance of amino acids which have side chain amino groups.

Table 2. Amino Acid composition of the Glycopeptide. %

Amino acid asparagine aspartic acid alanine arginine isoleucine glycine glutamine glutamic acid cystine serine tyrosine

— 13.0 4.1 0.7 0.9 11.6

—

18.7 0.7 4.0 0.2

Amino acid

%

tryptophan threonine valine histidine phenylalanine proline methionine lysine leucine NH

—

3

2.7 1.9 1.1 0.8 1.9 0.1 0.6 1.7 35.2

The carbohydrate composition determined by the method of Clamp et al. (36) is shown in Table 3. The galactose content is very high (74%), whereas the levels of N-acetyl-D-glucosamine observed were very limited. The production of "OH in the reduction of 0 with the glycopeptide under various conditions is shown in Table 4. The generation of "OH was measured with dimethyl sulfoxide using the method of Fukui et al. (37). The assay for "OH, based on the conversion of dimethyl sulfoxide to methane sulfinic acid, and is specific for'OH (19). 2

Table 3. Carbohydrate Composition of the Glycopeptide (Sugar %). Fucose 5

Xylose 5

Mannose Galactose Glucose N-acetyl-glucosamine 5

74

11

0


147 The results shown in Table 4 suggest that the glycopeptide containing Fe(II) alone reduces 0 to "OH via 0 " and H 0 , and catalyzes the oxidation of 2

2

2

2

electron donors, such as N A D H , in the presence of 0 to produce "OH. 2

Table 4. Effects of 0 and N Atmospheres, and of H 0 , electron Donors, Superoxide Dismutase, Catalase and a Hydroxyl Radical Scavenger on the Generation of Hydroxyl Radicals by an Extracellular One-Electron Oxidation Substance Isolated from T. palustris.


2

2

Additions to reaction mixtures

2

Atmospheres

None 3xl0- molH O 3xl0 mol N A D H 3xl0- mol NADH,0.3mg S O D 3xl0" mol NADH,0.3mg cata 3xl0' mol ascorbic acid 3xl0" mols N A D H and D M N A 6

2

2

_6

6

b)

6

c)

6

6

d )

100%O 100%N 100%O 100%O 100%O 100%O 100%O

2

2

2

2

2

2

2

2

Hydroxyl radical generation x IQ^moltth Relative values(%) 0.6 6 4.0 44 9.2 100 3.6 39 0.9 10 28.2 307 3.3 36

a)

All reaction mixtures contained 3.0mg of the extacellular substance^xlO^mol dimethyl sulphoxide , and acetate buffer (40mM, pH 4.5) to total 2ml in a 33ml test tube. Test tube were purged with 100% 0 or N . Each value represents the average of a triplicate experiment. 2

b)

c)

d)

2

Superoxide dismutase,2,670unit/mg, Catalase,42,000unit/mg. N,N-dimethyl-4-nitrosoaniline.

The ferric reducing ability of the glycopeptide is shown in Figure 2 which indicates that 1 mg of the glycopeptide reduces 1.7 u M of Fe(III) to Fe(II). that is, a single mole of the glycopeptide reduces about 20 moles of Fe(III) to Fe(II), provided the molecular weight is 12,000. The Fe(III)- and Cu(II)-reducing ability of the glycopeptide, and the content of aldehyde and carbonyl groups, excluding the peptide linkages in the glycopeptide, are shown in Table 5. The



148

Fig. 2. Absorption spectra of solution of Fe(II) and ferrozine, Fe(III) and ferrozine, the glycopeptide and ferrozine, and the glycopeptide, Fe(III) and ferrozine. A solution (500 ju 1) of Fe(III) (20ppm) was added to solutions of the glycopetide (0.066mg, 0.033mg or 0.0165mg ) and the solutions were permitted to stand for 24 hours at 28°C. Ferrozine was added to the solutions, and the solutions of Fe(II)(10X 10" g), Fe(III)(10X lO^g) or the glycopeptide. Then the reaction mixtures were allowed to stand for 2 days and the absorption spectra were measured. A l l reaction mixtures contained 0.4% ferrozine to total 1.5ml. A : 6.7 ppm Fe(II), B : 0.066 mg of the glycopeptide and 6.7 ppm Fe(III), C: 0.033 mg of the glycopeptide and 6.7 ppm Fe(III), D: 0.0165mg of the glycopetide and 6.7 ppm Fe(II), E: 0.066mg of the glycopeptide, F: 6.7 ppm Fe(III). 6


149 amount of carbonyl groups in the glycopeptide, excepting those of the peptide itself, measured with 2,4-dinitrophenylhydrazine is 0.8 fimol carbonyl groups/mg of glucopeptide, that is, about 9 mol/mol provided the molecular weight is 12,000. The aldehyde group in the glycopeptide was determined with thiobituric acid or methone (dimethyldihydroresorcin) which stoichiometrically condenses with aldehyde groups with the loss of water. The aldehyde group is less than 2.8x10" mol/mg or 0.04 mol/mol provided the molecular weight is 12,000. The Cu(II)-reducing ability of the glycopeptide measured by the method of Somogi-Nelson (38) is 1.6 pmol/mg or 19 mol/mol provided the molecular weight is 12,000. This method is used for the measurement of aldehyde and a -hydroxyketone or endiol groups. These results mentioned above indicate that the glycopeptide is a glycosylated peptide and has about 10 mol of glycosylamine/mol provided the molecular weight is 12,000.


9

Table 5. Fe(IH)- and Cu(II) Reducing Ability of the Glycopeptide,and Contents of the Carbonyl Group (Except that of Peptide Linkage in the Glycopeptide), pmol/mg Fe(III)-reducing ability Cu(II)-reducing ability Carbonyl group-content Aldehyde group-content

1.7 1.6 0.8 0.0

mol/1.2x1 (fg 20 19 9 0

The side chain amino groups of proteins will condense nonenzymatically with the aldehyde groups of a carbohydrate to give a glycosy1 amine (Schiff base) which undergoes nucleophile-catalyzed rearragements to 1-amino -1deoxy-D-fructose derivatives (an Amadori compound). Amadori compounds reduce 0 to H 0 and Fe(III) to Fe(II) (39). Thus glycosylated peptides containing iron ions can reduce 0 to "OH as shown in Figure 3. Extracellular glycopeptides have been isolated from wood-degrading cultures of the white-rot fungi, P. chrysosporium, Irpex lacteus and Trametes versicolor, and the brownrot fungus G. trabeum. The glycopeptides are essentially identical to the glycopeptide of T. palustris with regard to their physical and chemical characteristics. 2

2

2

2



150

Ri 1 N Ri

vC

I

HoO

I

i

H—C—OH 1

HO O—C — H H O — CI — H

/

fc.

I

f

I

N N

I

NH I H—C

II

H —C

NH I

H - -C—H

1

OH H—C — H O — C1 — H

-»>

C—OH

I

HO—C—H

I

I

R3

H—C —O—R 2

I

-•

C=0

I

HO—C—H

I

R3

R3

CH OH 2

H 0 2

Ri H2°2.

Fe(H)

I

2

2 F e (A)

NH I H —C—H

o

2

2 F e (HI)

I NH I

•H- -C—H I C —OH

I

C

II

-OH+ • OH

Fe(fll)

C=0 I R3

electron donor (NADH)

electron C—OH acceptor I (NAD*) R3

Fig 3. Proposed mechanism for the generation of • O H and the reduction of Fe(M) to Fe( II) by the glycated peptide of T.palustris.


151

Glycopeptide Proposed Mechanism Based on the results discussed above, we propose the following mechanism for wood degradation by fungi . In wood decay by brown-rot fungi, during the initial fungal attack on the wood cell wall, low-molecular-weight glycosylated peptides are secreted by fungal hyphae into the wood cell lumen. This effector is able to reduce the Fe(III) in wood to Fe(II), chelate the Fe(II), and penetrate or diffuse into the wood cell wall. Together with the bound Fe(II), the effector catalyzes the oxidation of electron donors present in the presence of 0 within the wood cell wall to produce "OH via H 0 . The "OH produced in the wood cell wall attacks chemical constituents of the wall and causes the depolymerization of both crystalline and noncrystalline cellulose. Modification of the lignin also occurs and the fungus then transforms the cell wall layer by cutting channels through the S layer for enzyme-diffusion. After that, enzymes such as endoglucanases can penetrate the cell wall to act on the cellulose and hemicellulose. The proposed mechnism is consistent with the rapid increase of the solubility of wood in various solvents, and the rapid initial decrease in average degree of depolymerization (DP) of the holocellulose in wood during decay by brown-rot fungi (1). In wood decay by white-rot and soft-rot fungi, hydroxyl radicals produced in wood cell walls by low-molecular weight glycosylated peptides modify lignin, resulting in new phenolic, benzyl radical, and cation radical substructures that are susceptible to attack by phenol oxidases. The modified part of lignin is completely depleted by phenol oxidases such as LiP, MnP or laccase. The hydroxyl radicals also depolymerize polysaccharides. The depolymerized and exposed polysaccharides are efficiently hydrolyzed and soon depleted by a complete cellulase system and hemicellulase. Some white-rot fungi lack exo1,4- j3 -glucanase (40, 4) and such species seem to decompose the lignin component in preference to the cellulosic component (41,4). The gradual decrease in average DP of the holocellulose in wood, and the very small change of the solubility of wood in various solvents during decay by white-rot fungi (1) can be interpreted in this proposed mechanism for white-rot.


2

2

2

3

References 1. 2. 3. 4.

Cowling, E. B. US Dep. Agric. Tech. Bull. 1961, No. 1258, 1-75. Kirk, T. K.; Highley, T. L. Phytopathology 1973, 63, 1355-1342. Highley, T. L. Mater. Org. 1987, 22, 39-45. Enoki, A . ; Tanaka, H.; Fuse, G. Holzforschung 1988, 42, 85-93.


152 5. 6. 7. 8. 9.


10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32. 33. 34.

Rätto, M.; Ritschkoff, A . C.; Viikari, L.Appl.Microbiol.Biotechnol 1991, 48, 53-57. Gierer, J.; Yang, E.; Reitberger, T. Holzforschung 1992, 46, 495-504. Chirat, C.; Lachenal, D. Holzforschung 1997, 51, 147-154. Espejo, E.; Agosin, E.; Vicuña, R. Arch.Microbiol.1990, 154, 370-374. Bacha, S.; Gierer, J.; Reitberger, T.; Nilsson, T. Holzforschung 1992, 46, 61-67. Koenigs, J. W. Wood Fiber, 1974, 6, 66-80. Itakura, S.; Hirano, T.; Tanaka, H.; Enoki, A . Res. Group Wood Preserv. IRG Secretariat, Stockholm, 1994; Doc. No. IRG/WP/94-10062. Enoki, A . ; Tanaka, H . ; Fuse, G. Wood Sci. Technol. 1989, 23, 1-12. Highley, T. L . ; Murmanis, L. Holzforschung 1985, 39, 73-78. Kuo, M . ; Stokke, D. D.; MacNabb, H. S. Jr. Wood Fiber Sci. 1988, 20, 405-414. Flournoy, D. S.; Kirk, T. K . ; Highley, T. L . Holzforschung 1991, 45, 383-388. Srebotnik, E.; Messner, K . Holzforschung 1991, 45, 95-101. Enoki, A . ; Hirano, T.; Tanaka, H. Mater. Org. 1992, 27, 247-261. Hirano, T.; Tanaka, H.; Enoki, A. Mokuzai Gakkaishi 1995, 41, 334-341. Hirano, T.; Tanaka, H.; Enoki, A . Holzforschung 1997, 51, 389-395. Hirano, T.; Enoki, A . ; Tanaka, H. J. Wood Sci. 2000, 46, 45-51. Tanaka, H.; Enoki, A . ; Fuse, G. Mokuzai Gakkaishi 1985, 31, 935-945. Tanaka, H.; Enoki, A . ; Fuse, G. Mokuzai Gakkaishi 1986, 32, 125-135. Tanaka, H.; Itakura, S.; Enoki, A. Holzforschung 1999, 53, 21-28. Tanaka, H.; Hirano, T.; Enoki, A . Mokuzai Gakkaishi 1993, 39, 493-499. Tanaka, H.; Itakura, S.; Enoki, A. J. Biotechnol. 1999, 75, 57-70. Tanaka, H.; Itakura, S.; Enoki, A . Mater. Org. 1999/2000, 33, 91-105. Martinez-Inigo,M.J.; Kurek, B. Holzforschung 1997, 51, 543-548. Srebotnik, E.; Messner, K . ; Foisner, R. Penetrability of white-rot degraded pine wood by the lignin peroxidase of Phanerochaete chrysosporium.Appl.Environ.Microbial.1988, 54, 2608-2614. Blanchette, R. A.; Kruger, E. W.; Haight, J. E.; Akhtar,M.;Akin, D. E . J. Biotechnol. 1997, 53, 203-213. Goodell, B.; Yamamoto, K . ; Jellison, J.; Nakamura, M.; Fujii, T.; Takabe, K . ; Hayashi, N . Holzforschung 1998, 52, 345-350. Tanaka, H.; Itakura, S.; Hirano, T.; Enoki, A . Holzforschung 1996, 50, 541-548. Tanaka, H.; Itakura, S.; Enoki, A . Holzforschung 2000, 54, 463-468. Enoki, A . ; Fuse, G.; Tanaka, H. Inter. Res. Group Wood Preserv. IRG Secretariat, Stockholm, 1991; Doc. No. IRG/WP/1516. Lowry, O.H.;Rosebrough, N.; Farr, A . L.; Randall, R. J. J.Biol.Chem. 1951, 193, 265-275.


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35.


36. 37. 38. 39. 40. 41.

Dubois, M.; Gilles, K. A . ; Hamilton, J. K . ; Rebers, P. A . ; Smith, F. Anal. Chem. 1956, 28, 350-356. Clamp, J. R.; Bhatti, T.; Chambers, R. E . "Glycoproteins", Gottschalk, A . Ed. Elsevier, Amsterdam, 1972; p. 300-321. Fukui, S.; Hanasaki, Y.; Ogawa, S. J. Chromatogr. 1993, 630, 187-193. Somogyi, M. J. Biol. Chem. 1951, 195, 19-23. Oak, J. H.; Nakagawa, K.; Miyazawa, T. FEBS Lett. 2000, 481, 26-30. Sethuraman, A . ; Akin, D. E . ; Eriksson, Karl-Erik, L.Biotechnol. Appl. Biochem. 1998, 27, 37-47. Srebonik, E. ; Messner, K . Appl. Environ.Microbiol.1994, 60, 13831386.


Physical and Chemical Characteristics of Glycopeptide from Wood

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