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HONDA, Yoichi

KUWAHARA, Masaaki

MIKAME, Keigo

WATANABE, Takashi

Kyoto University Research Information Repository

Title<Preliminary>Structure and Microbial Decolorization ofXylanase-resistant Chromophoric Xylans Isolated fromUnbleached Kraft PulpAuthor(s)MIKAME, Keigo; WATANABE, Takashi; HONDA, Yoichi;KUWAHARA, MasaakiCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (1995), 82: 28-30Issue Date1995-09-30URL http://hdl.handle.net/2433/53227RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityPreliminaryStructure and Microbial Decolorization ofXylanase-re~istant.Chromophoric XylansIsolated frqm Unbleached Kraft PUlp*lKeigo MIKAME*2, Takashi WATANABE*2,Yoichi HONDA*2 and Masaaki KUWAHARA*2(Received May 31, 1995)Keywords: kraft pulp, biob1eaching, xy1an,. LCC, lignin permcidaseOne of the most characteristic features of kraft pulp is that uronic acid and arabinoseside chains of xylans are cleaved during the cooking process, and the xylans are redepositedon the cellulose fibrils as the alkaline concentration of the cooking liquor decreases1,2). Theredeposited xylans are known to be categorized into two types, (I) xylans extractable withsolvents which are associated weakly with· cellulose fibrils and resistant to a xylanase-digestion, and (2) alkali-resistan,t xylans co-crystallizing with cellulose3-7). In this study, awater-soluble xylanase-resitantchromophoric xylan fraction (LF-D) and a residual ligninfraction (HF-P) were separated from a hard wood unbleached kraft pulp (UKP) by acellulase/xylanase-membrane bioreactor (Fig. I). Chemical and spectroscopic analysesrevealed that this chromophoricxylanase-resitant fraction (LF-D) contained a large amountof unremovable inorganic atoms including Si, Na and S, together with non-branched f3-I, 4-linked xylan chain. Although: storong UV absorption was observed in LF-D, a signaloriginating from methoxyl group of lignin nuclei was below the back ground noise level in its13C_NMR spectrum. A nucleus exchange reaction of LF-D also revealed that a part oflignin nuclei in LF-D had been demethylated during the course ofkraft pulping because LF-D produced a large amount oLcatechol and pyrogallol in a nucleus exchange reaction atlIO°C. A nitrobenzene oxidation of LF-D gave vanillic acid but yields of vanillin andsyringaldehyde were very low, suggesting that quinoid structures are involved in LF-D.On the other hand,.a higher molecular weight residual lignin fraction (HF-P), which wasobtained from an impermeable part of the enzymatic digests, was found to contain muchdiphenylmethane structure based on the nitrobenzene oxidation and nucleus exchangereaction.• 1 A pa~t of this work was presented at the 39th Lignin Symposium at Fukuoka, October, 1994, and at the 6thICBPPI at Vienna, June, 1995.• 2 Laboratory of Biomass Conversion.- 28-MIKAME et al.: Structure and Microbial Decolorization of Xylanase-resistant XylansUnbleached kraft pulp (100%)Membrane bioreactor (purified cellulase! xylanase)(95.2%)Silica gel columnchromatographyHigher molecular weightfraction (HF)centrifuged (4.8%)Supernatant(HF-S)(4.7%) (0.1%)LF-A LF-B LF-C ••(4.9%) (77.7%) (10.0%) (2.7%)Fig. 1. Enzymatic degradation and fractionation of unbleached kraft pulp.LF-D was more effectively decolorized by bacterial microflora from soil rather thanselected white rot fungi including Phanerochaete chrysosporium and Bjerkandera adusta which canpartly decolorize the original unbleached kraft pulp (UKP). However, CorioIus versicolorwas found to decolorize not only the UKP but also the isolated xylanase-resistantchromophoric xylan fraction. However, activities of extracellular lignin peroxidase, Mn-peroxidase, laccase and xylanase in the culture filtrates of C. versicolor and the bacterialmicroflora were very low during the cultivation with the chromophoric xylans. In contrastwith the oxidizing enzymes, reducing activity of sodium 2, 6-dichlorophenol-indophenol wasdetected in the cultures of the bacterial microflora. Thus, chromophores of the xylanase-resistant xylans are different from typical residual lignin in UKP, and their biochemicaldecolorization needs enzymes different from the extracellular ligninolytic enzymes.Although the xylanase-resitant xylans can be removed by D-E-D process from UKp4), theirmicrobial decolorization is important for environmentally-safe high yield bleaching8 ).Because p-etherified benzyl ether bonds between lignin and carbohydrates have beenconsidered to be one of the major cause for bleaching resistance of UKP, screening ofmicroorganisms which effectively cleave the benzyl ethers was studied. When a purifiedlignin peroxidase (H8) from P. chrysosporium was reacted with a p-ethylated {3-0-4 LCCmodel bearing methyl {3-glucoside at its a-position under the condition that veratryl alcohol- 29-WOOD RESEARCH No. 82 (1995)HO~~ yo~eHO OH CH20H'OMsScheme 1was oxidized to veratraldehype, methyl f3-g1ucoside was not detected in the reactionproducts by HPLC9). Therefo:r:e, screening of microorganisms which effectivery split off theben~yl ether model compound was carried out9). As a result, sev~ral bacterial singlecolonies which emit fluorescence during the cultivation were isolated. Extensive studies onthe screeneing and characterization of those benzyl ether-cleaving microorgansisms wouldi .also contribute to an environmentally safe biobleaching system of UKP.AcknowledgementThe authors are indebted to Dr. Masamitsu Funaoka, Mie University for the technicaladvice and discussion on the nucleus exchange reaction. This study was supported by aGrant-in Aid No. 05660189 for: Scientific Research from the Ministry of Education, Scienceand Culture of Japan.References1) j.K. HAMILTON, E,V. PARTLOW and N.S. THOMPSON : Tappi, 41, 803-811 (1958).2) S. YLLNER and B. ENSTROM: Svensk Papperstidn~, 59, 229-232 (1956).3) F. MORA, K. RUEL, j. COMTAT and j.P. jOSELEAU: Holiforschung, 40, 85-91 (1986).4) S. SKJOLD-j0RGENSEN, N. MUNK and L.S. PEDERSON: "Biotechnology in Pu1p and Paper Industry" ed byKuwahara, M. and Shimada, ~., Uni Publishers, Tokyo, 93-99 (1992).5) A. ISHIZU: Mokuzai Gakkaishi, ~9, 1335-1340 (1993).6) A. KANTELINEN, B. HORTLING,M. RANUA and L. VUKARI: Holiforschung, 47, 29-35 (1993).7) A. KANTELINEN, B. HORTLING,j.SUNDQUIST, M. LINKO and L. VUKARI: Holiforschung, 47,318-324 (1993).8) T. WATANABE: Trends in G?Jcosci. G?Jcotechol., 7, 57-68 (1995).9) K. MIKAME, T. WATANABE, Y. HONDA and M. KUWAHARA: Proc. of39th Lignin Symp., 79-82 (1994).- 30-

<Preliminary>Structure and Microbial Decolorization of Xylanase-resistant Chromophoric Xylans Isolated from Unbleached Kraft Pulp

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<Preliminary>Structure and Microbial Decolorization of Xylanase-resistant Chromophoric Xylans Isolated from Unbleached Kraft Pulp

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