9 pages, 3 figures, 18 references.-- Simposium celebrado del 27-30, agosto, 1985, en Budapest, Hungría.The excretion of extracellular enzymes and the degradation
of indulin (pine kraft lignin) by the fungus Coriolopsis gallica
were studied. By using a lignin-impregnated glass fibre disc
which simulated natural conditions, the fungus excreted phenol
oxidases during the log phase of growth and reached two activity
maxima in the autolytic phase. However, in absence of indulin
the fungus had a different behaviour with respect to phenol oxidases.
It was concluded that in the extracellular enzymatic
system at least two enzymes, laccase and peroxidase, were involved
in the indulin degradation. Evidence of structural changes
in the indulin molecule was obtained by infrared spectrophotometry.Peer reviewe

Gómez Alarcón, Gonzalo

Lahoz, Rafael

O'Connor, A

Sáiz-Jiménez, Cesáreo

Digital.CSIC

Akadémiai Kiadó Budapept, 198'1 IToc. 01 the 9th Int . Symp . on SoU Biol.. and Conservo of the Biosphepe (Ed. J . SzegiJ CHEMICAL CHANGES OF KRAFT LI GNIN AND SOME ENZYMES PRODUCED BY THE WH ITE-ROT FUNGUS CORIOLOPS IS GALL ICA GÓMEZ-ALARCÓN, G .1, SA1Z - JIMÉNEZ , c ., LAHÓ Z, R.' and O' CONNOR , A. 1 Agency of Environment, Junta de Andalucía Sevil1e, Spain 'Institute of Microbio1ogy "Jaime Fer rán " , CSIC Madrid, Spain INTRODUCTION A great variety of soi1 microorganisms, among which bacteria and fungi are included, take part in the transformation and degradation of p1ant materia1s . White - rot fungi are we11 known degraders of wood structura1 components, inc1uding 1ignin . Using Phanerochaete chrysosporium it has been shown that the 11gnino-1ytic activity takes place during secondary metabo1ism , when cultures starve of nitrogen, su1phur and carbohydrates ( Jeffries et al ., '981: Kirk, 198J) , this occurs mainly in fungi during the auto1ytic period o Lignin is a comp1ex molecule, highly res1stant to biogical degradation, constituted by pheny1propane units linked by carbon-carbon and diaryl ether bonds. A complex enzyrnatic system con-stituted by 1ntra and extracellu1ar enzyrnes is involved in lign1n degradation . In the present work, the wh1te- rot fungus Coriolopsis ga1lica was grown for a long period of time in a liquid rnedium w1th and without indulin with the airn of studying the activities of sorne extracellular enzymes more or less im-plicated in the 11gnin degradation , as well as the resultlng chemieal changes in the molecule of this po1ymer usin9 lnfrared spectrophotometry. MATERIAL ANO METHODS Organism o ~. galliea B- J was isolated from eucalyptus trunk tree by Dr. C . Saiz - Jiménez. ." Lignin. Indulin (p ine kraft l ignin ) was used in these experi-ments . It was repeatedly washed with distilled water in arder to eliminate soluble low molecular weight compounas and other impurities. Mediu m and cu l tur e conditions. The fu ngus was grown in a modi -fied medium (Reye s a nd Byrde , 1973) as fo11ow5 (g/l): glucose (anhydrous), 2 . 5 ; a mmonium tartrate, 0 . 5 ; KH 2P04 , 1.0; MgS04 .7H 20, 0 . 5 ; KCl, 0 . 5; yeast extract, 0 . 5; trace elements , 1 mI . The medium wa s distributed in 20 ml-amounts i n conical flasks of 100 mI capacity , and steam sterilized three consecu-tive days , 20 min each day . Each flask was inoculated with a circular fragment of a 10-12 days old culture of C. galilea grown on malt - agar at 2S oC . The flask s were statlonary i ncubated at 25 oC . This wa s repeated twice, a nd culture fluid separated from myce l i um by filtration through Whatman No. 1 filter paper on a Büchner funnel and the mycelium washed with diatilled water and dried at 70 Oc to constant weight. Culture fluid was made up to initial volume by the additlon of distilled water and analysed. Fungal culture on inert support in the presence of indulln. The technique of Ander et al . (1981) slightly modified was used . Aliquots of 20 mI of culture medium were poured into Petri dishe s . Separately , each inert glass fibre disc was impregnated with 20 mg indulin dissolved in 2 mI of a mixture acetone: water (80 : 20, v/v), the acetone being evaporated at 4 5 Oc and the support disc placed in the Petri dish , inoculating it afterwards with a 10 mm dia~eter fungal plug. Two Petri dishes were taken each time. The experiment was r epeated twice . Analyt ical methods. Total reducing substances were determined according to Somogyi (1945) i n conjunction with that of Nelson (1944). Soluble proteins in culture fluid were estimated by the Lowry t!t al . (1951) method . Enzyme aasays. Lacease (E . C .l.10 . 3 . 2 . ) activlty in culture fluid was determined with 0. 1 M guaiacol (Merck) at 30 Oc in presence o f 0 . 05 M citra te - phosphate- borate buffer pH 5 . 0 by the method of Klrk and Kelman (1965 ). One enzyme u n it ls the amount of enzyme that ox.id izes 1 "urno l guaiacol per min and samp l e · . • $amp~B . The amount of au~tU1'6 fluid aontai716d in a f~a6k al' Petri dish Peroxidase (E . C . l.11.1.7 . ) activity was measured in the same way as lacease, except that the assay mixture also contained 1 mI of H20 2 (final concentration 15 mM) without buffer (Ander and Eriksson, 1975). One enzyme unit is the amount of enzyme that oxidizes l,.umol guaiacol per min and sample. 1,3-¡3- g1ucanase (E.C . 3.2.1.6.) was measured by a technique based on the method of McLelland et al. (1970), using as stlbstrate a solution of 1 mg larninarin/rnl at 37 Oc . One unit Df enzyrne activity was de-fined as the arnount which libera tes 1 ,.urnol glucose per min and sarnple at 37 oC. The specific activity for each one of the studied enzyrnes expresses the enzyrne activity per mg of culture-filtrate protein . Degree of autolysis. The degree of autolysis is defined as the 1055 ( %1 in the myeelium dry weight between the incubation day of maximum growth to the day in which the sample ls taken . IR spectroscopy . Infrared speetra were obtalned in KBr discs using a Perking Elmer 377 spectrophotometer. RESULTS ~. galIica obtained its maxlmum weight at the 13th day of ineubation, eoinciding with the exhaustion of the carbon source. The degree of autolysis amounted to 85%, 76 days after inocu-lation (Fig. 1). Small changes in the value of the pH were ob -served during incuhation time, the lower value coinciding with the initiation of the autolytic phase of growth. Proteins in culture fluid slightly increased their content at the end of the period of time here studied. Lacease in flasks culture (without indulin) was only detect -ed in small arnounts in the first 17 days of incubation; reaching thereafter the highest aetivity (25 . 1 mU/mg) at 48 days of in -cubation . On the other hand, lacease in Petri dish cultures could al so be detected during the first days of the fungal growth , until reaehing two peaks of activity during autolysis (Fig . 2a ). Peroxidase, fraro flask cultures, could only be detected in very sroall amounts until the 26th day of incubation , its activ-ity being around the value 2 mU/rng throughout the period studied . In the Petri dishes (in presenee of indulin), the syn-;, '-100 '" '" 80 ~ ¡e ::l 60 <t "-O W 40 W '" '" W 20 O • • • Kl 20 • • • • ,o 40 • ._---• • 50 60 70 80 T IME OF INCUBATION (DAYSJ Fig.l .Var~ations in the degree of autolysis o f C . galIiea myceli-um durlng 76 days , in flask culture 40 ~ E , ::l ' o E >-~ 20 >-U <t ~ "-G W 10 "-'" ° • \j\ , , , , Ó , , , , , • , " • ,o' • , o' , ,-10 20 'O 40 50 TIME OF INCUBAT ION 60 (DAYSJ 70 80 Fig . 2a Changes i n the specif lc ac t ivity o f l a cease in the cul -ture f luid of C . galIlea during 76 days of lncubation . - 0 - 0 - , flask culture ; - e - . -. Pet r i dishes eac h containing a glass fibre dlsc i mpregn a ted wlth 20 rng indultn • ~ • E , , :;, E • >- 4 t: ~ , º Le 2 [j W a. '" 10 20 '0 40 50 60 70 80 TIME OF INCUBATION (DAYSJ Fig . 2b Changes 1n the specific activity of peroxidase in the culture fluid of C. galIlea during 76 days of incubation. -0-0-, flask culture; . -e- , Petri dishes (with indul!n) ~ 2000 E , => .5 >-~ ¡:: ~ 1000 " ¡¡: ¡; w "-'" ,0, .' , , , ° , , , , , , , , , , , , , t \/' , , , , , , , , , ' I ', o 8 ' ..... v., I 0.J-é"· ~ ~--------~ .- ~~. 10 20 '0 40 '0 60 70 TIME OF INCUBATION (DAYS) ea Fig . 20 Changes in the specific activity of 1 .. 3 -¡1-g1ucanase in the culture fluid of C. galliea during 76 days of incubation . -0-0-, flask culture; - . - .... Petri dishes (with indul!n) thes1s of perox1dase 1s an 1nduced one. reach1ng two max1ma ( 2 1 and 48 days of 1ncubat1on) dur1ng the autolyt1c per10d t F1g. 2b) . Both kinds of cultures, flasks and Petr1 d1shes, produced a low 1 , 3 -~ -glucanase act1vity prior to autolysis, the former cultures attaincd their maximum production (2005 mU/rng) at the 42 nd day of ineubation , the latter being 1192 mu/rog at the 60th day tFig . 2e) . The speetrum of i~dulin after 38 daya of culture with ~ . galliea showed differenees with regard to the sound lignin. Thus, the lower intensity of the skeletal rings breathing modes in-dieates a deereased eontent of aromatic rings (absorption bands at 1595, 1510 and 1420 cm- 1). The indulin after 76 days of culture display s a spectrum with a strong absorption at 1725 em- 1 due to C;O of earboxyl groups . Skeletal ring breathing modes also decreased with regard to the sound indulin. Further, ab-sorption s of alcoholic hydroxyls are weaker (Fig. 3). A .~ e e 1-,roo "ro 100 GOO 400 200 Wove number (cm- I) Fig. J Infrared spectra of indulin. Sound lignin. A ; Lignin aEter 38 days and 76 days oE in-cubation with C . galIiea B and e respeetiveIy DISCUSSION Several studies have con firmed that 11gn1n and 11gn05ul-phonates are degraded by wh1te-rot fung1 (Ander and Eriksson, 1975; Hiroi and Erikason , 1976) and phenol oxidases are impor -tant ln this process (Ander and Eriks50n , 1978). In cultures of C . galIlea wlthout 11gn1n we have observed that the seeretlon of lacease and peroxldase proqresslvely 1ncreases during auto -lY51s, until a maximum value 15 reaehed . In Petri dlsh cul tures In which the production of pheno1 oxIdases ls induced , their appearance takes place during the log and stationary phases , the two highest values of aetivity appearing during autolysis (Fig . 2) . Thia bimodal distribution , prev1ou51y observed by Ferm and Cowllng (1972) suggests that, at least in a first step , they oxidize the free phenolie hydroxyl groupa of 11gnin , later , the action of other enzymes acting on the oxid1zed substrates would induce a further increase in the phenol - oxidizing enzymes se-cretion (Fig . 2 ) . In this sen se it ia posaible to indicate that a laccase-glucose quinone oxido-reductase cycle (Green, 1977) and laccase-cellobiose quinone oxidoreductase cycle (Westermark and Eriksson, 1974) have been postulated in white-rot fung! (Iwahara, 1983). 1 , J -¡S-glucanase is excreted by many white-rot fungi (Reese and Mandels, 1959) . Thls enzyme 15 present in autolyzed cultures , as well as in the cell wall of several fungi (Pérez-Leblic ~ al. (1982). We have observed in ~. ga111ca that 1,3-f3-g1ueanase ls present in small amounts ln culture fluid from the fi r st days of lneubation , the actlvlty lncreasing as autolysls proceeds , colncidlng with the exhaustlon of the earbon souree (Fig . 2e ) . Th1s lnerease ln activity eould be attributed to the faet that the fungus synthetizes a greater amaunt af th1s enzyme in arder ta degrade the 1,3-~-gluean of the funga1 ce1l wall, therefare being used as energy sauree . It is known that the easily metaba11zable carbahydrates can aet as repressers for the hydrolytic enzyme synthesis (Ander and Eriks$on, 1975) . The appearance of 1 , 3 - p - glucanase in cultures of ~. galliea in the pre-autolysis wauld indica te that glueose does not eompletely repress the action of the enzyme . Taklng into account the complexlty of the 11gnln mo l ecu1e , lt ls reasonable to aS$ume that besldes the enzymes studied here , sorne other enzymes wi11 take part 1n the splitting of aryl - alkyl ether 11nkages, ring eleavage, etc., participating in the dcgradat1ve process. Infrared spectrophotometry has been found useful for the characterization of lignins oE diverse origin , as we11 as to deteet changes in the chemical strueture of 1ign1ns after degra-datlon by fungl (Klrk and Chang, 1974). In general, from the infrared speetra data it can be concluded that a degradation of indulin occurs during the autolytic phase of growth. Main alter-ation of the lignln molecule i5 oxldatlon of the side-chaln, whlch explains the decrease of the alcohol1c hydroxyl absorption and the increase of carboxyl , and cleavage of arcmatic rings. In a similar way , an oxidation af the C3-alkyl chaln at the C~ and ~ positions, and cleavage ln the side - chain , malnly between CA and ~ has been suggested for the biodegradation of lignln by Coriolus versicolor (Saiz-Jiménez and de Leeuw, 1984). Further studies on the ehemical characterization cf saund and degraded 11gn1ns are in progress. SUMMARY The excretion of extraeellular enzymes and the degradation cf indulln (pine kraft lignln) by the fungus Coriolopsis galliea were studied . By using a lignin- impregnated glass fibre disc which simulated natural eonditions, the fungus excreted phenol oxidases ducing the lag phase of growth and reached two activity maxlma in the autolytlc pha5e. However, in absence of indul!n the fungus had a different behavicur with respect to phenol oxi-dases . It was concluded that in the extracellular enzymatie system at least two enzymes, lacease and peroxidase , were in-volved in the indulin degradaticn . Evidence of struetural changes in the indulin moleeule was obtained by infrared speetrophoto-metry . .. , REFERENCES Ander, P. and EriJtsson , K. E. (1975) : Svensk. Papperstidn. 78, 643-652. Ander , P. and Eriksson , K. E. (1978): Lignin Oegradation and Utilizatlon by Microorganisms . In : Progre ss ln Industrial Microbiology, XIV. Bull. M.J. (ed.) EIsevier, Amsterdam, 1-58 . Ander, P ., Eriksson, K.E . , Mansson, P . and Pettersson , B . (1981): Lignin Degradat!on by Sporatrlchum pulverulentum . A new cul -tivation method to study fungal lignin degradatlon. In: The Eckman-Days , 1981, 111 , Stockholm, 71 - 74. Ferm, R. and Cowling , E . B. (1972): Svensk . Papperstidn. 75, 767-772 . Green, T.R. (1977): Nature, 268, 78-80 . Hiroi, T . and Eriksson , K. E. ( 1976) : Svensk . Papperstidn . 21 . 157- 161. lwahara, S. (1983): Metabolism of Lignin-related Aromatic Com-paunds by Fusarium Species. In: Recent Advances in Lignin Biodegradation Research . (Hlguchl , T . , Chang , H. 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Chemical changes of kraft lignin and some enzymes produced by the white-rot fungus Coriolopsis Gallica

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