ON TH E OCCURENCE OF LACCASE IN THE PR O CESS O F H U M IFIC A TIO N
D epartm ent of Biochem istry, M arie C urie-Skłodow ska U niversity, Lublin Head — Doc. Dr. J. Trojanowski
In th e process of th e m icrobiological decom position of lignin in p la n t m a te ria l resid ues in th e soil, its basic bu ild ing stones, m o nom ethoxyphenols w ith a pro p an ol side chain, o r re la te d com pounds such as feru lic acid [2, 3, 7], vanillic acid [2, 3, 7], v a n illin [1, 7] appear.
Com pounds of th is ty p e m ay be oxidized in th e presence of th e enzym e p -d ip h en o l oxidase (laccase), secreted in to th e m edium by fu n gi of th e
H y m e n o m y c e te s group, th e m y celiu m of w hich also grow s in soils [4].
B y p o ly m erization an d condensation of th e o x y d atio n p ro d u cts w ith am ino acids o r peptides, th e y m ay be con v erted into hum ic substances.
T he view th a t phenolic com pounds are th e p rec u rso rs of h u m u s is now w id e ly accepted [5, 6, 8, 9]. H ow ever, u n til now w e have lacked d ire c t evidence th a t p -d ip h en o l oxidase (laccase) re a lly p lay s a p a rt in th e process of th e n a tu ra l hu m ificatio n of dead p la n t residues.
O at roots w ere ta k e n from stu b b le a t th e end of S ep tem b er and dried a t room te m p e ra tu re . A fte r rin sin g and chopping, th e root's w ere placed in 500 gm. p ortion s in glass co n tainers of 10 1 volum e and soaked w ith th e follow ing n u trie n t solution:
The p ro p o rtio n of added n itro g e n to th e ro o t m ass was 0,4%,. The co n ten ts of th e co ntain ers w ere in o cu lated w ith a suspension p rep a re d fro m th e soil of th e stu b b le field. A fte r 35 days th e liquid was p o ured
MATERIALS AND METHODS
14,68 g. K H2P 04 3,10 g. M g S 04 • 7H20 1,86 g. KC1 24,83 g. N H4NO3 in 10 1 H 20 (pH - 5,5)
o ff and th e roots w ere tre a te d w ith a solution of am m onium n itr a te (3g/litre). By fre q u e n t shaking th e roots w ere k e p t c o n sta n tly m oist. T h e lev el of th e liquid n u trie n t w ith in th e glass co ntainers w as 3 cm.
RESULTS
No phenol oxidases w ere found in th e fluid from th e jars for th e firs t 60 days of in cu b atio n a t room te m p e ra tu re .
The laccase a c tiv ity a p p eared in th e fluid a fte r 90 days, b u t o nly in those jars, to w hich a b u ffe r of pH v alu e 5,5 had b een in tro du ced, and w h ere care had been ta k e n to m a in ta in th e pH v alue a t th is level. T he presence of p -dip h enol oxidase (laccase) w as established b y W a rb u rg ’s tech n iq u e in the presen ce of p -p h en y len ed iam in e or hy dro q u in o n e as specific su b stra te s for th is enzym e. The enzym e w as p re c ip ita te d fro m 100 m l. of th e fluid m oistening th e roots. In o rd e r to achieve this, th e c e n trifu g a te d flu id w as p o u red into a 3-tim es g re a te r volum e of cooled acetone. T he p rec ip ita te d enzym e, a fte r w ashing w ith w a te r, w as dissolved in 15 m l of b u ffe r pH 5,5. In th e m ain vessel of W a rb u rg ’s a p p a ra tu s w as placed 2,3 m l. of th is enzym e solution, in th e side a rm — 10 mg. of su b s tra te in 0,5 m l. H20 . T he re s u lts of th e estim atio n are given in Tab. 1. T a b l e 1 The oxygen uptake in m ic r o litr e s during the f i r s t 15 mins. o f the oxydation o f the su b strate
by the enzyme p r e c ip ita te d from the f lu id m oistening the r o o ts. The r e s u lts obtained in r e p e titio n s of the estim ation are given- sid e by sid e . Substrate - p-phenylenediamine
Time of incubation of roots in days
90 120 150 180
Solution of enzyme + su b str. Boiled s o lu tio n of enzyme + su b str.
26, 25 0 43, 37 0 17, 15, 20 0 26, 25, 18, 17 0
We also con firm ed th e p resen ce of in tra c e llu la r laccase both in th e hu m ified roots an d in th e a ir d ried roots. T he estim ations w e re m ade b y colorim etric tech n iq u e, a t 420 m|A, using p -p h en y ien e d iam ine as su b stra te , a fte r 20 m in u te s’ rea c tio n a t 25°C. F or th e colorim etric estim ation, a hom ogenate from 6,7 g. of d ry m ass of ro o ts in 100 m l. p h o sp h a te b u ffe r pH 5,5 w as p rep a re d . T he hom ogenate w as ce n trifu g e d a t 4000 r.p.m ., an d a p rec ip ita te w as se p a ra te d from th e s u p e rn a ta n t co ntaining th e to ta l a c tiv ity of th e laccase. T he s u p e rn a ta n t w as th e n tre a te d w ith 3-volum e acetone in th e cold. T he p re c ip ita te w as im m ed ia te ly c e n trifu g e d an d e x tra c te d w ith 10 m l. of p h o sp h ate b u ffe r pH 5,5. F o r th e colo rim etric m e a su re m e n t 3 m l. of th is e x tra c t w as used, adding 10 m l. o f 2%, solutio n of p-p h en y len ed iam in e. C ontrol sam ples w e re also p re p a re d in w hich th e
enzym e h ad b e e n in ac tiv a ted by boiling. T he re s u lts a re given in Tabs. 2 an d 3.
T he e x tin c tio n foun d in th e co ntro l te sts w ith th e boiled enzym e arises fro m th e endogenous colouring of th e sam ples.
T a b l e 2 R esu lts of the co lo rim etric determ inations of the a c t iv ity of laccase iso la te d from roots a fte r a 60-day period of h u m ification . The time of reaction of 3 ml.
of enzyme extract with 10 ml. 2% so lu tio n of p-phenylenediamine (su b stra te) was 20 minutes at pH 5,5» The r e s u lts of r e p e titio n s are given in the
table sid e by sid e
Laccase a c t iv ity (e x tin c tio n )
1) Enzyme + substrate 0,82 0,60
2) Enzyme in a ctiv a ted by b o ilin g + substrate 0,32 0,20
D ifference between 1) and 2) 0 ,50 0 ,40
T a b l e 3 R esu lts of the co lo rim etric determ ination of la cca se iso la te d from a ir dried r o o ts, Which had not been humified. The time of reaction o f 3 ml. of enzyme ex tra ct with 10 ml,
2% so lu tio n of p-phenylenediamine (su b stra te) was 20 minutes at pH 5*5- The r e s u lts of r e p e titio n s are given in the table sid e by sid e
Laccase a c t iv ity (e x tin c tio n )
1) Enzyme + substrate 0,45 0,42 0,56
2) Enzyme in a ctiv a ted by b o ilin g , added substrate 0,25 0,20 0 ,37
D ifferen ce between 1) and 2) 0,20 0 ,2 2 0,19
T he colorim etric re s u lts w ere conform ed by m easu rin g th e u p tak e of o x y g en b y th e W a rb u rg te c h n iq u e in th e presen ce of h y d roq uin on e. T he enzym e p re p a ra tio n w as p re p a re d from th e roots in th e sam e w ay as for colo rim etry . T he u p tak e of oxy g en by a q u a n tity of enzym e corresp o ndin g to 1 g. of to o t d ry m ass d u rin g th e firs t 15 m in u tes of rea c tio n w ith h y d ro q u in o n e is given in Tab. 4.
T a b l e 4 Oxygen uptake in Warburg v e s s e ls during the oxid ation of hydroquinone in
the presence of laccase iso la te d from the roots a fte r the process of h u m ification . The r e s u lts of r e p e titio n s are given side by sid e
Sample M ic r o litr e s O2 per 15 min.
Enzyme is o la te d from the roots 17 21 25
CONCLUSIONS
D u rin g th e process of h u m ificatio n of o a t ro ots in conditions sim ilar to n a tu ra l, th e occurence of active laccase (p-diphenyloxidase) w as dem o n strated . The a c tiv ity of th is enzym e w as estab lished b y th e reactio n w ith su b stra te s specific for laccase (p-p h en y len ed iam in e and h y d ro - quinone) using th e W a rb u rg tec h n iq u e or colorim etric test. T he activ ity of th e laccase w as stab le for 4 m o n th s a t room te m p e ra tu re .
REFERENCES [1] B l a n d E.: Holzforsch., 12, 19, 1958.
12] B ö r n e r H.: Naturw iss. 42, 1955, 583, also 43, 1956, 129. [3] B ö r n e r H.: Beitr. Biol. Pflanz., 33, 1956, 33.
[4] F a h r a e u s G., L j u n g g r e n H.: Biochim. Biophys. Acta, 46, 1961, 22. £5] F l a i g W.1,: Rapport S ixièm e Congrès de la Science du Sol, Paris, vol. II,
10, p. 471, 1956.
16] F l a i g W.: Verh. der II und IV Komm. der Intern. Bodenkundl. G esellschaft, Hamburg, vol. II, p. 11, 1958.
[7] M a e d e r H.: Doctoral dissertation, J. Liebig Univ. Giessen, 1960. [8] T r o j a n o w s k i J.: Studies about humus. Prague, p. 329, 1962. {9] Z i e с h m a n n W.: Brennstoff-C hem ie, 44, 53, 1963.
J . T R O J A N O W S K I , J . M A T W I J O W
FESTSTELLUNG DER AKTIVEN LAKKASE IM HUMIFIKATIONSPROZESS
L e h r s t u h l f ü r B i o c h e m i e a n d e r U n i v e r s i t ä t M a r ie C u r i e - S k ł o d o w s k a i n L u b l i n
Z u s a m m e n f a s s u n g
Als w ich tigste K om ponente für die Bildung der H um instoffe w erden die phenolischen Verbindungen angesehen [5, 6, 8, 9], zum B eispiel die Zersetzungs produkte des Lignins — die M ethoxyphenole [1, 2, 3, 7].
D ie M öglichkeit der Oxydation von M ethoxyphenolen in A nw esenheit von Laccase w urde schon früher festgestellt [4]. Das Enzym Laccase konnte aber bisher im Boden nicht gefunden werden.
In der vorliegenden Arbeit haben w ir die A nw esenheit der Laccase w ährend der Verrottung von H aferwurzeln in einer Klim akam m er festgestellt. D ie Wurzeln w urden m it einer N ährlösung (pH-Wert = 5,5) versetzt und m it Bodenm ikroflora (Bodensuspension) geim pft. D ie L accaseaktivität in den Ansätzen erreichte ein M aximum nach 4 M onaten Inkubation, nahm später um die H älfte ab und blieb in den nächsten 2 Monaten nahezu gleich.
Die Laccase wurde m it Hilfe von spezifischen Substraten. (p-Phenylenediam in und Hydrochinon) nachgew iesen. Die quantitativen Bestim m ungen wurden nach der m ikrom anom etrischen W arburg-M ethode durchgeführt und die Ergebnisse durch kolorim etrische M essung gestützt.
J . T R O J A N O W S K I , J . M A T W I J O W
DÉTECTION DE LACCASE ACTIVE D A NS LE PROCESSUS D ’HUMIFICATION
C h a i r e d e B i o c h i m i e à l ’U n i v e r s i t é M . C u r i e - S k ł o d o w s k a à L u b l i n R é s u m é
La conception de la biogenèse de l ’hum us à la base de phénol [5, 6, 8, 9] admet que le noyau de la m olécule des substances hum iques peut se form er par voie de polym érisation des produits d’oxydation de certains phénols. Comme substrats naturels sont à nomm er les m éthoxym onophénols prenant leur naissance lors de la décom position de la lignine [1, 2, 3, 7]. L’oxydation de ce type de substances est catalysée par la laccase [4]. Jusqu’à présent on nâ pas découvert de laccase dans les sols.
D ans notre travail nous avons dém ontré la présence de la laccase dans le m ilieu, où se passait l ’hum ification des racines d’avoine inoculées de m icroorga nism es de sol, à pH 5,5 dans les conditions de laboratoire ressem blant aux naturelles. L’activité de la laccase persistait quatre m ois dans ce m ilieu. L’enzym e à été id en tifié par la réaction avec les substrats spécifiques: p-phénylènediam ine et l ’hydroquinone. Les m esurages ont été effectués selon la m éthode colorim étrique et celle de Warburg.
J . T R O J A N O W S K I , J . M A T W I J O W
STWIERDZENIE AKTYW NEJ LAKAZY W PROCESIE HUMIFIKACJI
K a t e d r a B i o c h e m i i U n i w e r s y t e t u M . C u r i e - S k ł o d o w s k i e j , L u b l i n
S t r e s z c z e n i e
K oncepcja fenolow ej biogenezy hum usu [5, 6, 8, 9] zakłada, że rdzeń cząsteczki zw iązków hum inow ych może pow staw ać na drodze polim eryzacji produktów u tle niania pew nych fenoli. N aturalnym i substratam i mogą być m etoksym onofenole po w stające przy rozkładzie ligniny [1, 2, 3, 7]. U tlenianie tego typu zw iązków k atali zuje lakaza [4]. D otychczas nie w ykryto lakazy w glebach.
W naszej pracy w ykazaliśm y obecność lakazy w środowisku, w którym prze biegała hum ifikacja korzeni owsa, zaszczepionych m ikroflorą glebową, przy pH 5,5, w w arunkach laboratoryjnych zbliżonych do naturalnych. A ktyw ność lakazy utrzy m yw ała się 4 m iesiące. Enzym zidentyfikow ano na podstaw ie reakcji ze specyficz nym i substratam i: p-fenylenodw uam iną i hydrochinonem.
Pom iary wykonano m etodą Warburga oraz kolorym etryczną.
E . Т Р О Я Н О В С К И , Я . М А Т В И Е В ОБНАРУЖ ЕНИЕ АКТИВНОЙ Л А К К А ЗЫ В ПРОЦЕССЕ ГУМИФИКАЦИИ К а ф е д р а Б и о х и м и и У н и в е р с и т е т а и м . М а р и и К ю р и - С к л о д о в с к о й , Л ю б л и н Р е з ю м е По принципу фенолового биогенеза гумуса [5, 6, 8, 9] ядро молекулы гуми- новых соединений может образоваться путем полимеризации продуктов оки сления некоторых фенолов. Естественным субстратом могут быть метокси-мо-4 R o c z n i k i G l e b o z n a w c z e
но-фенолы , образующиеся при разлож ении лигнина [1, 2, 3, 7]. Окисление со единений этого типа катализирует фермент лакказа [4]. Лакказа до сих пор не была обнаруж ена в почве. Автором установлено в лабораторном опыте наличие лакказы в почвенной среде, в которой при pH 5,5 происходила гумификация корней овса зараж енны х почвенными микробами. Активность лакказы сохранилась в течение 4 месяцев. Фермент был идентифицирован по реакции с п-фенилендиамином и гидрохи ноном. Измерения проводились при помощи аппарата Варбурга и по колориметри ческому методу.
