APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 2971-2976 Vol. 60, No. 80099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Effect of Penicillium chrysogenum on Lignin TransformationA. RODRiGUEZ,1 A. CARNICERO,' F. PERESTELO,1 G. DE LA FUENTE,2 0. MILSTEIN,3

AND M. A. FALCON'*Departamento de Microbiologia y Biologia Celular, Facultad de Farmacia, Universidad de La Laguna,' and Institutode Productos Naturales Orgdnicos y Agrobiologia de Canarias del Consejo Superior de Investigaciones Cientificas, 2La Laguna, Tenenife, Spain, and Forstbotanisches Institut der Universitait Gottingen, D-3400 Gottingen, Germany3

Received 22 December 1993/Accepted 1 June 1994

A strain of Penicillium chrysogenum has been isolated from pine forest soils in Tenerife (Canary Islands).This strain was capable of utilizing hydroxylated and nonhydroxylated aromatic compounds, in particularcinnamic acid, as its sole carbon source. In an optimum medium with high levels of nitrogen (25.6 mM) andlow levels of glucose (5.5 mM), it was able to decolorize Poly B-411 and to transform kraft, organosolv, andsynthetic dehydrogenative polymerisate lignins. After 30 days of incubation, the amount of recovered kraftlignin was reduced to 83.5 and 91.3% of that estimated for uninoculated controls by spectrophotometry andklason lignin, respectively. At the same time, the pattern of molecular mass distribution of the lignin remainingin cultures was changed. The amount of organosolv lignin recovered from cultures was reduced to 90.1 and94.6% of the initial amount as evaluated by spectrophotometry and klason lignin, respectively. About 6% oftotal applied radioactivity of 014CH3-organosolv lignin was recovered as 14CO2 after 30 days of incubation, and18.5% of radioactivity from insoluble 014CH3-organosolv lignin was solubilized. After 26 days of incubation,2.9%o of 14C_P-dehydrogenative polymerisate and 4.1% of 14C-ring-dehydrogenative polymerisate evolved as14co2.

A significant part of lignin biosynthesis takes place in annualplants, and the breakdown of their lignocarbohydrate com-plexes in soil litter is just as important in global carbonturnover as that of woody plants. Wood-rotting fungi areconsidered the most efficient lignin degraders (1, 7, 23, 27), butsoil microorganisms presumably able to actively transformlignin in their ecological niche have been studied insufficiently(22). Particularly penicillia, normally soil inhabitants (33), havereceived little attention in lignin degradation studies, despitethe abundance of such fungi in agricultural wastes in differentstages of decomposition (3). Only a few reports have describeddifferent Penicillium strains as potential degraders (4, 19, 20,28, 31) or as able to degrade compounds with related ligninstructures (10, 11, 42). In general, most of the ligninolytic fungidescribed require a high level of consumption of easily metab-olized cosubstrate to carry out ligninolysis. Studies with basid-iomycetes have shown cosubstrate consumption levels of up to20 times (by weight) the amount of degraded lignin (24).

In this work, we report the isolation of a Penicillium chrysoge-num strain capable of attacking kraft, organosolv, and syntheticdehydrogenative polymerisate lignins in a medium with a lowconcentration of carbon source and a high level of nitrogen.

MATERIALS AND METHODS

Lignins. Kraft pine lignin polymer (PKL) (Indulin AT;Westvaco Co., Charleston, S.C.) was purified by the method ofKadam and Drew (19) with minor modifications. These con-sisted of repeated washings with ethyl acetate until no com-pounds were detected by thin-layer chromatography with ethylacetate-methanol (9:1) as the solvent system and Folin-Ciocal-teu as the developing reagent. Finally, PKL was washed withacidified water at pH 2.5 with 0.1 N HCI and vacuum dried toconstant weight. Beech organosolv lignin from MD-Nicolaus(Munich, Germany) was subjected to the same purification

* Corresponding author. Phone: 34-22-603655. Fax: 34-22-630095.

process as described above and denominated polymeric or-ganosolv lignin (POL).The dehydrogenative polymerisate (DHP) of the specifically

labeled coniferyl alcohol and 0'4CH3-organosolv lignin wereprovided by J. Trojanowski, Forstbotanisches Institut derUniversitat Gottingen, Gottingen, Germany, and prepared byfollowing the method described by Freudenberg and Neish(13) with minor modifications. "4C-I (side chain)-DHP lignin(specific activity, 0.02 ,uCi/mg), '4C-ring-DHP lignin (specificactivity, 0.05 ,uCi/mg), and 014CH3-organosolv lignin (specificactivity, 0.005 ,uCi/mg) were used as radiolabeled substrates.

Microorganisms. A strain of P. chrysogenum was isolatedfrom pine forest soil from the island of Tenerife by using amineral medium containing PKL as the sole carbon and energysource and was solidified with agar. This fungus was identifiedby the Laboratoire de Mycologie Systematique et Applique,L'Universite Catholique de Louvain, Louvain, Belgium, andcatalogued as MUCL 31363. One milliliter of spore suspensionwas used to inoculate 25 ml of medium. Spores were obtainedby stirring a 1-cm2 plug from an agar colony into 5 ml of 0.01%Tween 80 (vol/vol).

Characterization of ligninolytic potential. Sundman's testwas performed as described by Sundman and Nase (40) withPKL on agar plates. The Bavendamm test and extracellularoxidase, laccase, tyrosinase, peroxidase, esterase, and amylasetests were performed according to the methods of Rayner andBoddy (35). Endo- and exoglucanase assays were performedwith carboxymethyl cellulose and cellulose CF-11, respectively,stained with aniline blue black (21).Dye decolorization. Cultures in 50 ml of medium (500-ml

Erlenmeyer flasks), according to the method of Haider andTrojanowski (16), were supplemented with Poly B-411 (0.02%[wt/vol]) and incubated with shaking (Kottermann) (reciprocal50 strokes per min) for 28 days at 28°C. Decolorization wasmeasured as the rate of decrease in the A593/A483 ratioaccording to the method of Glenn and Gold (14).Media and culture conditions. Throughout the study, except

2971

2972 RODRIGUEZ ET AL.

for decolorization test studies, the media used were thosepreviously employed by Janshekar et al. (18). PKL and POLwere at 1-mg/ml concentrations in cultures. 0O4CH3-organo-solv lignin or DHPs specifically "C labeled in C-, or C-ringwere added to cultures to give 0.04 to 0.08 pCi per flask.D-Glucose was added as an easily metabolized carbon source ata final concentration of 5.5 or 55.5 mM.To study the effect of different C/N ratios on lignin trans-

formation, various nitrogen concentrations added as L-aspara-gine, NH4NO3, or a combination of both, all with glucose, weretested. The following C/N ratios were used: 0.65, 1.3, 2.5, 2.7,13, 25, 200, and 1,000. Cultures were incubated in 250-mlErlenmeyer flasks containing 25 ml of medium with shaking(Kottermann) (reciprocal 50 strokes per min) at 28°C for 30days. Uninoculated controls were incubated and treated iden-tically with inoculated lignin-containing cultures. Both culturesand controls were performed in triplicate, and additionalcultures without lignin (inoculated control flasks) were per-formed in duplicate.

Utilization of aromatic compounds. P. chrysogenum wasallowed to grow for 4 days in modified Janshekar medium (18)containing glucose (5.5 mM) and a high concentration ofnitrogen (25.6 mM; L-asparagine-NH4NO3). Culture mediawere then aseptically removed by decantation, and myceliawere washed twice with sterile medium without a carbonsource. Mycelial pellets were used to inoculate 25 ml of culturemedium containing vanillin (3.3 mM) or ferulic (2.6 mM) orcinnamic (3.4 mM) acid with or without the addition ofglucose. The percentage of degradation for each compoundwas measured by the reduction in the characteristic absorbancemaximum of each tested aromatic compound (284, 288, and279 nm for cinnamic and ferulic acids and vanillin, respective-ly).

Recovery of tested lignins. At the end of incubation, 10 ml of0.1 M NaOH was added to the entire contents of flasks tofacilitate the solubility of lignin adsorbed onto mycelia, and themycelia were then homogenized (Omnimixer) (15 s). Theresulting chopped mycelia were then subjected to ultrasonictreatment (12 ,um; 50 W; 20 s) to form a homogeneoussuspension according to the method of Janshekar et al. (17)and were centrifuged. Sediment was repeatedly washed with0.1 M NaOH until no A280 was detected in supernatants. Thisprocedure ensured that lignin entrapped or adsorbed bymycelia during incubation was also solubilized.These soluble lignins, evaluated spectrophotometrically at

A280 as total lignin in cultures, are designated alkali-solublelignin (ASL) in Fig. 1. Afterwards, ASL was precipitated with5 N HCl and called acid-precipitable lignin (APL) (Fig. 1).APL dissolved in 0.1 M NaOH (25 ml) was characterized byspectrophotometry and gel permeation chromatography onSephadex G-100. The amount of lignin in APL was alsoanalyzed by klason lignin content.Measurement of lignin degradation. The remaining PKL

and POL from cultures were measured by spectrophotometryat 280 nm (17) and also by residual acid-insoluble klason ligninand acid-soluble lignin after H2SO4 hydrolysis according to themethod of Crawford and Pometto (8). The change in lignincontent was expressed as the relative decrease in lignin, withthe amount in uninoculated flasks as 100%.

Radiorespirometric assay. 14CO2 evolved from metabolized'4C-lignin or 14C-DHP was trapped with 10% NaOH asindicated by Haider and Trojanowski (16), and radioactivitywas measured in a liquid scintillation counter (Optiphase III;Kabi Pharmacia) (combining 2 ml of sample with 4 ml ofscintillation liquid). Mineralization of lignin was expressed asthe percentage of added radioactivity recovered as 1 CO2. The

HOHOG NI XZw AND S0NXCATED CULTURES

STIR (30 m1n)

C3MTRIFuGAT3ON (18,000xg, 15 min)

-8WPERNATANT CES IDUE

WA8H WITH 0..I NaOHON

CITRIFUOATION

SUPERNATANT RZE IDUE

4ALALI SOLURLE LIGNIN

SPECTRUN 200-500 runEVALUATION AT 280 nm.

pH 3 WITH HC1coIa.

OVERNIGHT PRECIPITATION

CERTRIFuGATION (15,OOxg, 10 min)

SUPERNATANT RSXIDUE

WASH WITH ACID WATERt

C7RTIU4GATrXON (15 , oooxg)

SUPERNATANTRESIDT. IE

ACID WATERS VACUUE DRY AT 37fQC

4,ACID PRECXVPITA3LE LIGNXN

(DRY WEIGHT)

t]ISSOLVE IN 25 ml O.2.1i NzOH

8PECRUE 200-500 nmEVALUATION AT 280 nmSEPHAD3MC rH-60 AND G-100CHROHATOGRAPHY

pH 2-3 WITH HC! conc.

C3ERTRFUGATTrIOJ (15, OOOxg)

RES IXDU

VACiM DRY

XLASON TREATH]IT

A8WWP3AWAN

FIG. 1. Experimental procedure for estimating PKL and POLcontents in cultures after incubation.

amount of water-soluble lignin in culture media filteredthrough a Millipore filter (0.45-pum pore size) was determined.

Gel permeation chromatography. Gel permeation chroma-tography was performed with a Sephadex G-100 gel in acolumn (2 by 45 cm) with 0.1 M NaOH as eluent. APLrecovered from tested flasks and uninoculated controls wereredissolved in 0.1 M NaOH (25 ml), and 1.4-ml aliquots weresubjected to chromatography.

Glucose content. Glucose concentrations in supernatantswere determined with Sigma Diagnostics from a glucoseoxidase determination kit.

RESULTS AND DISCUSSION

Characterization of the ligninolytic potential of P. chrysoge-num. A strain of P. chrysogenum was isolated from andic brownsoil developed on basaltic tephra in Tenerife (Canary Islands)by using media containing PKL as the sole carbon source. It

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EFFECT OF P. CHRYSOGENUM ON LIGNIN TRANSFORMATION 2973

was submitted to different tests to characterize its presumedligninolytic potential (35).The experimental results for the Sundman test and extracel-

lular amylase, oxidase, laccase, peroxidase, and esterase testswere positive. However, the Bavendamm test and tyrosinase,endoglucanase, and exoglucanase tests were negative. As iswell known, Sundman's test shows a direct visualization oflignin degradation and conforms well with the Bavendamm testin experiments with basidiomycetes (40). However, a poorcorrelation between these tests has been reported for ascomy-cetes and imperfecti fungi (44). On the other hand, inconsis-tent results for the Bavendamm test and extracellular oxidaseand laccase tests with P. chrysogenum could be related to thepoor sensitivity of the Bavendamm test in detecting low phenoloxidase activity (39). Furthermore, ferulic acid and vanillinhydrolysis tests carried out according to prescription on petridishes were negative.

Figure 2 shows the ability of P. chrysogenum to utilizecinnamic or ferulic acid or vanillin as the sole carbon andenergy source. After 72 h of incubation, decreases in the ferulicand cinnamic acid UV-visible light absorption maxima of 41and 47.8%, respectively, were observed. In the case of vanillin,the change in the absorbance spectrum was produced concom-itantly with a decrease of 15.8% in the maximum of theabsorbance spectrum (Fig. 2). This provided additional evi-dence of the poor sensitivity of the Rayner and Boddy tests,since they could not show utilization of ferulic acid and vanillinon petri plates, although it has been proved in liquid cultures(Fig. 2). Despite the fact that the microorganisms catabolizingthese model compounds are not necessarily lignin degraders,lignin-degrading organisms are able to utilize low-molecular-weight lignin model substrates (9). Furthermore, several low-molecular-weight aromatic compounds are also metabolizedduring the ligninolytic phase of Phanerochaete chrysosporium(25). Moreover, Chen et al. (5) have clearly demonstrated thatpart of lignin degradation proceeds via low-molecular-weightaromatic acids.

Cultures of P. chrysogenum were also able to decolorize PolyB-411. As shown in Fig. 3, a 28% decrease in the A595/A482ratio was observed after 10 days of incubation. Decolorizationof Poly B-411 could not be related specifically to eitherdemethylation or ring degradation, and apparently the dyeserves as a substrate for fungal enzymes possibly related tolignin degradation (6, 14).

Elfect of culture conditions on lignin degradation. Almostall of the physiological studies related to fungal lignin metab-olism have been performed with the white rot fungus Phanero-chaete chrysosporium. Several nutritional and cultural param-eters are important for lignin degradation by this fungus. Kirket al. (24) have established optimal culture conditions for itsligninolytic activity: pH of 4.5, high oxygen tension, growth-limiting amounts of nutrient nitrogen, and high levels ofgrowth substrate. In addition, Reid (36) has reported that theC/N ratio of the medium may be a better predictor of lignindegradation than the absolute carbohydrate and nitrogenlevels.With all of the above taken into consideration, three param-

eters of the media, in particular, the C/N ratio, pH, andnitrogen sources and their concentrations, were changed toevaluate the optimal conditions for PKL degradation by P.chrysogenum. The greatest lignin decrease, evaluated as ASL,was 16.5% for the medium containing a C/N ratio of 1.3 and apH of 4.5. Under these culture conditions, the loss of 8 mg inthe klason lignin content of PKL was found to be concomitantwith the utilization of about 100 mg of glucose (Table 1). Asthe C/N ratio increased, the degradation of lignin decreased.

q)

5L

0,8

-20.6e~~~

<0.4

0.2-

o225 250 275 300 325 350 375 400

Wavelength (nm)FIG. 2. Changes in the UV spectra of the following aromatic

compounds before (solid lines) and after (dashed lines) incubationwith P. chrysogenum for 72 h: cinnamic acid (A), vanillin (B), andferulic acid (C).

Thus, in media containing a C/N ratio of 13, ASL degradationwas 8% (spectrophotometrical assay). In experiments withhigher C/N ratios of 25, 200, and 1,000, degradation was nil or

negligible (Table 1).As has been reported, different nitrogen sources can pro-

duce different effects on microbial ligninolytic catabolic sys-tems (23, 24, 37). Consequently, the effects of organic andinorganic nitrogen sources on lignin transformation by P.chrysogenum were also tested. No degradation of ASL in mediawith either L-asparagine or NH4NO3 at 25 mM nitrogen was

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2974 RODRIGUEZ ET AL.

2,5'0 4 8 12 16 20 24 28

Time (days)FIG. 3. Changes in the A593/A483 ratio of Poly B-411 in cultures of

P. chrysogenum. The insert shows changes in the absorbance spectra ofthe dye in cultures of P. chrysogenum (dashed line) and uninoculatedcontrol (solid line) after 28 days of incubation.

observed. However, the C/N ratio of 1.3 was optimal (Table 2).Furthermore, when L-asparagine was supplied as the solenitrogen source at 13.4 mM nitrogen (C/N ratio, 2.5), P.chrysogenum reached degradation values near those of theoptimum medium. However, when L-asparagine was replacedby NH4NO3 (12.2 mM N), lignin degradation rates decreased(Table 2). Ligninolytic activity of the fungus was inhibited byhigher concentrations of L-asparagine and NH4NO3 and incombination (Tables 1 and 2). These results suggest thatnitrogen metabolic regulation of P. chrysogenum can affectlignin degradation, although in a different manner from that ofPhanerochaete chrysosporium. Nitrogen concentrations whichstrongly repress the ligninolytic system of Phanerochaete chry-sosporium stimulated PKL degradation in P. chrysogenum.

However, there is evidence that the influence of the C/N ratioon ligninolysis is not an obligatory phenomenon in fungi (12,26, 41).

Only a few reports have discussed lignin degradation byimperfecti fungi. Kadam and Drew (19) reported that an

Aspergillusfumigatus strain mineralized 14C-labeled kraft ligninby 38%, whereas mineralization rates for two Penicilliumstrains were <2%. Besides this, the decrease in kraft lignin inculture media reported for another Aspergillus strain wasentirely due to cell surface binding (34).

P. chrysogenum changed the pattern of molecular mass

distribution ofAPL after 30 days of incubation, but only minordifferences were observed (Fig. 4). After 30 days of cultivation,11.7% of the lignin recovered was eluted in a volume of Kavbetween 0 and 0.057 corresponding to high molecular mass

(ovalbumin [45 kDa] was eluted in the same region), attenu-ating the same portion of lignin from uninoculated control byabout 3%. After 30 days, 64.1% of the lignin was eluted in a

volume corresponding to a lower molecular mass (Kav between0.057 and 0.46) (region where cytochrome c [12.5 kDa] was

eluted under the same conditions) compared with 69.6% of thelignin from uninoculated culture. On the contrary, 24.2% ofthe incubated lignin eluted in the region with a Kav coefficientbetween 0.49 and 0.75, the region where glucagon (3.48 kDa)was eluted under the same conditions, compared with 15.8% ofthe lignin from uninoculated culture. Thus, P. chrysogenumafter 30 days of incubation caused a transformation of thepattern of molecular mass distribution of lignin remaining inmedia after a certain amount of the initial polymer was

degraded.On the other hand, our strain of P. chrysogenum was also

able to degrade POL by 9.9% as measured by spectrophotom-etry. In this case, the decrease in klason lignin content wasestimated to be 5.4%. Modifications of organosolv lignin bywhite rot fungi have been reported (15, 29, 43); however, as faras we know, organosoluble-lignin degradation by imperfectifungi has not yet been described.

Incubation ofP. chrysogenum with "'C-labeled substrates. P.chrysogenum mineralized 4.1 and 2.9% of 14C-ring-DHP and14C-_ (side chain)-DHP, respectively, after 28 days of incuba-tion. These mineralization rates are higher than most of thosereported for 40 wood-inhabiting fungi (43). In relation to DHPdegradation by imperfecti fungi, a Fusanium solani strainshowed mineralization rates similar to those described for P.chrysogenum (32); however, Fusanium oxysporum releasedhigher rates of 14C02 (4). Moreover, several strains of imper-fecti fungi belonging to the Aspergillus and Penicillium genera

TABLE 1. PKL degradation after 30 days of incubation with P. chrysogenum at pH 4.5 with different concentrations of nitrogen and glucose

PKL degradation (%)'Concn KASLbAPLb Kason Total

lignin ligninC

C/N, 13; glucose, 55.5 mM; N (Asn),d 13.4 mM; N (NH4NO3),12.2 mM 8.0 ± 1.1e 9.2 ± 0.9 11.5 ± 1.5 10.6 ± 1.6C/N, 200; glucose, 55.5 mM; N (Asn),d 0.86 mM; N (NH4NO3), 0.78 mM NDef NDf 0.1 ± 2.0 0.7 ± 1.8C/N, 0.65; glucose, 5.5 mM; N (Asn),d 26.8 mM; N (NH4NO3), 24.4 mM 8.8 ± 1.5e 0.3 ± 0.7 NDf NDfC/N, 1.3; glucose, 5.5 mM; N (Asn),d 13.4 mM; N (NH4NO3),12.2 mM 16.5 ± 0.8e 12.6 ± 0.7 8.7 ± 1.5 9.2 ± 1.6C/N, 200; glucose, 5.5 mM; N (Asn),d 0.086 mM; N (NH4NO3), 0.078 mM 0.3 ± 0.4e 0.3 ± 1.2 NDf NDfC/N, 1,000; glucose, 5.5 mM; N (Asn),d 0.017 mM; N (NH4NO3) 0.016 mM 2.7 ± 0.7e NDf NDf NDf

a Values of degradation were calculated by taking the lignin content in uninoculated flasks as 100%. Data are means ± standard errors for three replicate cultures.b Determined by spectrophotometry.c Acid-insoluble lignin + ASL after H2SO4 digestion.d L-Asparagine.e Background due to fungal metabolites was subtracted from the absorbance levels of lignin-containing cultures.fND, no degradation.

APPL. ENVIRON. MICROBIOL.

EFFECT OF P. CHRYSOGENUM ON LIGNIN TRANSFORMATION 2975

TABLE 2. PKL degradation after 30 days of incubation with P. chrysogenum and different concentrations and sources of nitrogen and with5.5 mM glucose

PKL degradation (%)'Concn

ASLb APLb Klason lignin Total ligninC

C/N, 25; N (Asn),d 1.34 mM 2.7 ± 1.1e NDf 3.4 ± 2.5 0.9 ± 2.3C/N, 2.5; N (Asn),d 13.4 mM 8.4 ± 0.7e 7.3 ± 1.7 10.1 ± 1.5 10.2 ± 1.3C/N, 2.7; N (NH4NO3), 12.2 mM 4.4 ± 0.4e 3.0 ± 0.3 5.8 ± 2.0 5.4 ± 2.1C/N, 1.3; N (Asn),d 25.6 mM 3.3 ± 0.0e 1.0 ± 0.8 0.1 ± 2.4 0.2 ± 2.4C/N, 1.3; N (NH4NO3), 25.6 mM 0.4 ± 0.Se 0.2 ± 0.7 NDf NDf

a Values of degradation were calculated by taking the lignin content in uninoculated flasks as 100%. Data are means ± standard errors for three replicate cultures.b Determined by spectrophotometry.c Acid-insoluble lignin + ASL after H2SO4 digestion.d L-Asparagine.e Background due to fungal metabolites was subtracted from the absorbance levels of lignin-containing cultures.f ND, no degradation.

mineralized lower amounts of specifically labeled DHPs thandid P. chrysogenum (4, 20, 30). Furthermore, after 30 days, P.chrysogenum mineralized 5.8% of 0"4CH3-organosolv lignin.At the same time, 37.9% of initial radioactivity was recoveredas water-soluble compounds from cultures, compared with19.4% from uninoculated controls.This finding proved that P. chrysogenum was able to miner-

alize part of the industrial and synthetic lignins to 14C02 Someconversion of lignins to 14C-water-soluble intermediates wasalso observed. Soluble compounds as lignin degradation inter-mediates of metabolized lignin have also been found previ-ously in white rot fungus cultures (2, 38).

In conclusion, our results show that this newly isolated P.chrysogenum strain has a certain lignin degradation and trans-formation capacity, although to a lesser extent than thatreported for white rot fungi. Moreover, our strain of theimperfecti fungus P. chrysogenum performs lignin transforma-tion at a low cosubstrate requirement and could be potentiallyinteresting for the development of economically feasible ligninmodification processes.

0 0.2 0,4 0.6 0.8

Partition Coefficient (Kav)

FIG. 4. Effect of P. chrysogenum on the pattern of molecular massdistribution of PKL after 30 days of incubation. Lignin altered by thefungus (dashed line) and lignin from uninoculated culture (solid line).Calibration standards: ovalbumin (45 kDa) (a), cytochrome c (12.5kDa) (b), glucagon (3.483 kDa) (c), and anisoin (0.272 kDa) (d).

ACKNOWLEDGMENTSThis work was partially financed by the Spanish DGICYT (BIO-

880712) and the Gobierno Aut6nomo de Canarias (PI 91/16). O.M.also thanks the Spanish DGICYT for a sabbatic grant (SAB 93-0117).

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