Ihochimica el Bioph).s:ca Acta. 10"3 (1991) 114-119 r, 1991 ElsevierSciencePublishersB.V.(BiomedicalDivision)030a-41¢.5/91/$03.50 ADONIS (304,116591000728

114

BBAGEN 23428

Purification and characterization of a verztryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus G i o v a n n i S a n n i a l, P a o l a L i m o n g i 2, E n n i o C o c c a 1, F r a n c e s c o B u o n o c o r e 1 Gianpaolo Nitti 3 and Paola Giardina 1 i Dtpartimento dl Chtrnwa Organica e Bwlogtca. Unit,ersit~Jdi Napoli. Nafles (Italy). " Istituto Biochimica Proteine ed EnzirnologioL C.N.R.. Naples (hal)9 and J Dipartimento Bwtecnologie. Farrnitalia Carlo Erba. Mdano (ltalv)

(Rc.eivcd 14 Junc 1990) Key words: Lignindegradation; Veratrylalcoholoxida~; Basidiomycete;I P. o.streatrt~) A veratryl alcohol oxidase (VAO) enzyme was di~overod in cultures of PleuroUt~ ostreatus. T h e enzyme, which oxidizes veratryl alcohol to veratraldehyde reducing O, to H 2 0 2, was purified to homogeneity and its main structural and catalytic properties have been determined. The enzyme is a glycoprolein and contains FAD as a prosthetic group. The amin~ acid composition and carhoxy- and amino-termi-al sequences were determined. Primary aromatic alcohols with methoxy substituents in position four are good substrates for VAO; cinnamyl alcohol is the substrate which is oxidized faster whereas coniferyl alcohol is oxidized at a slower rate. The enzyme is moderately thermostable (tl/~ssocl about 1.5 h, apparent melting temperature about 60 ° C). The enzyme stability in 50% water/organic .~olvents mixtures has also been studied. Introduction Recently, great efforts have been made to understand lignin degradation mechanisms [1,2]. Lignin, the second most abundant renewable carbon source on earth, is a polymer of phenylpropanoid units interconnected by several types of carbon-carbon and carbon-oxygen bonds [3]. The most effective lignin degraders in nature are white-rot fungi which belong to the basidiomycete family; amongst these, P h a n e r o c h a e t e chrysosporium is the most widely studied species ]4,5]. This fungus produces extracellular peroxidases (ligninases) which catalyze, in the presence of hydrogen peroxide, one-electron oxidation in various lignin-related and other aromatic compounds, yielding radical cations which undergo further non-enzymatic reactions. Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is synthesized de novo from L-pbenylalanine by Ph. chrysosporium, and its formation coincides with the onset of ligninolytic activity ]1,6]. Veratryl alcohol is a substrate for ligninase, which catalyzes its oxidation to veratraldeAbbreviations: VAO, veratryl alcohol oxidase- DAB, diaminobenfidine. Correspondence: G. Sannia. Dipartimento di Chimica Organica e Biologica, L'niversit/idi Napoli. via Mezzocannone.16, 80134. Italy.

hyde. The possible role of veratryl alcohol in lignin degradation by Ph. chrysosporium is unclear [7,8]. H202 might be supplied by several different oxidases, both intracellular [9-11] (although it is not known whether this H202 is then excreted by the cells) and extracellular [12.13]. The white-rot fungus Pleurotus ostreatus has been shown to degrade lignin [14] and to produce laccase, a copper-containing phenol oxidase [15]. Efforts to detect the presence of iigninascs produced by P. ostreatus in several culture media and growth conditions have not succeeded. However, a further oxidase, which utilizes veratryl alcohol as a substrate, has been detected in fungal cultures under nutrient-limited conditions. This paper describes the purification and characterization of this enzyme called, in agreeni,;nt with Bourbonnais and Paice [16]. veratryl alcc,hd oxidase (VAO). Materials and Methods E n z y m e production P. ostreatus (strain Florida) was maintained, through

periodic transfer, at 4 ° C on agar-potato dextrose (Difco Laboratories, Detroit, MI) plates in the presence of 0.5% yeast extract (Difco). Incubations were carried out at 25°C by pre-inocdating 250 ml of potato-dextrose broth containing 0.5% yeast extract in shaken 500 ml flasks with the P. ostrea-

115

n~; mycelia. 50 ml of a 5-day-old culture were trans-

Molecular weight determination

ferred into 1 liter shaken flasks containing 450 ml broth (~tY7 rev.,/,,~ir~). O.'. the 20th d-?y of inc,:hafion the medium was collected and filtered through gauz,:. In order to improve VAO productior., various growiag conditions were tested: 5 mM ",eratryl alcohol. 0.05 ¢z (w/v) olive oil; 0.05% ( w / v ) T w e e n 80, 0.1% ( w / v ) Indulin AT and 0.5% (w/v) sawdust were added to culture broths containing potato-dextrose and yeast ~xtract.

Gel-filtration chromatography was performed using ,'i~her a Sephadex G-150 superfine or a Bio-Ge[ P-150 column (120 x 1 cm) at a flow rate of 4 ml/h. 60 p.g of the sample were loaded after calibration with alcohol dehydrogenase (M, 150000). cytoplasmatic aspartate aminotransferase (M, 92000), bovine serum albumin (M r 68000). ovalbumin (M r 45 000) and cbymotrypsinogen ( M r 25000).

Electrophoresis Enzyme purificatDn Proteins were precipitated by the addition of (NH4),SO 4 up to 80% saturation at 0 ° C and centrifuged at 10000 x g for 30 min. The precipitate was resuspended in 50 mM imidazole (pH 6.0) and extensively dialyzed against the same buffer. The sample was again centrifuged and the supernatant was loaded on a DEAE-Sepharose (Pharmacia, Uppsala, Sweden) column (2.5 x 40 cm) equilibrated with the imidazole buffer. The column was washed at a flow rate of 15 m l / b with 300 ml buffer, then a 0-0.5 M NaCI linear gradient (1.3 liter) was applied. Active fractions were pooled, concentrated and equilibrated in buffer A (10 mM imidazole, 0.125 M NaCI, pH 6.0) on an Amicon PM-10 membrane. The sample was loaded onto a Mono-Q column (FPLC system, Pharmacia) equilibrated in buffer A. The enzyme was eluted with a NaCI gradient (buffer B, 10 mM imidazole (pH 6.0) + 0.5 M NaCI; gradient: t = 0, %B = 0; t = 10 min, %B = 0; t = 4 0 rain, %B = 15; t =45 min, %B = 100). The active fractions were pooled and desalted.

Polyacrylamide (10%) slab gel electrophoresis in 0.1% sodium dodecyl sulphate (SDS-PAGE) was carried out as described by Laemmli [19]. For molecular weight determination, the gel was calibrated with phosphorylase b ( M, 94000), bovine serum albumin (M, 68000), ovalbumin ( M, 45000), carbonic anh3drase ( M, 30000), soybean trypsin inhibitor (M, 20000)and B-lactalburain (M, 14200). Electrofocusing in the pH range 2.57.0 was performed on a polyacrylamide gel slab in a Multiphor apparatus from LKB following manufacturer's instructions. The gels were stained using silver staining methods [20]. The staining of the H20,-producing enzyme was carried out under the conditions described previously for the peroxidase-coupled assay [17].

.4,ni,~o acid analysis Amino acid compesition was determined hydrolyzing protein samples in vacuum at I I 0 ° C for 24, 48 and 72 h in 6 M HCI and analyzing them using a Beckman 119CL apparatus.

A mino- and carboxy-terminal sequences Enzyme assay VAO activity was assayed by measuring the increase in absorbance at 310 nm due to the oxidation of veratryl alcohol to veratraldehyde. The assay mixture (1 ml) contained 10 mM sodium phosphate (pH 6.5) and 4 mM ~eratryi alcohol. To analyze substrate specificity, O z uptake during substrate oxidation was measured using an Orion oxygen electrode. The reaction mixture was the same as in the spectrophotometric assay, but the reaction volume was 10 ml. H202 production was determined using a peroxidase-coupled assay with diamincbenzidine (DAB) as the peroxidase substrate [17]. The reaction mixture contained 0.1 M sodium phosphate (pH 7.0), 0.3 mU horse-radish peroxidase, 0.36 mM DAB, 4 mM veratryl alcohol and 0.066 U VAO in a total reactior, volume of 1 ml. The increase in absorb~nce at 460 nm due to the oxidation of DAB was monitored.

Protein determination Protein concentration was determined using the BioRad Protein Assay, with bovine serum albumin as a standard [181.

Automated N-terminal sequence analysis was performed on a Model 470A gas-phase protein sequencer (Applied Biosystem, CA, U.S.A.) equipped with an on line 120A PTH-analyzer and a 900A data module [21,22]. All sequencing materials and reagents were purchased from Applied Biosystems. BioBrene Plus (3 mg) was applied onto the TFAetched glass fiber filter and subjected to three precycles of Edman degradation prior to sample application. The samples were analyzed using the 'RUN470-1" program as the normal cycle, with a "BGN470-1" as the first cycle providing an additional coupling with PITC when starting the sequencing run. In one case the sample was analyzed with a modified program using a lower temperature in the reaction cartridge and a reduced time for the cleavage step with TFA; the run was started with a "begin" cycle without a TFA wash. A time-course study of carboxypeptidase Y digestion of VAO was performed at room temperature in 4 M urea, 0.1 M pyridine acetate buffer (pH 5.5), using an enzyme/substrate ratio of approx. 1:50 ( w / w ) [23,24]. 1 nmol of protein was incubated for 2 h with 1.5 #g

116 of carboxypeptidase Y and 20-100 ttl aliquots were withdrawn at various time intervals, acidified with 20/xl of 20% trifluoroacetic acid to terminate the digestion. and dried in vacuum using a Speedvac centrifuge. The amino acids released during digestion were identified and quantified after automated derivatization with PITC on a Model 420A Derivatizer (Applied Biosystems) and subsequent HPLC analysis with on-line Model 130A analyzer. Data were normalized using N-Leucine as an internal standard. Separation of the derivatized PTCamino acids were carried out using a PTC-CI8 microbore column (220 × 2.1 mm, 5 p., Applied Biosystems) eluted with a 25 min gradient of solvent B (30 mM sodium acetate (pH 6.1)/70% acetonitrile) in solvent A (50 mM sodium acetate, pH 5.4).

02 oxidizes 1 mol of veratryl alcohol producing 1 tool of veratraldehyde. Production of H202 during the reaction was demonstrated using a peroxidase-coupled assay with diaminobenzidine as a peroxidase substrate Quantitative analysis of H202 production demonstrated that VAO produces 1 tool of H,O., per mol of O 2 reduced. It has also been shown that phenol oxidases are produced by ihe fungus [15,281, but ao lignin peroxidases could be detected when the mycelia wc,c grown in different culture media or in differently aged cultures. In particular, when P. ostreatus mycelia were grown under conditions similar to those described to induce ligninase production in Trametes versicolor and Ph. chrysosporium [29.30] no H202-dependent veratryl alcohol oxidation was detected.

Sugar content determination In order to establish the presence of a carbohydrate moiety in the protein, 2 /zg of purified enzyme were treated wi~h 50 mU of endoglycosidase F and subjected to SDS-PAGE together with untreated samples. Quantitative sugar determination on 60 and 120 p.g protein samples was performed using the phenolsuiphuric acid reaction [25]. The standard curve was prepared using 10-70/.tg glucose samples.

Purification of VA 0 P. ostreatus mycelia begin to secrete VAO after 10 days incubation; the enzyme production reaches its maximum after 20 days incubation. DEAE-Sepharose chromatography of secreted proteins from differently

Coenzyme determination 2 ml hot methanol were added to 8 p.g VAO it, 0.5 ml imidazole buffer. The solution was kept at I I 0 ° C for 30 min and then centrifuged at 18000 × g for 15 min at 4 °C. The supernalant was evaporated under a stream of N 2 and the residue dissolved in imidazole buffer. Flavin concentration was calcdlated either from absorbance at 450 nm ( e = 11300 M -~ . c m - t ) or from the fluorescence intensity ~,'xcitation 450 rim, emission 530 nm) using commercial riboflavine as standard [26}. In order to distinguish between FAD and FMN the flavin sample was analyzed by reverse-phase high-pressure liquid chromatography [271 using a Nucleosil 5Ci8 column and 5 mM ammonium acetate (pH 6) and methanol as buffers A and B, respectively (gradient used was: t = 0, %B = 10; t = 5 min. %B = 10; t = 10 min, %B = 30; t = 35 min, %B = 50; t = 40 min, %B = 100).

o sr

f

o

~o aoys

_J

2o

y

.o

~o

~o

,oo

°'r

~,o".'o

180 20

aays

180 20~

t

20

0.2

Results and Discussion

Production of veratryl alcohol omdase P. ostreatus produces veratryl alcohol oxidase (VAO) under nutrient-limited conditions. This enzyme oxidizes veratryl alcohol I,~ veratraldehyde with the concomitaat reduction of 02 to H202. Its prodection does not seem to be induced by the presence of vera,.ryl alcohol, olive oil. saw,lust, Indulin AT or Tween 80 it. the culture broth. When the O~ consumption wa.,, measured during the VAO catalyzed reaction, it was fot:nd that 1 mol of

Froct ,ons

Fig. 1. DEAE-Sepharose chromatography nf exlracdlular proteins from cuhures of P. ostrectus. Culturc~were prepared as dcs,'ribed in the text and filtered after t0 and 20 days incubation. The enzymes were eluted at plf 6 with a linear sodiumchloridegradient(from 0 to 0.5 M). For experi:ner.tal details see the MaLerial and Methods section. - - , Absorbance at 280 nm; II-..... II. phenol oxidase acli~lty(U/ml): [:]. . . . . . O, VA(.)activity(U/ml x 10).

117 TABLE l Purtfwatton of P. o~treattt~ VA 0

1.2 liters o1"20-day-old liquid cultures were purified, Fer experimental details ~ec Maler;,ds Jnd Methods Purifica|ion step

Culture broth (NH.D2SO.4 precipitation

Total ptot.++in

Total activity

(rag)

(UI

105

211

Specific acti',ity ~U/mg~ 2.0

60

191

3.::

l.b

°,0.5

Punfication factor

Yield (c[)

1120

DEAE-Sepharosechromatograph,,'

3.7

150

40.5

20.2

71 1

Mono-Qchromatography

0.78

S~

10?g

53.9

39.9

aged P. ostreatus culture broths, shows the presence of many phenol-oxidases, but only one single peak was observed for veratryl alcohol oxidase (Fig. 1). Enzyme purification was achieved as shown in Table I. E n z y m e characterization

The enzyme was homogenous when analyzed by SDS-PAGE showing an apparent molecular weigh', of 72 500. The electrophoretic mobility ot the enzyme increased after incubation with endoglycosidase F, thus suggesting the presence of carbohydra:es. Quantitative analysis was performed using the phenol-sulphuric acid reaction which indicates that the enzyme sugar content is about 25% (w/w). Gel filtration chromatography of the native enzyme on Sephadex G150 and on Biogel P150 produced a symmetric peak with an elution volume corresponding to apparent molecular weights of 92000 and 85000. respectively. These results do not clarify satisfactorily the oligomertc nature of the protein. The isoclectric point of VAO, determined by means of isoelectrofocusing, is 4.0. The amino acid composition, expressed as mol of each amino acid per 72 500 g of protein, was: Asx, 81: Thr, 40; Ser, 46; Glx, 56; Pro, 52; Gly, 68; Ala, 60; Val, 52; Met, 9; lie, 42:. Leu, 53;

',ooo~°=t

,~oo

~

L

~

~

-~

_i.__~G,o

i .oo s

'°i!! 0

.

20

.

40

.

.

.

.

60 8o Time (rn,n)

.

.

100

.

120

"4O

Fig. 2. Anuno acid recoveries after timed hyclrulysis by carboxypcptidasc Y. CarboxypcptidaseY digestion of VAO w:~spcrt,.,rmcd as d-scribed in tbe text. The C-terminal sequencewPs determined on the basisof the kineticso~aminoacid relea.~.

ryr. 9: Phe. 29: Lys. 16: His, 13" and Arg. 27. Tryptophan and cysteine were not determined. W h e n subjected to N-terminal ~equcnce analysis, the purified VAO was revealed to be heterogeneous. Three samples of the purified protein (300 pmol each) from different preparations were analyzed by automated gas-phase Edman degradation, obtaining in each run an unusuall)' high amiiio acid background throttg,,hout the sequencing. Apart from this, more than one amino acid c+mld be identified at each cycle: however, by comparison of the results obtained in different runs, two overlapping N-terminal sequences were identified as follows: I S _. H_~N- L y s - P r o - T h r " A l a - A s p - P h e - A s p - T y r !0 15 ICe-Vat-VaL - Gt y - / ~ L a - G L y - . ~ , s n - A L a - GL y 2O A S h - ,:aL-VaL- k l a - T ~ , , r - k r g - . ....

AS indi,:ated in the figure, the two N-terminal sequences zre thought to Ice generated from the same polypeptide chain. The major sequence (60-70%) starts with Ala ;n position four, with a shift of three amino acids from the other sequence (as indicated by the arrow). In order to exclude the possibility of hTd,'ohsis of the protein during the cleavage step of the Edman degradation, in a different trial, a sample was sequenced under mild+-r conoitions, using a lower temperature (40°C instead of 45°C) and a reduced time for the c!eavage with TFA, but no improveinent was obtained in terms of amino acid background and multiple sequences were observed as in the other runs. The obs~:rved heteruge,'~eity could be e~,plaineti by the existence of N-te:'mimd processing eccurring in vivo or during the purification procedure. C-ternfinal sequence analysis of VAO treated with carboxypeptidase Y in the presence t,¢ 4 M urea showed a rapid release of amino acios as illustrated in Fig. 2. On the basis of the~e data the as+~gnment of the Cterminal sequence is • - -(Thr, Val~-GIn-Aia-Leu.

118 TABLE I1

~00~ i

Rclam'e rate o~ oxtdatmn o/uarmu.s alcohols ht- V,40 Measurements were performed as described in Materials and Methods. O, consumption during the reaction was expressed as the percentage of activity observed with v,-ratryl alcohol. Substrate

Relative activity (~)

3.4-Dimcthoxybenzylalcohol Cinnamyl alcohol 3-t-I)d foxy 4-methoxybenz)I alcohol 4-Methoxybcnzylalcohol 2,4-Dimethoxybeazylalcohol Conlfcryl alcohol 3-Methoxybenzylalcohol 3.5-Din.ethoxybenzylalcohol 4-HydJoxy 3-methoxybenzylalcohol 2.3-I)imethoxyhenzylalcohc! benzyl alcohol AII)I alcohol 2.5-Dimethoxybenzylalcohol 2-Methoxybenzylalcohol

100 217 144 103 89

o-~-0-~

.-L=~--.-

8oP '

~ 60;.-

7

2O

,

°o

....

L__. 30

I

.

Z .

So TemDeroturc ('C)

.

. ,o

~__ 90

Fig. 4. Thermal inactivation of VAO. Residual VAO activity after 10 rain incubations at different temperatures.

3~6 30 25 19 1I 6 .~ 4

The enzyme is active within a large p H range (3.0-8.5) with maximum activity az p H 6.5. Fluorescence spectra indicate that VAO possesses flavin as a prosthetic group. The coer -'.yme, which is not covalently bound to the protein, was identified as F A D by means of reverse-phase high-pressure chromatography and the a m o u n t present was 0.8 mol per mol of protein.

Catalytic properties The apparent Km for veratryl alcohol, determined using the Lineweaver-Burk plot, is 0,32 mM. In order to study the enzyme specificity, V A O activity was tested on a number of different substrates by measuring O 2 consumption d u r i n g the reaction and the activity was expressed as a percentage of that with veratryl alcohol (Table II).

Primary a r o m a t i c alcohols with methoxy substituents in position four are good substrates for VAO and additional substitutions in position two or three d o not seem to influence the susceptibility to oxidation. As opposed to this. methoxy substituents in positions 2-3, 2 - 5 and 3 - 5 strongly decrease the susceptibility to oxidation of p r i m a r y a r o m a t i c alchols. C i n n a m y l alcohol seems to be the substrate oxidized faster, whereas coniferyl alcohol is oxidized at a slower rate.

Enzyme stability The enzyme is stable at 5 0 ° C (98% residual activity after 4 h incubation) (Fig. "~) and has a tt/2~ssoc~ of a b o u t 1.5 h. The a p p a r e n t melting temperature of the enzyme, d e t e r m i n e d m e a s u r i n g the residual activity after 10 rain i n c u b a t i o n at different temperatures, is 6 0 ° C (Fig. 4). Stability of the enzyme in several solutions containing 50% organic solvents was also tested. The e n z y m e is stable when i n c u b a t e d at 2 5 ° C in a 50% w a t e r / a c e t o n e solution, but is less stable in methyl alcohol (tt/2 a b o u t 2 h), acetonitrile and d i m e t h y l f o r m a m i d e (tt/2 a b o u t 1 h) (Fig. 5) 50% solutions.

40"C 251~

°

'~

-o,

"

50"C o~--~------

r '

loo

~50

20c~ ' 2 5 0

3c0

350

4 o

T,m~* (r,~,n) Fig. 3. TI".rmal slabili{yof VAO. Residual VAO activity after incubattons al ~0, 50, 55 and 60°C.

°~

~'o-~-~

~

.~-¢o~ --.'~o

Time (mln) Fig, 5. Stabihty of V A O in organic solvents. Residual V A O activity after timed incubations at 25 °C in mixtures containing 50~ (v/v) of

differ,:ntorganic solvents.

119 Conclusions

P. os.'rean~s h a s b e e n p r o v e d to p r o d u c e p h e n o l o x i d a s e s [15,28] a n d v e r a t r y l a l c o h o l oxidase, b u t n o e n z y m e o f the ' l i g n i n a s e " t y p e w a s f o u n d in c u l t u r e b r o t h s of m y c e l i a g r o w n u n d e r d i f f e r e n t c o n d i t i o n s . T h e s e findings suggest that, at least ia s o m e species of fungi, a ligoin m e t a b o l i s m significantty d i f f e r e n t f r o m t h a t r e p o r t e d in o t h e r b a s i d i o m y c e t e s m a y exist. V A O a c t i v i t y h a s b e e n r e p o r t e d so far o n l y in Pleurotus sajor caju [311 a n d in Polyslicus cersicolor [32]. M o r e recently, p u r i f i c a t i o n ancl c h a r a c t e r i z a t i o n of V A O h.:s b e e n r e p o r t e d in P. sajor c~qt+ [16]; this e r t z y m e s h o w s c a t a l y t i c a n d s t r u c t u r a l p r o p e r t i e s "~ery s i m i l a r to those r e p o r t e d for the e n z y m e d e s c r i b e d in this p a p e r , g i v i n g m o r e s u p p o r t to the h y p o t h e s i s of t h e e x i s t e n c e o f d i f f e r e n t lignin m e t a b o l i s m s . T h e e n z y m e d e s c r i b e d in this p a p e r h a s b e e n c h a r a c terized f r o m the s t r u c t u r a l a n d c a t a l y t i c p o i n t s o f view. M o r e o v e r . its stability h a s b e e n i n v e s t i g a t e d in o r d e r to e x p l o i t its p o s s i b l e b i o t e c h n o l o g i c a l utilization. T h e m e t a b o l i c role o f V A O still r e m a i n s u n c l e a r , b u t its r e m a r k a b l e stability a n d b r o a d specificity s u g g e s t t h a t it p l a y s a c e n t r a l role in the b i o d e L r , ; d a t i o n o f lignin by P. ostreatus. Acknowledgements T h i s w o r k w a s s u p p o r t e d b y g r a n t s f r o m the M i n i s t e r o P u b b l i c a l s t r u z i o n e ( R o m e ) ( P r o g e t t i di R i l e v a n t e l n t e r e s s e N a z i o n a l e ) . 3niversitb. di N a p o l i and C.N.R. (Rome) Comitato Nazionale Biotecnologie e Biologia M o l e c o ! a r e .

References I Kirk, T.K. and Farrel. R.L. (1987) Annu Rev. Microbiol. 41, 465-505. 2 Kirk, T.K. (1988) ISI Atlas of Sciences: Biochemistry I(I). 71-76. 3 Sarkanen. K.V. and Ludwig. CH. (1971) I.lgnins: O~currenc¢. Formation. Structure and Reactions. pp. 1-18, J. Wiley & Sons. New York. 4 Tien. M. (1987) CRC Critical Reviews J. Microbiol. 15. issue 2. pp. 141-168, CRC Press. Boca Raton. FL.

5 Kirk. r K,. ricn. M.. Kersten, PJ.. Mozuch. MD. and Kat'vanaraman. g J (ItlS6) Biochem. J. 236. 279-297. 6 LundquTsl. K and Kirk. T.K. (19"/g) Phytt~hemistry 17. |676. 7 Fa~son. BD.. Kirk. T.K. and Farrell, R.L. 1198o) Appl. Environ. Mtcrobiol. 52. 251-254. S Har'.e'.. P J, Schoemaker H.E. and Palmer. J.M. (1986"~ FEBS l.ett lqS. 242-246. q Kelley. R.L. and Reddy. CA+ (1986) Arch. Microbiol. 144. 2a'S253 !t) Enksson. K.F,. Petterson. B.. Vole. J. and Musilek. V, 11~86) Appl. Mi,:robiol. Biotechnol. 23. 257-262. 11 Greene. R.V. and Gould. J.M. (1984) Biochem Biophys. Res. Commun. 118. 437-443. 12 Paszczynski. A. Huynh, V.B. and Crawford. R L (1986) Arch Bile:hem. Biophys. 244. 750-7o~,. 13 Kersten, J P. and Kirk. T.K. (1987) J. Bacteriol. 169. 2195-2201. 14 Agosin. E.. Daudin, JJ and Ogler. E. (1985) Appl. Microbiol. Biotechnol 22. 132-13,~. 15 Sannia. G . Giardina. P.. Luna, M.. Rossi. M. and Buonocore. V. (1986) Biotechnol. Len. 8, 797-800. 16 Bourbonnais. R, and Paice. G. (1988) Biochem. J. 255. 445-450. 17 Cohen, HJ. (1973) Anal. Biochem. 53, 208-222. 1,'4 Bradford, M.M. (1976) Anal. Biochem. 72. 248-254. 19 Laemmli. M. (1970) Nature 227, 680-6R5. 20 Merril. CR. ¢.;tddman. D. and Kenrcn. M. (1983) Methods Enzymol 96. 230-239. 21 Hunkapillar. M. and Hce'd, I.. 11983) Science 219, 650-65 t~ 22 Hunkapillar. M. (1986) in Methtxls in Protein Sequence Analysis (Walsh. K.. ed.). pp. 367-384, Humana Pre,~, Clifton, NJ. 23 Jones. B.N. 0986) in Methods of Proteins Microcitaracterization (Shively. J.E.. ed.). pp. 337-361, Humana Press. Clifton. NJ. 24 Klemm, P (19841 in Methods in Molecular Biology: Proteins (Walker, J.M., ed.), pp. 255-259, Humana Pres,~. Clifton. NJ. 25 Dubois. M.. Gilles. K.A.. Hamilton. J.K., Rebers, P.A. and Smith. F. (1956) Anal. Chem. 28. 350-356. 26 Singer, T.P.. Salach, J.. Hemmerich+ P. and Ehremberg. A. (19711 Methods Enzymol. lgB, 416-427. 27 Hausinger, RP., Honek, S.F. and Walsh. C. (1986) Methods Enzymol. 122, 199-209. 28 Giardina. P.. Buonocore. V.. Cannio, R., Fazzo, E. and Sannia. G. (1988t hal. J. Bi(x:hem. 37. 345-346. 29 Jonsson. L.. Johansson, T.. Sjostrom. K. and Nyman, P.O. (1987) Acta Chem. Stand. Bit. 766-769. 30 Kirk, T.K., Croan, S., Tien, M.. Murtagh. K.E. and Farrel. R.L. (1986) Enzyme Microbiol. Technol. 8, 27-32. 31 Fukuzumi. J. (1987) in Ligain Enzynuc and Microbial Degradation (Ogler, E.. ed.), pp. 17'7-141, INRA Publications, Pans. 32 Farmer. C.. Henderson. N.E.K. and Ru~sel. D.J. (1960) Bit,chem. J. 74. 257-262.