APPLIED MICROBIOLOGY, Sept., 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 5Printed in U.S.A.

Effect of Culture Conditions on the Production ofD-Galactose Oxidase by Dactylium dendroides'

Z. MARKUS, G. MILLER,2 AND G. AVIGAD

Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel

Received for publication 22 March 1965

ABSTRACT

MARKUS, Z. (The Hebrew University, Jerusalem, Israel), G. MILLER, AND G. AVIGAD.Effect of culture conditions on the production of D-galactose oxidase by Dactyliumdendroides. Appl. Microbiol. 13:686-693. 1965.-The effects on enzyme production ofinoculum size and age, medium composition, and culture conditions were studied inshake flasks and in a pilot-plant fermentor. Using a medium consisting of glucose,yeast extract, and inorganic salts in deionized water, we found that the addition ofCu++ was essential for the formation of active enzyme. Cultures grown in the absenceof added copper produced an inactive enzyme protein which could be activated by103 M Cu+. Thiamine fulfilled all requirements for exogenous vitamins for growthand enzyme production. Glucose concentrations higher than 1% markedly suppressedenzyme formation. The mycelium inactivated the enzyme on prolonged incubation ofthe culture. Mycelial autolysates and sonic extracts were found to contain a thermo-stable and slowly dialyzable galactose oxidase-inactivating factor. The experimentssuggest that this factor operates as a chelating agent which forms complexes with thecopper of the enzyme. Copper ions (10I8 M) prevented enzyme inactivation and re-stored activity to samples previously inactivated by this factor.

Production of D-galactose oxidase by thefungus Polyporus circinatus Fr. was first reportedby Cooper et al. (1959). Avigad et al. (1962)studied this enzyme in detail and developed aprocedure for its purification. They found thatD-galactose as well as certain D-galactosides areoxidized at the C-6 position, and suggested thatthis enzyme requires a metal cofactor. Amaral etal. (1963) later characterized a crystalline prep-aration of the enzyme as a copper-containingmetalloprotein. Nobles and Madhosingh (1963)re-examined the enzyme-producing organism andidentified it as Dactylium dendroides, rather thanP. circinatus.

Galactose oxidase has been found to be a help-ful analytical tool for the specific determinationof D-galactose in blood plasma (DeVerdier andHjelm, 1962), in plant extracts (Rorem andLewis, 1962), and in phospholipids (Agranoff,Radin, and Suomi, 1962; Bradley and Kanfer,1964), and was highly useful for the characteriza-tion of terminal D-galactoside units in severalpolymers (Avigad et al., 1962; Blumenfeld et al.,1963; Barker, Pardoe, and Stacey, 1963; Osbornet al., 1964; Robinson and Pierce, 1964).

1 Taken in part from a Ph.D. Thesis to be sub-mitted to the Hebrew University by Z. Markus.

I Permanent address: Israel Institute for Bio-logical Research, Ness-Ziona, Israel.

In view of the increasing interest and uses ofthis enzyme, we have conducted a more detailedstudy of the pattern of its appearance in fungalcultures under various conditions.

MIATERIALS AND METHODS

Propagation of the fungus, and purification andconcentration of the enzyme were carried out asdescribed by Avigad et al. (1962). However, toobtain reproducible yields of enzyme, the modifi-cations described below were introduced.

Stock cultures. Stock cultures were maintainedon slants of the following composition: glucose,2%; yeast extract (Difco), 0.2%; peptone (Difco),0.5%; CaCO3, 0.2%; agar (Difco), 2.0%; in de-ionized water. Transfers were carried out once in3 months. Slants were incubated at 30 C for 3days and then kept at 4 C.

Culture medium. The culture medium, hence-forth called medium G, was basically similar tothat used by Avigad et al. (1962), and contained1.0% glucose, 0.2% (NH4)2SO4, 0.1% NH4NO3,0.1% yeast extract (Difco), 0.9% KH2PO4, 0.8%Na2HPO4, 400 mg per liter of MgSO4*7H20,2.0 mg per liter of MnSO4.H20, and 2.5 mg perliter of CuSO4.5H20, dissolved in deionized water.To avoid carmelization and formation of pre-cipitates, medium G was sterilized in 3 separatefractions: solution A containing glucose; solutionB containing MgSO4, MnSO4, and CUSO4; andsolution C containing yeast- extract and the other

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PRODUCTION OF GALACTOSE OXIDASE

ingredients. The sterile solutions were combinedaseptically before inoculation.

In certain shake-flask experiments, and in allpropagations in the fermentor, the following anti-foams were applied: Silicone Emulsion RD (Hop-kin & Williams Ltd., Chadwell Heath, U.K.), andM-8 and KG-1 (Hodag Chemical Corp., Skokie,Ill.). The antifoams were added to solution Cbefore sterilization.

Alternative media compositions investigatedare described in Results.

Inocula for shake-flask experiments were pre-pared as follows. A piece of mycelium was trans-ferred into a 500-ml Erlenmeyer flask containing100 ml of medium G and was incubated for 3 daysat 28 to 30 C on a rotary shaker (model G10;excentricity, 1 inch; New Brunswick ScientificCo., New Brunswick, N.J.) at 240 rev/min; 2 mlof culture were transferred into a 1-liter Erlen-meyer flask having three indentations (height ofindentations, 13 cm) and containing 100 ml ofmedium G. Propagation was carried out for 24 hras in the previous step. This culture was used asseed for the shake-flask experiments with aninoculum size of 1 to 2% (v/v).For experiments in the fermentor, an additional

step was included: inoculum (2%0, v/v) was trans-ferred into 2-liter Erlenmeyer flasks with sideoutlet, containing 300 ml of medium G, and waspropagated for 24 hr as described before.

Shake-flask experiments. Shake-flask experi-ments were carried out in duplicate series of500-ml Erlenmeyer flasks containing 100 ml ofmedium. Shake flasks were closed by cotton plugsand incubated on the shaker, usually for 72 hr.The mycelium was separated by filtration throughtwo layers of gauze, and the filtrate was kept at4 C. Results given in this report are average valuesof two parallel experiments.

Growth in fermentor. Batches of 30 liters werepropagated at 28 to 30 C in a 65-liter stainless-steel(type 316) fermentor (Palbam Ltd., Ein Harod,Israel), with aseptic techniques. The baffles ofthe fermentor were removed to avoid excessivefragmentation of the mycelium, which was foundto decrease enzyme production. The culture wasagitated by a turbine impeller at 210 rev/min.Sterile air was sparged at the rate of 10 liters permin. Propagation was carried out at atmosphericpressure. After 50 hr, the culture was cooled to5 C. The mycelium was then separated by a basketcentrifuge. The supernatant fluid was kept at 4 C,until further utilization and purification of theenzyme.Assay of galactose oxidase activity. The chro-

mogen reagent of Huggett and Nixon (1957) con-tained 5 mg of O-dianisidine per 100 ml. Theenzyme was determined by the peroxidase-chro-mogen colorimetric method (Avigad et al., 1962).The assay mixture consisted of 1.5 ml of reagent,0.3 ml of a 3% galactose solution (to give a finalgalactose concentration of 2.5 X 10-2 M), and0.2 ml of a properly diluted galactose oxidasesolution containing 0.1 to 1 enzyme unit. The

reaction was allowed to proceed for 10 min at30 C, and was stopped by addition of 0.1 ml of40% KOH. Optical density was read at 420 my inthe Zeiss model PMQ II spectrophotometer withthe use of cells of 1-cm light path. A unit ofenzyme was defined as by Avigad et al. (1962).Specific activity is expressed as units of enzymeper milligram of dry mycelium.

Glucose. Glucose was determined colorimetri-cally (Sols and de la Fuente, 1957) with the use ofa commercial glucose oxidase reagent (Worthing-ton Biochemical Corp., Freehold, N.J.).Dry mycelium. Dry mycelium was obtained by

filtration of 100 ml of culture through two layersof gauze. The mycelium was washed with de-ionized water, squeezed lightly, and dried at105 C for 3 to 4 hr in an oven with forced aircirculation (A.E.W. Ltd., Edgware, MiddlesexU.K.).

Preparation of mycelial extracts. Mycelial sonicextracts were prepared by the method of Sih andKnight (1956). Fresh, filtered mycelium waswashed with deionized water and, upon additionof alumina, was disintegrated in a 10-kc sonicoscillator (Raytheon Manufacturing Co., Wal-tham, Mass.; model DF 101) for 30 min. Thesupernatant fluid collected by centrifugation at8,000 X g for 15 min was used for enzyme-inacti-vation experiments.

Mycelial autolysates were obtained by incu-bating shake-flask cultures in medium G for 1week at 30 C, and then separating the myceliumby filtering through two layers of gauze.

RESULTSInoculum. As stated previously, the last stage

of seed preparation was carried out in indentedshake flasks where only a limited degree of frag-mentation of the mycelium occurs. By this tech-nique, a homogeneous inoculum was obtained,which could be accurately pipetted.The effect of inoculum size on enzyme pro-

duction was investigated in shake-flask cultureswith medium G (Fig. 1). Highest yield of activeenzyme in growth on glucose (about 40 units perml) was obtained with small inocula (0.2 and0.5%, v/v; Fig. 1). Larger inocula resulted inlower yields of enzyme. However, in most of theshake-flask experiments reported later, a 2%inoculum was used to ensure a higher accuracy ofduplication in transfer of mycelia suspensions toseries of experiments. It is seen (Fig. 2) that,whereas the growth rate was higher with thelarger inocula, the final mycelial yield was similarin all cases. It thus appears that yield of enzymeachieved by a slowly growing mycelium is higherthan that obtained by a rapidly growing one.The effect of inoculum age on enzyme activity

was studied in shake-flask experiments withmedium G. For this purpose, a homogeneousinoculum was kept at 4 C for periods as indicated

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MARKUS, MILLER, AND) AVIGAI)

00 0.2% V/V inoculum

6-6 0.5%*- 2.0°/.0-0 5%andlO% V/V inoculum

0-0 02% V/V inocutum&-6 0.5%¶

0-O 2 0% -

O-- 5.0% -

A.-i10 0% *

-

E

E

0z

Propagation hours

FIG. 2. Effect of inoculum size on mycelial growth.

on enzymie for-

in Table 1. It was fotnd that, though the growthcapacity of the seed was not affected by storage,its calpacity for enzyme production decreased sig-nificantly.

Composition of medium. The effect of variousmetal ions in different combinations on activeenzyme formation was studied in shake-flask ex-

periments as shown in Table 2. In these experi-ments, stock solutions of each metal salt were

autoclaved separately and combined asepticallywith solutions A and C of medium G.

MIaximal enzyme activity in glucose medium,usually about 40 units per ml, was obtained withthe combination Mg4W,Mn4+ , and Cu+ . Thiscombination of cations was, therefore, adoptedfor routine enzyme production in later studies.It should be indicated that neither mycelialgrowth nor enzyme activity was affected by theincrease of CuS04 concentration ul) to 10-3 M.

The effect of copper can be seen also in kineticexperiments (Fig. 3). The presence of 10-5 M

copper facilitated the appearance of much more

active enzyme, the bulk of which was producedduring mycelial growth. In these experiments(10-5 M CuS04 in the medium), when shakingwas continued beyond the stationary phase(above 70 hr of incubation), a decrease in enzymeactivity ensued, in part, presumably, because ofinactivation due to autolysis of mycelia, as willbe discussed later.The effect of different glucose and galactose

concentrations added to medium G was investi-

TABLE 1. Effect of inocuiurm age on growth andenzyme production

Age ofinoculum* Dry myceliumt Enzyme activityt Specific

days mg/ml units,/ml

0 6.0 36.0 6.08 5.7 20.0 3.515 7.0 15.0 2.1

* Storage at 4 C.t Shake-flask cuilttures at 72 hr.

gated (Table 3). Maximal yields of galactose oxi-dase were obtained with 0.5 to 1% glucose.Higher glucose concentrations resulted in in-creased mycelia lproduction, but lowered the levelof enzyme in the medium; i.e., productivity ofthe mycelium decreased. At 10-3 M copper and5% glucose, the yields of enzyme were somewhatimproved but still did not reach the maxima]value obtained with 1%c/. glucose. At 5 and 10%galactose, no enzyme could be found in the me-

dium, though an abundant growth of myceliumoccurred. An increase in Cu' concentration inthis case facilitated the appearance of only a

trace amount of enzyme.In other experiments, several alternative car-

bon compounds were investigated (Table 4) inaddition to those reported earlier (Avigad et al.,1962). With L-sorbose, enzyme yields were higherthan those obtained on glucose. This confirmsobservations by C. Asensio (personal communica-tion), who is studying this effect in detail. Theother carbon sources used were equal or inferiorto glucose in promoting enzyme production. Theeffect of different concentrations and ratios of

24 48 72 96Propagation hours

FIG. 1. Effect of inoculum sizenitation.

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TABLE 2. Effect of metal ions on enzyme synthesis in Dactylium dendroides cultures

Salt*

MgSO4o7H20

+

MnSO4u FeSGo ZnSO4 CaS04H20 7H20 SO CaO

+

+

* MgSO4- 7H20 at 400 ,ug/ml and the other salts at 2 ,ug/ml were added to medium solutions A + C.

Luc

z

0-0 Enzyme, Medium G without Cu- with16'M Cu

0--0 Mycelium - without Cue

8-_a - with Cu'+

3 =-E

3 2:,i

Propagation hours

FIG. 3. Effect of copper on enzyme formation.

ammonia, nitrate, and urea on galactose oxidaseformation is shown in Table 5. High enzyme

activity could be obtained with urea as the miuiro-

gen source when it was supplementing glucose.Urea by itself was not efficient as a sole carbonsource, either for growth or enzyme production.

Shake-flask experiments were undertaken tostudy the replacement of yeast extract in mediumG by several B Vitamins (Table 6). It is clearthat thiamine is an essential growth factor forthis organism arid( 5 ,ug of thiamine per ml com-

pletely replaced yeast extract for growth andenzyme production.The addition of 100 units per ml of sodium

penicillin G and of 100 ,ug/ml of streptomycinsulfate did not affect enzyme production in me-

dium G in standard shake-flask experiments.Addition of several antifoams to medium G in

TABLE 3. Effect of different concentrations ofglucose, galactose, and copper on enzyme

activity in culture

Carbon source En-Cu++ zyme Dry my- Produc-

P concn activ- celium tivitytCompound cent ity

M untts/ mg/ml

Glucose 0.2 10-5 7 1.7 4.10.5 10-5 18 2.4 7.51.0 10-5 33 4.9 6.72.0 10- 29 9.2 3.15.0 10-5 5 17.0 0.310.0 10-5 4 17.4 0.21.0 10-3 33 5.6 5.95.0 10-3 16 16.5 1.0

Galactose 1.010°- 27 4.3 6.25.0 10- 0 12.7 010.0 10- 0 16.3 05.0 10-3 12 16.0 0.8

* All other ingredients as in medium G, withcarbon source as indicated.

t Units of enzyme per milligram of dry my-celium.

shake-flask experiments showed that siliconeemulsion RD or antifoam M-8 at 0.05 to 0.1%,as well as KG-1 at 0.1 to 0.3%, did not suppressgrowth and enzyme formation.

Effect of oxygen absorption rate (OAR). At-tempts were made to propagate the organism atdifferent OAR, with medium G. For this purpose,a series of shake flasks calibrated to OAR in therange of 0.3 to 1.5 mmoles of 02 per liter per min,as described by Corman et al. (1957), were used.Equal mycelial growth and enzyme production

CuS04-5H20

++

Combination

1234567891011

Units ofenzyme/mlof super-natant

0282727104062025

Drymycelium(mg/ml)

5.55.25.55.46.05.76.05.95.66.15.8

Units ofenzyme/mg

of drymycelium

05.44.95.01.77.01.00.300.30.9

+

+

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TABLE 4. Effect of various carbon sources onenzyme activity in culture

Carbon source * Enzyme Dry my- Specificactivity celium activity

units/ml mg/ml

Glucose ................. 32.0 5.3 6.0Glycerol ................ 2.0 2.0 1.0Sodium citrate .......... 2.0 2.0 1.0Sodium lactate .......... 2.0 4.0 0.5Sodium acetate.. ............... 2.0 2.90.7Sodium succinate ....... 0 2.4 0L-Sorbose ............... 70.0 4.9 14.3Sorbitol ................. 18.0 3.1 5.8D-Mannitol .............. 18.0 5.2 3.5Fructose ................ 20.0 5.0 4.0D-Mannose .............. 32.0 5.0 6.4Sucrose ................. 33.0 4.9 6.7Maltose ................. 6.0 3.0 2.0Inulin .................. 12.0 2.7 4.1Starch, soluble .......... 6.0 6.7 0.9Corn steep liquor . 0 4.9 0

* All other ingredients as in medium G, butwith carbon source as indicated, at 1% concen-tration.

TABLE 5. Effect of different nitrogen sources onenzyme activity in culture*

Glu- Nitrogen source Enzyme Dry my- Specificcose activity celium activity

% units/ml mglml1.0 (NH4)2SO4t, 0.2%7o 37.0 6.3 5.9

NH4NO3(2), 0.1%

1.0 (NH4)2SO4, 2.0% 24.0NH4NO3, 1.0% 7.0 3.4

5.0 (NH4)2SO4, 2.0%NH4NO3, 1.0% 0 20.3 0

Ureat, 1.0% 0 2.1 01.0 Urea, 0.1% 19.0 7.0 2.71.0 Urea, 0.5% 24.0 6.9 3.51.0 Urea, 1.0% 32.0 7.0 4.61.0 Urea, 5.0% 7.0 3.1 2.2

* All other ingredients as in medium G.t Concentrations as in medium G.t Sterilized by Seitz filtration.

were obtained by propagations conducted at thisrange of OAR.

Pilot-plant experiments. Typical results ob-tained in the fermentor and in parallel shake-flask cultures are given in Fig. 4. Although more

mycelium was produced in the shake flasks thanin the fermentor, the final yields of active enzymewere similar (about 25 units per ml). In the fer-mentor, glucose utilization, mycelial growth, and

enzyme formation proceeded in the same patternsobserved in the shake flasks. Growth in the fer-mentor resulted in significant foam production.Addition of 500 ppm of antifoam M-8 or 1,500ppm of antifoam RD was found to be sufficientto supress foam during the whole propagationperiod.

Inactivation and reactivation of enzyme in cul-ture. Whole cultures grown in shake flasks or inthe fermentor in the presence of 10-5 M CuS04,which contained 25 to 40 enzyme units per ml,were stored at 4 C in the presence of the myce-lium. Culture samples were taken at differenttime intervals, the mycelium was removed bycentrifugation, and the supernatant fluid wasstored at 4 C. It was found that 2 weeks of stor-age of whole cultures resulted in almos a com-plete loss (96%) of enzyme activity. On the otherhand, in the supernatant fluids separated ini-tially, the galactose oxidase activity was retainedcompletely during this period. In several cases, itwas also observed that in stationary cultureskept at 30 C, after the cessation of mycelialgrowth, a complete inactivation of enzyme oc-curred within 72 hr. This enzyme inactivation,which occurred during storage in the presence ofthe mycelium but not in the freshly isolated su-pernatant fluids, was prevented by the additionof excess copper (10-3 to 10-2 M CuS04) to theculture prior to storage. It was also found that,even after enzyme inactivation in whole cultures,enzyme activity could be restored completely by24 hr of incubation at 30 C after the addition ofCUSO4 to 103 M. When mycelial autolysates (0.5ml) or sonic extracts (0.5 ml, 1.75 mg of protein)were incubated with 20 units of purified galactoseoxidase in 1.5 ml of reaction mixture at pH 7.0,complete inactivation of enzyme resulted after4 hr of incubation at 30 C. Addition of CuSO4 to10-3 M to the reaction mixture prevented enzymeinactivation or restored complete activity in in-activated systems, after an overnight incubationat 30 C. Mycelial sonic extracts boiled for 10min or dialyzed against deionized water for 24hr at 4 C still retained most of their enzyme-inactivating capacity.

Activation of enzyme in copper-free medium. Aspreviously shown (Table 2), no enzyme activitywas found in the cultures on medium G withthe omission of heavy metal cations (solution Bomitted), but abundant growth occurred. Whenthe supernatant fluid obtained from such culturesafter 72 hr of growth was subsequently supple-mented with CUSO4 to 1073 M and incubatedovernight at 30 C, galactose oxidase activityappeared in an amount similar to that obtainedin control cultures simultaneously grown in the

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TABLE 6. Replacement of yeast extract by B vitamins

Expt no. Vitamin source* Concn Enzyme Dry Specificactivity mycelium activity

igiml units/ml mg/ml

a Yeast extract (Difco) 1,000 39 5.7 6.8b Thiamine-HCI 5 29 5.4 5.3c Thiamine-HCI, biotin, Pyri- 5 28 5.9 4.7

doxine, calcium pantothenate,p-aminobenzoic acid and nico-tinic acid (each)

d Controlt 10 3.6 2.8

* Medium composition: (a) medium G; (b to d) medium G without yeast extract, andadded as indicated.

t Without yeast extract and vitamins.

O-O Enzyme in fermentor

&--M MyceeiumiinD0 Enzyme in shake flasks

0- --Mycelium in ..

A--A Residual gtucose in fermentor658 _ *-* pH

I 64 -

6.0_

Propagation hours

FIG. 4. Enzyme formation and glucose utilizationin fermentor and shake-flask cultures.

complete medium G (30 to 40 units per ml indifferent experiments). This interesting phenom-enon of the appearance of an inactive enzymeprotein in cultures grown without copper andits process of activation is currently beingstudied in detail in our laboratory.

DIscussIoN

The main objectives of the present study were

directed towards the elucidation of factors lead-ing to optimal yields of galactose oxidase by D.

with vitamins

dendroides in shake-flask cultures and in a pilot-plant fermentor.

In looking for a convenient way to obtain seedcultures which are convenient for handling andmeasuring, the use of indented shake flasks wasfound to be advantageous. For optimal yields ofenzyme, the seed culture should be fresh (3 to 7days), as older mycelia initiated growth of lessproductive mycelia with respect to enzyme for-mation.

It is clear that the presence of Cu+ in themedium is obligatory for the appearance of activeenzyme in growing cultures. Negligible or only arelatively low level of enzyme activity could bedetected in culture supernatant fluids of thefungus grown in a medium prepared with deion-ized water but without any addition of Cu*+.Mg and MnH alone at the concentration usedin medium G usually facilitated appearance of alow level of enzyme activity which might be at-tributed to the presence of contaminants (unpub-lished data). Elimination of Mg+ and Mn++ aswell as Cu++ resulted in no galactose oxidaseactivity, though normal growth of mycelium wasfacilitated. The fact that an overnight incubationof these inactive culture filtrates with 10-3 MCuH resulted in the dramatic appearance of themaximal enzyme activity expected (by compari-son with control cultures grown in the presenceof copper) clearly suggests that an inactive"apoenzyme" protein had accumulated in themedium. The slow process of activation of thisprotein by CuH presumably results from a slowformation of the active configurational form ofthe metalloprotein. This behavior of the enzymeis similar to the reactivation by Cu++ of the ga-lactose oxidase which was previously treated withmetal chelates to remove the Cu+ from the na-tive enzyme (Amaral et al., 1963). From thepresent studies, it can be assumed that the ap-pearance of active enzyme in media without the

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extraneous addition of the cation (Avigad et al.,1962) was probably due to copper contaminantsin the ingredients or the distilled water used forgrowing the fungus.The finding that mycelia contain an enzyme

inhibitor which is counteracted by excess Cu+further shows the advantage of using a relativelyhigh concentration of CuSO4 in the culture me-dium to ensure the persistence of maximal levelsof active enzyme. Whereas the nature of the in-hibitor found in the mycelium is yet unknown,the fact that excess CuH can dissociate it fromthe inhibited enzyme, with the concomitant res-toration of galactose oxidase activity, suggests achelation type of reaction between the copper-enzyme protein and the inhibitor. This phenom-enon is currently being studied in this laboratory.

Considering the high viscosity attained alreadyat early growth of the fungus, it is not surprisingthat in shake flasks calibrated to various OARlevels no significant differences in mycelial growthand enzyme yields were found. In fact, OAR de-terminations by the sulfite method are apparentlynot applicable to dense cultures of filamentousorganisms (Lockhart and Squires, 1963). Thus, itmay be of importance to re-examine the effect ofOAR level on enzyme formation with the use ofdifferent methods of agitation and aeration. Asnoted above, however, excess fragmentation ofmycelia by strong mechanical agitation mightcause a drastic decrease in enzyme productionwhich could be explained only partly by therelease of the enzyme inhibitor from the mycelia.From the other nutritional studies reported in

this work, several additional important pointsshould be mentioned. Concentrations of glucoseor galactose higher than 2% usually retardedformation of enzyme even in the presence of ex-cess Cu*+, though they promoted intensivegrowth. At present, however, it is difficult tospeculate on the mechanisms of this repression ofenzyme formation by excess sugar in the medium.Certain aspects of this phenomenon, however,could be related to the well-known "catabolite"repression of enzyme formation in microorgan-isms (Magasanik, 1961). Urea could be sub-stituted for NH4+ and NO3- as a nitrogen sourcebut could not replace sugar as a carbon sourcefor enzyme formation. Thiamine could satisfy thevitamin requirements of D. dendroides; thus, bysubstituting yeast extract, a completely syntheticmedium was established.

It should also be noted that different peroxi-dase-chromogen preparations might give differ-ences as great as 50% in measurements of galac-tose oxidase activity (see Avigad et al., 1962).It seems, therefore, that enzyme yields reported

by different laboratories might not be completelycomparable because of different reagents used.We could indeed confirm the observation thatmost of these differences presumably arose fromthe fact that the o-dianisidine routinely used asthe chromogen in this reagent competes withgalactose and inhibits the galactose oxidase (J.Mager, personal communication). A similar ob-servation was recently made by Fischer andZapf (1964) and Roth, Segal, and Bertoli (1965),who suggested the use of another chromogen forthe galactose oxidase assay.

ACKNOWLEDGMENTSWe wish to thank J. M. Sudarsky, Wasco,

Calif., for a grant which in part supported thisinvestigation. This research was also supportedby grant FG-IS-14I from the U.S. Department ofAgriculture.

LITERATURE CITEDAGRANOFF, B. W., N. RADIN, AND W. SUOMI. 1962.Enzymic oxidation of cerebrosides: studies on

Gaucher's disease. Biochim. Biophys. Acta.57:194-196.

AMARAL, D., L. BERNSTEIN, D. MORSE, ANDB. L. HORECKER. 1963. Galactose oxidase ofPolyporus circinatus: a copper enzyme. J. Biol.Chem. 238:2281-2284.

AVIGAD, G., D. AMARAL, C. ASENSIO, AND B. L.HORECKER. 1962. The D-galactose oxidase ofPolyporus circinatus. J. Biol. Chem. 237:2736-2743.

BARKER, S. A., G. I. PARDOE, AND M. STACEY.1963. Sequential enzyme induction: a new ap-proach to the structure of complex mucopro-teins. Nature 197:231-233.

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BRADLEY, R. M., AND J. N. KANFER. 1964. Theaction of galactose oxidase on several sphingo-glycolipids. Biochim. Biophys. Acta 84:210-212.

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