Comparatively little attention has been devoted to the enzymes capable of hydro- lyzing nucleosides. These, and the related nucleoside phosphorylase and deoxyribonu- cleoside transferase, are described in the book by Colowick and Kaplan (4). Nucleo- sidases have been reported from Escherichirl coli (3, 4, 5, 9), Bacillus cereus (8), Lncto- bacillus pentosus (3, 6 ) , L . rl~~lbrueclcii (13), yeast (2, 3, 4, 6), fish muscle (14), and nlung bean seedlings (1). We now report on a number of filngal enzymes, active only ori the purine p-ribof~~ranosides. At least two separate types exist: nucleosidase g, which is more active on guanosine than on adeno- sine, and nucleosidase a, which is more active on adenosine. Both types are N- ribosyl-purine ribohydrolase of the Enzyme Commission non~enclature, EC 3.2.2.1.

Extracellular purine p-ribosidases from fungi

E. T. REESE Pioneering Research Laboratory, United States Army Natick Laboratories, Nntick, Massnch~rsetts

Received September 27, 1967

Purine P-ribonucleosidases are inducible enzymes secreted into the growth medium by several species of Asper-gillus and Penicillilltn. They act on purine B-N-ribosides to produce purine and ribose, but do not hydrolyze pyrimidine P-N-ribosides, deoxyribonucleosides, or P-0-ribosides. Nucleosidase g has properties different from nucleosidase n, one of which is the added ability to hydrolyze adenosine 5'-phosphate to adenine and ribose 5'-phosphate.

Canadian Journal of Microbiology, 14, 377 (1968)

the yields in preliminary tests, they both have been routinely incorporated in the medium, each at 0.05%.

Enzjltne pr-epnrntiotrs (1 1) were made by removal of the organism on a filter, and precipitation of the enzyme from the filtrate (pH 4.7) with two volumes of cold (0 "C) acetone. In general, recovery of activity was not good (15-60s). Somewhat better recoveries were obtained by dialysis of a concentrated filtrate, followed by lyophilizing in the presence of celite.

All of the enzynles which were prepared as indi- cated above were of the nucleosidase a type. The nucleosidase g type was found at low concentrations in a colnnlercial preparation, "Cellulase 9X", a product of Asper-gilllls niger grown in tank culture (L. Underkoflcr, Miles Laboratories, Inc., Elkhart, Indiana). Attempts to increase the specific activity of nucleosidase g were unsuccessful, but, since it is clearly different from the other fungal enzymes, it is worthy of further study.

Methods Gr-otvtl~ nnd Enzytne Pro(/~iction (1 1)

The organisms (QM Culture Collection, Natick, Mass.) were grown on a basal medium containing (per liter): KH2P04, 2.0 g; MgS04.7H20, 0.3 g; CaCI2, 0.3 g; trace elements (Fe++, 1.0 mg; Mnt+, 0.5 mg; Co++, 0.5 mg; Zn++, 0.8 mg); proteose peptone 0.05 g; and yeast extract, 0.05 g. The initial pH was 4.1. Most of the nitrogen was supplied as part of the substrate, e.g., RNA (yeast, Cal. Biochem.) 8 g/l. When non-nitrogenous carbon sources were used, nitrogen was added as (NH4)2S04, 1.4 g/l. The cultures were incubated 011 a reciprocal shaker at 29" for 10-12 days, during which period the culture filtrates were assayed for activity.

Earlier experience (unpublished) that sucrose monopalmitate or Tween 80 (ca. 0.1 yo) often enhances enzyme production has been confirmed. Tween 80 is preferred for some organisms, sucrose mono- palmitate for others. In some cases, neither compound is beneficial; but since in no case did they inhibit

Assay Proceclloe (1 1) A mixture of 0.5 ml adenosine solution (4 mg

adenosine in 0.05 M citrate pH 4.0) and 0.5 ml enzyme (suitably diluted in the same buffer) was incubated for 30 minutes at 40'. Reducing sugar was determined by the dinitrosal~cylic acid method of Sumner (12). A unit of activity is defined as the amount of enzyme required to produce I pmole (0.15 mg) ribose under the assay conditions. Since adenosine is the substrate for these tests, the activity is generally referred to as an adenosinase unit (p).

C/7romntogrnp/ly Two solvent systems proved useful for the separa-

tion of products. The first is that used primarily for sugar separations (isopropanol : acetic acid : water : : 54:s: 18). The second is often used for nucleosides (water plus NH4OH to pH 10.0). The bases and nucleosides were detected by short wavelength ultraviolet light, and can be observed on sheets developed in both solvents. The benzidine - trichloroacetic acid spray for ribose and ribose 5'-phospllate (11) can be used only after the first solvent system.

The following abbreviations have been used: A = adenine; G = guanine; Hx = hypoxanthine; R = ribose; P = phosphate.

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378 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

Results 1. Search for Purine Ribonucleosiclases

Over 200 cultures of fungi and bacteria were tested for their ability to produce N-glycosidases when grown on a medium containing yeast n~icleic acid. These rep- resented genera from all of the major groups of fungi, and a few bacteria and Strepto- inyces spp. Many grew on the RNA, but did not secrete enzyme. The only active cultures were species of Aspergillus and of Penicilliurn. A further and more thorough investigation revealed that only a few of the species of these genera produce and secrete nucleosidase.

A. versicolor group: A. anzbiguus (57 ,u /ml) and A. nlicrocysticus (91 p/ml). These are two closely related species.

A. glaucus group: A. clzevalieri (3 p/ml) A. restrictus group: A. restrictus (5 p/ml) A. Jlavipes group: A. niveus (two strains;

12-15 ,u/ml) A. terreus group: A. terreus (eight strains;

8-41 ,u /ml) Many species (e.g. 32) of Penicilliuin have

been found active, but these represent but a small portion of all Penicillium species.

Fifty commercial enzyme preparations were tested for nucleosidase. The best of these, Cellulase 9X (Miles Lab.), contained 1.7 y adenosinase/mg. Most others were

inactive. The dry preparations obtained in the current work are 10-40 times this value.

The most active cultures, i.e. those pro- ducing over 30 p of adenosinase/ml, are listed in Table I. I t is interesting that the Cellulase 9X is derived from A. niger, but none of the strains of A. iziger used by us produced the enzyme.

The following Penicilliunz species also produced adenosinase but at less than 30 ,u /ml of culture medium : P. botryosuin, brefeldianuin, cari~7iizoviolaceuin, clzermesinum, cinerascens, citrinwn, corylophilum, daleae, fellutanwn, Iqidosunz, Ievitunz, lilacino-echi- nulatwn, IiviCIUin, iniczynslcii, nigricans, novae- zeelandiae, paxilli, piscarium, rugulosum, ter- liko~vskii, and ~vestlingi.

2. Inducible Nature of Purine Ribonucleo- sidases

In the preliminary tests, many organisms were grown on media containing various carbon sources: starch, yeast cells, dextran, deoxyribonucleic acid, glucose, or glucose plus adenosine. Nucleosidases were produced by only a few organisms and in very small amounts. When yeast ribonucleic acid was used as a substrate, the enzyme was induced in some organisms. In general, the yields increased with the concentration of the RNA

TABLE I

Nucleosidase a production by fungi grown on various carbon sources*

Maximum units per milliliter, when grown on:

Adeno- A.R. A.R. G.R. QM RNA slne 5'P 3'P 3'P Ribose Glucose

Aspergillus ambiguus -4. microcysticus A. terreus Penicilliurn adametzi P. atrovenetum P. biforme P. brasilianum P. frequentans P. frequentans P. herquei P. janthinellurn P. javanicum P. ochrochloron P. parvum P. simplicissimum P. vit~aceum

*We are measuring here only enzyme that has been secreted by the fungi.

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REESE: B-RIBOSIDASES FROM FUNGI 379

i n the medium (up to 35; tested). The extracellular enzyme concentration reached a peak value in 3-6 days, after which it (usually) fell rapidly to zero. This coincided with consumption of substrate, and an increase in pH to above 6.5.

We belicve that the RNA acts indirectly as an inducer of the nucleosidase because it is hydrolyzed by the organism to provide the nucleoside (the direct inducer) slowly over a period of time. Substitution of nucleo- side (adenosine, g~~anosine) for RNA has given very poor yields (Table I). Usually no more than traces of enzyme were pro- duced. This appears to be attributable to the rapidity with which the pH rises during incubation on these substrates. S~ibstit~itioil of a nucleoside phosphate (A.R.SIP, A.R.3'P, G.R.3'P) for RNA gives good yields of enzymes. As with RNA, these systems seem to operate through the slow release of the substrate-inducer, i.e., of the nucleoside.

Ribose-a product of nucleosidase action- may act as an inducer (Table I). The best results have been obtained with P. parvum. With ribose as substrate, the pH of the medium tends to remain low. Higher yields are obtained when ribose is added in small

amounts periodically to a glucose culture, but in no case do these approach the values obtained using yeast RNA.

At no time in these experiments, even with DNA or deoxyribonucleosides as substrates, have we been able to detect any cleoxyribonu- cleosidases, pyrimidine ribosidases, or 0 -0- ribosidases. DNA was used for growth by some of the fungi tested, but no deoxyribonu- cleosidases could be detected in these.

3. Properties The pH optimum for the fungal nucleosi-

dases (Fig. 1) acting on adenosirle is 4.1 f 0.4, with at least 50% of maximum activity over the range 3.5-5.5. There is very little activity above 6.0, or below pH 2.5. The pH curves for activity on other purine ribosides (inosine, xanthosine, guanosine) are generally similar to those on adenosine. Several minor deviations have been observed, the most fre- quent being a shift to higher pH for optimal action on guanosine (Fig. 1) by some of the preparations. Other curves (not shown) sug- gest the possibility of two nucleosidases in a culture.

These enzymes are not as heat stable (Fig. 1) as other carbohydrases. Inactivation is

P H Temperature

FIG. 1. A. Effect of pH on activity of A. atnbiglllrs (QM 8155) nucleosidase; a, vs. adenosine, g, vs. guanosine. Temperature 40 "C. B. Effect of temperature on inactivation of nucleosidase. Con- ditions: pH 4.0, citrate M/20, 1 h. Then to assay at 40' for 30 min with adenosine; W, Penicilli~on llerquei, V , Asper~ill~rs microcystic~rs, L, A. niger (Cellulase 9X, Miles Labs.), K, P. parv~rttr.

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380 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

detectable a t temperatures as low as 40" (pH 4.0, 1 hour), but enzymes of different organ- isms vary appreciably. For routine assays, 40" is acceptable and convenient.

The effect of substrate concentration has been determined for several enzymes. There is relatively little increase in rate of hydrol- ysis as the substrate concentration increases over the range 1-10 mg/ml reaction mixture. Above 10 mg/ml, substrate iilhibitioil may occur. K, values of the several enzymes tested fall in the range 0.3-0.7 X 10-3M for adenosine, and 0.4-1.2 X 10-3 M for ino- sine.

There appear to be no cofactor require- ments for these enzymes. Dialyzed enzyine solutions and acetone-precipitated ellzyrnes retain their activities. Phosphate and acetate buffers can replace citrate. With some dry enzyme preparations, however, there is diffi- culty in obtaining activity. Reconstitution of these in water gave no activity, but the same enzyme taken up in 0.05 M buffer (pH 4 + 1) was highly active. Sodium chloride was less effective than buffer. Addition of buffer t c inactive aqueous suspensions did rzot restore activity.

4. Specificit)) The enzymes under iiivestigation are purine

P-ribofuranosidases (Table 11). All that we

have produced show nearly the same re- sponse (at p H 4.1) to thedifferent substrates, e.g. :

Inosine Adenosine Xanthosine Guanosine HxR > AR > X R > G R 100 : 59 : 42 : 1 4 .

These are average values (Table 11) from which individual preparations show some deviation. Relative susceptibility varies with pH (Fig. 1). At higher pH values (e.g., 5.6), guanosirle is more susceptible than xantho- sine. At lower p H values (e.g., 3.3), adenosine is sometimes as susceptible as inosine.

Some of these enzyme preparations hydro- lyze A.R.5'P (Table II), but this is due to tlie presence of a phospliatase (optimum pH 4 +), and not to thc ilucleosidase itself (un- published data). Ribose 5'P was never a product with ilucleosidase a. The activity 011

A.R.5'P is correlated with the amount of phosphatase present.

Nucleosidase g of A. iiiger has a different specificity (Table 11).

HxR > G R > AR > X R 1 0 0 : 36 : 20 : 1

Furthermore, the nucleotides A.R.S'P, Hx. R.5'P, and G.R.5'P are substrates of this enzyme, the rates of hydrolysis being ~icarly equal to the rates on the correspondi~ig nucleosides. Nucleotides having tlie phos- phate at C2 or C3 are not hydrolyzed.

TABLE I1 Specificity of purine /3-ribosidases of fungi (relative activity at pH 4.1)

Deoxy- Ino- Adeno- Guano- Xantho- A.R. adeno- Thymi- Uri-

QM slne sine sine slne 5'P sine dine dine

Nucleosidase n Aspergillrrs nnibiglrris A. nricrocysticris A. terrerrs Perzicillirrrrz ndnn~etzi P. ntrove?~etrriiz P . biforme P. brasiliarlrmz P . freqlrentnrzs P . /ieqrrer~tnns P . herqrrei P . jnvnnicriin P . ochrochloron P . parvrrrn P . sin~plicissirnutii P . vinncerrrr~

Nucleosidase g Cellulase 9X ( A .

niger) Miles Labs.

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REESE: 8-RIBOSIDASES FROM F U N G ~ 381

All preparations are essentially devoid of activity on (a) deoxyribonucleosides, (b) pyrimidine ribonucleosides, and (c) the 0-0-ribosides. Both methyl 0-ribofuranoside and methyl 0-ribopyranoside (courtesy H. Fletcher) were tested and found inert. The two modified adenosines (5'-chloro-5'-deoxy-; and 7-0-)-wliicli are good inhibitors (to be published)-are resistant to hydrolysis. (Long incubation did yield 5'-cliloro-5'-deoxy ri- bose.) Deoxyinosine on long hydrolysis showed a trace of breakdown. 0-C-ribosides are resistant.

Several nucleosides were subject to hydrol- ysis by dilute acid (0.15 N HCl) at 100' for 5 minutes. Inosine was most rapidly hydro- lyzed, followed by guanosine and adenosine. Deoxyadenosine was very slowly hydrolyzed, and uridine was completely resistant. This order of susceptibility is roughly similar to that of the substrates to the various enzymes. While adenosine and guanosine are equally susceptible to acid liydrolysis, most of thc enzymes hydrolyze adenosine much more rapidly. But here again, the A. niger enzyme is an exception.

5. Products of Hydrolysis Purine 0-ribofuranoside + HOH 5 purine

+ ribose. Both nucleosidases a and g act on the

nucleoside to give the expected products. Nucleosidase g had, also, the ability to hydrolyze 5'-nucleotides. For example:

adenosine 5'-phosphate --t adenine + ribose 5'-phosphate (Rribose = 0.30).

The enzyme is unusual in that substit~ltion with phosphate on thc sugar liloicty a t C5 does not affect the rate of hydrolysis.

No deaminases were observed in the nu- cleosidase preparations. A deaminase was found, however, in an A. lucl~uensis phospha- tase solution that we were using to remove the phosphate from nucleotides. Acting on A.R.SIP, this preparation gave first adenosine, then, on longer incubation, inosine.

Phosphatases (optimum pH 4.5) contamin- ate some of the preparations. The one present in A. luc/luensis was tested 011 various nucleo- tides and found to remove the phosphate from both the 3' and 5' positions. It did not affect the cyclic 3'-, 5'-monophosphate. All phos-

phatases tested acted on ribose 5'-phosphate and onp-NO2 phenyl phosphate. The level of phosphatase in nucleosidase a preparations determines the extent of their action on adenosine 5'-monophosphate (Table 11).

6. Production of Guanosiize ,from Yeast RNA Some fiuigi (Mj)rot/zeciul?? verrucaria, Rhi-

ZOPUS orrkizus) were observed to grow poorly on the yeast RNA and to produce no nucleo- sidase. When culture filtrates of these were kept in the refrigerator a precipitate appeared. This product moves at the rate of guanosine on chromatogranis in two solvent systems. It is hydrolyzed to guanine and ribose by nucleosidase, and the liydrolysis rate is the same as that of guanosine. The aliiount of this product approaches the quantity of guanosine present in the RNA used as substrate. It appears that these fungi possess the necessary enzymes to hydrolyze RNA to the n~lcleoside stage, but that they lack the ability to grow well 011 the products. While guanosine is the major in.soluble product, there are also soluble products which have not been investigated.

Discussion There appears to be a requirement for

enzynles capable of hydrolyzing all four kinds of nucleosides.

Purine 0-ribosides Fungi, yeast, bacteria, beans, fish

Pyrimidine 0-ribosides Bacterial and yeast enzyme

Purine 0-deoxyribosides ? Pyrimidine 0-deoxyribosides ? Ribonucleosidases of both the purine and

pyrimidine types are now known. Perhaps some enzymes hydrolyze both. Some pyrimi- dine P-ribosidases appear to be specific for uridine (2, 8). Cytidine must be dean~inated before it can be hydrolyzed by these enzymes. No deoxyribonucleosidases are known. Per- haps only a purine 0-deoxyribosidase is re- quired, as an enzyme is known which trans- fers deoxyribose from a pyrimidine to a purine base (4). The disruption of the purine deoxyri- boside might involve a phosphorylase, but

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382 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

neither hydrolytic nor phosphorolytic systems have been reported.

Nuclcosidase a is readily produced when certain fungi are grown on an inducer. While the "true" inducer appears to be a purine riboside (the enzyme substrate), RNA, or purine nuclcotides, give the best yields of enzyme. This reseiubles the cellulase system, wherecellobiose slowly released fi-om cellulose is the inducing agent (10). High concen- trations of inducing agents may act simul- taneously as repressors. It is, therefore, im- portant to supply the inducer at a low level over a long time. This is acco~nplished when the organism acts 011 RNA or on a nucleotide to form the inducing nucleoside.

Unlike bacterial, or yeast, purine ribosi- dases, the optimal pH for activity of the fungal nucleosidases is low (pH 4.1 k). The fungal enzymes are liberated into the medium so that no autolysis or sonication is required for their extraction. The fungi are thus readily available sources for this enzyme.

The purine (3-ribonucleosidases so far reported show differences in their specific- ities:

fungal nucleosidase N, pH 4.1 inosine > adenosine > xanthosine > guanosine;

A. niger nucleosidase '7, pH 4.1 inosine > guanosine > adenosine > > xanthosine;

Lnctobacillus delbrueckii, pH 7 + (13) in- osine = adenosine 5 guanosine > cyti- dine > uridine > > > xanthosine:

The nucleosidase g of A. iziger differs in several respects from the nucleosidase n of the other fungi in:

(1) its order of specificity on purine nu- cleosides (xanthosine poorly attacked);

(2) its ability to hydrolyze A.R.SfP to ribose 5'-phosphate ;

(3) its response to various inhibitors (to be published).

The purpose of this paper has been to show that many filngi produce purine ~lucleosidases and secrete them into the medium. There is some evidence to indicate that a few f~ l i~gi may be producing more than one such enzyme. Our specificity data, however, indicate very siinilar patterns of activity within nucleosi- dase n, and a marked difference between nucleosidase n and nucleosidase g. Our results also indicate the absence of many enzymes (phosphatases, deaminases, phosphorylases, etc.) in these extracellular fluids that often contaminate nucleosidases originating from cell extracts. Essentially what we are saying is that even in the absence of extensive purifi- cation, these fungal ribonucleases are rela- tively free of competing systems.

Acknowledgments 1 thank Miss Anne Maguire for technical

assistance, F. W. Parrish for samples, and H. S. Levinson for revision of the manu- script.

fish musc1e7 pH 5.5 (I4) guanosine > I. ACHAR, B. S. and VAIDYANATHAR, C. S. 1967. adenosine = inosine > xanthosine > Uridine hydrolase from mung bean seedlings. cytidine; Arch. Biochem. Biophys. 119: 356-362.

2. CARTER, C. E. 1951. Uridine nucleosidase from fish muscle, PH 8.6 (14) inosine (others not "east. J. Am. Chem. Soc. 73: 1508-1510.

hydrolyzed) ; 3. ~ O H N , W. E. 1960. Pseudouridine. J. Biol. Chern. 235: 1488-1498. yeast, pH 7'4 > adenosine > > 4. CoLowIcK, S. P. and KAPLAN, N. 0. 1955.

guanosine; Methods in enzymology. Vol. 2, 3. Academic L. pentosus, pH 7.5 (7) inosine 5 adeno- Press, N.Y.

-.-- 5. Fox. J. J.. CODMGTON. J. F.. YUNG. N. C.. >ll lC. KAPLAN, L:, and LAMPEN, J. 0. 1958. ~Gimid ine

The significance of thesedifferencesin specific- nucleosides. IV. Svnthesis of I-B-D-lvxofurano- - - ~ ~ - - - - J - - ~ - ---- - - - 8 - - ~- - - - ~ - -

ity is hard to evaluate. It has been that sylthymine. J. ~ r n . Chem. SOC. 80: 5155-5160. 6. HEPPEL, L. A. and HILMOE, R. J. 1952. Phos- a reversal in order can be obtained by chang- phorolysis and hydrolysis of purine ribosides

ina the concentrations of substrate used by enzymes from yeast. J. Biol. Chem. 198: (I?), as well as by changing the pH (14).

The requirement for a ribofuranosyl (rather than a ribopyranosyl moiety) has been shown (6, 13), and it is clear that no other sugar tested can replace ribose (5).

683-694. 7. LAMPEN, J. 0. and WANG, T. P. 1952. Mechanism

of action of Lactobacill~cs pentoslcs nucleo- sidase. J . Biol. Chem. 198: 385.

8. LAWRENCE, N. L. and TSAN, Y. 1962. Reactions of spores and cells of Bacil l~s cereus with pyrimi- dine ribosides. J. Bacteriol. 83: 228-233.

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REESE: 8-RIBOSIDASES FROM FUNGI 383

9. LEHMAN, I. R. Nucleases of E. coli. bz Progress ogy. Vol. VIII. Edited by Colowick and Kaplan. in nucleic acid research. Vol. 2. Edited by J. N. Academic Press, N.Y. p. 607. Davidson and W. E. Cohn. Academic Press, 12. SUMNER, J. R. and SOMERS, F. 1944. Laboratory N.Y. pp. 83-123. experiments in biological chemistry. Academic

10. MANDELS, M. and REESE, E. T. 1960. Cellobiose Press, IIIC., N.Y. as an inducer of cellulase. J. Bacteriol. 79: 13. TAKAGI, Y. and HORECKER, B.L. 1957. Bacterial 816-826. riboside hydrolase. J. Biol. Chem. 225: 77-86.

11. REESE, E.T. and MANDELS, M. 1966. P-Gluca- 14. TARR, H.L.A. 1955. Fish muscle riboside nases other than cellulase. In Methods in enzymol- hydrolases. Biochem. J. 59: 386-391.

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