The constitutive nature of bacterial cellulases

The ConstitutiveNature of Bacterial Cellulase@ .R. A. Hammerstrom, IL D. Claus, Jane W. Coghlan and R, H, McBee From the Department of Botany and Bacteriology, Montana State College,Bozeman, Montana Received October 4, 1954 INTRODUCTION

Cellulaseis commonly assumedto be an adaptive enzymeformed only in the presenceof cellulose (1). The possibility that the cellulase produced by some microorganisms could be a constitutive enzyme is revealed in the work of Reeseand Levinson (2) in which actively cellulolytic filtrates were obtained from cultures of Aspergillus luchuemis grown on glucose, glycerol, cellobiose, and starch. None of the other numerouscultures of fungi tested by them producedmeasurableamounts of cellulaseunder similar conditions. It must be assumed,therefore, that A. luchuensisis an exceptional microorganism and that in general t’he fungal cellulasesare truly adaptive in their nature, not being formed except in the presenceof cellulose. Enebo (3) noted the formation of a cellulasewhen the thermophilic cellulose-fermentingbacterium Clostridium thermocellulaseumwas grown on a variety of substrates,including sucroseand xylose. Enebo did not comment on this observation, so his opinion as to whether bacterial cellulasesare adaptive or constitutive was not apparent. Our own work over a period of severalyears has led to the conclusion that bacterial cellulase is not necessarily an adaptive enzyme, but in some instances may be like the pectinase describedby Phaff (4) which was formed when the hydrolytic products of its substrate were present in the culture medium or may, on the other hand, be a truly constitutive enzyme. 1This investigation was supported in part by a grant from the National Science Foundation. * Now a l.ieutenant in the Medical Service Corps, U. 8. Army. 123

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HAMMERSTROM,

CLAUS,

MATERIALS

COGHLAN

AND McBEE

AND METHODS

Cultures Several cellulose-digesting cultures representing two genera have been tested for cellulase production. These include strains 167, 661, and EB of C. thermocellum (5); two Cellulomonas sp., 11A and llC, isolated by Nishio (6); and Cellulomonas jlavigena (QM 528). All of the cultures were grown on a mineral-base medium consisting of 0.5% Na2HP04.12He0, 0.4yo NaCl, 0.1% KHSPOI , O.O3o/o (NHd)&O, , 0.01% MgSOa, and 0.01% CaC12 made up in tap water. To this was added O.Ol-O.l’% yeast extract, 0.21.0% of the substrate; and for the anaerobes, 0.02Q/o sodium thioglycolate. The aerobic organisms were sparged w-ith compressed air, shaken continually, or simply shaken at infrequent intervals. The culture procedures were not uniform because the work was done by four different persons over a period of nearly 5 years.

Cellulase Preparations Culture fluids containing cellulase were centrifuged to remove the majority of the bacterial cells. They were usually reduced in volume by vacuum evaporation at a temperature below 30°C. Toluene, chloroform, or Merthiolate (O.Oloj,) was added as a preservative when necessary. Storage, when required, was at -10°C.

Substrates Enzyme activity was tested cotton (7), cotton precipitated (9), and carboxymethylcellulose~

on a variety of materials, including from 70% HlSOa (S), cuprammonium (10).

ball-milled cellulose

Sugar Determinations Changes in concentrations Malmros method following the Folin-Wu method after determined manometrically measured as glucose following biose was made on the basis

of reducing sugars were determined by the Folinprotein precipitation with trichloroacetic acid or by tungstic acid precipitation of proteins. Glucose was by means of glucose oxidase (11). Cellobiose was acid hydrolysis. Identification of glucose and celloof their osazones. RESULTS

The bacterial cultures were grown on cellulose and also on those hydrolytic products of cellulose and other carbohydrates which would support their growth. The culture fluids were then examined for their ability to hydrolyze cellulose or carboxymethylcellulose (CMC) . Thermophilie Cellulose-Fermenting Bacteria Strains 157, 651, and EB (5) of Clostridium thermocellum (12) produced an active extracellular cellulase when grown in medium containing 3 Carboxymethylcellulose Powder Co., Wilmington,

50T was obtained Delaware.

through

the courtesy

of Hercules

BrZCTERIAL

125

CELLULASES

cellulose. This could be readily demonstrated in the cell-free culture fluid concentrated to one-tenth of its original volume. The pH was adjusted to 5.5 with an acetate buffer, and sufficient 5% ball-milled cotton was added to give a concentration of 0.1% cellulose. Toluene was added as a preservative, and the tubes were incubated at 35°C. They were examined daily for reducing sugars by testing small portions with Benedict’s qualitative reagent. After 3 days incubation, this test was positive and continually increased in strength for the additional 3 days of the experiment. A similar experiment was set up using the fluid from a culture of strain 157 which had been grown on cellobiose. The results were identical, the first detectable amount of sugar appearing on the third day. Cultures of strain 157 grown on cellulose, cellobiose, xylose, and a hemicellulose from oat hulls were tested for cellulase activity on CMC using a method similar to that of Reese and Levinson (2). This was carried out with a citrate buffer (pH 5.5), at a temperature of 45°C. using 0.1% CMC and Merthiolate as a preservative. After an incubation period of 2 hr., the increase in reducing sugar concentration was calculated as milligrams glucose/ml. A cellulase was present (Table I) after growth on all four of these materials, representing all of the carbohydrates that culture 157 is known to utilize. Similar but less extensive experiments have been carried out with several cultures of the thermophilic cellulose-fermenting bacteria. Cellulomonas Strains 11A and 11C of CeEZuZomonassp. (6) were cultivated in both glucose and cellobiose media. The culture fluids were tested for cellulase activity on ball-milled cotton without concentration, concentrated two times and ten times. Reducing sugars were formed only in the test with the tenfold concentration of enzyme, although the enzyme-substrate mixtures were incubated for as long as 30 days, indicating that a certain enzyme concentration was necessary before measurable hydrolysis of TABLE Cellulase

Production

Test conditions:

I

by Clostridium

thermocellum

5O”C., pH 5.3; CMC,

Culture grown on

GlUCOSe

mg.fmi.

Cellulose, 0.1570 Cellobiose, 0.15% D-Xylose, 0.1% Oat hull hemicellulose,

0.1%

(167)

0.5%; 2 hr.

0.35 0.58 0.10 0.10

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COCHLAN

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McBEE

cellulose could ensue. This cellulase was also tested against cellulose precipitated from sulfuric acid and from cuprammonium solution and was found to be as active on these as on ball-milled cotton, although no more so, despite their probably lower degree of polymerization. In one experiment cellulase from a culture of 11C grown on cellobiose was allowed to act on 0.1% ball-milled cotton at a pH of 5.5 for a period of 10 days at 35°C. The supernatant fluid gave a strong test for reducing sugars when boiled with Benedict’s reagent. A manometric determination of glucose by means of glucose oxidase showed less than 0.1 mg. glucose/ml. An osazone preparation revealed the presence of cellobiose but not glucose. Therefore, the manometric determination of glucose was repeated after the sugar solution had been made 1 N with HCl and heated to boiling for 1 hr. The glucose concentration following this hydrolysis was 3.0 mg./ml. indicating that 2.8 mg. cellobiose/ml. had been present originally. Apparently very little, if any, extracellular P-glucosidase was formed by these organisms. Culture fluids from Cellulomonas fiavigena (QM 528) grown on glucose and also on cellobiose were concentrated to one-tenth of their original volume and tested for their ability to hydrolyze CMC at 45°C. The increases in reducing value after 1 hr. (Table II) show that a cellulase was present in both the glucose and the cellobiose media. Although this demonstrated that cellulase could be formed in the absence of cellulose, it was not clear whether the enzyme was truly constitutive or was being formed in response to hydrolytic products of cellulose. To clarify this situation, the same organism was grown not only on glucose and cellobiose but also on CMC, starch, xylose, mannitol, and glycerol. The cultures were grown for 48 hr. at 35°C. with cons ant shaking. The initial substrate concentration was 0.1%. The presence in the cell-free culture fluid of a cellulase active on CMC was determined at 50°C. using an acetate buffer of pH 5.5 and an incubation TABLE II by Cellulomonas flavigena (QM 6.98) on Glzlcose and Cellobiose Culture fluid concentrated to %O the original volume. Test conditions: 45”C., pH 5.0; CMC 5OT, 0.5%; 2 hr. Culture grown on Glucose Cellulase

Production

mg.jml.

Glucose, 0.1% Cellobiose, 0.1% CMC 5OT, 0.1% Cellulose, 0.1%

0.65 0.62 0.60 0.37

BACTERIAL

TABLE Cellulase

Production

127

CELLULASES

III

by Cellulomonas flavigena Substrates

(QM 6.28) on Various

Culture fluid not concentrated. Test conditions: 5O”C., pH 5.5; CMC 5OT, 0.5%; 2 hr. Cultures incubated 2 days 3 days

Culture grown on

GIUCCW

GhOXe

mg./ml.

Glucose, 0.1% Cellobiose, 0.1% Glycerol, 0.1% D-Xylose, 0.1% Mannitol, 0.1% Starch, 0.1% CMC 5OT, 0.1% Cellulose, 0.1% TABLE Cellulase Cultures

Production incubated

0.21 0.08 0.02 0.10 0.10 0.20 0.42

IV

by Cellulomonas flavigena Xylose 3 days.

mg./ml.

0.05 0.21 0.01 0.01 0.09 0.10 0.23 0.26

(QM 628) on Glycerol

Hydrolysis

products

5O”C., pH 5.5; CMC

5OT, 0.5%.

determined

and

at 2, 7, and 23

hr. Test conditions:

Culture grow* on

Glycerol D-Xylose

hr. mg./d. 2

0.12 0.02

Glucose 7 hr. mg./ml.

0.21 0.04

23 hr. mg.lml.

0.30 0.09

period of 2 hr. Some cellulase was formed in each of the cultures (Table III). Since the amount of sugar produced by the xylose and glycerol culture fluids was so small, these two were tested again for a longer period of time which revealed their cellulase activity more clearly (Table IV). The possibility that the enzyme active on CMC would not be active on cellulose was next examined. The fluid from a culture of Cellulommas Jlavigena grown for 48 hr. on a mannitol medium was chosen for this test since the cellulase activity as demonstrated on CMC was greater when mannitol was the substrate for growth than when the organisms were grown on any of the other nonglucogenic compounds. The culture fluid was tested without concentration and also following a threefold concentration. Sufficient 5 % ball-milled cotton was added to give 0.125 % cellulose. The mixture was overlaid with toluene and stoppered, and duplicate experiments were incubated at 35 and 50°C. Reducing sugars

128

HAMMERSTROM,

CLAUS,

COGHLAN

TABLE

AND

McBEE

V

Cellulaee Production by Cellulomonas Hydrolysis

products Test

50°C. Culture Coned.

determined

flavigena (QM 638) on Mannitol at 3, 6, and 9 days. Glucose, mg./ml.

Conditions

Initial

3 days

6 days

9 days

fluid cult. fluid

0.01 0.01

0.02 0.05

0.03 0.07

0.03 0.07

fluid cult. fluid

0.00 0.01

0.05

0.10

0.11

0.14

0.17

0.18

35°C. Culture Coned.

were determined initially and at 3-day intervals for 9 days (Table V). The presence of a cellulase is clearly demonstrated. It is also apparent that concentration of the culture fluid gave a greater activity and that the enzyme is more active at 35°C. than at 5O”C., or is more rapidly inactivated at the higher temperature. DISCUSSION

The production of an extracellular cellulase is no longer a controversial matter. The factors which influence this production, however, are incompletely understood. This is especially true with regard to the effect of the substrate upon cellulase production. It is difficult to understand how the presence of a material as highly insoluble as cellulose could stimulate the formation of an adaptive enzyme. It appears to be more logical that the products of celhllose hydrolysis should lead to the production of cellulase since these soluble materials could enter the cell and exert an influence there. This effect upon enzyme formation has already been reported by Phaff (4). A third possibility is that cellulase might be a truly constitutive enzyme, that is, present regardless of the substrate upon which the microorganism is grown. The present state of our knowledge, however, does not permit us to rule out any of these three possibilities. The experimental evidence using cultures of Cellulom~as and Clost&Gum thermocellum shows quite clearly that the cellulase produced by these organisms is a constitutive enzyme. The stimulation of its production only by the hydrolytic products of cellulose is ruled out in the Cellulomonas species tested by the fact that cellulase was present when the bacteria mere grown on xylose, mannitol, and glycerol. Since C. thermocellum will not grow on the majority of the carbohydrates, the production of cellulase when cultured on xylose and hemicellulose is the best evidence that its cellulase is also constitutive. The observation by

BACTERIAL CELLULASES

129

Enebo (3) that Clostridium thermocellulaseum also produced a cellulase when grown on xylose would indicate that this situation was not a unique character of CZ. thermocellum. It is possible, however, that the examination of other cellulolytic bacteria may reveal that not all bacterial cellulases are constitutive. The situation found in the fungi may be paralleled in the bacteria although there is presently no evidence for such a hypothesis. The failure to demonstrate the presence of cellulase in a culture fluid may be a result of the test methods employed. For example, we have found that in some instances a cellulase cannot be demonstrated unless the culture fluid has been concentrated to one-third or one-tenth of its original volume. The test substrate is also very important since an enzyme preparation that will cause significant hydrolysis of CMC in 2 hr. may require several days to hydrolyze ball-milled cotton sufficiently to give measurable amounts of reducing sugars. This situation may be complicated further by the observation of Reese (2) that there is more than one component to cellulase. Some of these may be constitutive wherea,s others are adaptive. SUMMARY

A cellulase capable of hydrolyzing both cellulose and soluble cellulose derivatives has been found in cultures of Clostridium thermocellum grown on cellulose, cellobiose, xylose, or a hemicellulose. A similar enzyme is produced by members of the genus Cellulomonas when grown on cellulose, cellobiose, glucose, starch, mannitol, and glycerol. These enzymes were found to be constitutive rather than adaptive, as had formerly been believed. REFERENCES 1. SW, R. G. H., “Microbial Decomposition of Cellulose.” Reinhold Pub. Corp., New York, 1951. 2. REESE, E. T., AND LEVINSON, H. S., Physiol. Plantarum 6,345 (1952). 3. EN~CBO, I,., Thesis. Royal Institute of Technology, Stockholm, Sweden, 1954. 4. PKAFF, H. J., Arch. Biochem. 13, 67 (1947). 5. MCBEE, R. H., Bacterial. Revs. 14, 51 (1950). 6. NISHIO, J., Thesis. Montana State College, 1952. 7. HUVGATE, R. E., Bacterial. Revs. 14, 1 (1950). 8. SCALES,F. M., Zen&. Bakteriol. Parasitenk. II, 44,661 (1916). 9. KELLERMAN, K. F., AND MCBETH, I. G., Zentr. Bakteriol. Parasitenk. II, 34, 485 (1912). 10. LEVINSON, H. S., MANDELS, G. R., AND REESE, E. T., Arch. Biochem. and Biophys. 31, 351 (1951). 11. KEXLIN, D., AND HARTREE, E. F., Biochem. J. 42,230 (1948). 12. MCBEE, R. H., J. Bacterial. 67, 505 (1954).