Purification and properties of cellulases from the termite Macrotermes mülleri (termitidae, macrotermitinae) and its symbiotic fungus termitomyces sp.

Comp. Biochem. PhysioL Vol. 91111,No. 3, pp. 449-458, 1988

0305-0491/88 $3.00 +0.00 Pergamon Press pie

Printed in Great Britain

PURIFICATION AND PROPERTIES OF CELLULASES FROM THE TERMITE MACROTERMES MULLERI (TERMITIDAE, MACROTERMITINAE) AND ITS SYMBIOTIC FUNGUS TERMITOMYCES SP. C. ROULAND,* A. CIVAS,~"J. RENOUX* and F. PETEK'[" *Laboratoire de Biologie des Populations et de Zoologie, Universit6 Paris Val de Marne, 94000 Cr6teil, France; tLaboratoire de Biochimie, CNRS ERA no. 396, Facult6 de Pharmacie, 92260 Ch~tenay-Malabry, France (Received 6 November 1987)

A~tract--l. From termite Macrotermes mfilleri, two cellulases (celluases Ir and II), respectively, a 1,4-fl-glucan glucanohydrolase (EC 3.2.1.4) and a 1,4-fl-glucan cellobiohydrolase (EC 3.2.1.91) were purified by procedures including hydroxyapatite adsorption chromatography, ion exchange chromatographies on DEAE Sepharose and Mono Q~, and molecular sieving column Superose ® 12. 2. The 1,4-fl-glucan glucanohydrolase (cellulase IF isolated from mycot~tes of termite symbiotic fungus Termitomyces sp.) was also purified. The comparison of kinetic data and physical properties suggested that cellulases lx and IF were identical. 3. The apparent molecular weights of cellulases I and II as determined by SDS-PAGE were respectively 34,000 and 52,000. 4. The pH optimum of both enzymes was 4.4 and K,, values determined with CMC as substrate were 0.75% for cellulase I and 10% for cellulase II. 5. These enzymes differed also by their thermal stability, their optimal activity temperature and the effect of cations on their activity.

INTRODUCTION Termites, because of their number and their nutrition mode have a great effect on the organic matter turnover (Boyer, 1973; Mielke, 1978) and on the humus formation (Mishra and Sen-Sarma, 1980). These insects which eat plants present different nutrition modes: xyiophageous termites sensu stricto eating wood; soil-feeding termites, feeding upon humus, which sometimes are even geophageous and fungus growing termites which have a symbiotic relationship with fungi on degradation of plant material. The fungus growing termites in the sub-family Macrotermitinae present the most important and active colonies (Matsumoto, 1978; Lepage, 1981). It was also pointed out that fungus growing termites reprocess more than 90% of dry wood in arid tropical areas (Buxton, 1981). The degradation of plant material by Macrotermitinae is, to a great extent, due to their double digestive symbiosis: ectosymbiosis with Termitomyces sp., endosymbiosis with gut microflora. The ectosymbiotic fungus which grows on structures built by termites, called fungus combs (Grassr, 1982) is found as mycelium in the fungus comb and as white round structures on the comb surface named mycot&es (Helm, 1977). Electron microscopic studies have shown that pectocellulosic membranes and polyphenol-proteins (tanins) were extremely degraded from the upper part to the basis of the fungus comb (Rouland et al., 1984). The inferior part of fungus comb, degraded by fungus, is eaten up by termites for their nutrition. Previous works have shown that Macrotermes miilleri (Termitidae, Macrotermitinae) had to be dis449

tinguished from other termite genera owing to the important levels of osidasic activities detected in its digestive tract (Rouland et al., 1986a). Whether these enzymes are produced or not by termite cells and/or by their endosymbionts remains unknown as yet. These osidasic activities can also be produced by symbiotic fungus and can be ingested by termites when they eat fungus comb (Martin and Martin, 1979). The mechanism of the symbiotic relationships could only be established if all the enzymatic components which are involved in the process were identified. The presence of cellulases has already been detected in different termite species (Potts and Hewitt, 1974a, b; O'Brien et al., 1979; Martin, 1983; Schultz et al., 1986), but until now, none had been purified from fungus growing termites. In a previous paper, we have characterized the fl-glucosidase (fl-glucosidase A) from the gut of M. miilleri and the ff-glucosidase (fl-glucosidase B) we reviewed as distinct from fl-glucosidase A produced by its symbiotic fungus (Rouland et aL, 1986b) and described a procedure for their purification. This report deals with the purification of endo and exocellulases from M. miilleri digestive tract and its symbiotic fungus Termitomyces sp. MATERIALS AND METHODS

Insects and fungus origin Macrotermes miilleri workers and mycotrtes of Termitomyces were collected from the Mayombe forest in the Congo. Termites and mycot&es were taken directly out of their nests and then frozen at -20°C for transport and storage.

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Step Crude extract

Table 1, Purification of cellulase Ir from M. miilleriworkers Volume Protein Activity* Specific activity Purification (ml) (mg) (units) (units/rag protein) (fold) 40 61.6 74 1.2 1

Hydroxyapatite 90 2.7 14.4 DEAE Sepharose 100 0.09 4.2 Mono Q 6 0.032 2.1 Superose 12 4 0.005 1.8 *With CMCellulose as substrate. One unit is a ~M of equivalent glucose per minute.

Measurement of enzymatic activity Protein concentration was determined according to the Sedmak and Grossberg (1977) technique using bovine serum albumin (Koch-Light Laboratories, Coin Brook, U.K.) as standard. Powdered microcrystalline cellulose (Whatman), sodium carboxymethylcellulose (CMC, Pronoval, France) with a degree of substitution determined by the chromotopic acid (DS: 0.71) according to the technique of Courtois and Bui (1965) were used as substrates. The reaction medium contained either CMCellulose (8mg/m]), or cellulose ( 10 mg/ml) with 30 mM Macllvaine (1921) buffer of suitable pH. After 30 rain incubation at + 37°C, the reducing sugar was determined by the Somogyi-Nelson (Somogyi, 1945; Nelson, 1944) micromethod (Williams et al., 1978). Specific activity was expressed as the amount of glucose equivalent (#M) liberated per rain (=unit) per mg of protein.

Chromatography The oligosaccharides released by the hydrolysis of cellulose were identified by thin layer chromatography. Thin layer chromatography on silica gel sheets (Schleicher & Schull) was carried out using n-propanol-ethanol-ethyl acetate-water (7:3:2:3; v/v) as developing solvent. After migration, the gel was dried at 100°C and oligosaccharides were revealed with an alcoholic solution of fl-naphtylamine, followed by 2% sulphuric acid in ethanol (Petek, 1962). Hydroxyapatite was prepared in our laboratory according to the modification by Levin (1962) of the method by Tiselius et al. (1956). DEAE Sepharaose, Mono Q~ and Superose "~ 12 for FPLC system were purchased from Pharmacia.

Polyacrylamide gel electrophoresis Electrophoresis was performed on 7.5% acrylamide separating gel according to the technique described by Maizel (1964). Experiments were carried out at +4°C with a current of 6 mA per gel. The gels were stained with Coomassie Brilliant Blue (0.25% in methanol-acetic acid-water, 50:10:40, v/v) for 2hr and bleached at +40°C in a 25% methanol and 7% acetic acid solution. SDS-PAGE was carried out on 7.5% acrylamide gel containing 0.1% SDS following the procedure described by Weber and Osborn (1969). A current of 3 mA per gel was used. Electrophoresis was performed overnight at room temperature. The gels were stained under the above conditions. Phosphorylase (M, 94,000), bovine serum albumin (Mr 68,000), ovalbumin (Mr 43,000), pepsin (Mr 35,000) and trypsine (M r 23,000) were used as molecular weight markers. RESULTS

Purification of cellulolytic enzymes from digestive tract o f the termite Macrotermes mi.illeri Macrotermes mfilleri workers (15 g) were mixed in 60 ml 0.9% (w/v) NaCI and disrupted with sonication during 4 × 30see with a Branson sonifier B15. The suspension was centrifugated at 20,000g for 20 min

5.3 46.6 65.6 360

4.4 38.8 54.6 300

Yield (%) 100 19.4 100 29 14.5 13.3

at + 4 ° C . The supernatant was precipitated with a m m o n i u m sulphate at a 80% saturation at + 4 ° C . The precipitate obtained by centrifugation as above was taken up in 40 ml of distilled water, dialysed in pig gut membrane (Naturin) against distilled water for 24 hr. The dialysed solution constituted the crude extract. The crude extract was adsorbed on to a hydroxyapatite column previously equilibrated with 2 r a M potassium phosphate buffer pH 5.3. The elution with the same phosphate buffer provided a peak with cellulase activity (cellulase IT) (fraction 1). A second cellulase activity (cellulase II) was recovered with 10 m M phosphate buffer (fraction 2). Later steps o f cellulase Ir purifieation. This cellulase activity was never retained on any tested ionexchange chromatography supports ( D E A E acrylamide, C M acrylamide, D E A E sepharose, C M sepharose, Agarose Blue, M o n o S ~' and M o n o Q~) irrespective of the pH and of the ionic strength of the buffer; best results for purification were obtained as follows: The fraction I was chromatographed on a D E A E Sepharose column previously equilibrated with a 2 m M potassium phosphate buffer pH 7.7. The cellulase activity was detected in the fraction (fraction la) eluted by 5 m M o f the same phosphate buffer. After dialysis and freeze-drying, fraction la was taken up in 10 ml of 20 m M phosphate buffer pH 8 then applied on to a M o n o Q column equilibrated with a 20 m M potassium phosphate buffer pH 8. The cellulase activity was eluted with the equilibration buffer (fraction lb). Fraction lb was freeze-dried. The residue was taken up in 0.5 ml of distilled water, dialysed in the above conditions and chromatographed on a gel filtration column Superose 12. The column was eluted with 50 m M NaCI. At this last step the enzyme is purified 300 fold (Table 1). Polyacrylamide gel electrophoresis showed only one protein band possessing cellulase activity (Fig. 1). The purified enzyme presents a specific activity of 360 U / m g protein on CMCellulose. Later steps o f cellulase H purification. After dialysis, fraction II was adsorbed on to a D E A E Sepharose column which was equilibrated with a 1 0 m M potassium phosphate pH7.7. The column was eluted stepwise with the same buffer containing increased NaC1 concentration, namely 25 m M NaCI, 50raM NaC1 and 100mM NaC1. The cellulose activity was detected in the fraction eluted by 10 m M + 100 m M NaCI buffer. The homogeneity of the protein fraction was checked by analytical electrophoresis on polyacrylamide gel

Purification and properties of termite cellulases

451

® @® Fig. 1. Polyacrylamide (7.5%)-gel electrophoresis of cellulase Ix at successive stages of purification. (1) Crude extract; (2) hydroxyapatite column; (3) DEAE Sepharose column; (4) Superosd 2 column.

(Fig. 2). After the last purification step (Table 2), cellulase II was purified approximately 240-fold and presented a specific activity of 290 U/mg protein on CMCellulose.

Purification of cellulolytic enzymes from symbiotic fungus Termitomyces sp.

Termitomyces mycot~tes (10 g) were homogenized in 0.9% NaC1 (w/v). The procedure used was the same as described for obtaining the termite crude extract except for a longer time of sonifying (8 x 30"). After an a m m o n i u m sulphate precipitation stage, the pellet was suspended in 40 ml of distilled water, then dialysed. The dialysed solution was the crude extract.

Step Crude extract

Fig. 2. Polyacrylamide (7.5%)-gel electrophoresis of cellulase II at successive stages of purification. (1) Crude extract; (2) hydroxylapatite column; (3) DEAE Sepharose column.

The crude extract was adsorbed on to a hydroxyapatite column previously equilibrated with 2 m M potassium phosphate buffer pH 5.3. Stepwise elution with increased buffer concentration from 2 m M phosphate buffer to 1 M, after the elimination of a protein fraction without any cellulase activity with the equilibration buffer, led us to obtain the active fraction as a second peak with the same buffer. No later fractions eluted were active on CMCellulose nor on microcrystalline cellulose. Later steps of cellulase lr purification. The cellulase IF was not retained on different chromatographic supports tested (DEAE and CM acrylamide, D E A E and CM sepharose, Mono Q, Mono S) as previously

Table 2. Purificationof cellulase II from M. miilleri workers Volume P r o t e i n Activity* Specific activity Purification (ml) (mg) (units) (units/mg protein) (fold) 40 61.6 74 1.2 1

Hydroxyapatite 115 2.1 21.4 DEAE Sepharose 140 0.06 17.4 *With CMCelluloseas substrate. One unit is a/zM of equivalentglucose per minute.

5.9 290

4.9 242

Yield (%) 100 29 100 81

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This procedure required the use of three columns after hydroxyapatite: DEAE Sepharose, Mono Q and Superose 12. After the last chromatographic column, polyacrylamide gel electrophoresis presented only one protein band possessing cellulase activity (Fig. 3). The enzymatic protein which was purified 210-fold, had a specific activity of 378.4 U/mg protein on CMC (Table 3).

Properties of the three purified cellulases Molecular weights. The apparent molecular weights

® ®® ® Fig. 3. Polyacrylamide (7.5%)-gel electrophoresis of cellulase I F at successive stages of purification. (1) Crude extract; (2) hydroxyapatite column; (3) DEAE Sepharose column; (4) Superose12column.

noted for cellulase IT. We therefore attempted to apply the same procedure established for the purification of cellulase I T for cellulase IF purification.

determined by the method of Hedrick and Smith (1968) were the following: cellulases IT and I~. Mr 34,000; cellulase II Mr 52,000. These Mr values were confirmed by SDS-PAGE which also showed the monomeric nature of these three cellulases. Glycoprotein nature. The three enzymes were tested for carbohydrate by periodic acid/Schiff staining on polyacrylamide disc gel electrophoresis. Only cellulases Iv and IF presented a positive response to periodate fuschine suggesting a glycoprotein nature for these enzymes. Enzyme specificity. Three enzymes hydrolyse CMCellulose, cellulose microcrystallin, avicellulose and Cellulose E but they did not present activity on eellobiose and on ONP fl-D-glucoside (Table 4). Cellulase II hydrolysed cellulose releasing only cellobiose and cellotriose, whereas cellulase I T and I v action on cellulose led to the formation of cellobiose, cellotriose and higher homologues in equal amounts. These results suggested that cellulases IT and IF were 1,4-fl-glucane glucanohydrolase (EC 3.2.1.4) then cellulase II was a 1,4-fl-glucancellobiohydrolase (EC 3.2.1.91). Kinetic properties. Rate curves of the hydrolysis of CMC by the three cellulases showed linearity up to 20 min. Then, for all the other experiments, an incubation time of 20 min was used. Effect of pH on rates of CMC hydrolysis. The effect of pH on the activity of all the cellulases was studied by using MacIlvaine buffers of pH 2.8-8 at +37°C (Fig. 4). Cellulases I T and I v showed the same pH activity profile for CMC hydrolysis with a maximum

Table 3. Purification of cellulase I v from the fungus Termitomyces sp. Step Crude extract Hydroxyapatite DEAE Sepharose Mono Q Superose 12

Volume (ml)

Protein (mg)

Activity* (units)

Specific activity (units/mg protein)

Purification (fold)

Yield (%)

40 140 80 10 4

56 4.9 0.12 0.061 0.025

102.2 49 23 17.2 9.46

1.8 10 191.6 281.9 378,4

1 5.5 106.4 156.6 210.2

100 47.9 22.5 16.8 9.25

*With CMCellulose as substrate. One unit is a / z M of equivalent glucose per minute. Table 4. Specific activity values of cellulases I v, II and I v determined with different celluloses as substrates and expressed as #tool of glucose equivalent per min per mg of protein at + 37~C (the results are averages of 10 measurements) Substrates

Cellulase IT

Enzymes Cellulase I F

Cellulase II

CMC Microcrystalline cellulose Avicellulose a Cellulose Z

360 _+ 8.2 21 _+ 2.7 15.4 +_ 1,8 18.1 4- 2,3

378 4- 10.4 24.6 + 2.8 18 + 2.1 20.2 + 2.2

274 + 6.2 36.2 _+ 3.8 34.8 _+ 2.5 37.6 +_ 3.1

Purification and properties of termite cellulases

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at pH 4.4. The pH activity profile of cellulase II was clearly different but showed the same maximum between pH 4.3 and 4.5. The experiments on the pH stability of the enzymes were carried out by incubating the enzymes at +37°C at pH values ranging from 2.8 to 8 for 1 hr before the assay at their optimum pH. These experiments showed that cellulase Ix and IF were stable over the pH range 2.8-5.5 but their stability rapidly decreased to basic pH. Thermal denaturation. The' three cellulases remained stable at +4°C for nearly a month. They were also proved to be stable enough to freeze. To study the effect of temperature on the reaction rate of cellulases I r, I F and II incubations were carried out (at p H 4 . 4 ) a t +25°C, +30°C, +37°C, +40°C, ~45°C, +50°C, +55°C, +60°C, +65°C for 20min (Figs 5a, 6a and 7a). The initial velocity increase was maximum at + 55°C for cellulases I T and I F, cellulase II presented a maximum initial velocity at about + 37°C. The stability of the purified enzymes was examined in the temperature range of 30-60 ° by incubating equal a m o u n t s of enzymes (Figs 5b, 6b and 7b). Three cellulases were heat ' sensitive but cellulase II was the most thermolabile. Preincubation at + 50°C C.RP. 91/3B--E

for 5 min caused 100% loss of activity for cellulase II while under the same conditions, residual activity for cellulases I r and I F was 60%. Effect of substrate concentration. The effect of CMC concentration on the velocity of hydrolysis was investigated with the use of varying concentrations of CMC (4--10-2%) in 50 mM Macllvaine buffer pH 4.4 (Fig. 8). Stock solutions of the CMC used as substrate were made up by percentage weight (w/v) since the initial degree of hydration and exact molecular weight were unknown. The Lineweaver-Burk plots showed straight line relationships, giving K~, values of 10% for cellulase II and 0.75% for cellulases I T and I F . Effect of various cations. With salts up to 2 x 10-2M, the activity of enzyme was measured under the standard assay conditions. No significant alteration of three cellulases activities was observed with Mg 2+, Cd 2+ and Co 2+. Under the same conditions, cellulase II was inactivated by Mn 2+, Fe :+ and Ag + and presented a slight activation with Mo 2+ and Ca 2+, cellulases I T and I F were only inhibited by M n 2+ and Ag +. EDTA over the concentration range of 10 mM had no effect on cellulases IT and Iv activity whereas cellulase II lost 88% of CMCase activity. This activ-

454

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ity was entirely recovered by adjunction of Mo 2+ and Ca + ÷ in the reaction medium DISCUSSION

Ceilulase I T purified from the gut of Macrotermes miilleri and cellulase I F isolated from its symbiotic fungus are in fact the same enzyme as produced by

Termitomyces sp. and ingested by the termite. This finding is supported by the comparison of different properties of their purified forms. As shown in the present paper, cellulases IT and IF presented the same elution profile on all chromatography supports tested and they were purified by using the same procedure. Furthermore, the specific activity values determined either on microcrystaUine cellulose (21 U and 24 U,

Purification and properties of termite cellulases

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respectively) or CMC (360 U and 378 U, respectively) were almost identical. The identity of Km values calculated for both enzymes when CMC was used as substrate and the absence of any distinguishable difference in TLC pattern of oligosaccharides, released during eellulolysis, suggested a common mechanism of action. The effect of pH, temperature and substrate concentration on their rate of hydrolysis was also similar.

The action of this enzyme on microcrystalline cellulose (leading to the production of various oligoglucosides) and the high ratio of hydrolysis obtained with CMC/Avicellulose showed that cellulase I was a 1,4-fl-glucan glucanohydrolase (EC 3.2.1.4). The apparent molecular weight determined according to the method of Hedrick and Smith (1968) and confirmed on SDS-PAGE in reducing conditions revealed that cellulase I was a monomeric protein of M,

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34,000. The relative thermostability of this enzyme can be explained by its glycoproteic nature which was suggested by its positive reaction with periodic acid/Schiff reagent. Cellulase II, produced by the termite and isolated from the gut, hydrolysed microcrystalline cellulose and CMC. Cellulolysis led to the formation of cellobiose and celiotriose, and it appeared that cellulase II can be considered as a 1,4-fl-glucan cellobiohydrolase. However the CMC/Avicellulose hydrolysis ratio

was very high in comparison to that obtained with other exoceilulases but a similar activity was described for a cellulase component of Streptornyces flavogriseus (MacKenzie et al., 1984). It appeared that two cellulase specific activities of 274 U/mg protein and 370 U/mg protein, respectively on CMC and of 36U/rag protein and 24U/mg protein on microcrystalline cellulose are very important because they represented 10 times the specific activities of other cellulolytic fungi (Sternberg and

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Mandels, 1980; Vodjani, 1981; Mishra et al., 1985) and of other xyiophageous insects (Chararas et aL, 1983). Several authors noticed that some purified cellulase presented mannanasic activity (Clarke and Stone, 1965), lichenasic activity (Hurst et al., 1978) or xylanasic activity (Toda et aL, 1971; Potts and Hewitt, 1974b; Schikata and Nisizawa, 1975; Kanda et al., 1976). Cellulases I and II are both inactive on all the other tested polyosides. Cellulase I and II molecular weights, of 34,000 and 52,000 respectively are comparable with the M, values determined on purified cellulases from other organisms (Berghem et al., 1976; Chararas et al., 1983; Kittsteiner-Eberle et al., 1985). Optimum pHs (4.2-4.5) were also comparable with those described for cellulases but we must notice the great stability of endocellulase whose activity remained constant from pH 2.8 to 5.5. Cellulase II was quite sensitive with an optimal temperature at + 37°C whereas cellulase I remained active even at high temperature with an optimal temperature at +55°C. This last result was similar to the optimal temperature of thermophilic fungi (Eriksen and Goksoyr, 1977). The Km values calculated with CMC as substrate were 0.75% for cellulase I and 10% for cellulase II. These results showed that cellulase II had a weak

457

affinity for this substrate. It would be interesting to determine Km values with cellotriose, cellotetraose, cellopentaose etc. as substrates to determine the mechanism of action of the two enzymes. Both cellulases were inactivated by Ag + but they were not activated by Zn 2+ like cellulases of Rhagium inquisitor (Levatidou et al., 1971) or Microtermes traghardi (Abushama and Kambal, 1975). Only cellulase II lost 60% activity with EDTA. This activity was re-established with Ca 2+ and Mo 2+, suggesting that bivalent cations may interfere on its active site. The presence of several cellulolytic enzymes has already been observed in insects (Chararas and Chipoulet, 1982; Chararas et aL, 1983; Chipoulet and Chararas, 1985; Schultz et al., 1986) but for the first time, the coexistence, in the digestive tract, of enzymes produced by two different organisms was elicited. The presence of cellulase I in the gut of termites could result from the ingestion by termites of fungus comb (degraded plant material containing Termitomyces mycelium) and/or Termitomyces mycot&es. Experiments with Termitomyces sp. growing in liquid medium in the presence of mineral supplemented only with cellulose as carbon source, showed that cellulase I was secreted in the culture medium (Rouland et al., 1988). We conclude therefore that this enzyme produced and secreted by fungus cells in the comb could be simultaneously ingested by M. miilleri workers with partially degraded cellulose. The secretion of this enzyme seems to be essential for its "appropriation" by the termite. This hypothesis was strengthened by the fact that fl-glucosidase B, previously purified from Termitomyces sp. extracts (Rouland et al., 1986b), was not secreted in the culture medium and was never detectable in the termite digestive tract. The relative stability of cellulase I may be important during its transfer from the fungus cells through the fungus comb to the termite digestive tract. Degradation of cellulose can therefore be achieved in the gut of Macrotermes miilleri workers by the combined action of its cellulase II, its fl-glucosidase A (previously described in Rouland et al., 1986b) and its symbiotic fungus cellulase I. REFERENCES

Abushama F. T. and Kambal M. A. (1975) The role of zinc in the activation of the enzymes cellulases in termites. Experiemia 32, 19-20. Berghem L. E. R., Petterson L. G. and Axi6-Frederiksson U. B. (1976) The mechanism of enzymatic cellulose degradation. Isolation and some properties of a fl-glucosidase from Trichoderma viride. Eur. J. Biochem. 46, 295-305. Boyer P. (1973) Action de certains termites constructeurs sur l'6volution des sols tropicaux. Ann. Sci. Nat. Zool. Paris 15, 329-498. Buxton R. D. (1981) Termites and the turn-over of dead wood in an arid tropical environment. Oecologia 51, 379-384. Chararas C. and Chipoulet J. M. (1982) Purifications by chromatography and properties of a/%glucosidase from the larvae Phoracantha semipunctata. Comp. Biochem. Physiol. 7211, 559-564. Chararas C., Eberhard R., Courtois J. E. and Petek F.

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