Some properties and reaction characteristics of the partially purified cellulase from the termite Trinervitermes trinervoides (Nasutitermitinae)

Comp. Biochem. Physiol., 1974, Vol. 47B, pp. 327 to 337. Pergamon Press. Printed in Great Britain

SOME PROPERTIES AND REACTION CHARACTERISTICS OF THE PARTIALLY PURIFIED CELLULASE FROM THE TERMITE T R I N E R V I T E R M E S T R I N E R V O I D E S (NASUTITERMITINAE) R. C. P O T T S * and P. H. H E W I T T Department of Entomology, University of the Orange Free State, Bloemfontein, Republic of South Africa (Received 18 April 1973)

A b o t r a c t m l . Purified cellulase from the workers of Trinervitermes trinervoides

readily attacks insoluble cellulose and the effects of the physical state of the substrate on its hydrolysis were investigated. 2. The hydrolysis was "random" and cellotriose was the main product. 3. The cellulase appears capable of hydrolysing xylan. 4. Laminaranase (fl-(1-3)-glucanase) was found in worker homogenates. INTRODUCTION MOST work on the characterization of cellulases and their reactions have been confined to enzymes derived from fungi and other micro-organisms. No previous attempts have been made to characterize insect cellulases, probably because of the need to remove all cellobiase activity. T h e elimination of cellobiase was achieved in the purification of the T. tn'nervddes ceUulase and permits a closer examination of the reactions of the enzyme. T h e present work looks at the influences of the physical and chemical state of the substrate on its rate of hydrolysis by the T. trinervoides cellulase. T h e specificity of the enzyme is studied in terms of different substrates and the hydrolytic mechanism (i.e. " r a n d o m " or "endwise acting") is examined, by analysis of hydrolysis intermediates and products, and by changes in viscosity. MATERIALS AND METHODS Enzyme preparations

The source of enzyme and the purification procedure were as described previously (Ports & Hewitt, 1973b). Estimation of reducing sugars

Reducing sugars were estimated by the modified 3,5-dinitrosalicylic acid (DNSA) method as described by Potts & Hewitt (1973). Details of the experimental conditions will be described in their relevant sections. * Present address: Department of Zoology, The University, Sheffield $10 2TN, U.K. 327

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R. C. POTTS AND P. H. HEWITT

Cellulose substrates T h e cellulose substrates used in studying the influence of the physical structure of the cellulose o n the hydrolytic activity were prepared as described below. (a) Phosphoric acid swollen cellulose. This was prepared by thoroughly wetting 20 g of cotton wool with cold (4°C) 85% w/v phosphoric acid and allowing it to stand, with occasional stirring, for 6 hr at 4°C. T h e mixture was then added slowly, with rapid mechanical stirring, to 2 1. of cold water. T h e cellulose was collected on a Biichner funnel, the acid washed out and the cellulose finally suspended in distilled water. (b) Raw cotton. Cotton fibres were removed from unopened bolls, boiled and washed repeatedly in 50% aqueous ethanol, then water and finally suspended in distilled water. (c) Cotton wool. D r y cotton wool fibres (10-mg portions) were placed in test-tubes and 1"0 ml water was added to each tube. (d) Cellulose powder. Whatman CC31 cellulose powder was added to water to make a 1% (w/v) suspension. (e) DEAE-cellulose (Whatman DE 11). This was prepared as for ion-exchange chromatography and made up to a 1% (w/v) suspension.

Substrates for testing specificity Pustulan (/~-(1-6)-glucan) and crude laminaran (/3-(1-3)-glucan) were obtained from Dr. H. J. Potgieter (Department of Microbiology, U.O.F.S.). T h e laminaran was purified by precipitation from aqueous solution with ethanol (4 vol.). /~-(1-4)-Xylan was obtained from the Nutritional Biochemicals Corporation. These substrates were used at a concentration of 1% b y weight.

Paper chromatography Unless otherwise stated Whatman No. 1 paper was used. For the separation of mono-, di- and trisaccharides, iso-propanol-n-butanol-HaO ( 7 : 1 : 2 v/v/v) was used as the developing solvent. In mixtures which contained sorbitol, iso-propanol-H~O (4 : 1 v/v) was used. Higher oligosaccharides were separated with an n-butanol-pyridine-HzO (6 : 4 : 3 v/v/v) solvent system. All sugars were detected with a silver nitrate reagent made up as follows: (a) AgNO8 saturated solution in H~O, 1 vol and acetone, 400 vol. and (b) NaOH, 0.5% in ethanol. T h e papers were dipped in (a), dried in an air stream and then dipped in (b). After drying they were drawn through 2 N aqueous ammonia and then washed in running tap water. Sorbitol was detected by spraying the ehromatograms with a 1 : 1 (v/v) mixture of 1% vanillin in ethanol-3% aqueous perehloric acid (mixed just before use) and heating the papers at 85°C for 3-4 min. Quantitative estimation of the products of cellulase action were made by scanning the paper chromatograms with a Photovolt scanning densitometer connected to a Varicord variable response recorder and Integraph automatic integrator. T h e densitometer was calibrated and the standard curves drawn by developing chromatograms carrying known amounts of glucose, cellobiose and cellotriose standards. These chromatograms were treated in the same way in all respects as the experimental chromatograms.

Preparation of cellotriose standard Cellotriose was obtained by hydrolysis of cellulose with sulphuric acid and fraetionation of the hydrolysate on a charcoal column b y a method based on that of Miller (1963). T h e purity of the peaks was checked by paper chromatography. It was found that all the peaks, especially the higher oligosaccharides, were contaminated by lower homologues. T h e cellotriose standard was purified by chromatography on Whatman 3 M M paper (n-butanolp y r i d i n e - H 2 0 , 6 : 4 : 3 v/v/v, as solvent), elution and freeze-drying.

CELLULASE FROM THE TERMITE TR1NERVITERME..~ TRINERVO]DES

329

Preparation of reduced cellodextrin Cellodextrin was prepared by sudden dilution of a solution of cellulose powder in 66% HsSO~ and dialysing the resulting fine suspension against distilled water. Fifty ml of cellodextrin suspension at a concentration of 19 mg/ml was treated with 0"715 g potassium borohydride for 2 days at room temperature. The reaction mixture was acidified with acetic acid until gas evolution ceased, dialysed against distilled water and concentrated slightly to 21"6 mg/ml. The product possessed only very slight reducing power, most of the terminal (reducing) glucose residues having been reduced to sorbitol. The cellodextrin formed a stable suspension in water at room temperature.

Viscometry Because of the need for the rapid introduction of well-mixed samples and for short efflux times a simple viscometer, consisting of a thick-walled capillary tube 118 cm long and 1"02 mm i.d. with two timing marks 57 cm apart marked on it, was used. A rubber tube connected to the top was used to draw the sample into the capillary from the sample container which was immersed in a constant temperature water-bath. The effects of slight differences in temperature between the water-bath and the capillary were ignored because these were assumed to be constant from one minute to the next. Assays were of 10 rain duration. Other methods will be described in their relevant sections. RESULTS

Influence of the physical structure of the cellulose on its susceptibility to attack by cellulase Solutions or suspensions of C M C , cotton wool fibres and W h a t m a n cellulose powder were made up to contain 10 mg/ml. T h e cotton fibres, the H3PO4-swollen cotton and the DEAE-cellulose, which were all in aqueous suspension, were prepared to approximately 10 mg/ml. T h e concentrations of the latter were determined b y drying small samples and then adjusted to concentrations of 10 mg/ml. All these were quite finely divided (especially the DEAE-cellulose) and pipetted samples contained reasonably reproducible quantities of substrate. Buffered enzyme (0.5 ml) was added to 1.0 ml substrate and incubated for 1 hr at 40°C. T h e r e were seven replicates for each substrate. T h e degree of hydrolysis was measured b y the increase in reducing power using the D N S A reagent. T h e results are shown in T a b l e 1. TABLE 1 - - T H E QUANTITIES OF REDUCING SUGARS FORMED (AS ~ g GLUCOSE) FROM THE ENZYMIC HYDROLYSIS OF DIFFERENT CELLULOSE SUBSTRATES (MEANS OF SEVEN REPLICATES). FOR CONDITIONS SEE TEXT.

Substrate CMC HsPO4-swollen cotton Raw cotton (undried) Cotton wool Whatman cellulose powder Swollen DEAE-cellulose

Products (as pg glucose) 540.0 211 "7 135"3 135.6 145"6 100.1

R. C. POTTS AN]) P. H. HEWITT

330

An analysis of variance of the data (excluding CMC) showed highly significant differences (F = 306.0, P < 0.01). Tukey's multi-range test showed that all the substrates, with the exception of raw cotton vs. cotton wool, differed significantly from each other.

Specificity Reconstituted acetone powder, prepared from crude homogenates, hydrolysed xylan and laminaran fairly rapidly (about 25 per cent of the rate of hydrolysis of cellodextrin) but pustulan was not hydrolysed at all. Electrophoresis (Fig. 1) of purified cellulase showed that the fractions which hydrolysed CMC and xylan coincided but no hydrolytic activity towards laminaran was detected. Electrophoresis was carried out as described by Potts & Hewitt (1974). The purified cellulase preparation contained no amylase activity with soluble starch (1 per cent) as substrate. 2.5-

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FIG. 1 E]ectrophoretogram showing the coincidence o f ce]lulase activity ( and xylanase activity ( . . . . . . ).

Analysis of the products of cellulase hydrolysis of reduced cellodextrin Cellodextrin was selected as substrate since it is unsubstituted and resembles native cellulose in this respect. Furthermore, its rate of hydrolysis by cellulase is sufficiently rapid to yield reliable results. A small quantity of purified freeze-dried ceUulase was dissolved in 0.2 ml water and 2.0 ml reduced ceUodextrin (see Materials and Methods section) was added. The mixture was incubated at 30°C and aliquots of 0.2 ml were removed at intervals (see Table 2) for analysis. The reactions were stopped by heating the

CELLULASE FROM THE TERMITE T R I N E R V I T E R M E S

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samples at 96°C for exactly 5 min. Samples of each (10 ~I) were spotted on Whatman No. 1 paper. Development, detection and quantitative estimation were carried out as described previously. The results, on a molar basis, are shown in Table 2. The relative rate of production of cellotetraose is also shown but since no cellotetraose standard was used arbitrary units are given. TABLE 2--THE

RELATIVE MOLAR CONCENTRATIONS OF THE PRODUCTS OF THE ENZYMIC HYDROLYSIS OF CELLODEXTRIN AT THE TIMES S H O W N

Products ~moles/ml) Incubation time (min) 0 10 20 30 45 60 90 120 240

Glucose 0"05 0"12 0-13 0"15 0-13 0"20 0"28 0"38 0"53

Cellobiose

Cellotriose

Cellotetraose (arbitrary units)

0"12 0-42 0"55 0.55 0-80 0"98 1"53 1"70 2.85

2"75 4"25 4.80 6.25 8"60 9"80 13"30 15"00 18.50

0 1 1 2 4 6 10 12 14

No sorbitol, glucosyl-sorbitol or other oligosaccharide containing sorbitol could be detected on the chromatograms. A slight positive reaction could be seen at the origins. Although the quantities of glucose and cellobiose are very much lower than the cellotriose, these quantities could be determined accurately because glucose reacts more strongly with the AgNO8 reagent than does cellobiose and cellobiose reacts more strongly than cellotriose and so on.

"Randomness" of hydrolysis estimated by viscometric/reductimetric comparisons The changes in specific viscosity of CMC+H~SO4 and CMC+cellulase mixtures were measured concurrently with the increase in reducing power. The specific viscosities, ~sp, were calculated from the equation

B,p = ( t - to)/to, where t is the efflux time, in sec, of the hydrolysis mixture and t o is the efflux time of a solution of similar composition containing glucose in place of CMC (i.e. corresponding to 100 per cent hydrolysis). Acid hydrolyses were conducted as previously described (Potts & Hewitt, 1974). The results are given in graphical form (Fig. 2) where log (specific viscosity x 100) is plotted against the log of the concentration of reducing sugars (as #moles/ml glucose x 100). DISCUSSION The influence of the physical (and chemical) structure of the cellulose on its susceptibility to enzymic attack is very marked (Table 1). With CMC the high

332

R.C. POTTS AND P. H. HEWITT 2

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FIo. 2. Plots of log specific viscosity against log reducing sugar concentration (as glucose) for the hydrolysis of CMC by HsSO, and by ceUulase. rate of hydrolysis is due entirely to the solubility of the substrate (see Cowling, 1963). Highly substituted CMC is rather resistant to attack because of the protection of the bonds by adjacent carboxymethyl groups. As the degree of substitution is decreased the substrate becomes more susceptible to hydrolysis until this is offset by decreasing solubility (HaUiweU, 1963; Eriksson & Hollmark, 1969). The phosphoric acid-swollen cotton was much more readily attacked than the other insoluble substrates. The acid treatment probably swelled the fibres beyond the natural swollen state of the raw cotton (Rollins & Tripp, 1963). A considerable amount of degradation undoubtedly took place in the acid treatment (Jayme & Lang, 1963). The fact that the DEAE-cellulose was attacked more slowly than raw cotton or cotton wool is probably the result of protection by the diethylaminoethyl substituents. Although the degree of substitution is very low it is not known how this substituent affects the susceptibility to enzymic attack. It is worth noting that the DEAE group is considerably larger than the carboxymethyl group. Despite the low rate of hydrolysis of DEAE-cellulose, this was sufficiently high to create problems in ion-exchange chromatography of the cellulase on this adsorbent (Potts & Hewitt, 1974). The fact that there was no significant difference between the rate of hydrolysis of raw cotton and cotton wool is of some interest. Reese (1963) mentioned that undried cotton from unopened bolls was very susceptible to enzymic attack and

CELLULASE F R O M T H E T E R M I T E

TR1NERVITERMES TRINERVOIDES

333

underwent extensive hydrolysis. The figures given by Reese (1953) show that the extent of hydrolysis varies with the enzyme source but does not show a close correlation with activity towards CMC. The significance of the latter is obscure but unfortunately comparative data for the activity towards dried cotton fibres were not given. Reese & Levinson (1952) found that the bacterium Sporocytophaga myxococcoides, a powerful cellulolytic organism, was indifferent to the physical properties of different celluloses. It is possible that the T. trinervoides cellulase is more affected by the true crystalline structure of cellulose and not so much by the "false" crystallization produced by the physical changes during drying (Green, 1963). The treatment of dried cotton fibres with concentrated phosphoric acid would not only reverse the drying changes, but would also disrupt the true crystalline structure of the cellulose (Jayme & Lang, 1953; Rollins & Tripp, 1953). Whatman cellulose powder (finely powdered cotton linters) was attacked at a slightly higher rate than dried cotton fibres and this is probably due to the greater surface area available to the enzyme. It has often been said that an enzyme should not be called a cellulase if it does not hydrolysc "native" cellulose (i.e. cotton fibres), but should be called a carboxymethyl-cellulase if it attacks only CMC, e.g. the enzyme from Poria vaillantii (Sison et al., 1958). With the T. trinervoides cellulase the ratio of the cotton fibre hydrolysis rate to the CMC hydrolysis rate is about 1 : 4 which is very high even when compared with accepted fungal cellulases. Thus this enzyme amply qualifies as a genuine cellulase. The T. trinervoides cellulase appears to be capable of hydrolysing xylan. It is possible but extremely unlikely that two enzymes, a specific xylanase and a specific cellulase, were present but were not separated during the purification procedure and migrated together under electrophoresis. It has long been thought that xylan and cellulose are hydrolysed by a single enzyme. Some cellulases which may hydrolyse xylan have been described by Bishop & Whitaker (1955), Thomas (1956) and Kooiman (1957). Xylanases have been obtained free of cellulase activity (Reese & Mandels, 1959; Lyr, 1950) but in only a few cases have cellulases been obtained free of xylanase activity (Myers & Northcote, 1959; Pettersson, 1959). The amylases of certain micro-organisms appear to be very similar to the cellulases in physical structure and separation of the two activities is very difficult. In one case (Hashimoto & Nisizawa, 1953) it was not possible to remove the amylase activity from a crystalline cellulase. The T. trinervoides amylase appears to be more closely related to the a-glucosidases and its separation from the cellulase is comparatively easy. The presence of a fl-(1-3)-glucanase (laminaranase) in T. trinervoides is interesting and since the laminaranase activity is not present in the purified cellulase this must be due to a separate enzyme. The fl-(1-3)-glucans are found predominantly in marine algae but they also occur in callose from pollen and woody tissues of higher plants. Clarke & Stone (1953) mention the fairly widespread occurrence of these glucans in cereal plants and grasses. They are, however, largely concentrated in the endosperm of the seeds. Enzymes capable of hydrolysing fl-(1-3)12

334

R. C. POTTS AND P. H. ~ T T

glucans have been reported from only two animals, a marine mollusc (Huang & Giese, 1958) and from the terrestrial snail Helixpomatia (Myers & Northcote, 1959). These enzymes are, however, quite common in bacteria, fungi and higher plants. It has often been proposed that there may be endo- (random acting) and exocellulases (endwise hydrolysing) as there are endo- and exoamylases. Although several claims have been made that truly endwise degradation occurs (King, 1963) the random-acting cellulases are by far the most common. The cellulase from T. trinervoides appears to be no exception. Firstly, the products of hydrolysis by an exocellulase would be glucose (or cellobiose) only and there would be no intermediates. Glucose, cellobiose, cellotriose and cellotetraose all appear as products from the hydrolysis of insoluble cellodextrin by the T. trinervoides cellulase (Table 2). Higher homologues were probably also present but these did not move from the origins of the paper chromatograms (even the cellotetraose was very close to the origin). This analysis is only possible with ceUulases from which all cellobiase activity has been removed. The cellobiase would hydrolyse the lower oligosaccharides to glucose as soon as they were formed and the higher oligosaccharides (on which this enzyme acts very slowly) would not be detected by paper chromatography because of their low mobilities. However, the fact that there are intermediates is good evidence for random cleavage. With the cellulase from T. trinervoides, cellotriose appears to be the final hydrolysis product and no hydrolysis of this sugar could be detected reductimetrically. Since the cellulase displays zero or negligible activity towards it, the glucose and cellobiose (not in equimolar concentration) were probably derived from the hydrolysis of oligosaccharides higher than cellotriose. This is similar to the random acting cellulase from the bacterium Pseudomonas fluorescens (Nisizawa et al., 1963). In fact the molar ratio of cellotriose to cellobiose is much higher in T. trinervoides hydrolysates than in those from P. fluorescens although the cellobiose : glucose ratio is about the same. The formation of compounds with greater paper-chromatographic mobilities than authentic 3-(1-4)-oligosaccharides showed that transglycosylation occurred in the P. fluorescens preparations. When 14C-labelled glucose was used as the acceptor these spots were strongly radioactive and the difference in mobility of the transglycosylation products implied the formation of bonds other than 3-(1-4) (Nisizawa et aL, 1963). No compounds with intermediate mobilities were found in the T. trinervoides hydrolysates and this indicates that if transglycosylation did occur, all the new bonds would have to be 3-(1-4). Further evidence for the random nature of the hydrolysis catalysed by the T. trinervoides enzyme is provided by comparing the reduction in viscosity with the increase in the number of reducing end groups. The results for acid and cellulase hydrolysis are shown in Fig. 2. A rapid reduction in viscosity, and hence degree of polymerization (D.P.) (Swenson, 1963), with a relatively slow increase in reducing power indicates random attack. This would appear as a line of large (negative) slope in Fig. 2 and the acid hydrolysis represents the completely random condition (slope = - 1.50). The reason the slope for cellulase hydrolysis is not as

CELLULASE F R O M T H E ,TERMITE T R 1 N E R V I T E R M E S T R 1 N E R V O I D E S

335

great (-0"61) as that for acid is probably a function of the crystallinity of the substrate. The acid at the high concentration employed is a solvent for the cellulose and all bonds become equally accessible to hydrolysis. However, in the case of the enzyme the crystallinity of the substrate and the larger size of the protein molecules would limit the number of accessible bonds. This introduces a considerable non-random effect. A strictly endwise acting enzyme would yield an almost negligible slope under similar conditions. For example, the successive endwise removal of 100 glucose units from a polymer of D.P. 5000 would result in a 10,000 per cent increase in reducing power but only a 2 per cent reduction in D.P. Any enzyme which produces an appreciable slope under the conditions given in Fig. 2 would have to be "random", as far as substrate crystallinity permits (Nisizawa et al., 1963). The "random" action of the T. trinervoides cellulase probably accounts for the absence of detectable quantities of sorbitol or glucosyl-sorbitol among the hydrolysis products of reduced cellodextrin. In addition, Whitaker (1956) found that the linkages at the chain ends were more resistant to random action than the other linkages. As mentioned earlier the T. trinervoides cellulase is very active and also displays exceptionally high activity towards insoluble celluloses (ratio of cotton-fibre--CMC hydrolysis rates = 1:4). From the data of Kovoor (1970) it appears that the ratio of the reaction rates for the hydrolysis of cellulose powder and CMC is in the region of 1 : 20 to 1 : 30, using homogenates from the termite Microcerotermes edentatus. When assayed with CMC the M. edentatus cellulase is also much less active than the T. trinervoides cellulase. Incubation mixtures containing equal volumes of 0"5 per cent CMC and M. edentatus homogenates (37"5 guts/ml) released about 1300/~g glucose/ml in 42 hr at 40°C. When T. trinervoides preparations were assayed under similar conditions, 1 gut/ml released 190/~g glucose/ml in 15 min at 30°C. Although it is almost certain that the hydrolysis described by Kovoor (1970) was not linear over 42 hr, the activity was still very low. The M. edentatus cellulase assays were conducted at pH 4.6 and the T. trinervoides cellulase was assayed at pH 5.8 but assuming that the two enzymes are similar in physical properties, the difference in activity could not be accounted for by pH effects alone. The activity of the T. trinervoides cellulase was decreased by only 10 per cent when the pH was lowered from 5.8 to 4-6 (Potts & Hewitt, 1974). The relatively high in vitro activity of the T. trinervoides cellulase is probably sufficient to account for the hydrolysis of most of the cellulose ingested. Fibrous matter is still visible in all parts of the hindgut and this undigested material is presumably lignified plant tissue containing some cellulose and possibly other polysaccharides (Noirot & Noirot-Timoth6e, 1969). T. trinervoides usually feeds on the grass Eragrostis lahmanniana. Beukes (1969), using in vitro rumen techniques, determined the percentage of the total dry matter of E. lehmanniana potentially available for digestion. Only 49.04 per cent of the grass collected in winter and 53.1 per cent of the summer-collected grass was digestible.

336

R. C. POTTS AND P. H. HEWITT

T h e T. trinervoides cellulase shows m a n y of the features displayed b y cellulases of microbial (especially fungal) origin. T h i s is not surprising since only the cellulases produced b y micro-organisms have been studied to any extent and these properties m a y prove to be c o m m o n to all cellulases. I t will only be possible to make meaningful comparisons w h e n cellulases f r o m other animals, especially arthropods, have been studied. Acknowledgements--This work was supported by funds supplied by the Council for Scientific and Industrial Research, Republic of South Africa, and by the Department of Agricultural Technical Services, Government of the Republic of South Africa.

REFERENCES

BEUKm H. (1969) 'n Studie van die voedingswaarde van grasse en bossies. Study report for the Agricultural Technician's Diploma, Department of Pasture Science, University of the Orange Free State, South Africa. BmHOP C. T. & WHITAKmt D. R. (1955) Mixed arabinose-xylose oligosaccharides from wheat straw xylan. Chem. Ind. 1955, 119. CLARKE A. E. & STONE B. A. (1963) Chemistry and biochemistry of ~-l,3-glucans. Rev. pure appl. Chem. 13, 134-156. COWLING E. B. (1963) Structural features of cellulose that influence its susceptibility to enzymic hydrolysis. In Enzymic Hydrolysis of Cellulose and Related Materials (Edited by REmE E. T.) pp. 1-32. Pergamon Press, Oxford. ERIKSSON K.-A. & HOLLMAag B. H. (1969) Kinetic studies of the action of cellulase upon sodium carboxymethyleellulose. Archs Biochem. Biophys. 133, 233-237. G R ~ J. W. (1963) Drying and reactivity of ceUulose. In Methods in Carbohydrate Chemistry (Edited by WHISTLERR. L.), Vol. III, pp. 95-103. Academic Press, New York. HALLIWELLG. (1963) Measurement of ceUulase and factors affecting its activity. In Enzymic Hydrolysis of Cellulose and Related Materials (Edited by REESE E. T.), pp. 71-92. Pergamon Press, Oxford. HASHIMOTO Y. & NISIZAWA K. (1963) Purification of cellulase and related enzymes. In Enzymic Hydrolysis of Cellulose and Related Materials (Edited by REESE E. T.), pp. 93114. Pergamon Press, Oxford. HUANG H. & GIESE A. C. (1958) Digestion of algal polysaccharides by marine herbivores. Sdence, Wash. 127, 475. JAYME G. & LANG F. (1963) Cellulose solvents. In Methods in Carbohydrate Chemistry (Edited by WHISTLERR. L.), Vol. III, pp. 75-83. Academic Press, New York. KING K. W. (1963) Endwise degradation of cellulose. In Enzymic Hydrolysis of Cellulose and Related Materials (Edited by R~v~E E. T.), pp. 159-170. Pergamon Press, Oxford. KOOIMAN P. (1957) Some properties of cellulase of Myrothecium verrucaria and some other fungi--II. Enzymologia 18, 371-384. KOVOORJ. (1970) Prrsence d'enzymes cellulolytiques dans l'intestin d'un termite suprrieur Microcerotermes edentatus (Was.). Annls Sci. natn. Zool. 12, 65-71. LYR H. (1960) Die Bildung yon Ektoenzymen durch holzzerst/Srende und holzebewohnende Pilze auf verschiedenen Niihrbtiden--IV. Mitteilung (Schluss) : ein komplexes Medium als C-Quelle. Arch. Mikrobiol. 35, 258-278. MILLER G. L. (1963) Cellodextrins. In Methods in Carbohydrate Chemistry (Edited by WHmTLER R. L.), Vol. III, pp. 134-139. Academic Press, New York. MYERS F. L. & NORTHCOTED. H. (1959) Partial purification and some properties of a cellulase from Helix pomatla. Biochem. ft. 71, 749-756.

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NISIZAWA K., HASHIMOTOY. & SHIBATAY. (1963) Specificities of some ceHulases of the "random" type. In Enzymic Hydrolysis of Cellulose and Related Materials (Edited by RmmE E. T.), pp. 171-184. Pergamon Press, Oxford. NOIROT C. & NOIROT-TIMOTH~ E. (1969) The digestive system. In Biology of Termites (Edited by KRISHNA K. & WEESNER F. M.), Vol. I, pp. 49-88. Academic Press, New York. PETTERSSON G. (1969) Studies on cellulolytic enzymes--VI. Specificity and mode of action on different substrates of a cellulase from Penicillium notatum. Archs Biochem. Biophys. 130, 286-294. POTTS R. C. & HEWITT P. H. (1973) The distribution of intestinal bacteria and cellulase activity in the harvester termite Trinervitermes trinervoides (NasutitermJtinae). Insectes Soc. (In press.) POTTS R. C. & HEWITT P. H. (1974) The partial purification and some properties of the cellulase from the termite Trinervitermes trinervoides (Nasutitermitinae). Comp. Biochem. Physiol. 4713, 317-326. Ry~p E. T. (1963) See discussion to paper by HALLIWELLG. (1963). REESE E. T. & LEVINSON H. S. (1952) A comparative study of the breakdown of cellulose by micro-organisms. Physiologia Pl. 5, 345-366. RF~E E. T. & MANDELSM. (1959) Use of enzymes in isolation and analysis of polysaccharides. Appl. Microbiol. 7, 378-387. ROLLINSM. L. & TRIPP V. W. (1965) Light microscopy of cellulose and cellulose derivatives. In Methods in Carbohydrate Chemistry (Edited by WHISTLER R. L.), Vol. I I I , pp. 335356. Academic Press, New York. SISON B. C., SHUBERT W. J. & NORD F. F. (1968) On the mechanism of enzyme action--LXV. A cellulolytic enzyme from the mold Poria vaillantii. Archs Biochem. Biophys. 75, 260-272. SWENSONH. A. (1963) Intrinsic viscosity and its conversion to molecular weight. In Methods in Carbohydrate Chemistry (Edited by WHISTLERR. L.), Vol. III, pp. 84--91. Academic Press, New York. THOMAS R. (1956) Fungal cellulases--VII. Stachybotrys atra: production and properties of the cellulolytic enzyme. Aust..7. biol. Sci. 9, 159-183. WaITArma D. R. (1956) The steric factor in the hydrolysis of/g-l,4"-oligoglucosides by Myrothecium cellulase. Can.J. biochem. Physiol. 34, 102-115.

Key Word Index--Cellulase ; termite; laminaranase; xylanase; Trinervitermes trinervoides.