β-Giucosidase ‘EC 220.127.116.11’ and endo-β-1,4-gIucanase ‘EC 18.104.22.168’ activities were measured in the wood-eating higher termite Nasutitermes takasagoensis. β-Glucosidase activity was present mainly in the salivary glands (66.7%) and midgut (22.2%), whereas endo-β-1,4-glucanase activity was detected mainly in the midgut (90.1%). Specific activity of endo-β-1,4-glucanase was also the highest in the midgut, indicating that cellulose is digested in the midgut. The major endo-β-1,4-glucanase component of N. takasagoensis was purified from whole termites by gel filtration on Sephacryl S-200 HR, Superdex-75 and hydroxyapatite column chromatography. Subsequently, the endo-β-1,4-glucanase activity from a crude midgut extract was eluted in an identical volume (Kd=0.68) to that from whole termites, suggesting the purified endo-β-1,4-glucanase is identical to that in the midgut. The molecular weight of the purified endo-β-1,4-glucanase was 47 kDa, and its specific activity was 1,200 units/mg. The optimal pH and temperature were 5.8 and 65°C, respectively. The Km and Vmax values on carboxymethyl cellulose were 8.7 mg/ml and 2,222 units/mg, respectively. The purified endo-β-1,4-glucanase hydrolyzed cellopentaose to cellotriose and cellobiose, and cellotetraose to cellobiose and a trace of cellotriose and glucose, but cellotriose and cellobiose were not hydrolyzed. The activity and stability on pH and temperature of the purified endo-β-glucanase are prominent among those from various organisms.
Cellulose is one of the most abundant renewable biomasses in the biosphere. It is a polysaccharide composed of β-D-glucopyranosyl units joined by 1,4-glycosidic bonds. There are three major types of enzyme involved in hydrolysis of cellulose (Wood, 1985). Endo-β-1,4-glucanases ‘EC 22.214.171.124’ hydrolyze mainly amorphous cellulose, resulting in a rapid decrease in chain length together with a slow increase in reducing sugars. β-Glucosidases ‘EC 126.96.36.199’ hydrolyze cellobiose and soluble cellooligosaccharides to glucose. Cellobiohydrolases ‘EC 188.8.131.52’ degrade cellulose by splitting off cellobiose units from the non reducing end of cellulose chain, but not attack substituted cellulose like carboxymethyl cellulose. Native cellulose has been supposed to be hydrolyzed by cooperative action of the enzymes. However, insects never appear to be capable of forming cellobiohydrolases except the possible case of the silverfish (Prins and Kreulen, 1991).
Cellulolytic systems of insects have been mainly studied in lower termites and wood-eating cockroaches which were closely related to each other phylogenetically (Kambhampati, 1995). It has been widely known that lower termites and wood-eating cockroaches have numerous protozoa within an enlarged part of their hindgut, so-called “paunch”. In early studies on cellulose digestion in termites, Cleveland (1923, 1924, 1925, 1934) proposed that intestinal protozoa were essential for the lower termite Reticuiitermes flavipes and the wood-eating cockroach Cryptocercus punctulatus, Yokoe (1964) first demonstrated in Leucotermes (=Reticuiitermes) speratus that termites have their own cellulase activity. Subsequently, Yamaoka and Nagatani (1975) demonstrated that cellulolytic activity is present in both the salivary glands and hindgut of R. speratus. Similar reports suggesting the presence of the salivary and hindgut cellulases were obtained in Mastotermes darwiniensis (Veivers et al., 1982), Neotermes bosei (Mishra, 1980), Coptotermes lacteus (O'Brien et al., 1979) from the late 1970s to the beginning of 1980s. Therefore, Yamaoka (1989) assumed in R. speratus that cellulose was degraded in food vacuoles of the symbiotic protozoa by synenergistic action of both protozoan C1-cellulase (cellobiohydrolase) and endogenous Cx-cellulase (endo-β-1,4-glucanase) from the salivary glands. In 1994, Scrivener and Slaytor separated and characterized endogenous endo-β-1,4-glucanase and β-glucosidase components from the wood-eating cockroach Panesthia cribrata. Subsequently, Watanabe et al. (1997) reported purification and properties of endogenous endo-β-1,4-glucanase components from the lower termite R. speratus. Thus, enzymatic properties of cellulases in lower termites and xylophagous cockroaches have started to be clarified.
In higher termites, it has been considered that their cellulolytic systems differ from that of lower termites because higher termites have no symbiotic protozoa in spite of numerous bacteria within their gut (Bignell et al., 1979,1980a, b, 1983; Czolij et al., 1985; Anklin-Muhlemann et al., 1995). Until the beginning of 1980s, it had been assumed that hindgut bacteria of higher termites substituted for protozoa of lower termites in the role of cellulose digestion (Breznak, 1982). However, several investigations indicated that a large amount of cellulase activity was present in the midgut (Potts and Hewitt, 1973; Martin and Martin, 1979; O'Brien et al., 1979; Malaka, 1986; Hogan et al., 1988; Chararas and Noirot, 1988; Veivers et al, 1991). In addition, there are no affirmative reports on bacterial cellulose degradation except one study which suggested the possible role of bacteria in cellulose degradation of wood-eating termites (French, 1975). The presence of a large amount of cellulase activity independent of any symbionts has led to the proposal that cellulose is digested in the midgut by endogenous cellulases in xylophagous higher termites although it is controversial on the role of fungus in cellulose digestion of fungus-growing termites which ingests fungal nodules growing on special faeces of termites (Martin, 1991; Slaytor, 1992). Cellulases were not fully purified from a xylophagous higher termite although a few of them were partially purified from Trinervitermes trinervoides (Potts and Hewitt, 1974a, b) and Nasutitermes walkeri (Schulz et al., 1986). Characterization of the enzymatic properties is a key to understand the cellulolytic systems of termites and cockroaches. Since higher termites are more evolved species than R. speratus or P. cribrata, and are the most successful species in the tropical area (Wood, 1988), the clarification of the cellulolytic system in the higher termite is of importance in understanding not only cellulolytic system of termites but also the evolution of the interaction between termites and their intestinal symbionts.
In the present study, we report cellulose digestion, purification and properties of the major endo-β-1,4-glucanase component from the wood-eating higher termite, Nasutitermes takasagoensis (Shiraki) (Isoptera: Termitidae; Nasutitermitinae).
MATERIALS AND METHODS
Arboreal nests and logs inhabited by Nasutitermes takasagoensis (Shiraki) were collected at Iriomote-island in Okinawa prefecture located in the subtropical region of Japan. Termites were kept at room temperature with nest materials.
Preparation of extracts for enzyme assays
0.1 M Sodium acetate buffer, pH 5.5, was used in the preparation of all extracts and in all assays, unless otherwise indicated. All procedures were carried out at 4°C.
The salivary glands and whole gut were removed from 20 termites and divided into the foregut, midgut, mixed segment, first proctodeal segment, paunch and colon with the rectum. The salivary glands and each of gut sections were homogenized in 10 μl of buffer in microcentrifuge tubes (1.5 ml) and centrifuged at 20,100 × g for 20 min. Supematants were collected and diluted to 600 μl with buffer, then which were referred to as enzyme extracts.
Enzyme extract (25 μl) was incubated with 200 (μl of 2% (w/v) sodium carboxymethylcellulose (CMC; standard molecular weight: 250,000, degree of carboxymethyl substitution: 0.7 (w/v); Aldrich) in buffer at 37°C for 30 min. Reducing sugars were detected with tetrazolium blue (Sigma) (Jue and Lipke, 1985) and expressed as glucose equivalents.
The protein contents of samples were determined by the direct UV method of absorbance at 280 and 260 nm (Layne, 1957) or at 215 and 225 nm (Murphy and Kies, 1960) using bovine serum albumin as a standard.
Definition of enzyme unit
One unit of enzyme activity is defined as the amount of enzyme which produced 1 μmol of reducing sugar (glucose equivalents) or glucose/minute. Specific activity is defined as units/mg protein.
Worker and soldier termites (15 g) were homogenized in 150 ml of distilled water, and centrifuged at 24,000 × g for 30 min. The supernatant was recovered as a crude extract. Ammonium sulfate was added to the crude extract and centrifuged at 24,000 × g for 30 min to collect precipitation. Precipitation from 35 to 70% (w/v) ammonium sulfate solution was collected and dissolved in 30 ml of 0.3 M ammonium acetate buffer, pH 5.0. This is referred to as 70% precipitation and was applied to column chromatography.
All chromatography was carried out at 4°C. Fractions containing endo-β-1,4-glucanase activity were concentrated using an ultrafiltration cell (Model 8003, 8010 or 8050; Amicon, Grace Japan K.K.) with a UK-10 membrane (PVDF, molecular cut off 10,000, Advantec TOYO). Protein was monitored at 280 nm using a UV-1 monitor (Pharmacia).
The 70% precipitation (5 ml at once) was applied to Sephacryl S-200 HR (Pharmacia) gel filtration column (26 × 900 mm), equilibrated and eluted with 0.3 M ammonium acetate buffer, pH 5.0, at a flow rate of 1.0 ml/min, with the collection of 5 ml fractions. Fractions containing endo-β-1,4-glucanase activity from 6 replications of the gel filtration were concentrated to 1 ml using the ultrafiltration cell, and then chromatographed on a HiLoad 16/60 Superdex-75 prep grade column (Pharmacia). The column was equilibrated and eluted with 0.3 M ammonium acetate buffer, pH 5.0, at a flow rate of 0.6 ml/min, with the collection of 1.2 ml fractions. Active fractions were recovered, desalted and concentrated to 1 ml using the ultrafiltration cell with distilled water. The concentrated sample was adsorbed on a hydroxyapatite (DNA grade, Bio-Rad) column (10 × 50 mm). The column was washed with 40 ml of starting buffer (20 mM sodium phosphate buffer, pH 5.5), and eluted with a 50 ml linear gradient to 1 M sodium phosphate, pH 5.5, at a flow rate of 0.2 ml/min, with the collection of 1.0 ml fractions. Active fractions were desalted and concentrated to 1 ml using the ultrafiltration cell, and again adsorbed on hydroxyapatite, then eluted by the same procedure.
Midguts from 100 workers were homogenized in 1 ml of buffer in a microcentrifuge tube, and centrifuged at 20,100 × g for 30 min. The supernatant, which is referred to as a crude midgut extract, was similarly chromatographed on the Superdex-75 column.
Activity during the purification steps of endo-β-1,4-glucanase was measured using 25 μI of appropriately diluted sample and 200 μI of 2% (w/v) CMC at 37°C for 10 min.
The purified endo-β-1,4-glucanase was examined by SDS-PAGE according to the method of Laemmli (1970). The sample and protein standards (GIBCO BRL, Life Technologies, Inc.) were run on a 10% (w/v) acrylamide gel at 15 mA. Proteins were detected with silver stain kit (Bio-Rad).
Characterization of the purified endo-β-1,4-glucanase
Optimal temperature and pH.
Activity of the pure endo-β-1,4-glucanase was measured at pH 5.5 over the range from 20 to 70°C with 5°C intervals for 5 min to determine the optimal temperature. To evaluate thermal stability, enzyme samples were incubated at constant temperature from 20 to 70°C with 5°C intervals for 30 min and then assayed at 37°C for 5 min. In order to measure optimal pH, 2% (w/v) CMC in three kinds of buffers was used for enzymatic assays: 0.04 M phosphoric acid, 0.04 M acetic acid and 0.04 M boric acid mixture −0.2 M NaOH buffer (pH 1.8 to 12.0); citrate-phosphate buffer (pH 2.2 to 8.0; Mcllvaine, 1921); 0.2 M boric acid and 0.2 M potassium chloride −0.2 M sodium carbonate buffer (pH 7.4 to 11.0).
Hydrolytic products from cellodextrins by TLC.
Samples (10 μl) were incubated with 10 μI of 60 mM solutions of cellopentaose, cellotetraose, cellotriose, cellobiose (Sigma) at 37°C. Aliquots (2 μl) were collected at 0 time and 30 min for estimation of kinetic constants, and 0 time and 18 hr for analysis of hydrolytic products. The products were analyzed by TLC as described by Hansen (1975). The TLC plates for hydrolytic product analysis were scanned by a GT-5000 scanner (EPSON). The density of each spot was estimated using NIH Image software on Macintosh computer (Apple Computer Inc.).
General view of the alimentary canal
Figure 1 shows a schematic drawing of the alimentary canal and the salivary glands in N. takasagoensis. The alimentary canal was composed of the foregut, midgut, and hindgut. The midgut elongated toward the hindgut, and formed the mixed segment. The hindgut was further subdivided into five segments; the first proctodeal segment (P1), enteric valve (P2), paunch (P3), colon (P4), and rectum (P5).
Cellulase activity in the alimentary canal
The distribution of cellulase activity through the gut of N. takasagoensis is shown in Table 1. β-Glucosidase activity was dominant in the salivary glands (66.7%), while 22.2% of (3-glucosidase activity was detected in the midgut. The level of β-glucosidase activity was lower in the mixed segment (1.2%) than that in the midgut, and it was hardly detected in the first proctodeal segment. Only a small amount of β-glucosidase activity was detected in the paunch (4.9%). β-Glucosidase activity was not detected in the colon and rectum. Specific activity of β-glucosidase was the highest in the midgut, and was slightly higher than that in the salivary glands.
Distribution of cellulase activities in the gut of N. takasagoensis
More than 90% of endo-β-1,4-glucanase activity was localized in the midgut. The level of endo-(3-1,4-glucanase activity was much lower in the foregut (3.6%) and mixed segment (2.3%) than in the midgut, and it was hardly detected in the salivary glands and first proctodeal segment (0.4%). Endo-β-1,4-glucanase activity was absent in the paunch, where was considered as the main site of cellulose digestion in lower termites. A small amount of endo-β-1,4-glucanase activity was detected in the colon and rectum (3.2%). Specific activity of endo-β-1,4-glucanase was the highest in the midgut. It was 2.9 and 240 times higher than those of the foregut and salivary glands, respectively.
Purification of the major endo-β-1,4-glucanase component
A major endo-β-1,4-glucanase component was purified from the whole termites. The major endo-β-1,4-glucanase activity was eluted on Sephacryl S-200 HR at 365 ml (Kd=0.75). However, the protein profile did not show any peaks at a corresponding volume to the activity peak because the sample contained only a small amount of endo-β-1,4-glucanase protein (Fig. 2). Active fractions in each replication were combined and chromatographed on a Superdex-75 column (Fig. 3). The endo-β-1,4-glucanase activity was eluted at 87.6 ml (Kd=0.68). The protein profile also showed an identical peak to the endo-β-1,4-glucanase activity. In hydroxyapatite chromatography, the endo-β-1,4-glucanase activity was eluted at 49 ml (175 mM sodium phosphate) (Fig. 4). Active fractions were again applied to the same hydroxyapatite column. Then, the endo-β-1,4-glucanase activity was also eluted at 49 ml (175 mM sodium phosphate). The purified endo-β-1,4-glucanase was pure after the duplicated hydroxyapatite chromatography because it was detected as a single band on SDS-PAGE by silver staining (Fig. 5). The molecular weight of the major endo-β-1,4-glucanase component was estimated to be 47 kDa by comparing its mobility with standard proteins on SDS-PAGE. The purification of the major endo-β-1,4-glucanase component was summarized in Table 2. The endo-β-1,4-glucanase was purified 6,000-fold from the crude extract.
Purification of the major endo-β-1,4-glucanase component fromN. takasagoensis
The crude midgut extract was also chromatographed on Superdex-75 (Fig. 6). Endo-β-1,4-glucanase activity was eluted in an identical volume (Kd=0.68) to that from whole termites.
Characterization of the purified endo-β-1,4-glucanase
Figure 7 shows the effect of pH on activity of the major endo-β-1,4-glucanase component. The major endo-β-1,4-glucanase component had optimal activity at pH 5.8, and retained more than 60% of the maximal activity from pH 5.0 to 9.2. However, the activity dropped sharply in more acidic or alkaline ranges. The activity was lost at pH 4.0 and 11.8.
Hydrolytic products by the major endo-β-1,4-glucanase component were examined using cellodextrins (Table 3). Cellopentaose was hydrolyzed to equimolar amounts of cellotriose and cellobiose. Cellotetraose was hydrolyzed mostly to cellobiose, and to a trace of cellotriose and glucose. However, the major endo-β-1,4-glucanase component did not hydrolyze cellotriose or cellobiose.
Kinetic constants and hydrolytic products for the major endo-β-1,4-glucanase component
Kinetic constants on CMC, cellopentaose and cell-otetraose were shown in Table 3. Km value on cellopentaose was smaller than that on cellotetraose. Km value on CMC, 8.7 mg/ml, is almost equivalent to 34.8 μM according to the average molecular weight of CMC. Thus, the Km value on CMC was extremely smaller than those on cellopentaose and cellotetraose. In contrast, Vmax value on cellopentaose was larger than that on cellotetraose. Similarly, Vmax value on CMC was much larger than those on cellopentaose or cellotetraose.
β-Glucosidase activity was found in the salivary glands and midgut. Specific activity was also high in the salivary glands and midgut. This is the first report suggesting that the salivary glands are the major secretion site of β-glucosidase as well as the midgut in higher termites.
The midgut had more than 90% of endo-β-1,4-glucanase activity, whereas a small amount of the activity was detected in the hindgut. This result is consistent with the case of other xylophagous higher termites (Potts and Hewitt, 1973; O'Brien et al., 1979; Hogan et al., 1988; Chararas and Noirot, 1988), supporting that the midgut is the main site of cellulose digestion in N. takasagoensis. Gel filtration profile from the crude midgut extract showed that the major endo-β-1,4-glucanase activity was eluted in a corresponding volume with that from whole termites, suggesting that the major endo-β-1,4-glucanase component was identical to that in the midgut. Although absence of symbionts was reported in the midgut of termites belonging to Nasutitermitinae (Potts and Hewitt, 1973; Czolij et al., 1985), the specific activity in the midgut was higher than upstream and downstream parts of the alimentary canal (i.e. salivary glands, foregut and hindgut). These results indicate that endo-β-1,4-glucanase is secreted in the midgut tissue. Worthy to note, the main secreting site of endo-β-1,4-glucanase is the salivary glands in lower termites (Yokoe, 1964; Yamaoka and Nagatani, 1975; O'Brien et al., 1979; Mishra, 1980; Veivers et al., 1982; Mednicova and Tiunova, 1984; Watanabe et al., 1997). It is probable that secretion sites of cellulase have been changed in the course of evolution of termites. In the present study, no endo-β-1,4-glucanase activity was detected in the paunch, where is considered as the main site of cellulose digestion in lower termites, suggesting that the paunch is not the site of cellulose digestion in N. takasagoensis. Although no bacterial cellulase was found in the hindgut of N. exitiosus (Hogan et al., 1988), the presence of bacterial cellulase is not deniable in the hindgut of N. takasagoensis because the small amount of endo-β-1,4-glucanase was detected in the colon and rectum, where have no secretory cells but are associated with intestinal bacteria (Noirot and Noirot-Timothee, 1977; Yamaoka and Nagatani, 1978; Bignell et al., 1980c; Czolij et al., 1984).
In spite of numerous studies on purification and properties of cellulases from fungi, bacteria and plants, only a few insect cellulases have been purified and characterized. Endo-β-1,4-glucanase components have been purified from four species of insects; the fungus-growing higher termite Macrotermes mulleri (Rouland et al., 1988), the lower termite R. speratus (Watanabe et al., 1997), the wood-eating cockroach P. cribrata (Scrivener and Slaytor, 1994) and the wood-eating larvae of the longicorn beetle Ergates faber (Chararas et al., 1983). Table 4 summarizes enzymatic properties among endo-β-1,4-glucanase components from different organisms. In the present study, molecular weight of the major endo-β-1,4-glucanase component from N. takasagoensis was estimated to be 47 kDa. This value is similar to the previously reported molecular weights from the insects, and is within the range of that of most fungal and plants endo-β-1,4-glucanases (Wood, 1991; Maclachlan and Carrington, 1991).
Comparison of enzymatic properties among purifiedendo-β-1,4-glucanase components from various organisms
Specific activity of the major endo-β-1,4-glucanase component from N. takasagoensis is higher than those from other organisms. Since CMC which has universally used as substrate has varied in degree of polymerization and substitution among lots or suppliers, enzyme affinity and reaction velocity are affected by degree of them. Even if differences of enzyme affinity and reaction velocity are taken into account, specific activity of the major endo-β-1,4-glucanase component from N. takasagoensis is considered to be prominent among those from different organisms (Table 4).
Optimal pH of the major endo-β-1,4-glucanase component from N. takasagoensis is identical to that of T. trinervoides (Potts and Hewitt, 1974a), and is similar to those of R. speratus (Watanabe et al., 1997), N. exitiosus (Schulz et al., 1986), most anaerobic bacteria (Rapp and Beermann, 1991) and plants (Maclachlan and Carrington, 1991). Although activities of endo-β-1,4-glucanase components from the other insects decreased in alkaline pH, more than 60% of activity of the purified endo-β-1,4-glucanase from N. takasagoensis was retained even in pH 9.0. Optimal temperature of the major endo-β-1,4-glucanase component from N. takasagoensis was 65°C and stable at 55°C at least 30 min. The optimal temperature and high thermostability are similar to those of N. exitiosus (Schulz et al., 1986) and T. trinervoides (Potts and Hewitt, 1974a), but the major endo-β-1,4-glucanase component from N. takasagoensis is stabler than those of other insects.
Hydrolytic properties of the major endo-β-1,4-glucanase component from N. takasagoensis are similar to those of partially purified endo-β-1,4-glucanase from N. exitiosus, which hydrolyses cellotetraose to cellobiose, but does not hydrolyze cellotriose or cellobiose (Schulz et al., 1986). In R. speratus, YEG1 also shows similar properties to the major endo-β-1,4-glucanase component from N. takasagoensis though YEG2 hydrolyses cellotriose (Watanabe et al., 1997). In P. cribrata, EG1 and EG2 also do not hydrolyze cellotriose or cellobiose (Scrivener and Slaytor, 1994). A trace of glucose production from cellotetraose by the major endo-β-1,4-glucanase component from N. takasagoensis was presumably due to transglycosidation as is reported in the case of EG1 of P. cribrata (Scrivener and Slaytor, 1994). As the Km value decreased with higher polymerization degree of substrate, the purified endo-β-1,4-glucanase is considered to increase affinity to the substrate when polymerization degree become higher. The Vmax values indicate that specific activity also become higher when polymerization degree become higher. Similar tendency was reported for endo-β-1,4-glucanase components from R. speratus (Watanabe et al., 1997) and P. cribrata (Scrivener and Slaytor, 1994). Therefore, endo-β-1,4-glucanase activity from these species depends on the length of cellulose chains.
Our results demonstrated prominent efficiency of the major endo-β-1,4-glucanase component from N. takasagoensis in cellulose digestion among those from various organisms. This higher efficiency seems to compensate the lack of protozoan cellulases because higher termite lost cellulolytic protozoa in the course of evolution. The wood-eating higher termites must have increased adaptability to cellulose diet and have flourished by acquisition of highly efficient endo-β-1,4-glucanase. This higher efficiency also seems to partially explain why higher termites play more significant role than lower termites in keeping carbon balance in nature (Wood and Johnson, 1986).
The authors would like to thank Dr. A. M. Scrivener of NISES for his technical advice, Dr. M. Sakakibara of JIRCAS for providing the termites, Dr. K. Kurata of NISES for many helpful supports and Dr. I. Yamaoka of Yamaguchi University for his useful suggestions.