Abstract: Endo-β-1, 4-glucanase (EC 3.2.1.4) activity was measured in the gut fluid of phytophagous insect Podontia quatuordecimpunctata Linnaeus (Coleoptera: Chrysomelidae) in different days of development. The eight day-old larva showed maximum activity with 1.73 U mg-1 of protein, which was confirmed by gel zymography. In zymogram, using Carboxymethyl Cellulose (CMC) as substrate, four distinct cellulolytic protein bands were detected in leaf borer gut fluids through out of its development. The optimum temperature and pH were 60°C and 5.0, respectively. This endo-β-1,4-glucanase showed maximum stability at 20-45°C with ~ 20% remaining activity. Zymography also showed complete loss of endo-β-1,4 glucanase activity at 55°C. This is the first report that the cellulolytic enzyme is produced in the gut of P. quatuordecimpunctata through the whole developmental stages, from the 1st instar to the adult, except for pupae.
INTRODUCTION
Cellulose is the main structural polysaccharide of the plant material and is the most globally abundant biopolymer. These polymers are comprised of sugar units, usually glucose, joined by β-1,4 and β-1,3 glycosidic bonds (Watanabe and Tokuda, 2001). Biological cleavage of these bonds require the synergistic action of three types of glycoside hydrolases (GH): endo-β-1,4-glucanases (EG; EC. 3.2.1.4), exo-β-1,4-cellobiohydrolases (CBH; EC. 3.2.1.91) and β-glucosidases (EC. 3.2.1.21) (Clarkel, 1997). EG, hydrolyzing internal β-1,4-glycosidic bonds of cellulose polymers of four or more glucose units, shortens and dissolves cellulose polymer, thus provides substrate for CBH (Scrivener and Slaytor, 1994; Tokuda et al., 1997; Watanabe et al., 1997). Moreover, through catalyzing transglycosylation reaction it can also rejoin the glucose oligomers (Lindner et al., 1983). CBH releases cellobiose from the nonreducing ends of cellulose. CBH is also thought to disrupt the hydrogen bonding in crystalline cellulose, thus allowing the EG to endo-depolymerize. β-glucosidases hydrolyse cellobiose or longer cellulose chains to release glucose.
Although cellulases have been extensively used in different industries including textile, laundry, brewery, wine, pulp, paper and agriculture (Bhat and Bhat, 1997; Bhat, 2000; Ohmiya et al., 1997) for long time but the most recent interest in cellulase research is due to its ability to degrade lignocellulose materials of plants thus to produce biofuel. Although bacterial and fungal cellulases have been used for the last two decades in industrial bioethanol technology, more efficient cellulases are needed to reduce cost in biofuel production (Wyman, 2007) and also for applications in other industries.
Insects are very efficient in degradation of biomass with their highly efficient gut systems which can truly be considered as highly efficient natural bioreactors. Previously, enzymatic activity against cellulose substrates was detected in digestive fluids of insect species belonging to ten insect orders (Martin, 1983; Watanabe and Tokuda, 2001; Willis et al., 2010a). These activities were historically attributed to gut symbiotic flora, until the first insect cellulase was described in Reticulitermes speratus (Watanabe et al., 1998). Various studies have reported endogenous insect cellulase enzymes in the orders of Blattaria, Coleoptera, Hymenoptera, Hemiptera, Phthiraptera and Orthoptera (Watanabe and Tokuda, 2001).
Larva and adults of defoliator, Podontia quatuordecimpunctata L. (Coleoptera: Chrysomelidae), feed on the leaves of the Otaheite apple Spondias dulcis tree. There are only a few reports on cellulolytic activity in the Coleoptera (Oppert et al., 2010; Willis et al., 2010a), only in single species, Aulacophora foveicollis, from Chrysomelidae family has been shown to have a cellulolytic system (Sami and Shakoori, 2008). The purpose of this study was to investigate the presence of cellulolytic activity in P. quatuordecimpunctata.
MATERIALS AND METHODS
Insect collection: Podontia quatuordecimpunctata larva of different ages (2, 3, 6, 8, 10 and 12 days) and adults were collected from leaves of an infested hog plum tree during April-June from Fatehpur village near the Chittagong University campus, Bangladesh. Until dissection, insects were kept on or in close proximity to plant host tissues where insects were actively feeding.
Gut dissection and fluid collection: Insects were dissected on the same day of collection. After immobilizing on ice for around half an h, the larva were kept abdomen side up on a petri dish which was on ice and dissected to remove the entire, intact guts. The multiple guts of different day’s larva and adult were taken separately in micro centrifuge tubes. Dissected guts were cut into small pieces, homogenized by vortexing to ensure fluid extraction and centrifuged at 10,000 rpm for 10 min at 4°C. The resulting supernatants were transferred to new 1.5 mL micro centrifuge tubes and stored at -20°C until use. All the biochemical analyses with this supernatant were carried out in triplicates.
Plate assay for cellulolytic activity: A modified substrate-agar plate assay (Teather and Wood, 1982) was used to assay endo-β-1,4-D-glucanase activity qualitatively in the gut fluid of eight day’s larva. To observe the clear zone formation around the sample well against a red-staining background on agar plates, 1% CMC and 3% agar were mixed with 0.1 M Na-acetate buffer, pH 5.3 and poured in petri dishes to solidify the gel. 220 μg of gut protein in water was loaded in the hole punched in the center of plate. After overnight incubation at 37°C, the plates were stained with 1% Congo red (Sigma-Aldrich) for 10-15 min before de-staining with 1.0 M NaCl solution for 15-20 min for several times.
Measurement of endo-β-1,4-D-glucanase activity: Endo-β-1,4-D-glucanase activity was quantified as the rate of production of reducing sugars from the substrate, carboxymethyl cellulose (BDH chemical Ltd pool England), using a modified 3,5-dinitrosalicylic acid (DNSA) assay method (Miller, 1959). Gut fluids (33 μg of protein) of each respective day’s larva and adult mentioned above and 235 μL of 1% CMC suspended in 0.1 M Na-acetate buffer, pH 5.3 were mixed separately and incubated for 30 min at 37°C. The reactions were stopped by adding 450 μL DNSA solution followed by heating in boiling water for 10 min. After adding 40% Rochelle salt (MARK), the reaction mixers were cooled at room temperature for 5 min and centrifuged at 10,000 rpm for 5 min. Supernatants were transferred to new microcentrifuge tubes to measure absorbance at 540 nm on a UV spectrophotometer (Shimadzu).
| Fig. 1: | Standard calibration curve for the determination of reducing sugar released during endo-β-1,4-D-glucanase assays |
A blank was prepared with substrate and DNSA solution before adding gut juice so that enzymes present in the juice could not hydrolyze CMC and the subsequent process same as described above. One unit of cellulolytic activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per min at 37°C and pH 5.3. A standard curve of absorbance against amount of glucose (50-300 μg) was constructed to enable calculation of the amount of reducing sugar (glucose equivalents) released during endo-β-1,4-D-glucanase assays (Fig. 1). In all following experiments, enzyme activity was measured using this method and the extract of 8 days larvae was used.
Effect of temperature and pH on enzyme activity: The effects of temperature and pH on enzyme activity of gut fluids were estimated at eleven temperatures and nine pH values. The effect of temperature on endo-β-1,4-D-glucanase activity was examined by incubating the reaction mixture (in 0.1 M Na-acetate buffer pH 5.3) over a temperature range of 20 to 70°C (using 1% CMC as substrate). The thermal stability of endo-β-1,4-D-glucanase was determined by pre-incubation of the sample at 20, 25, 30, 35, 40, 45, 50 and 55°C for 30 min, followed by measurement of activity under the standard test conditions as mentioned before. Activity at optimum temperature was taken as 100.
Optimal pH for the enzyme activity was determined by measuring the hydrolysis of CMC in a series of buffers at various pH values ranging from pH 2 to 10. The buffers used were KCl-HCl buffer 0.1 M pH 2.0, K-hydrogen pthelate HCl buffer (0.1 M) pH 3, Na-acetate buffer (0.1 M) pH 4 and 5, phosphate buffer (0.1 M) pH 6 and 7, tris-HCl buffer (0.1 M) pH 8, glycine-NaOH buffer (0.1 M) pH 9 and 10.The pH at which activity was highest was considered as 100%.
Zymography: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) were performed with some alterations to detect activity of endo-β-1,4-D-glucanase through zymogram techniques (Schwarz et al., 1987). For the CMC zymogram, SDS-10% PAGE resolving gel was prepared by adding 0.2% CMC slowly to prevent aggregation. After dissolving CMC, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were added and gels were incubated at 45°C for 2 h or until polymerization. Defoliator gut fluids (8 μg) of different aged larva and adult were solubilized in 1 volume of sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.01% bromophenol blue) and loaded onto the gel. Proteins in samples were separated at a constant 100 V at 4°C for approximately 4 h or until dye reached the bottom of the gel.
After electrophoresis, the gel was washed with 50 mL wash buffer (0.1 M Na-succinate pH 5.8 and 0.1% β-mercaptoethanol) at room temperature for five times (each was for 30 min) but last wash was at 30°C to allow for enzyme activity against the substrate. Then, the remaining CMC in the gel was stained with 0.1% Congo red for 30 min and destained by washing in a solution of 1 M NaCl at room temperature to reveal zones of clearing where CMC had been degraded by enzyme. For improving visualization of clearing area, 100 μL of glacial acetic acid was added after destaining the gel (Waeonukul et al., 2007). Zymography was also carried out with 8 days’ larval gut protein after heating the solubilized sample at 45 and 55°C for 30 min.
Measurement of protein in gut fluid of P. quatuordecimpunctata: The concentrations of protein in samples of gut fluid were measured using FCR method (Lowry et al., 1951). In brief, different aliquots of standard Bovine Serum Albumin (BSA) solution (250 μg mL-1) were pipetted out in different tubes along with the aliquots of samples in separate tubes. Equal amount of alkaline copper sulphate was added in each tube and then allowed to stand for 15 min. Equal amount of Folin-Ciocalteu reagent was added in each tube and the tubes were left for 30 min. Blue color was formed which was measured at 650 nm against a proper blank where no protein solution was used. The protein concentration of the extract was calculated from the standard curve (Fig. 2).
| Fig. 2: | Standard calibration curve for the determination of total protein in gut juice |
RESULTS
Qualitative measurement of cellulolytic activity: Substrate-agar plate assay showed the presence of cellulase activity by forming the cleared zone (yellow color) around the sample well against a red-stained background on agar plates (Fig. 3). The cellulase activity digested the CMC in area that did not retain the Congo red dye on destaining.
Endo-β-1,4-D-glucanase activity during P. quatuordecimpunctata development: Activities of endo-β-1,4-D-glucanase enzymes were assayed from gut fluids of six different aged larva (2, 3, 6, 8, 10 and 12 days after their hatching) on their development, pupa and adult of P. quatuordecimpunctata. Larval extract of 8th day showed maximum enzyme activity which was 1.73 U mg-1 protein and calculated as 100%. Activity was decreased at both sides gradually before and after of this developmental stage (Fig. 4a). The last instar (12 days) larva and adult exhibited only 9.0 and 7.0% of maximum activity, respectively, but pupa showed no activity.
To evaluate the activity of different components of endo-β-1,4-D-glucanase system during P. quatuordecimpunctata development, zymogram analysis using CMC as a substrate was used. Zymography showed that at least four active bands are responsible for the measured cellulase activity (Fig. 4b). No differences in number and positions of the cellulolytic protein bands in the gel were observed among different larval stages and adult. The two of the four bands were very prominent and close compare to the remaining two other bands as visualized on the gel zymography.
Effect of temperature on endo-β-1,4-D-glucanase activity: The optimum temperature for the hydrolysis of carboxymethyl cellulose was 60°C and another broad range temperature was 35-45°C at which around 90% of highest enzyme activity was found (Fig. 5a).
| Fig. 3: | Presence of cellulolytic activity in substrate-agar plate |
| Fig. 4 (a-b): | (a) Comparison of cellulolytic activity (with standard error bars) in gut fluids from different larval developmental stages (2, 3, 6, 8, 10 and 12 days after hatching), pupa and adult containing 15 μg of proteins and (b) Zymogram (10% PAGE+0.2% CMC) depicting corresponding active cellulase protein profile in gut fluids (8 μg of total gut protein per lane) from different life stages of P. quatuordecimpunctata development. Arrows indicate the four cellulase activity bands detected |
| Fig. 5 (a-c): | Endo-β-1,4-D glucanase activity (a) At different temperatures between 20-70°C without pre-meubation, (b) With pre-incubation at different temperatures between 20-55°C, Error bars represent the standard deviation and (c) Conformation of temperature stability of endo-β-1,4-D-glucanase in gel zymography. Sample without (—) and with pre-incubation at 45 and 55°C for 30 min prior to load in the gel. Arrows indicate the four cellulase activity bands detected |
The activity of endo-β-1,4-D-glucanase was decreased gradually to zero at 75°C. After pre-incubation for 30 min at different temperature, the enzyme was stable at 20-50°C. The enzyme retained only around 20% of its activity in the range of 20-50°C but out of this range, it lost its activity completely (Fig. 5b). Remaining activity is measured as the percentage of enzyme activity compare to the activity assayed under the standard test conditions. In zymogram analysis, only one out of four isoforms of enzyme retained little activity at 45°C but activity was completely lost at 55°C (Fig. 5c).
Effect of pH on endo-β-1,4-D-glucanase activity: The effect of the pH on defoliator larval endo-β-1,4-D- glucanase activity was examined at various pH ranging from pH 2.0 to pH 12.
| Fig. 6: | Optimum pH of endo-β-1,4-D-glucanase, Error bars represent the standard deviation |
The optimal pH for the enzyme activity was found at pH 5.0 which is the sharp peak on the graph. The activities were decreased rapidly on both sides and fell to around zero at pH 2.0 and pH 9.0 (Fig. 6).
DISCUSSION
Many insects especially phytophagous and or xylophagous insects are solely dependent on cellulose as a major food source for available energy/nutrients and use cellulolytic enzymes for their degradation to simple useable form. Therefore, cellulases are believed to be present in insects that have nutritionally poor diets such as termites and cockroaches (Terra and Ferreira, 1994). Previously, insects’ cellulolytic activities have been confirmed in some species belonging to ten taxonomic orders (Martin, 1983; Watanabe and Tokuda, 2001; Willis et al., 2010a). Cellulases can be produced from insect endogenously [Coleoptera (Genta et al., 2006), Isoptera (Martin and Martin, 1978; Slaytor, 1992; Bignell et al., 1994), Thysanura (Treves and Martin, 1994), Orthoptera (Willis et al., 2010b), Blattodea (Scrivener et al., 1989; Genta et al., 2003), Hemiptera (Adams and Drew, 1965) and Phasmatodea (Cazemier et al., 1997)] and also from symbiotic organisms harboring in the insects’ gut (Ohtoko et al., 2000) or both (Breznak and Brune, 1994; Watanabe et al., 1998; Ohtoko et al., 2000).
In this study, the data have reported the presence of endo-β-1,4-D-glucanase enzyme in the gut of phytophagous insect P. quatuordecimpunctata L. (Coleoptera: Chrysomelidae). This reports for the first time that the cellulolytic enzyme produced in the gut throughout the whole developmental stages of P. quatuordecimpunctata, from 1st instar to adult excluding pupa. Qualitative assay carried out by substrate-agar plate assay showed the presence of endo-β-1,4-D-glucanase activity in the gut of larva (Fig. 3).
Among all developmental stages, 8 days’ larva showed the maximum activity (1.73 U mg-1 protein). The endo-β-1,4-D-glucanase activity was found to increase with the larval development up to the eight days but after that the activity decreased gradually (Fig. 4a). Previously, cellulase activity reported for lepidopteran larvae showed increased activity with advancing larval development (Nakonieczny et al., 2006; Pant and Ramana, 1989). In contrast, Willis et al. (2010b) did not detect significant differences in cellulolytic activity during development of Dissosteira carolina.
Highest endo-β-1,4-D-glucanase activity may co-relate to highly feeding of larva and increased expression of endo-β-1,4-D-glucanase gene. These different activities of endo-β-1,4-D-glucanase enzyme in different developmental stages of insect may be due to the quantity of enzyme produced as same amount of gut proteins (15 μg) from all larval developmental stages were used for assay. These had been confirmed by gel zymography in which 8 μg of gut proteins were used (Fig. 4b). In the zymography, the intensities of bands produced by enzymatic hydrolysis of CMC were varied during different developmental stages similarly to the enzymatic activities determined optically showed in Fig. 4a. So the data suggest that the amounts of enzymatic proteins were different in the same amounts of gut proteins used in both enzyme assay and zymography.
Zymograms were performed also to assess the number of enzymatic isoforms responsible for the observed activities. The clear zones on zymographic gel indicated multiple forms of endo-β-1,4-D-glucanase enzyme in different developmental stages (Fig. 4b). Although intensities of the bands on zymographic gel were different, there were at least four cellulolytic isoforms of endo-β-1,4-D-glucanase enzyme in each developmental stage of P. quatuordecimpunctata (pupa was not included in this experiment due to unavailability of sample). Multiple forms of cellulases have been previously reported for some insect orders: Coleoptera (Girard and Jouanin, 1999; Sami and Shakoori, 2006, 2008; Li et al., 2008; Rehman et al., 2009a; Geib et al., 2010; Oppert et al., 2010; Sami et al., 2011), Orthoptera (Sami and Shakoori, 2006; Oppert et al., 2010; Willis et al., 2010b) and Decapoda (Xiao et al., 1999). There are several possibilities to the presence of multiple forms of cellulase enzyme in the insects. Firstly these could represent cleavage products of large enzymatic protein, as suggested by Wood et al. (1995), proteolysis by proteases originated either from the insect digestive tract or from the symbiotic microbes. Secondly, it could be because of separate gene products or gene duplication even single gene where the gene products could be glycosylated differentially as post translational modification. Girard and Jouanin (1999) suggested that beetle Phaedon cochleariae, has at least 3 cellulase genes, which probably encode divergent enzymes. Different levels of glycosylations was suggested for appearance of more than one form of cellulase activity bands as glycosylation was required for the activity of cellulases of beetle, Apriona germari (Wei et al., 2005). However, another possibility of cellulases of microbial origin produced by the microbes residing in the digestive tract of insects cannot be ruled out unless further work is carried out to investigate the reason for the multiplicity of cellulases in P. quatuordecimpunctata. There are very few, if any more, report on activity and multiplicity of cellulase enzyme throughout developmental stages of insects. Willis et al. (2010b) showed similar banding pattern of cellulase in zymography although the cellulase activities changes randomly throughout the developmental stages of Dissosteira carolina whereas in the present study the cellulase activity correlated well when measured in gel and by DNS method at different developmental stages of P. quatuordecimpunctata.
P. quatuordecimpunctata endo-β-1,4-D-glucanase showed the highest activity at the temperature of 35-45°C and 60°C (Fig. 5a) but it was stable up to 50°C and completely unstable at 55°C (Fig. 5b). The broad range in optimum temperature endo β-1,4-D-glucanase activity and the adverse effects of temperature on its stability may be due to inactivation of some multiple forms of enzymatic proteins at different temperatures. Zymography showed only one band with decreased intensity compared to control (without preincubation) when enzyme was preincubated at 45°C for 30 min before loading in gel and interestingly, preincubation at 55°C, no band appeared which coincided the instability of enzyme at that temperature (Fig. 5c). 60 and 35-45°C as optimum temperatures of endo-β-1,4-D-glucanase are also comparable to the optimal temperature of longicorn beetle, A. germari at 45-60°C (Lee et al., 2005; Wei et al., 2005) and cricket Teleogryllus emma at 40°C (Kim et al., 2008), respectively. Ni et al. (2007), Zhou et al. (2007) and Kim et al. (2008) also support the temperature stability of P. quatuordecimpunctata endo-β-1,4-D-glucanase at 20-50°C (Fig. 5b).
The acidic pH at which P. quatuordecimpunctata endo-β-1,4-D-glucanase exhibited maximum activity (pH 5.0) correlated well with those reported for other insects (Sugimura et al., 2003; Lee et al., 2005; Wei et al., 2006; Zhou et al., 2007; Kim et al., 2008; Rehman et al., 2009b). It has been reported that the treatment of cellulose with acids lead to generate ends and amorphous regions for enzymatic hydrolysis (Qian et al., 2003) and usually cellulases are present in the gut of insects which has acidic pH and the acidic pH optima could be related to the acidic environment of the gut (Zverlov et al., 2003). Because acid can keep some catalytic residues of Apriona germari endo-β-1,4-D-glucanase in an ionized form so that it can bind water molecule for the nucleophilic attack on β-1,4-glycosidic linkage of cellulose (Sami and Haider, 2007).
This study confirmed the presence of different cellulase (endo-β-1,4-D-glucanase) activities in the gut fluid of P. quatuordecimpunctata. Purification of individual cellulases and their characterization are necessary for better understanding of their properties. Further experiments are also necessary to determine whether these cellulolytic activities are endogenous to the insect or from other symbiotic organisms.
ACKNOWLEDGMENT
We are grateful to Mr. Santosh Mozumdar, M. Phil student, Department of Zoology, University of Chittagong for his assistance in identifying the insect.