Predominant contribution of an endogenous cellulase to the cellulolysis in the digestive system of grub of banana pseudostem weevil, Odoiporus longicollis (Coleoptera: Curculionidae)

doi:10.21203/rs.3.rs-38095/v2

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Predominant contribution of an endogenous cellulase to the cellulolysis in the digestive system of grub of banana pseudostem weevil, Odoiporus longicollis (Coleoptera: Curculionidae)

https://doi.org/10.21203/rs.3.rs-38095/v2

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published 14 Jun, 2023

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Insects have evolved with effective strategies to utilize cellulose as energy source by possessing novel cellulolytic enzymes which can be used as an optimal resource in the bioenergy sector. The study was aimed for evaluating the cellulolytic enzyme in the grub gut of banana pseudostem weevil, Odoiporus longicollis Olivier (Coleoptera: Curculionidae). Primarily, cellulase activity was localized in the gut system, in which the midgut showed the highest activity. The optimum temperature and thermo-tolerance of cellulase activity were found to be 60°C, and was stable at a pH between 5 and 6. Various concentrations of divalent cations (CaCl₂, MgCl₂, and CuCl₂) have differential enhancing and inhibitory effects on cellulase activity. The cellulase was purified using anion exchange chromatography. The specific activity of purified cellulase was 1190.46 U mg^− 1. The molecular weight of the cellulase was determined to be 47 kDa. The physicochemical parameters of purified enzyme were similar to that of enzyme activity of crude extract. Mass spectrometry results identified this cellulase was of GHF5 family. The gut microbial cellulase activity as exogenous source showed no competence comparatively.

Entomology

Animal Physiology

Cellulase

Coleoptera

Endoglucanase

Glycosyl hydrolase family 5 (GHF5)

Weevil

The predominance and successful distribution of plants are credited by their rigid cell wall composed of abundant cellulose polymers strengthened by hydrogen bonds and van der Waals forces. This complex glycoside is hydrolyzed into simple glucose by the action of cellulases such as endo-β-1,4-glucanases (EGs), cellobiohydrolases (CBHs) and β-glucosidases (BGs), among which EGs are common in nature (Lo et al. 2010). Interestingly, this hydrolysis mechanism has drawn attention in the bioenergy sector to produce alternate fuel by utilizing cellulose (Tomme et al. 1995), as current cellulolytic technologies in hydrolysing lignocelluloses demand improvement, especially in cost management. The limitations of the former are related to the enzyme stability and inhibitory agents, which opens up the search for novel enzymes (Kristensen et al. 2009). It is generally believed that only bacteria and fungi possess cellulolytic enzymes, especially gut symbionts, in eukaryotes. This old-fashioned thought eventually faded after the first publications of purified endogenous cellulase from the wood-feeding cockroach Panesthia cribrata and the termites Reticulitermes speratus and Nasutitermes takasagoensis that do not harbour the cellulolytic symbiont (Scrivener and Slaytor 1994; Tokuda et al. 1997; Watanabe et al. 1997). Recently, endogenous cellulases from the insect orders Coleoptera, Isoptera, Blattodea, Orthoptera, Lepidoptera, Diptera and Zygentoma have been reported (Pothula et al. 2019; Willis et al. 2010a). The glycosyl hydrolase family (GHF) is a major group in which different types of cellulases (endogenous and exogenous) are categorized accordingly. Insect cellulases belong to the four main GHFs such as, GHF1, GHF5, GHF9, and GHF45 (Calderón-Cortés et al. 2010; Tokuda 2019). The origin of endogenous cellulase in animals was explained by two hypotheses, vertical and horizontal transfer where genes are inherited from early metazoans and non-animal organisms, respectively. Symbiont-independent cellulose digestion is an ancestral mechanism for plant cell wall (PCW) digestion in insects, and this prospected them as an optimal source for novel cellulases (Martin 1991; Scrivener et al. 1989; Tokuda 2010; Watanabe and Calderón-Cortés et al. 2012). On the other hand, the use of cellulases as potential candidates for inhibition to control insect pests has also been in line of attention (Girard and Jouanin 1999).

Banana stem weevil or banana pseudostem borer Odoiporus longicollis Olivier (Coleoptera: Curculionidae), which is an economically important pest of banana plantations, feeds the plant during flowering and bunch formation stages (Padmanaban et al. 2001). It was first recorded in the Poonch and Rajouri districts of the Jammu region as a major pest of banana plants (Musa spp.) which is grown in patches. They breed throughout the year and likely do not undergo winter rest (Azam et al. 2010). Grubs are voracious feeders and tunnels into the pseudostem and feed on the internal content. The banana pseudostem is composed primarily of cellulose, followed by hemicellulose (Ma 2015). O. longicollis feeding such high dietary cellulose content must possess a cellulase enzyme either from endogenous, symbiont-dependent i.e., exogenous or from both the source to hydrolyse.

In this context, the present study is aimed at i) localizing the cellulolytic activity of both exogenous and endogenous sources from the gut of the grub of O. longicollis and ii) further purifying and characterizing the responsible enzyme, which may act as a novel source for applications in industry and other fields.

Insects

The fourth and fifth instar grubs were collected from the affected banana plants, in Pattambi, Palakkad, Kerala and suburban regions of Chennai, Tamil Nadu, India. The collected insects were identified using standard taxonomic keys.

Gut extracts

The collected larvae were wiped with ethanol and the head, whole gut, foregut, midgut and hindgut were separately dissected out in 0.9% physiological saline solution (Fig. 1). The gut contents, including food particles, were removed from the intestinal tract. For the assays and purification, dissected tissues treated with saline containing sodium azide (NaN₃) and ampicillin (1 mg/ml) was used. The dissected gut was extracted using a mortar and pestle with an equal volume (w/v) of sodium acetate buffer (pH 5.5). The extract was centrifuged at 12000 x g for 20 min at 4°C. The supernatant was collected and stored at -20°C. The microbe-free gut extracts upon validation in the Petri plates were used for the assays.

Protein estimation

The protein concentrations of gut extracts and purified cellulase were determined using the method established by Lowry et al. (1951) using bovine serum albumin as a standard.

Isolation of gut bacteria

The whole gut extract was streaked onto plates containing LB medium and incubated at 37°C for 24 h. The bacterial colonies were picked up, subcultured in LB medium broth, and restreaked for the isolation of a single colony. The obtained colonies were subjected for the DNA isolation and 16S rRNA amplification using the primers (Forward 5’-AGAGTTTGATCCTGGCTCAG-3’; Reverse 5’-ACGGCTACCTTGTTACGACTT-3’) for the species identity. The amplified products were sequenced and the sequences of identified bacterial isolates were deposited in NCBI and obtained with accession numbers. These bacterial isolates were used to check for the production of cellulase.

Cellulase-Congo red plate assay

The plates were prepared by adding nutrient medium containing NaNO₂, K₂HPO₄, MgSO₄.7H2O, KCl, FeSO₄, along with carboxy methyl cellulose (CMC) (HiMedia). Wells were made in the plate, and extracts of head, foregut, midgut, and hindgut were added to the wells and kept for 24 h of incubation. Simultaneously, the isolated bacterial colonies were spotted on cellulose plates and incubated at 37°C for 24 h separately. The plates were then stained with 1% Congo red for 20 min and destained with 1 M NaCl for 15 min. The clear zone observed was measured using the scale (Miller 1959).

Quantification of cellulase from gut extracts and isolated gut microbes

The isolated gut bacterial colonies were inoculated in the nutrient broth and incubated for 24 h and 48 h at 37°C. Then, the produced cellulase in the nutrient broth was separated from the bacterial colonies by centrifuging at 800 x g for 10 minutes. The supernatant was collected, and the activity of the produced cellulase was determined by the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). Gut extract (equal w/v) and bacterial colony-produced cellulase were mixed separately with an equal volume of 1% CMC (dissolved in sodium acetate buffer, pH 5.5) and incubated for 1 h at 60°C and 37°C, respectively. Then, an equal volume of DNS reagent was added to it. The reaction was ended by incubating the mixture at 90°C for 10-15 min. The dark brown colour was obtained and the amount of reducing sugars produced in these reactions was measured with a spectrophotometer at 540 nm using the DNS method. Various fixed concentrations of glucose were used as a standard.

Physicochemical characterization of gut extract

Optimum reaction temperature

To determine the optimal temperature, the whole gut extract of the larvae was incubated with 1% CMC in 0.1 M sodium acetate buffer (pH 5.5) at various temperatures ranging from 20°C to 80°C for 1 h using the DNS method (Miller 1959).

Thermal stability

Thermal stability was determined by preincubating the gut extract at various temperatures (20°C to 80°C) for 10 min. Then, the preincubated samples were determined for cellulase activity using the DNS method (Miller 1959).

pH optima

To determine the optimal pH, the gut extract of the larvae was mixed with 1% CMC in buffers of different pH values (4.0 - 8.0) and the activities of the solutions were measured using the DNS method (Miller 1959). The buffers used include acetate (pH 4.0-6.0), phosphate (pH 7.0) and Tris-HCl (pH 8.0).

Effect of divalent cations on cellulase activity

Divalent cations such as CaCl₂, MgCl₂, and CuCl₂ were individually applied at different concentrations of 5, 10, 15, and 20 mM. These additives were dissolved with 1% CMC assay buffer (0.1 M sodium acetate buffer, pH 5.5) and the amount of total reduced sugar was measured by the DNS method (Miller 1959).

Purification of cellulase

Batch assay

The binding ability of cellulase enzyme in the whole gut extract of O. longicollis was analysed with ion-exchange column matrices using batch assays to find a suitable matrix for the isolation of these molecules. The column matrices used were DEAE-Sepharose CL-6B and CM-cellulose. DEAE-Sepharose CL-6B (200 µl) was extensively washed with 20 mM phosphate-buffered saline (PBS) (pH 7.2), whereas CM-Cellulose was washed extensively using 20 mM sodium acetate buffer (pH 4.6). Then, an equal volume of gut extract was applied and incubated at room temperature for an hour. The residual cellulase activity was checked by the DNS method to determine the cellulase binding capacity of the column matrices used.

Anion exchange chromatography

Cellulase from the whole gut extract of O. longicollis was purified by anion exchange chromatography on DEAE-Sepharose CL-6B. A 13 × 1 cm diameter and 1 ml bed volume column of DEAE-Sepharose CL-6B was equilibrated with 20 mM PBS, pH 7.2, at a flow rate of 20 ml/h. A total of 750 µl extract diluted one-fold with equilibration buffer was loaded onto the column at a flow rate of 1 ml/h and after adsorption, the column was washed using 5 ml of 20 mM PBS, pH 7.2, at a flow rate of 5 ml/h to remove the unbound proteins. Bound proteins were eluted with stepwise sodium chloride elution (30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM) in 20 mM PBS, pH 7.2, at a flow rate of 20 ml/h. Fractions contained cellulase were eluted in 50 mM NaCl, and their absorbance were detected at 280 nm. The fractions were analyzed using Native-PAGE.

Molecular characterization of purified cellulase

Electrophoresis

The purified cellulase fraction was analyzed in both a native-PAGE Tris/glycine (pH 8.3) buffer system (Maurer 1971) and denatured SDS-PAGE (Laemmli 1970). After electrophoresis, the gel was stained with Coomassie brilliant blue-R250 and silver nitrate.

Zymogram

The zymogram was performed by following Schwarz et al. (1987) with minor modifications. Briefly, 12% SDS-PAGE with 0.5% CMC was used for zymogram. Samples were mixed with SDS sample buffer and kept at 4°C for 1 h for passive denaturation. Then, the samples were loaded and run for electrophoresis in a refrigerator. After running, the gel was kept in 20% isopropanol (20 ml isopropanol in 80 ml PBS buffer) for 20 min. It was then washed with PBS 6 times in 10 min intervals and incubated at 37°C in PBS overnight. The gel was stained with 0.1% Congo red dye at room temperature for 30 min and destained with 1 M NaCl at room temperature for 15-20 min. Finally, for an improved visualization, 0.2% acetic acid was added (Waeonukul et al. 2007).

Physicochemical characterization of the purified cellulase: Optimum temperature, thermostability and pH optima

The methods for optimal temperature, thermal stability and pH optima for purified cellulase were carried out as mentioned for the crude gut extract.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis

The isolated enzyme was analysed for peptide mass fingerprinting to yield peptide sequence using the standardized procedure for MALDI-TOF MS spectrometry (Wilm et al. 1996). The data obtained under the mass spectrum were subjected to the database (NCBI non-redundant/Swiss-Prot) search using MASCOT (http://matrixscience.com) analysis. The calibrated peptide masses were searched with 100-500 ppm mass accuracy.

Multiple sequence alignment using ClustalW

The multiple sequence alignment program, ClustalW (Thompson et al. 1994) was chosen for alignments of observed peptide sequences from the MASCOT search engine for the purified enzyme subjected to MALDI-TOF MS.

Statistical analysis

The experiments were executed in triplicates and their results were subjected to mean and standard error of the mean (x̅ ± SE) (Sokal and Rohlf 1973). The differences in mean were evaluated using two-way ANOVA followed by GraphPad Prism (Version 8.0.2). Tukey's multiple range analysis was employed to determine the statistical differences between control and test. The p-value p < 0.05 was set as a significant level.

Congo red plate assay

CMC degradation by different grub tissue extracts, such as the whole gut, head, foregut, midgut and hindgut, was tested in a CMC-agar plate with Congo red dye. CMC degradation was indicated by a clear zone around the well (Fig. 2A). The measurements of zone of activity are given in Table 1. CMC degradation by four gut bacterial isolates Enterobacter cloacae (OM630475), Alcaligenes faecalis (OM630484), Exiguobacterium acetylicum (OM630524) and Bacillus cereus (OM670085) were also tested for the production of cellulase. The obtained zone of activity is presented in Table 1. The above-mentioned identified bacteria obtained with NCBI accession numbers were initially involved in the process of 16S rRNA amplification that produced a 1.5 kb sized product followed by the sequencing.

Quantitative analysis of cellulase activity

The quantitative analysis of cellulase enzyme from head, foregut, midgut, hindgut and from the gut bacterial isolates E. cloacae (OM630475), A. faecalis (OM630484), E. acetylicum (OM630524) and B. cereus (OM670085) were performed using the DNS method. The highest cellulase activity was shown by the midgut as 2858 U min^-1mg^-1, followed by the foregut and head. The hindgut showed the lower activity of about 2479 U min^-1mg^-1,suggesting a 13.26% decrease in the activity of the hindgut in comparison to the midgut activity. The whole gut extract showed the activity of 2800 U min^-1mg^-1 which is slightly lower than the midgut activity due to the saturation point of the enzymatic activity in the whole gut extract. However, the difference between the activities of gut extracts and gut bacteria was marked accounting of about approximately 90%, implying the predominant contribution of cellulase activity by the gut. On the other hand, there was also a noticeable change in the fold of cellulase activity among the bacterial isolates E. cloacae, A. faecalis, E. acetylicum and B. cereus during various hours of incubation. A difference of 28.5%, 3.54% and 19.67% decrease in cellulase activity was demonstrated during 48 h of incubation when compared to 24 h in the case of isolates E. cloacae, A. faecalis, E. acetylicum, respectively. However, B. cereus failed to show activity in either case (Table 2).

Physicochemical characterization of crude extract

The optimum reaction temperature and thermal stability

Gut extract with the substrate was tested in the range of 20-80°C. The reaction mixture showed the highest activity of 2800 U min^-1mg^-1 at 60°C which was determined to be the optimum temperature for further studies. The thermal stability of the cellulase was tested by the preincubation without the substrate in the same temperature range as above. The activity was stable between 50°C and 60°C, with the highest activity of 2712 U min^-1mg^-1at 60°C (Fig. 2B).

Effect of pH

The effect of pH on the cellulase activity of the whole gut extract was determined at various pH ranged from pH 4.0 to 8.0. The cellulase activity was stable in the optimum pH range between 5 and 6 (Fig. 2B).

Effect of divalent cations

The metal ions examined have stimulatory and inhibitory effects on the cellulase activity of crude extract. The enzyme activity was increased by 35.71% over control by 15 mM Cu²⁺. While, 10 mM Mg²⁺ and Ca²⁺ stimulate the enzyme activity by 13.04% and 7%, respectively. However, in the presence of 20 mM of the metal cations, Ca²⁺ and Mg²⁺, an inhibitory effect was observed by 71.42% and 28.21%, respectively (Fig. 2C).

Purification of cellulase from the gut extract of O. longicollis

Batch assay

DEAE-Sepharose CL-6B and CM-cellulose were chosen as matrices for anion and cation exchange chromatography, respectively. The whole gut extract with cellulase activity of 2800 U min^-1mg^-1 was adsorbed with matrices to analyse the binding ability. The unbound and stepwise eluted fractions (100 mM to 500 mM NaCl) were checked for cellulase activity. The unbound fraction from the anion exchange matrix did not show any activity, followed by eluted fractions that were observed with cellulase activity of 1460 and 550 U min^-1 mg^-1in 100 mM and 200 mM and no activity in subsequent elution concentrations. The unbound fraction of the cation exchange matrix showed a cellulase activity of 2650 U min^-1mg^-1,which was almost equal to the activity of the applied sample, and the eluted fractions did not show any activity (Table 3). Overall, it was inferred that cellulase had an effective binding ability with the DEAE-Sepharose CL-6B matrix and showed no binding to CM-cellulose.

Anion exchange chromatography

Based on the batch assay results, DEAE-Sepharose CL-6B was found to be an ideal matrix for the purification of cellulase; therefore, anion exchange chromatography was adopted. A sample volume of 1.5 ml (3.468 mg of protein) with cellulase activity of 2800 U min^-1mg^-1was passed through the column. The unbound effluent was washed until the baseline value was zero at 280 nm, indicating that the matrix adsorbed (saturated) all the activity from the gut extract. The cellulase fraction was eluted at a 50 mM NaCl concentration. Three out of four fractions collected showed significant cellulase activity of 1166 U min^-1mg^-1. A typical elution profile depicting the isolation of cellulase is shown (Fig. 3A). As summarized (Table 4), this anion exchange chromatography method adopted for the purification of cellulase from the whole gut extract of the larva of O. longicollis resulted in about 41.66% recovery with specific activity increased up to 1190.46 U min^-1mg^-1.

Molecular characterization of purified cellulase

Analysis of the purified cellulase in native-PAGE

The protein profiles of both gut extract and purified cellulase from the grub of O. longicollis were analysed using discontinuous PAGE (8%) under non-denaturing conditions (Fig. 3B). The purified enzyme was visualized as a single protein band without any contamination in the polyacrylamide gel upon staining with Coomassie brilliant blue (for gut extract) as well as silver nitrate (for the purified enzyme).

Zymogram

The cellulase zymogram analysis was performed for whole gut extract and eluted fractions from 50 mM NaCl. The bands corresponding to enzyme activities appeared and visualised using 0.1% Congo red dye (Fig. 3B).

Analysis of the purified cellulase protein in SDS-PAGE

The protein profiles of both gut extract and purified cellulase from the grub of O. longicollis were analyzed using discontinuous PAGE (12 %) under reducing conditions in the presence of SDS and β-mercaptoethanol (Fig. 3B). A monomeric band without any polypeptide subunits appeared in the gel. The molecular weight of the band corresponded to approximately 47 ± 2 kDa. Hence, the molecular weight of the purified cellulase was determined to be 47 ± 2 kDa.

Physicochemical characterization of purified cellulase

The optimum reaction temperature and thermal stability

Purified cellulase with the substrate was tested in the range of 20 - 80°C. The reaction mixture showed the highest activity of 1166 U min^-1mg^-1 at 60°C which was determined to be the optimum temperature for cellulase activity. The thermal stability of the purified cellulase was tested by preincubation without the substrate in the same temperature range as above. The activity was stable around 40°C to 60°C, with the highest activity of 2800 U min^-1mg^-1at 60°C (Fig. 3C).

Effect of pH

The effect of the pH on the activity of purified cellulase was determined at various pH values ranging from pH 4.0 to 8.0. The cellulase activity was stable in the optimum pH range between 5 and 6 (Fig. 3C).

MALDI-TOF MS

The subjection of 47 kDa purified cellulase to MALDI-TOF MS analysis to find the relevant protein matching yielded homology to the known cellulases. The MALDI-TOF MS fingerprints of the tryptic-digested purified cellulase with different m/z ratios in “lift” mode were obtained (Fig. 4A). The acquired short peptide sequence “WLSGVDK” of a fragment (m/z ratio of 804.3890) identified significant homology to endoglucanase with 35% of protein coverage (Fig. 4B). The matched endoglucanase belonged to the cellulase family, glycosyl hydrolase 5 families (GHF5), which are also typically found in coleopterans. Hence, the various sequences of m/z 804.3890 were further extracted in all entries of the search and aligned with coleopteran GHF5 cellulases, which showed considerable homology (Fig. 4B).

The cellulase that hydrolyses the CMC is denoted as endoglucanase as it prevents them from attacking the chain ends where it is substituted with carboxymethyl groups in place of open- ended sugar terminals. Such limitation is similar to the native cellulose present in the diet (Watanabe and Tokuda 2001). Due to the presence of methylated hydroxyl groups in CMC facilitating water solubility, CMC has been widely used for the documentation of cellulase activity in assays (Willis et al. 2010a). The application of CMC in electrophoresis has its advantage over plate assays by visualizing the protein bands responsible for cellulolytic activity directly under partially denatured and low-temperature conditions (Schwarz et al. 1987). On the other hand, the crystalline part of cellulose, which is acted upon by cellobiohydrolase, is usually found in hardwoods that by digestion produce cellobiose (Yoshimura et al. 1996). The banana plant is known to have pseudostem and by the insect i.e., O. longicollis possessing mandibles which crush the stem into microparticles eliminates the assumption of crystalline cellulose presence and its testing in this study for O. longicollis. Hence, this study proceeded with the standard CMC as a substrate. An extensive study carried out with 68 phytophagous insects fitting to the 8 orders reported that gut fluids act more on CMC than crystalline cellulose. Moreover, the coleopteran order displayed low levels of activity against crystalline cellulose, unlike CMC, implying the existence of phylogenetic relationships rather than feeding habits (Oppert et al. 2010). The secretion of a cellulase completely by an insect evolved independently in phyla. This is reflected in the different types of cellulase in the divergent taxa (Liu et al. 2015). The observation of cellulase activities in insects that do not have symbiotic associations, symbiont-free sites such as the foregut and midgut, and sterile hepatopancreas of gastropods together concluded that cellulases are secreted endogenously (Watanabe and Tokuda 2001). The absence of the cellulase gene in some important model insects argues about the critical interpretation of genome sequences and sampling artefacts (Davison and Blaxter 2005). To overcome this, the pyrosequencing was approached to unravel the presence of 167 plant cell wall degrading enzymes (PCWDE) lying to eight different enzyme families in beetles (Pauchet et al. 2010). Willis et al. (2011) reported the first endoglucanase from an insect model organism Tribolium castaneum. The order coleoptera has been identified with the occurrence of endogenous cellulases (Martin 1991). Many endoglucanases have been studied exclusively in Coleopterans (Busconi et al. 2014; Calderón‐Cortés et al. 2010; Lee et al. 2004; Lee et al. 2005; Wei et al. 2006; Xia et al. 2013).

The cellulase characterized in this study showed higher activities in all the tested gut tissues, with midgut being highest followed by the foregut, head and hindgut. This could be due to the structural integrity of midgut that has numerous infoldings to increase the surface area for cellulolysis digestion (Shelomi et al. 2014). The foregut and midgut are the major sites for the expression of large quantities of endogenous cellulase in coleopteran species such as Apriona germari, Phaedon cochleariae and Psacothea hilaris (Girard and Jouanin 1999; Sugimura et al. 2003; Wei et al. 2006). Unlike the hindgut, where either bacteria- or protozoa-based cellulolysis occurs, the midgut majorly holds up one type of endo-β-1,4-glucanase (Tokuda et al. 1997; Tokuda et al. 1999). Cellulase expression was determined throughout the entire digestive tract of the cricket, including the hindgut (Kim et al. 2008). While focusing on relative endoglucanase expression, it was higher in head fluids than in gut tissue of T. castaneum whereas in Dissosteira carolina it was higher in foregut and midgut regions (Willis et al. 2010b; Willis et al. 2011). Contemplating both head and gut regions, highest cellulase activity for Zygentoma and termites was found in the head, foregut and midgut tissues (Pothula et al. 2019; Tokuda et al. 1997). But on the other hand, speaking of site-specificity, the foregut had predominant activity in the wood-eating cockroach P. cribrata (Scrivener et al. 1989). Overall, it implies that cellulase is differentially expressed in head, foregut and midgut of insects.

The highest crude cellulase activity in this study was 2858 U min^-1mg^-1 in the midgut. To date, in insects, the highest crude enzymatic activity ever reported in Zygentoma species was around 2500 (Units/g), suggesting it as an insect model for high cellulase activity (Pothula et al. 2019). O. longicollis showing similar higher cellulase activity could also be envisioned as a model for cellulolytic system.

Only a mere number of bacteria seemed to harbour the entire digestive tract of O. longicollis. The reason for it remains elusive and could be influenced by many unknown factors, however, gut regions displaying cellulolytic activity in Orthoptera and Phasmotodea also had low numbers of bacteria (Cazemier et al. 1997). The gut tissues used in this study for further assays (except for gut bacterial isolation) were extracted with aseptic measures such as the use of antibiotic and sodium azide. No bacterial growth was observed in plates with these extracts. In previous reports, aseptic conditions were maintained in the rearing of insects and removal of the gut to assure the elimination of cross-contamination of symbiont cellulase. The activity was not affected even after the removal of bacteria (Lasker and Giese 1956; Lo et al. 2010; Scrivener et al. 1989; Tokuda and Watanabe 2007; Yokoe and Yasumasu 1964). Attempts to isolate cellulose-degrading bacteria seemed to be unsuccessful or drawn mixed success in termites (O’Brein and Slaytor 1982). The isolated gut bacteria’s cellulase activity in the present study was extremely below par and negligible when compared to the gut extract activity, implying a clear pattern distinguishing endogenous from its counterpart. Nakashima et al. (2002) and Tokuda et al. (2007) in wood-feeding termites have proven the existence of a dual cellulose-digesting system where both endogenous and symbiotic cellulases contribute. As in this study, the contribution of cellulolytic digestion from symbionts seemed to be trace as mentioned. A study on a range of digestive enzymes from the phytophagous beetle P. cochleariae encountered the production of cellulases by microorganisms only in minor fractions, suggesting that endogenous cellulase played a major role in PCW degradation which is in accordance with O. longicollis (Girard and Jouanin 1999). Because the hindgut is highly unlikely to produce endogenous cellulase as it does not possess secreting cells, we cannot be conclusive about its cellulolytic function (Chapman 1998). With head, foregut, and midgut showing the enormous amount of cellulolytic activity in O. longicollis, we presume that a secreting out of enzyme to the hindgut from the upper digestive tract could be a reason that the hindgut showed some activity. At times, the endoglucanases present in the salivary glands or upper digestive tract are presumed to be secreted in the digestive tract and reach the hindgut, where the latter shows cellulase activity (Kim et al. 2008; Nakashima and Azuma 2000).

Ion exchange chromatography (IEC) has been used to purify cellulase primarily or with a combination of other strategies (Eriksen and Goksøyr 1977; Umezurike 1979). Anion exchange chromatography (AEC) was adopted to purify EG from the mulberry longicorn beetle A. germari, T. castaneum (Lee et al. 2004; Rehman et al. 2009) and glycosidases from the palm weevil Rhynchophorus palmarum (Yapi et al. 2009; Yapi et al. 2015). AEC was used for purifications of several other endoglucanases and glycosidases from insects, other invertebrates and even insect gut flagellates (Li et al. 2003; Li et al. 2005; Marana et al. 2000; Scrivener and Slaytor 1994; Willis et al. 2010b; Yang et al. 2004). The molecular weight of the purified cellulase of O. longicollis is 47 kDa. This is in agreement with molecular weights of cellulases from Coleoptera that were determined to be approximately 49.5 kDa, 29 kDa, 58.66 kDa, 25 kDa, and 47 kDa in T. castaneum, A. germari, Monochamus alternatus, Batocera horsfieldi and P. hilaris, respectively (Lee et al. 2004; Mei et al. 2016; Rehman et al. 2009; Sugimura et al. 2003; Wei et al. 2006; Willis et al. 2011; Xia et al. 2013); those of other order insects including P. cribrate, T. emma, and D. carolina were approximately 47-55 kDa (Kim et al. 2008; Scrivener and Slaytor 1994; Willis et al. 2010b) and those of other invertebrates (nematodes and gastropods) were approximately 44 to 50 kDa (Anzai et al. 1984; Anzai et al. 1988; Li et al. 2005; Pothula et al. 2019; Smant et al. 1998).

The specific activity of purified cellulase from O. longicollis is 1190.46 U/mg. In comparison with coleopterans and other insect orders, the specific activities of EGs seemed to be 12.11 U/mg, 992 U/mg, 812 U/mg, 928 U/mg, 1030.87 U/mg, and 1037 U/mg (Lee et al. 2004; Lee et al. 2005; Mei et al. 2016; Wei et al. 2006; Xia et al. 2013; Yang et al. 2004). The specific activities of multiple cellulases from other invertebrates were 7.60, 6.67, 6.45, and 0.30 U/mg (Yin et al. 2011).

The influence of divalent cations on cellulase activity of O. longicollis yielded differential patterns without proportionality in terms of activity. Cellulase from the beetle B. horsfieldi has also shown similar results, where ions and their concentration produced diverse effects on the activity (Mei et al. 2016), and in the case of the xylophagous larvae of Ergates faber (Coleoptera: Cerambycidae), no influence occurred (Chararas et al. 1983). However, cellulase activity from the red palm weevil Rhynchophorus ferrugineus was enhanced by the application of Ca²⁺ and Mg²⁺,which is quite similar to the results of this study (Vatanparast et al. 2014). The optimum temperature and thermal stability of a cellulase from O. longicollis were 60°C with an optimal pH between 5 and 6. Many insects and invertebrate cellulases reported to date have been stable at approximately 40-50°C with optimal pH values from 4 to 6.5 (Kim et al. 2008; Mei et al. 2016; Xia et al. 2013; Yin et al. 2011). However, in some cases, high stability was observed up to 55-60°C (Lee et al. 2004, Lee et al. 2005; Wei et al. 2006) with at certain instances even after 24 h of incubation (Li et al. 2005) and in alkaline pH (Willis et al. 2011). The optimal pH 5-6 determined in this study is close to the gut luminal pH of O. longicollis, such as 6.0-6.4 in the foregut, 6.8-6.9 in the midgut, and 6.1-6.4 in the hindgut (Singh and Prasad 2010). These physiological pH values, which were the same in experimental conditions, are necessary for the function of cellulases (Sugimura et al. 2003). The favourable pH in the hindgut of O. longicollis implies that cellulase with a stability range between pH 5 and 6 could effectively exhibit activity in the hindgut as well.

The partial peptide sequences obtained from the mass spectrometric analysis identified that the purified cellulase of O. loingicollis exhibited homology to endoglucanase belonging to the GHF5 family of cellulase. The first report of GHF5 (a 47 kDa endo‐β‐1,4‐glucanase) from arthropods was from a Coleopteran insect, P. hilaris (Sugimura et al. 2003). Endoglucanases belonging to GHF5 have also been reported in coleopterans such as A. germari (47 kDa) and Mesosa myops (Liu et al. 2015; Wei et al. 2006). GHF5 in coleopterans lies in a separate clade where nematode and beetle cellulase resides in a way proposing that GHF5 could have diverged between prokaryotic and early eukaryotic cellulases (Calderón‐Cortés et al. 2010; Kyndt et al. 2008). Cellulase sequences of nematode and longhorn beetle show considerable similarity in phylogeny analysis (Lo et al. 2003). Insect GHF5 and GHF45 have its place in very ancient gene families, and specifically, GHF5 is prospected to be evolved from a common ancestor (bacteria) through horizontal gene transfer (Shin et al. 2022; Watanabe and Tokuda 2010).

Industrially used cellulolytic enzymes must be stable in bioreactors by withstanding heat and acidic conditions (Willis et al. 2010a). The physicochemical parameters of cellulase from O. longicollis infer that it is a highly stable enzyme. On the other hand, a study assessing these digestive enzymes in coleopteran pests to assort them as potential inhibiting targets was also of interest (Girard and Jouanin 1999). Along with these commercial aspects, the role of endogenous cellulase in insects could also unearth new possible functions like substitutions in the cellulase sequence in deuterostomes might result in gaining new functions such as chitinases involved in innate immunity, no wonder that termites expressing cellulases in many organs might open up a new hypothesis (Davison and Blaxter 2005). The new findings of multifunctional cellulases in insects such as xyloglucanolytic cellulase and a specific ancestral cellulase that lacks cellulolytic ability in almost all insect orders necessitate critical investigation with expansion of the enzyme hypothesis (Shelomi et al. 2020). These divergent approaches to insect cellulases indicate their multifaceted potentialities that hold a strong stand in research pursuits.

The findings of this study demonstrate the presence of a stable endogenous cellulase belonging to the GHF5 family with predominant activity over an exogenous type, thereby signifying the independent mode of cellulose digestion in the gut system of O. longicollis. Moreover, characterisation in the perspective of commerciality in the foreseeable future, this cellulase would possess great potential in application prospects.

Conflict of interest

The authors declare that there are no competing interests.

Acknowledgments

The first author is gratified by the support of the University Grants Commission (UGC), India for the NET-SRF (CSIR-UGC) fellowship (Certificate Sr. No. 2121530460, Ref. No: 20/12/2015(ii) EU-V). The authors acknowledge the infrastructure support provided in the department established under the DST-FIST programme. We would like to thank the Molecular Biophysics Unit (Proteomics facility), Indian Institute of Science for the instrumental service of MALDI‐TOF‐MS analysis.

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Table 1 Analyses of cellulase activity (zone of activity) of gut extracts and gut bacterial isolates from the grub of O. longicollis by congo red plate assay

S. No.	Gut extract^a	Cellulase activity^b (Zone of activity in mm)
1.	Whole gut extract	27±2
2.	Foregut	27±1
3.	Midgut	28
4.	Hindgut	22
5.	Head	24
	Gut bacterial isolates^c
1.	Enterobacter cloacae	2
2.	Alcaligenes faecalis	3
3.	Exiguobacterium acetylicum	3
4.	Bacillus cereus	0

^a100 µl of gut extracts that contained 578 µg, 550 µg, 570 µg, 600 µg, 580 µg of protein in extracts of whole gut, foregut, midgut, hindgut and head, respectively. ^bData based on median value of three separate determinations. ^cBacterial isolates were spotted onto the plates. Sodium acetate buffer was used as a negative control.

Table 2 Quantitative analyses of cellulase activity of gut extracts and gut bacterial isolates from the grub of O. longicollis by DNS method

S. No.	Gut extract	Cellulase activity^a,b ( U min^-1mg^-1 )
1.	Whole gut extract	2800
2.	Foregut	2800
3.	Midgut	2858
4.	Hindgut	2479
5.	Head	2625
	Gut bacterial isolates	24 h	48 h
1.	Enterobacter cloacae	207	150
2.	Alcaligenes faecalis	311	300
3.	Exiguobacterium acetylicum	311	250
4.	Bacillus cereus	0	0

^aOne unit of cellulase activity is defined as the amount of enzyme that releases 1 µmol of glucose min^-1. ^bData based on the median value of three separate determinations.

Table 3 Batch profile of the binding ability of the column matrix to cellulase in gut extract^a

Gut extract	Cellulase activity^b in U min^-1mg^-1
	2800
	Unadsorbed	100 mM	200 mM	300 mM	400 mM	500 mM
Incubation with DEAE Sepharose CL-6B	0	1460	550	0	0	0
Incubation with CM cellulose	2650	0	0	0	0	0

^aData represent the mean values of three separate experiments.

^bOne unit of cellulase activity is defined as the amount of enzyme that releases 1 µmol of glucose min^-1.

Table 4 Summary of purification of cellulase from the gut extract of O. longicollis by anion exchange chromatography DEAE Sepharose CL-6B^a

Sample

volume

(ml)

Total protein (mg)

Total activity (units)^b

Specific activity

(units/mg of total protein)

Purification

fold^c

Recovery

(%)^d

Diluted crude gut extract

(one-fold)

1.5

3.468

800

230.68

100

Eluates

0.280

333.33

1190.46

5.1

41.66

^aData represent the mean values of three separate experiments.

^bOne unit of cellulase activity is defined as the amount of enzyme that releases 1 µmol of glucose min^-1.

^cPurification fold = Specific activity of total isolated protein/Specific activity of the crude protein.

^dRecovery of activity (%) = (Total activity of isolated protein/Total activity of crude protein) x 100.

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published 14 Jun, 2023

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Predominant contribution of an endogenous cellulase to the cellulolysis in the digestive system of grub of banana pseudostem weevil, Odoiporus longicollis (Coleoptera: Curculionidae)

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Abstract

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Introduction

Materials And Methods

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Discussion

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