cDNA cloning, expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

Gene 514 (2013) 62–68

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cDNA cloning, expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi Dingguo Xia a, b, Yadong Wei b, Guozheng Zhang b, Qiaoling Zhao b, Yeshun Zhang b, Zhonghuai Xiang a, Cheng Lu a,⁎ a b

Institute of Sericulture and System Biology, Southwest University, Chongqing 400716, China Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212018, China

a r t i c l e

i n f o

Article history: Accepted 24 August 2012 Available online 29 November 2012 Keywords: Batocera horsfieldi Beetle Cellulase cDNA cloning Insect Enzymatic activity

a b s t r a c t In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Cellulose, the most abundant organic compound mainly produced by plants (del Campillo and Bennett, 1996), is composed of repeating glucose units linked by β-1,4-glycosidic bonds (Carpita and Gibeaut, 1993). In recent years, cellulose has attracted worldwide attention as a major renewable resource and raw material that can be converted into bio-based products and biofuels (Taherzadeh and Karimi, 2008), such as ethanol and biogas. Although cellulose can be an energy and chemical resource, it is not useful in its polysaccharide form. It must first be biologically degraded by several cellulases and then converted into smaller molecules, i.e., glucose, ethanol, and other biochemicals (Li et al., 2009). Therefore, cellulose utilization as a source of bioenergy

Abbreviations: Ag-Egase I, β-1,4-endoglucanase I from the beetle, Apriona germari; Ag-Egase II, β-1,4-endoglucanase II from the beetle, Apriona germari; Bh, Batocera horsfieldi; Bh-EGase II, β-1,4-endoglucanase II from the beetle, Batocera horsfieldi; Bm, Bombyx mori; BmN cell, Bombyx mori N cell; BmNPV, Bombyx mori nucleopolyhedrovirus; BSA, bovine serum albumin; CMC, carboxymethyl cellulose; C. tremulae, Chrysomela tremula; DNS, dinitrosalicylic; D. ponderosae, Dendroctonus ponderosae; EGase, endoglucanase; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GHF, glycoside hydrolase family; G. zeae, Gibberella zeae; LB, Luria Bertani; L. decemlineata, Leptinotarsa decemlineata; M. circinelloides, Mucor circinelloides; O. albomarginata chamela, Oncideres albomarginata chamela; ORF, open reading frame; PBS, phosphate buffer solution; PFU, plaque forming units; RACE, rapid amplification of cDNA ends; R. oryzae, Rhizopus oryzae; R. speratus, Reticulitermes speratus; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SOC, super optimal broth with catabolite repression; S. oryzae, Sitophilus oryzae; TMB, tetramethylbenzidine. ⁎ Corresponding author. Tel.: +86 23 68250346; fax: +86 23 68251128. E-mail address: [email protected] (C. Lu).

and other bio-based products is restricted by cellulose degradation. Cellulose consists of composite forms of highly crystallized microfibrils among amorphous matrices, and is not amenable to enzymatic hydrolysis (del Campillo and Bennett, 1996). Cellulases are multisubunit complexes composed of endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and cellobiases (EC 3.2.1.21). Cellulose is efficiently hydrolyzed by the synergistic action of three major types of cellulolytic enzymes (Béguin and Aubert, 1994; Henrissat et al., 1985). The ability of insects to digest cellulose indicates that cellulases are produced by the insects themselves (Girard and Jouanin, 1999; Lee et al., 2005; Nakashima et al., 2002; Tokuda et al., 1999; Watanabe et al., 1998), from symbiotic organisms harbored in their guts (Ohtoko et al., 2000), or both (Breznak and Brune, 1994; Ohtoko et al., 2000; Scharf et al., 2003, 2005; Watanabe et al., 1998). Thus far, genes encoding endogenous insect cellulolytic enzymes have been isolated from three beetles (Girard and Jouanin, 1999; Lee et al., 2005; Sugimura et al., 2003) and a few termites (Girard and Jouanin, 1999; Nakashima et al., 2002; Tokuda et al., 1999; Watanabe et al., 1998). These cellulases belong to three glycosyl hydrolase families (GHFs), namely, GHF 45 (Phaedon cochleariae beetle), GHF 5 (Psacothea hilaris beetle), and GHF 9 (termites and cockroaches). These three GHFs are structurally unrelated and likely to have independent evolutionary origins (Sugimura et al., 2003). In the present paper, we report the gene structure, expression, and enzyme activity of a cellulase (Bh-EGase II) belonging to GHF 45 from Batocera horsfieldi. Bh-EGase II cDNA from B. horsfieldi was expressed functionally in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II was assayed for enzymatic properties.

0378-1119/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.08.044

D. Xia et al. / Gene 514 (2013) 62–68

2. Materials and methods 2.1. Animals Beetles (Batocera horsfieldi, Coleoptera: Cerambycidae) were collected in mulberry fields and reared on an artificial diet as described previously (Yoon and Mah, 1999). The silkworms (commercial name: 54A) used were maintained and preserved in laboratories in the Sericultural Research Institute, Chinese Academy of Agricultural Sciences. Silkworm larvae were reared on fresh mulberry leaves at 25 °C, 65% ± 5% relative humidity, and a 12 h light:12 h dark photoperiod. When the larvae grew to the fifth instar, they were divided into several groups for recombinant virus DNA injection before feeding.

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electrophoresis on 1% (w/v) agarose gel and then cloned into a pMD18-T Easy vector (TaKaRa, Japan). The nucleotide sequence of the cloned Bh-EGase II gene was determined by the dideoxy chaintermination method with a nucleotide sequencer.

2.4. Cloning of Bh-EGase II ORF The specific primers for the Bh-EGase II ORF, 5′-GCGGGATCCATGAA GGTATTGTTGGCA-3′ and 5′-GTCGAATTCTTAGTGATGGTGATGGTGATG TGAATAATTGCATCCGGA-3′ (six histidines are underlined), were designed using the first-strand cDNA from B. horsfieldi beetle midgut as the template. The Bh-EGase II ORF was cloned by PCR and subsequently cloned into a pMD18-T Easy vector (TaKaRa). The resulting plasmid was named T-Bh-EGase II and sequenced.

2.2. cDNA library screening, nucleotide sequencing, and data analysis Total RNA was extracted from B. horsfieldi beetle midgut with Trizol reagent (Invitrogen, USA), and the first-strand cDNA was synthesized using an M-MLV RTase cDNA Synthesis Kit (TaKaRa, Japan). According to the conserved sequences at both ends of insect cellulase genes (GenBank AY741064, AY451326, and GU001942), the B. horsfieldi EGase II gene was amplified with ExTaq DNA polymerase using the sense primer 5′-ACTGTTGCAAGCCATCATG-3′ and antisense primer 5′-GGGGATGGCGATGTCGA-3′ with the first-strand cDNA as the template. Amplification was performed under the following conditions: pre-denaturation for 5 min at 95 °C; 30 cycles of denaturation for 50 s at 94 °C, annealing for 50 s at 56 °C, and extension for 2 min at 72 °C; and a final extension for 7 min at 72 °C. The PCR product was confirmed by electrophoresis on 1% (w/v) agarose gel and subsequently cloned into pMD®18-T vector. The nucleotide sequence was determined using a BigDye Terminator Cycle Sequencing Kit and an automated DNA sequencer (model 310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Biosystems, Foster City, CA, USA). Based on the sequencing result, 5′ RACE specific primers (5′-CGCCTTCTTCACAAGCCGATTT-3′ and 5′-GTTGCTGATCGGAACACATGTA-3′) and 3′ RACE specific primers (5′-TTCAAGTGACCAACACAGGCAG-3′) were designed. The experiments for the full cDNA of the B. horsfieldi EGase II gene were carried out using a BD SMART™ RACE cDNA Amplification Kit according to the user's manual. GenBank databases were searched for sequence homology using the BLASTX and BLASTN programs of the NCBI (http://www.ncbi. nlm.nih.gov/BLAST). The ClustalW program of Molecular Evolutionary Genetics Analysis (MEGA) 4.0 (Tamura et al., 2007) was used to align the amino acid sequences of cellulases. Phylogenetic analysis was performed using MEGA 4.0 with the minimum evolution and adjacent merge algorithms. The accession numbers of the sequences in GenBank are as follows: Apriona germari cellulase Ag-EGase II (AY451326), A. germari Ag-EGase I (AY741064), the borer beetle Oncideres albomarginata chamela endo-beta-1,4-glucanase (GU001942), Leptinotarsa decemlineata (HM175845), Chrysomela tremula (HM175780), Sitophilus oryzae (HM175741), Dendroctonus ponderosae (HM175783), Rhizopus oryzae (AB056667), Gibberella zeae (AY342397), Mucor circinelloides (AB175927), and the protists of the termite Reticulitermes speratus (BAA98035, BAA98042, BAA98043, BAA98046, and BAA98049). 2.3. Genomic DNA isolation and PCR of cellulase genes in B. horsfieldi Genomic DNA was extracted from the fat body tissues of B. horsfieldi beetle using a universal genomic DNA Extraction Kit Ver. 3.0 (TaKaRa, Japan) according to the manufacturer's instruction. The primers used for the amplification of a genomic DNA encoding B. horsfieldi beetle Bh-EGase II cDNA were 5′-ATGAAACTCTTGTTGGCAATCGTT-3′ for the translational start sequence region and 5′-CGAATTATGAATAATTGCAT CCGGA-3′ for the 3′ coding region. The PCR product was confirmed by

2.5. Cell culture and virus A B. mori-derived cell line, BmN (conserved in our laboratory), was maintained at 27 °C in TC-100 medium (GIBCO, Invitrogen Corporation, USA) supplemented with 10% fetal bovine serum (GIBCO, Invitrogen Corporation, USA), as described by standard methods (O'Reilly et al., 1992). Wild-type B. mori Nucleopolyhedrovirus (BmNPV) and recombinant BmNPV were propagated in the BmN cells. The titer was expressed as plaque-forming units (PFU) per milliliter (O'Reilly et al., 1992). 2.6. Construction of recombinant donor plasmid and recombinant BmNPV virus The Bh-EGase II gene fragment was cut from T-BhEGase II by double digestion with BamHI and EcoRI, and inserted into the same restriction enzyme sites of pFast-BacHTb donor plasmid. The resultant recombinant plasmid was designated pFast-BacHTb-BhEGase II. The BmNPV/ Bac-to-Bac expression system (Motohashi et al., 2005) was applied to generate the recombinant bacmid as follows. About 1 ng of purified pFast-BacHTb-BhEGase II was transformed into 100 μl of DH10BmBac competent cells. The mixture was incubated at 37 °C for 4 h for the transposition of the Bh-EGase II gene into BmNPV Bacmid. The cells were serially diluted with super optimal broth with catabolite repression medium, and then 100 μl of each dilution was distributed evenly on Luria–Bertani agar plates containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/ml IPTG. After 48 h of incubation at 37 °C, the largest and most isolated white colonies were selected and inoculated in liquid culture containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, and 10 μg/ml tetracycline. Finally, the recombinant bacmid DNA was extracted according to the protocol for isolating large plasmids (>100 kb) (Invitrogen Instruction Manual of Bac-to-Bac systems), and then analyzed to verify successful gene transposition to the bacmid by PCR with the M13 primers and Bh-EGase II gene primers. Recombinant baculovirus harboring the Bh-EGase II gene was constructed by transfecting recombinant bacmid DNA into the BmN cells for homologous recombination. About 1 μg of recombinant bacmid DNA and 6 μl of Cellfectin reagent (Invitrogen, USA) were separately diluted into 100 μl of unsupplemented TC-100. The diluted recombinant bacmid DNA combined with diluted Cellfectin was mixed gently and incubated for 30 min at room temperature. The Cellfectin–DNA complexes were added dropwise to the medium covering the cells (1.0–1.5 × 10 6 cells per 35 mm cell culture dish). After incubation at 27 °C for 5 h, TC-100 medium containing antibiotics and 10% FBS was added to each dish and incubation at 27 °C was continued. At 4 days post-transfection, the supernatant was harvested, cleared by centrifugation at 2000 rpm for 5 min, and stored at 4 °C. Recombinant BmNPV plaque was purified on six-well plates seeded with 1.5 × 10 6 BmN cells as described by O'Reilly et al. (1992).

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2.7. Production of recombinant Bh-EGase II in silkworm larvae The recombinant virus and BmNPV bacmid virus were used to infect newly molted fifth-instar silkworm larvae. The larvae were anesthetized on ice for 10 min until they did not move actively. About 10 7 recombinant viruses were injected subcutaneously into each larva. About 30 min after injection, the larvae were fed with mulberry leaves and reared at 25–27 °C. The hemolymph of infected larvae were collected after 5 days and stored at − 20 °C until use. 2.8. SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis The cell lysates, culture supernatants and hemolymph were placed on a 12% slab gel for SDS-PAGE with an equal volume of sample buffer (10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue, and 0.125 M Tris–HCl, pH 6.8) and boiled for 3 min. After SDS-PAGE, the proteins were transferred onto a Protran® Nitrocellulose Transfer Membrane (0.45 μm, Whatman, Germany) under 110 mA for 1.5 h and blocked with blocking reagent (PBS containing 5% non-fat dried milk) for 2 h at room temperature. After three 10 min washes with PBS containing 0.05% Tween-20 (PBS-T), the membrane was incubated in blocking reagent containing 8000× diluted 6× His-antibody (THE™ Anti-His Monoclonal Antibody, 1 mg/ml, GenScript, USA) on a shaker for 1 h at room temperature. The membrane was washed three times for 10 min each with PBS-T, and then incubated for 1 h with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG polyclonal antibody from GenScript (5000× diluted in blocking reagent) at room temperature. After three 10 min washes with PBS-T, the antibody was detected with an HRP-DAB substrate coloration kit (Qiagen, Japan). 2.9. Determination of recombinant Bh-EGase II concentration by indirect ELISA The recombinant Bh-EGase II concentration was determined by ELISA. After BmN cells and silkworm larvae were infected with the recombinant BmNPV–Bh-Egase II virus, the supernatant of BmN cells and silkworm larva hemolymph were harvested at different hours. As protein standard, the concentration of 0.48 mg/ml of eGFP which includes six histidines was diluted to 6 gradients: 0.015 mg/ml, 0.03 mg/ml, 0.06 mg/ml, 0.12 mg/ml, 0.24 mg/ml and 0.48 mg/ml. Flat-bottomed 96-well plates were coated for 1 h at 37 °C with 100 μl of each eGFP standards and 10 μl of supernatant diluted in 90 μl of carbonate bicarbonate buffer (pH 9.6). After blocking with 1% BSA in PBS for 1 h, the wells were washed with PBS containing 0.05% (v/v) Tween-20 (PBS-T) and later incubated at room temperature for 1 h with 1:2000 dilutions of mouse anti-His primary antibody (GenScript, USA). The plates were then incubated at 37 °C for 1 h with HRP conjugated anti-mouse IgG (QIAGEN) and washed again. The peroxidase reaction was visualized using tetramethylbenzidine (Sigma) as substrate after incubation for 30 min at room temperature. The reaction was stopped by adding 2 M H2SO4, and the absorbance was read at 450 nm by a microplate autoreader (Xiang et al., 2011). 2.10. Determination of enzyme activity Recombinant Bh-EGase II mixed in substrate solution (2% (w/v) carboxymethyl cellulose (CMC, Sigma) in 0.1 M acetate buffer (pH 6.0)) was incubated at various temperatures ranging from 10 °C to 90 °C (10 °C intervals) for 1 h to determine the optimal temperature, and the remaining activities were measured. For the optimal pH, recombinant Bh-EGase II was treated at various pH ranges (pH 1.0–12.0) at 50 °C for 1 h, and the remaining activities were measured. The buffers used were acetate (pH 2 and 3), citrate (pH 4 to 6), phosphate (pH 7), Tris–HCl (pH 8 and 9), glycine–NaOH (pH 10), and

KCl–NaOH solution (pH 11 and 12). The amount of reducing sugars produced by these reactions was measured by a dinitrosalicylic (DNS) reagent method (Miller, 1959). One unit of enzyme activity was defined as the activity producing 1 μmol of reducing sugars in glucose equivalents per minute. 3. Results 3.1. Cloning, sequencing, genomic organization, and phylogenetic analysis of Bh-EGase II cDNA Using the 3′ RACE and 5′ RACE methods, the full-length Bh-EGase II cDNA sequence of 974 bp was determined from B. horsfieldi beetle midgut. It contained the 5′ UTR, 3′ UTR, and an intact ORF of 720 bp encoding 239 amino acid residues. The deduced amino acid sequence of the Bh-EGase II cDNA harbored three putative N-glycosylation sites at positions 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The calculated molecular mass and estimated pI of Bh-EGase II were 28 kDa (including six histidines, 0.84 kDa) and 7.55, respectively. A multiple sequence alignment of the deduced protein sequence of Bh-EGase II cDNA with available cellulase sequences is shown in Fig. 1. The Bh-EGase II sequence was closely related to Ag-EGase II (Lee et al., 2005), which belongs to GHF 45, and differed in only fifteen amino acids. The catalytic sites of GHF 45 (Bourne and Henrissat, 2001; Girard and Jouanin, 1999; Li et al., 2003) in Bh-EGase II were well conserved at positions 36–48 (K–T–T–R–Y–W–D–C–C–K–P–S–C). The Bh-EGase II alignment showed 14 conserved cysteine residues and three putative N-glycosylation sites. Among the three N-glycosylation sites of Bh-EGase II, a putative N-glycosylation site (N99–S100–T101) was commonly present in all four EGases. Similarly, the deduced protein sequence of the Bh-EGase II showed 93.72% protein sequence identity to the mulberry longicorn beetle Ag-EGase II, 73.64% protein sequence identity to Ag-EGase I, and 67.78% protein sequence identity to O. albomarginata chamela. To determine the genomic structure of the Bh-EGase II gene, a set of primers based on the Bh-EGase II cDNA sequences was designed and the resulting band was amplified from B. horsfieldi genomic DNA using this primer set. The PCR product was cloned and sequenced. The genomic PCR product sequences were 100% identical with Bh-EGase II cDNA in both size and sequence, indicating that Bh-EGase II is an intronless gene. Phylogenetic analysis using the deduced amino acid sequences of cellulases from beetles, fungi, and symbiont was performed. Cellulases including Bh-Egase II from beetles were found to belong to one subgroup, whereas cellulases from fungi and symbionts belonged to another subgroup. With the deduced amino acid sequence of the Bh-EGase II gene belonging to GHF 45 of beetle endogenous cellulase, the two beetle species A. germari and O. albomarginata chamela formed a subgroup (Fig. 2). 3.2. Construction of recombinant Bh-EGase II Bacmid and recombinant Bh-EGase II baculovirus To assess the Bh-EGase II gene, the 720 bp Bh-EGase II cDNA was inserted into a baculovirus transfer vector, and its large recombinant Bh-EGase II Bacmid was isolated using the alkaline lysis method followed by PCR analysis with M 13 primer and EGase II gene specific primers. The correct insertion of the Bh-EGase II gene into the BmNPV bacmid was confirmed. The purified recombinant Bacmid DNA was transfected into BmN cells using Cellfectin reagent. The cells were incubated in a 27 °C humidified incubator and visually inspected daily for signs of infection using an inverted phase microscope. Compared with the uninfected cells, the transfected cells had typically increased cell diameters and nuclei, stopped growing, became suspended, and exhibited lysis and conglobation 120 h post-transfection. At 24, 48, 72, and 96 h post-transfection, the culture supernatants were collected and stored at −80 °C for further analysis.

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B.horsfieldi EGase II A. germari EGase II A. germari EGase I O. albomarginata chamela

B.horsfieldi EGase II A. germari EGase II A. germari EGase I O. albomarginata chamela

B.horsfieldi EGase II A. germari EGase II A. germari EGase I O. albomarginata chamela

B.horsfieldi EGase II A. germari EGase II A. germari EGase I O. albomarginata chamela Fig. 1. Comparison of the amino acid sequences of EGases from B. horsfieldi and other known GHF 45 cellulases. The potential catalytic domains of GHF 45 are shown in solid boxes. The potential N-glycosylation sites are underlined below the alignment.

The recombinant P1 viral solution was collected from BmN cells 120 h post-transfection and stored at 4 °C protected from light. The P1 viral stock was further used to infect BmN cells to generate the high-titer P2 stock, which was used to infect the silkworm larvae by subcutaneous injection. 3.3. Expression of Bh-EGase II in BmN cells and silkworm larvae Given that the P2 generation BmN cells gradually demonstrated signs of infection due to recombinant baculovirus Bh-EGase II

propagation in vivo, Bh-EGase II was expressed simultaneously in cells. At 48 h post-infection, the infected cells were harvested and the cell lysates and supernatants were placed on 12% gel for SDS-PAGE and Western blot analysis. A ~ 28 kDa protein band was found in lane 1 (unglycosylated Bh-EGase II from cell lysates) and a ~ 33 kDa protein was found in lane 2 (glycosylated Bh-EGase II from cell supernatants) (Fig. 3A). This finding indicated the successful expression of Bh-EGase II in the BmN cells. To express Bh-EGase II gene in silkworm larvae, at 96 h postinfection the P2 generation recombinant Bh-EGase II baculovirus

R.speratus protist(1-16) R.speratus protist(6-47) R.speratus protist(2-54)

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R.speratus protist(8-44) R.speratus protist(7-10) G.zeae R.oryzae

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M.circinelloides S.oryzae C.tremulae D.ponderosae L.decemlineata A.germari EGaseI

Beetle

O.albomarginata chamela B.horsfieldi EGaseII A.germari EGaseII 0.1 Fig. 2. Relationships among the amino acid sequences of Bh-EGase II and the known cellulases. The minimum evolution were used for the amino acid sequences of Bh-EGase II and known cellulases. The sequence sources are described in Materials and methods. The tree was obtained by bootstrap analysis with the option of heuristic search, and the numbers on the branches represent bootstrap values for 1000 replicates.

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Fig. 3. Expression of recombinant Bh-EGase II in BmN cells and in silkworm larva hemolymph 96 h post-injection, as well as the enzymatic activity determined by a yellow halo zone. A) Western blot analysis of recombinant Bh-EGase II collected from cell lysates (lane 1) culture supernatants (lane 2) and recombinant Bh-EGase II collected from silkworm hemolymph 96 h post-injection(lane 3). B) SDS-PAGE analysis of recombinant Bh-EGase II collected from silkworm hemolymph 96 h post-injection(lane 2). Mock (lane 1). C) and D). The Bh-EGase II activity assay was performed with the culture supernatants by using CMC agar plate (Béguin, 1983). The Bh-EGase activity was detected by yellow halo zone.

(BmNPV bacmid virus as a negative control) was injected into the first-day larvae of the fifth instar before feeding by subcutaneous injection at about 10 7 particles per worm in a volume of 10 μl. Normal silkworm larvae were reared as a control. There was no clear difference among the three treatment groups in the early stage (1–3 days post-injection). The silkworms presented typical symptoms of NPV infection both in BmNPV bacmid baculoviruses and recombinant

Bh-EGase II baculoviruses from 72 h post-injection compared with the normal control, i.e., blackening of the body, loss of appetite, and growth retardation. The diseased larvae became obvious in the rearing trays due to their shiny white translucent skin, elevated intersegmental membrane, and swollen body segments 72–120 h post-infection. At the advanced stage of infection, the body wall ruptured, acute lethal

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Medium Fig. 4. Expression and enzymatic activity of recombinant Bh-EGase II in baculovirus-infected BmN cells 48, 72, and 96 h post-transfection, and in silkworm larvae 96 h post-injection. Green columns represent the productivity of secreted Bh-EGase II from recombinant baculovirus-infected BmN cells and recombinant baculovirus-injected silkworm larva hemolymph. BmN cells (0.5 × 106 cells/ml) were infected with the recombinant virus at an MOI of 5 PFU per cell. Newly molted fifth instar silkworm larvae were injected with 10 μl of 108 recombinant viruses per larva. Cyan columns represent the enzymatic activity of the secreted recombinant Bh-EGase II. The amounts of secreted recombinant Bh-EGase II collected from cell supernatants and hemolymph of injected larvae were measured by a DNS reagent method.

D. Xia et al. / Gene 514 (2013) 62–68

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infection developed, and the larvae died in the later period of the fifth-instar stage (about 120 h post-infection). Hemolymph was harvested at 96 h from ten silkworms and stored at −80 °C until use. They were examined using 12% SDS-PAGE analysis (Fig. 3B). Recombinant Bh-EGase II expressed by the Bh-EGase II cDNA was present as a single band of a ~33 kDa polypeptide in recombinant Bh-EGase II collected from silkworm hemolymph 96 h post-injection with the recombinant virus (lane 2), but not in silkworm hemolymph infected with the BmNPV bacmid virus (lane 1). The activity of recombinant Bh-EGase II was clearly detected in culture supernatant from the recombinant virus-infected insect cells and in silkworm larva hemolymph 96 h post-injection. The activity of unglycosylated Bh-EGase II from cell lysates and silkworm hemolymph infected with the BmNPV bacmid virus was not detected (Figs. 3C and D).

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The ELISA results showed that the yields of recombinant Bh-EGase II were 0.00123 μg/ml at 48 h, 0.00393 μg/ml at 72 h, and 0.0249 μg/ml at 96 h post-transfection in BmN cells, as well as 1150 μg/ml in silkworm larva hemolymph 4 days after newly molted larvae were injected at the fifth instar (Fig. 4). The activities of recombinant Bh-EGase II were 0.00114 U/mg at 48 h, 0.00365 U/mg at 72 h, and 0.0231 U/mg at 96 h posttransfection in BmN cells, as well as 1067 U/mg in silkworm larva hemolymph 4 days after newly molted larvae were injected at the fifth instar (Fig. 4). The enzymatic activity of recombinant Bh-EGase II was approximately 928 U/mg from BmN cells and larva hemolymph. To determine the optimum temperature of Bh-EGase II, recombinant Bh-EGase II was examined at various temperatures ranging from 10 °C to 90 °C. The optimum temperature for recombinant Bh-EGase II activity was 50 °C (Fig. 5A). The optimum pH of recombinant Bh-EGase II was determined to be from pH 1.0 to 12.0 by incubating recombinant Bh-EGase II at 50 °C for 1 h, and pH 6.0 was optimal (Fig. 5B).

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4. Discussion Cellulose, a linear homopolymer consisting of glucose units linked by β-1,4 bonds, is the most important energy source for herbivorous animals. However, animals generally absorb sugars in monomeric forms such as glucose and fructose (Levin, 1994). In many cases, the presence of symbiotic protozoa or bacteria in invertebrates and herbivorous animals has been used to explain cellulose digestion (Attwood et al., 1996). Recently, indications for the existence of cellulase genes from animals have been obtained by gene cloning and cellulase activity measurement in microbe-free digestive tissues of longicorn beetle (Lee et al., 2005; Ohtoko et al., 2000), termite (Tokuda et al., 1999), grass carp (Das and Tripathi, 1991), redclaw crayfish (Xue et al., 1999), and red flour beetle (Rehman et al., 2009). In the present study, Bh-EGase II from the B. horsfieldi beetle is proposed to belong to GHF 45, which presents the same conserved catalytic site of GHF 45 (Girard and Jouanin, 1999) and N-glycosylation site (Wei et al., 2006). More interestingly, the amino acid sequence of Bh-EGase II cDNA was very close to Ag-EGase II (93.72%). The Bh-EGase II gene structure was encoded by only one exon without any intron, but the Ag-EGase II gene consists of two introns and three exons. We also noted that the Bh-EGase II enzyme consisted of a single catalytic domain and lacked both a cellulose-binding domain as well as a spacer sequence, similar to the amino acid sequences from Ag-EGase I (Lee et al., 2004), Ag-EGase II (Lee et al., 2005), and R. speratus hindgut protein (Ohtoko et al., 2000). The Bh-EGase II cDNA was expressed in baculovirus-infected insect BmN cells and fifth instar larvae of silkworm. Western blot analysis showed bands of about 28 kDa from cell lysates and 33 kDa from cell supernatant as well as larva hemolymph. The different mobility on SDS-PAGE can be explained by a possible post-translational

Fig. 5. Enzymatic properties of recombinant Bh-EGase II expressed in baculovirusinfected silkworm larvae. (A) Optimum temperature of recombinant Bh-EGase II. The enzyme activity at the optimum temperature was determined by incubating the recombinant Bh-EGase II in acetate buffer (0.05 M, pH 6.0) containing 1% (w/v) CMC from 15 °C to 85 °C (5 °C increments), and by assaying the reducing sugars released using a DNS reagent method. (B) Optimal pH of recombinant Bh-EGase II. Recombinant Bh-EGase II was incubated in 0.05 M buffer for 1 h at 50 °C before measuring the residual EGase activity under standard assay conditions.

modification at three potential N-glycosylation sites, because they were expressed in vivo. We discovered that Ag-EGase II shared 93.72% identity with Bh-EGase II. Its N-linked glycosylation site at amino acid residues 99–101 (NST) was essential to its enzymatic activity, which is conserved in known beetle GHF 45. Our yellow halo zone experiment supported the fact that the secreted glycosylated recombinant Bh-EGase II had enzyme activity. In contrast, the recombinant unglycosylated Bh-EGase II from cell lysates lost its enzymatic activity. In conclusion, we have cloned a novel EGase gene belonging to GHF 45 from the beetle B. horsfieldi. This endogenous EGase gene consisted of only a single exon and had three potential glycosylation sites. We also expressed the Bh-EGase II cDNA in BmN cells and a fifth instar larvae of silkworm, as well as characterized the optimum temperature and pH of this enzyme. Our results strengthen the understanding of the gene structure and enzyme activity of insect cellulases. Bh-EGase II can be used as a candidate gene for transgenic silkworm research to improve food efficiency. Acknowledgments This project was supported by the National Natural Science Foundation of China (grant nos. 30871829 and 31072083). We thank Kirill

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Gorshkov (Johns Hopkins University) for critical manuscript revisions. We thank Prof. Shen Xingjia, Dr. Shengpeng Wang, and Dr. Shunming Tang (Sericultural Research Institute, CAAS) for thoughtful advice. We thank the kind support of Prof. Wenbing Wang (Jiangsu University) for providing the BmNPV bacmid. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.08.044. References Attwood, G.T., Herrera, F., Weissenstein, L.A., White, B.A., 1996. An endo-beta-1,4glucanase gene (celA) from the rumen anaerobe Ruminococcus albus: cloning, sequencing, and transcriptional analysis. Can. J. Microbiol. 42, 267–278. Béguin, P., 1983. Detection of cellulase activity in polyacrylamide gels using congo red stained agar replicas. Anal. Biochem. 131, 333–336. Béguin, P., Aubert, J.P., 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13, 25–58. Bourne, Y., Henrissat, B., 2001. Glycoside hydrolases and glycosyltransferases: families and functional modules. Curr. Opin. Struct. Biol. 11, 593–600. Breznak, J.A., Brune, A., 1994. Role of microorganisms in the digestion of lignocellulose by termites. Annu. Rev. Entomol. 39, 453–487. Carpita, N.C., Gibeaut, D.M., 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3, 1–30. Das, K.M., Tripathi, S.D., 1991. Studies on the digestive enzymes of grass carp, Ctenopharyngodon idella (Val.). Aquaculture 92, 21–32. del Campillo, E., Bennett, A.B., 1996. Pedicel breakstrength and cellulase gene expression during tomato flower abscission. Plant Physiol. 111, 813–820. Girard, C., Jouanin, L., 1999. Molecular cloning of cDNAs encoding a range of digestive enzymes from a phytophagous beetle, Phaedon cochleariae. Insect Biochem. Mol. Biol. 29, 1129–1142. Henrissat, B., Driguez, H., Viet, C., Schulein, M., 1985. Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Nat. Biotechnol. 3, 722–726. Lee, S.J., et al., 2004. cDNA cloning, expression, and enzymatic activity of a cellulase from the mulberry longicorn beetle, Apriona germari. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 139, 107–116. Lee, S.J., et al., 2005. A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 140, 551–560. Levin, R.J., 1994. Digestion and absorption of carbohydrates—from molecules and membranes to humans. Am. J. Clin. Nutr. 59, 690S–698S.

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