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Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic LibraryS
Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic LibraryS
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1196-1206
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 07, 2014
  • Accepted : July 11, 2014
  • Published : September 30, 2014
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About the Authors
Chang-Muk Lee
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
changmuk@rda.go.kr
Young-Seok Lee
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
So-Hyeon Seo
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
Sang-Hong Yoon
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
Soo-Jin Kim
National Agrodiversity Center, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
Bum-Soo Hahn
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
Joon-Soo Sim
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
Bon-Sung Koo
Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea

Abstract
A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens , a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii , displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50℃ and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50℃ and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.
Keywords
Introduction
Cellulose is a organic polymer composed of repeating cellobiose units attached by β-1,4-glucosidic bonds. Cellulose constitutes a major polysaccharide compound in plant cell walls and is considered as one of the most plentiful organic compounds on Earth [4 , 24] . In recent years, owing to the indiscriminate overuse of petroleum and fossil fuels produced by the old-fashioned industry, many serious problems of the exhaustible traditional fuels and environmental pollutions, including global warming and waste water, have been aggravated still further [33] . With a number of fundamental solutions being considered, the clean bioprocess technology of bioethanol production is being magnified as an environmentally friendly plan that can balance the development of natural resources with environmental preservation [26] . Cellulose has attracted worldwide interest in the way that has great potential as a renewable and an alternative natural resource for biofuels and other industrially important chemicals [14 , 26] .
Cellulases are differentiated from all other glycosyl hydrolases by the faculty of hydrolyzing β-1,4-glucosidic bonds between the anhydrous glucose residues and can be divided into the following three major classes in the enzymatic degradation of cellulose in nature: endoglucanases (E.C. 3.2.1.4); exoglucanases (E.C. 3.2.1.91); and β-glucosidases (E.C. 3.2.1.21) [3 , 25] . Cellulases have a tremendous potential in many kinds of industrial applications, including food, animal feed, textile, fuel, and chemical industries [4] . In addition, there are other application possibilities that involve the pulp/paper industry, detergents formulation, juice clarification, and waste/pollution treatment.
In these industrial applications, it is important for the biotechnological process to possess biocatalysts with broad ranges of substrate specificity, high enzyme activity, and enzyme stability against various severe conditions, including pH, temperature, organic solvents, salts, and so forth.
Because of the limitation of current cultivation techniques, no less than 99% of the microorganisms existent in a natural ecosystem are not easily culturable and therefore are not available for basic biological studies or biotechnology [1 , 15 , 28] . This unculturable and unidentified microbial diversity is treasured as an unexploited natural source of novel enzymes and the related metabolic pathways that can be applied in the fields of biotechnology and bioremediation [12 , 29] . The metagenomic strategy could be applied to screening candidates with high potential as novel biocatalysts for biotechnological applications without requiring any particular cultivation from uncultured microbial communities [13 , 15 - 17 , 27] .
Hermetia illucens ( H. illucens ), better known as the black soldier fly (BSF), belongs to the polyphagous insect group. These BSFs have an excellent digestive ability that can devour enormous amounts of garbage and food scraps in a matter of hours, with the result that they convert the wastes into organic material used as fertilizer [8 , 18 , 21] . In comparison with microorganisms, fungi, and other insect scavengers, the unequaled digestive capabilities of the BSF larvae for the inestimable environmental wastes suggested the scientific investigation about the biological features of metabolic enzymes in the BSF in playing a major role in many industrial applications [18 , 20] . In the gut of the BSF, there must be many extremophilic microbes that can thrive under harsh conditions, giving a positive perspective of the identification of unique enzymes with novel DNA sequences. In this study, we first report the construction of a metagenomic library from the gut microflora of H. illucens for screening the clones of various biocatalysts with unique and useful characteristics in industrial processes. From the metagenomic library, a gene with cellulase activity was screened and many different biochemical properties of the purified protein were characterized.
Materials and Methods
- Construction of Metagenomic Fosmid Library
The larvae of H. illucens were obtained with help of the Applied Entomology Division in the National Academy of Agricultural Science of the Rural Development Administration, Korea. The guts were removed with a pincette in a fixed state from more than thousand larvae, and appropriately crushed using a mortar and pestle, and then genomic DNA of the intestinal microorganism was extracted by means of a PowerSoil DNA isolation kit (MoBio, Carlsbad, CA, USA). The metagenomic fosmid libraries from these samples were constructed based on a previously described method [16 , 17] . To remove humic compounds in the samples, the extracted DNAs were further purified by two-step DNA purification with pulse-field gel electrophoresis (CHEF; Bio-Rad, Hercules, CA, USA). The DNAs were fractionated in a 1% low melting temperature agarose gel under the electrophoresis conditions of 4 V/cm electrical field at 14℃ for 12 h. The DNA fragments of approximately 100 to 190 kb were cut from the gel and processed with agarase (1 U per 100 mg slice; Takara, Tokyo, Japan). The Sau 3AI partially digested DNA fragments were again fractionized under the same conditions, and a gel slice containing DNA fragments above 40 kb was processed with agarase. The isolated DNA was end-repaired with an End-It DNA End-repair kit resulting in the DNA being blunt-ended and 5’-phosphorylated, and ligated into pEpiFOS-5 fosmid vector (Epicentre, Madison, WI, USA). In sequence, in vitro packaging of the fosmid vector constructs was accomplished with a MaxPlax lambda packaging extract kit (Epicentre). After the addition of lambda packaging extracts, the ligates were infected with a phage T1-resistant EPI300-T1 R Escherichia coli ( E. coli ) according to the manufacturer’s instruction. The E. coli transformants containing different fosmid vector constructs were transferred to 96-well microplates and finally stored at -80℃.
- Screening of a Clone with Cellulase Activity
The clone with carboxymethyl cellulose (CMC; Sigma Aldrich, St. Louis, MO, USA)-hydrolyzing activity was screened from the metagenomic fosmid libraries of gut microflora of Hermetia illucens , according to a Congo-red overlay method as previously described [16] . The fosmid libraries were replicated onto 96-well microplates containing Luria–Bertani (LB) broth added with chloramphenicol (12.5 mg/ml). After a cultivation at 37℃ incubator for 24 h, the fosmid libraries were replicated onto LB agar plates supplemented with 0.1% CMC and chloramphenicol (12.5 mg/ml) by the use of a 96-pin replicator. The reaction was further continued in a 28℃ incubator for 7 days, followed by flooding with 0.1% aqueous Congo-red for 10 min and washing with excess 1 M NaCl solution. A clearing zone around the colonies on the red background signifies the hydrolysis of CMC by cellulase activity, because Congo-red reagent interacts with (1→4)-β-D-glucans, (1→3)-β-D-glucans, and (1→4)-β-D-xylans.
The fosmids were isolated from the positive clones selected by the first screening of a previous Congo-red overlay test and transformed into E. coli DH5α, followed by re-examination of an occurrence of the zone by a repetitive Congo-red overlay test. Fosmids isolated from those positive clones were cut into the average piece of 4 kb by hydroshear (DigiLab, Holliston, MA, USA), cloned into a pUC118/ Hinc II vector after end-repairing, and transformed into E. coli DH5α to establish a shot-gun subclone library.
The final positive clone showing cellulase activity was selected by additional screenings of the same Congo-red overlay test for the subclone library.
- Primary Sequence Analysis of Deduced Amino Acids
The sequence homology search and conserved domain analysis of the deduced protein sequence were performed using the BLASTP program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) and CDART program ( http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi ) of NCBI, respectively. Multiple sequence alignments between protein sequences were performed with the ClustalW program, and the phylogenetic tree was reconstructed with the Molecular Evolutionary Genetic Analysis (MEGA) 5.1 software. The putative signal peptide sequence in the deduced amino acid sequence was predicted using the SignalP 3.0 server ( http://www.cbs.dtu.dk/services/SignalP-3.0/ ).
- Heterologous Expression and Purification of Recombinant Protein
The putative cellulase gene, removing its signal peptide sequence, was amplified from the HC3 fosmid library with the CMC-hydrolyzing clone by polymerase chain reaction (PCR) using the following forward and reverse primers: 5’-CAAATGGGTCGC GGATCC AATAACCCTATAGAGGGAGATAAG-3’ and 5’-GTGGTGGTG CTCGAG TTTGTTTTCTTTCTGCAATGTGTTTC-3’( Bam HI and Xho I restriction enzyme sites underlined). The amplified PCR product was ligated into the pET21a(+) expression vector (Novagen, Darmstadt, Germany) using the In-fusion HD cloning kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions, and the recombinant DNA was transformed into E. coli BL21 (DE3). The E. coli BL21 (DE3) harboring pET21a(+)- CS10 was grown at 37℃ in 100 ml of LB broth with ampicillin (100 μg/ml) until the optical density of the cell reached 0.6 at 600 nm, and was then induced by addition of 0.5 mM isopropyl-D-1-thiogalactopyranoside (IPTG). After incubation for 20 h at 18℃, the cells were harvested by centrifugation (8,000 × g , 20 min, 4℃) and resuspended in 50 mM HEPES buffer (pH 7.0) containing 0.1 M NaCl and 10% glycerol. The resuspended cells were lysed by sonication on ice and centrifuged at 13,000 × g for 30 min at 4℃. The resulting supernatants were purified to a column of TALON metal affinity resin (BD Biosciences, Clontech, Palo Alto, CA, USA) equilibrated by the previous resuspension buffer, according to the manufacturer’s instructions. After washing with 10 mM imidazole in 50 mM HEPES buffer (pH 7.0) containing 0.3 M NaCl and 10% glycerol, the bound proteins were eluted with the same buffer containing 300 mM imidazole.
- SDS-PAGE and Zymogram Analysis
The molecular mass and purity of the purified protein were examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions.
For zymogram analysis, the purified protein was applied to 10% SDS-PAGE gel containing 0.1% CMC incorporated directly into the resolving gel at 4℃, according to previously descrbed methods [7 , 22] . After electrophoresis, the gel was immersed in renaturation buffer (50 mM sodium phosphate buffer, pH 7.0, 2.5% Triton X-100) for 30 min, repeating several times to remove SDS. Followed by washing three times with 50 mM sodium phosphate buffer (pH 7.0), the gel was incubated in the same buffer at 40℃ for 1 h, stained with Congo-red (1%) for 30 min, and then destained with 1 M NaCl until a clear zone was visualized against the red background of the polyacrylamide gel.
- Enzyme Activity Assay
Cellulase activity was evaluated by measuring the amount of reducing sugars produced from carboxymethylcellulose (CMC) by the 3,5-dinitrosalicylic acid (DNS) method using glucose as a standard. The standard assay mixture (200 μl) contained 0.5% CMC and the properly diluted enzyme solution (1 μg) in 50 mM sodium phosphate buffer (pH 7.0) The enzyme reaction was conducted at 50℃ for 10 min, stopped by addition of DNS solution (600 μl), and boiled for 10 min in a water bath followed by cooling in ice for color stabilization. The amount of hydrolysis product of CMC was determined by measuring the absorbance at 540 nm using a GeneSys 20 spectrophotometer (Thermo Scientific, Waltham, MA, USA). One unit of enzyme activity was defined as the amount of the enzyme capable of liberating reducing sugars equivalent to 1 μmol of D-glucose per minute under standard assay condition. Additionally, the enzyme activity toward chromogenic sugar derivates substituted with the ρ-nitrophenyl group was measured by spectrophotometric method at 405 nm using a standard curve of ρ-nitrophenol (ρNP; Sigma Aldrich, St. Louis, MO, USA) after incubation under the same condition used in the standard a ssay o f cellulase a ctivity [16] . One unit of hydrolysis activity for ρ-nitrophenyl-sugar derivates was defined as the amount of protein required to release 1 μmol of ρNP per minute under standard assay conditions. The concentration of protein in solution was determined with the Bradford assay kit (Bio-Rad, Hercules, CA, USA) depending on bovine serum albumin as a standard.
- Biochemical Properties of the Recombinant CS10
The optimum temperature and pH of purified CS10 were determined by measuring the enzyme activity for 10 min at different temperature (20~70℃ with an interval of 5℃) and pH (4.0~10.0 with an interval of 0.5) values using the following buffers: 50 mM sodium acetate buffer (pH 4.0~6.0), 50 mM sodium phosphate buffer (pH 6.0~7.5), 50 mM HEPES buffer (pH 7.5~8.5), and 50mM CHES buffer ( pH 8 . 5~10. 0). The thermal stability was compared by pre-incubating the enzyme without substrate for 1 h at different temperatures ranging from 10 to 60℃, and then measuring the residual activity under the standard assay conditions. The pH stability was compared by pre-incubating the enzyme without substrate overnight in 4℃ at various pH values as above, and then measuring the residual activity under the same standard assay conditions. The effect of various organic solvents, metal ions, detergents, and other chemical reagents on enzyme activity was also examined. In all the tests, the enzymes had been preincubated with the compounds in 50 mM sodium phosphate buffer (pH 7.0) at 30℃ for 1 h and the residual activity was measured under the same standard assay conditions. The residual activity of CS10 performed in the absence of any additives was defined as the 100% level. To investigate the substrate specificity, hydrolytic activities of the enzyme toward other substrates were assayed under the above standard assay conditions by replacing CMC with the respective substrates. The substrates used in this study were barley β-D-glucan, CMC, 2-hydroxyethyl cellulose (2-HE), filter paper, Avicel, cellobioside, laminarin, birchwood xylan, oat spelt xylan, ρ-nitrophenyl-β-D-cellobioside, ρ-nitrophenyl-β-Dglucopyranoside, and ρ-nitrophenyl-β-D-xylopyranoside. For the insoluble substrates such as Avicel PH101 and filter paper (10 mm × 20 mm; a piece of Whatman no. 1 filter paper), 20 μg of enzyme was added into the same assay solution and the reaction was incubated in a w ater b ath shaker at 150 rpm f or 1 20min. The kinetics of CS10 was characterized in terms of Michaelis-Menten kinetic constants using Lineweaver-Burk plots. The K m and V max kinetic parameters were determined by assaying the enzyme activity at substrate concentrations ranging from 2.5 to 20 mg/ml in 50 mM sodium phosphate buffer (pH 7.0) at 50℃ for 10 min and analyzed using Graph Pad software. Data of the a ll s tudies were obtained from three independent experiments done on triplicate samples.
- Analysis of Hydrolysis Products by Thin-Layer Chromatography
To determine the action mode of cellulase activity, the hydrolysis products of cellooligosaccharides (cellobiose to cellopentaose; Sigma Aldrich, St. Louis, MO, USA) with CS10 were analyzed by thin-layer chromatography [19 , 24] . The hydrolysis was conducted in the enzyme reaction mixture (200 μl) of cellooligosaccharides (1%) and purified CS10 (1 μg) in 50 mM sodium phosphate buffer (pH 7.0) at 40℃ for 4 h. The reaction products were separated on Silica Gel 60 TLC plates (Merck, Darmstadt, Germany) using a solvent of n -butanol/acetic acid/water (2:1:1 (v/v/v)) and visualized by heating at 120℃ for 10min after the spraying of freshly prepared 10% (v/v) H 2 SO 4 in ethanol.
- Nucleotide Sequence Accession Number
The nucleotide sequence of the putative cellulase gene, CS10 , was deposited in the GenBank database under the accession number KC684420.
Results
- Construction of Metagenomic Library and Screening of a Clone with Cellulase Activity
The larvae of H. illucens , or a BSF, can surpass other chemical catalysts both in environmental remediation and economic feasibility, in the respect that the larvae are voracious feeders of environmental wastes such as organic wastes and food leftovers [8 , 18] . In consideration of all these characteristics, the larvae were expected to possess many kinds of novel microorganisms that have the distinctive features of capability to degrade various substrates, showing high resistance against very extreme conditions.
In this sense, a metagenomic library consisting of 92,000 individual fosmid clones was constructed from the gut microflora of H. illucens , as mentioned in Materials and Methods. A positive fosmid clone, HC3 , was identified by preliminary screening of Congo-red overlay test from the fosmid clones, showing obvious cellulase activity around the bacterial colony on LB plates containing CMC. To confirm whether it was formed by the false-positive by the E. coli host cell itself or the contamination of megagenomic fosmid libraries, the isolated fosmid was transformed into E. coli DH5α and the cellulase activity of the clone was repeatedly confirmed on LB plates containing CMC (data not shown). To identify genes responsible for cellulase activity, several positive clones were identified by further shotgun cloning and additional screening using the same Congo-red overlay test. Among these clones, we selected a single positive clone with relatively high cellulase activity on the plates (Fig. S1) .
- Amino Acid Sequence and Phylogenetic Analysis of CS10
Sequence analysis of the clone revealed the presence of an open reading frame of 996 bp (designated as CS10 ), encoding a single polypeptide of 331 amino acids with an estimated molecular mass of 38.064 kDa and a pI of 5.64. A search for the putative cleavage site of the signal peptide using the SignalP 3.0 program predicted that this protein possesses a signal peptide sequence of 25 amino acids with a cleavage site between Ser-25 and Asn-26. Comparison of the deduced amino acid sequence of CS10 with those of other proteins registered in GenBank showed the highest degree of sequence similarity to glycosyl hydrolase family 5 of Dysgonomonas mossii (GenBank Accession No. EGK03707.1) with 72% identity and 80% homology. Although a cellulose catalytic domain (Ser-51 through Tyr-294) of glycosyl hydrolase family 5 (GH5; pfam00150) was identified using the CDART program of NCBI, no cellulose binding domain (CBD) and its linker region were found in the amino acid sequence of CS10 ( Fig. 1 A). Multiple sequence alignment with the cellulose catalytic domain of different cellulases belonging to GH5 exhibited that CS10 shared amino acid residues with a characteristic feature conserved among the cellulases of GH5 ( Fig. 1 A). However, phylogenetic analysis of CS10 with many closely related enzymes in GenBank showed that the enzyme was classified from other members, forming a separate branch in the phylogenetic tree ( Fig. 1 B).
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Multiple sequence alignment (A) and phylogenetic tree (B) of CS10. (A) The deduced amino acids between CS10 and other cellulases belonging to GH5 were compared by multiple sequence alignmnet using the ClustalW program. (B) The phylogenetic tree of CS10 and other closely related enzymes was reconstructed using the neighbor-joining method (MEGA5.1 software). The protein sequences of related enzymes were retrieved from GenBank of NCBI. Bootstrap values at the nodes were supported on 1,000 replicates of the dataset.
- Purification and Biochemical Characterization of CS10
The CS10 gene without the putative signal peptide sequence was cloned into the pET21a(+) expression vector, overexpressed as a soluble protein in E. coli BL21 (DE3), and purified by metal-chelating chromatography under native condition, as described above.
SDS-PAGE analysis of the purified CS10 showed a single band with apparent homogeneity of approximately 40 kDa that correlated with the theoretical molecular mass of a C-terminal His-tagged protein ( Fig. 2 ). Activity staining of the purified CS10 on polyacrylamide gels by zymography revealed the active band of cellulase at a position corresponding to the molecular mass of the protein on SDS-PAGE.
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Purification of recombinant cellulase CS10 protein. Lane M, molecualr weight marker (Bio-Rad, Hercules, CA, USA); Lane 1, total cellular extracts before induction; Lane 2, total cellular extracts after induction; Lane 3, soluble fraction after induction; Lane 4, purified CS10 protein; Lane 5, zymogram analysis of CS10 protein on polyacrylamide gel incorporated by 0.1% CMC and stained by Congo-red.
The enzyme activity and stability of the purified CS10 were measured at different temperature and pH values. The purified CS10 was active in a pH range of 5.0~8.0, and in a temperature range of 30~45℃, with optimal activity at pH 6.0 and 50℃ ( Figs. 3 A and 3 C). Thermostability analysis of the purified CS10 revealed that the activity of this enzyme remained stable up to 50℃ and decreased rapidly above 50℃ ( Fig. 3 B). The purified CS10 was remarkably stable over broad pH values ranging from 4.0 to 10.0, retaining at least 70% of its maximum activity even under highly acidic and alkaline conditions ( Fig. 3 D).
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Effect of temperature or pH on the activity and stability of purified CS10. (A) The enzyme activity of CS10 at various temperatures was measured in 50 mM sodium phosphate buffer (pH 7.0) for 10 min. (B) The thermostability of CS10 was verified by measuring the enzyme activity in 50 mM sodium phosphate buffer (pH 7.0) at 50℃ for 10 min after preincubation at various temperatures in the same buffer for indicated intervals of time. (C) The effect of pH on enzyme activity was measured in the following 50 mM buffers. Sodium acetate (pH 4.0~6.0), sodium phosphate (pH 6.0~7.5), HEPES (pH 7.5~8.5), and CHES (pH 8.5~10.0). (D) To verify the pH stability of CS10, the enzyme was pre-incubated in the above 50 mM buffers at 4℃ for overnight and the residual activity was examined in the 50 mM sodium phosphate buffer (pH 7.0) at 50℃ for 10 min. Each assay for cellulase activity was performed by the DNS method using CMC as substrate. The error bars represent the SEM from three independent experiments each with triplicates.
The effects of organic solvents, metal ions, and detergents on the enzyme stability were examined. The purified CS10 was fairly stable against 10% concentration of hydrophilic organic solvents (dimethylsulfoxide, dimethylformamide, methanol, acetonitrile, ethanol, and acetone), displaying more than 80% residual activity, while partial inhibition was observed in the case of 20% concentration ( Fig. 4 A). The residual activity of CS10 was partially decreased by the addition of metal ions such as Cu 2+ , Co 2+ , and Fe 3+ , but ethylene diamine tetraacetic acid (EDTA) showed no obvious influence on the activity of CS10 ( Table 1 ). The activity of CS10 was highly decreased to 3% of the control by the addition of Hg 2+ . The addition of 1% non-ionic detergents (Tween 20, Tween 40, Tween 80, and Triton X-100) slightly increased the activity of CS10, whereas the cationic detergent cetyl trimethyl ammonium bromide (CTAB) decreased the residual activity to 60% ( Fig. 4 B). Although a complete loss of the CS10 activity was observed on the addition of 1% of the anionic detergent sodium dodecyl sulfate (SDS), the enzyme activity was retained up to 60% compared with control in the presence of 0.1% (data not shown). Reducing agents such as β-mercaptoethanol and dithiothreitol (DTT) had no significant effect on the activity of CS10 ( Table 1 ). CS10 still retained 100% of its original activity under denaturation conditions by guanidine-HCl or urea, requiring concentrations as high as 0.5 and 1.0 M, respectively.
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Inhibitory effects of different organic solvents or detergents on the stability of CS10. (A) To verify tolerance of CS10 against organic solvents, the enzyme was pre-incubated at 30℃ for 30 min under 50 mM sodium phosphate buffer (pH 7.0) with the concentration of 10% and 20% of different organic solvents, and the residual activities were measured at 50℃ for 10 min. (B) To verify the effect of detergents on the stability of CS10, the enzyme was pre-incubated in 50 mM sodium phosphate buffer (pH 7.0) with 1% detergents at 30℃ for 30 min, and the residual activities were measured at 50℃ for 10 min. The residual activity of CS10 in the absence of any additives was taken as 100%. DMF, Dimethylformamide; DMSO, Dimethylsulfoxide; CTAB, Cetyltrimethylammoniumbromide; SDS, Sodium dodecyl sulfate.
Effects of metals and other chemical reagents on the stability of CS10.
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CS10 was pre-incubated in 50 mM sodium phosphate buffer (pH 7.0) with metals or other chemical reagents at 30℃ for 30 min, and the residual activities were measured at 50℃ for 10 min. aAddition of other metals (Na+, K+, Mg2+, Zn2+, Ca2+, Ni2+) showed little effect on the stability of CS10 bThe residual activity of CS10 in the absence of any metals or chemical reagents was taken as 100%. The ± values represent the SEM from three independent experiments each with triplicates.
The substrate specificity of CS10 was investigated by incubation of the enzyme with different substrates under standard assay conditions. As shown in Table 2 , CS10 exhibited the highest activity toward barley glucan (β-1,3/1,4-glycosidic bonds, 203.3 ± 5.1 U/mg) followed by CMC (β-1,4-glycosidic bonds, 46.2 ± 2.4 U/mg) and 2-HE (β-1,4-glycosidic bonds, 10.3 ± 1.7 U/mg). However, no significant substrate hydrolysis by CS10 was detected on laminarin (β-1,3-glycosidic bonds), ρ-nitrophenyl-β-D-cellobioside, cellobiose, and Avicel.
Substrate preference of cellulase CS10.
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aSubstrate specificity of CS10 was determined by measuring the amount of reducing sugars produced from various substrates under standard assay conditions, using glucose as a standard. One unit of enzyme activity toward cellulose substrates was defined as the amount of protein required to produce 1 μmol of r educing sug ar p er minute under standard assay conditions. Substrate specificity of CS10 toward ρ-nitrophenyl-derivated chromogenic sugars was determined by measuring the amount of ρ-nitrophenol released under the same assay conditions, using the standard curve of ρ-nitrophenol. bND, not detectable.
The kinetic constants K m and V max for CS10 were calculated from a Lineweaver-Burk plot, using initial reaction rates for the different concentrations of CMC and barley glucan under standard assay conditions ( Fig. 5 ). The K m and V max values of CS10 were 8.7 mM and 160.6 μmol/min/mg toward CMC, respectively; and 6.1 mM and 364.3 μmol/min/mg against barely glucan, respectively.
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Michaelis-Menten data and Lineweaver-Burk reciprocal plots of CS10. Plot of initial velocity of reaction versus initial CMC concentration was performed with varying concentrations of CMC in 50 mM sodium phosphate buffer (pH 7.0) at 50℃ for 5 min.
The hydrolysis products of cellulooligosaccharides by CS10 were analyzed by thin-layer chromatography ( Fig. 6 ). Cellotetraose (G4) was hydrolyzed to release only a cellobiose (G2), whereas cellopentaose (G5) was degraded to cellobiose (G2) and cellotriose (G3). However, no hydrolysis by CS10 was observed for cellobiose (G2) and cellotriose (G3).
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TLC analysis of products upon hydrolysis of cellooligosaccharides by CS10. Each substrate was incubated with CS10 in 50 mM sodium phosphate buffer (pH 7.0) at 50℃ for 2 h and the hydrolysates were analyzed by TLC. TLC was performed using n-butanol/acetic acid/water (2:1:1 (v/v/v)) solvents. Lane M, standard oligosaccharides: G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; and G5, cellopentaose. Lanes 1-4: hydrolysates of G2-G5 treated with recombinant CS10.
Discussion
On the basis of the superior scavenging abilities of BSF, the BSF larvae were industrially applied for garbage disposal, environmental management, and recycling into the higher value-added resources such as natural manure [8 , 18 , 21] . In many countries, bioremediation using the BSF larvae has been spotlighted as a more cost-effective and eco-friendly improvement method to solve environmental problems such as soil contamination and water pollution. In spite of the potential industrial application of black soldier fly, the molecular and biochemical characteristics of their enzymes have not yet been studied properly and are little-known.
This report is the first outcome from the construction of a metagenomic library from the gut microflora of H. illucens and the identification of a clone, which we called CS10 , homologous with cellulase through massive screenings for cellulolytic activity. Based on homologies of the amino acid sequences, glycosyl hydrolases have been categorized into 90 families listed in the CAZy sever ( http://www.cazy.org/Glycoside-Hydrolases.html ) [6] . Most of the cellulases are classified to 14 families, although some families contain other enzymes such as xylanase and mannanase. Primary sequence analysis by the conserved domain database search and multiple sequence alignment suggested that CS10 belongs to glycosyl hydrolase family 5 with only a cellulose catalytic domain. Despite the high level of similarity in a cellulose catalytic domain with the other known cellulases of glycosyl hydrolase family 5, the phylogenetic analysis revealed that CS10 was definitely separated in the phylogenetic tree, implying that it may have been originated from a currently unidentified microorganism in the evolutionary aspect.
To investigate the biochemical properties of the enzyme under different conditions, the CS10 gene was functionally expressed in E. coli BL21 (DE3) and purified with a high purity. The purified CS10 showed optimal activity at 50℃ and was stable up to 50℃, reflecting the thermal properties of the typical mesophilic enzymes. Specifically, CS10 was highly stable after a pre-incubation under the broad pH ranges of 4~10, which inferred that the enzyme can exist in a stable form even under an extreme pH condition required to avoid biological contaminations in industrial processes [9 , 10 , 30] . The addition of EDTA and some divalent metal ions had no significant influence on the activity of CS10, implying that the enzyme is not a metalloenzyme. Complete loss of the activity of CS10 in the presence of Hg 2+ suggested that at least one sulfhydryl group is involved in the active catalytic site of the enzyme. Moreover, no obvious inhibitory effect on the activity of CS10 was observed under the presence of reducing agents such as β-mercaptoethanol and DTT, suggesting there exist thiol groups in the active site of the enzyme. Non-ionic surfactants have the capability to modify the cellulase surface property during hydrolysis and help minimize the irreversible inactivation of the enzyme, suggesting definite advantages in commercial applications of the paper industry [2 , 32] . In the presence of 1% concentration of all these non-ionic detergents, the activity of CS10 was slightly increased compared with the control. CS10 was definitely stable against almost all polar organic solvents, with logP ow ≤ -0.24 in the presence of 10% concentration. Although there are few studies in the effect of organic solvents on the enzyme stability of metagenomederived cellulases, the enzyme stability of CS10 against organic solvents was higher than that of any other known metagenome-derived cellulases [11 , 24 , 27 , 31] . CS10 had efficient catalytic activities toward most of the soluble celluloses with a β-1,4-glucosidic bond (barely glucan, CMC, and 2-HE), with the exception of β-1,3-linked laminarin, which suggested that the catalytic activity of CS10 was specific for the β-1,4-glucan linkage of the amophous region in celluloses. In general, a cellulase without the cellulose-binding domain can hydrolyze only the soluble crystalline form of cellulose, reflecting that the cellulose-binding domain is involved in the degradation of insoluble cellulose with a high crystalline index [5 , 23 , 25] . None of the cellulose-binding domains were found in the deduced amino acid sequence via CDART program of NCBI, and thus no enzyme activity of CS10 was shown on the insoluble Avicel with approximately 70% crystallinity index, indicating a consistent result with previous proven study. Product patterns of cellooligosaccharides for CS10 were obtained on the hydrolysis of cellotetraose and cellopentaose. Cellotetraose was hydrolyzed to release only a cellobiose, and cellopentaose was drgraded to cellobiose and cellopentaose, suggesting that CS10 randomly cleaves the internal bonds of the cellulose chain, like an endo-type glucanase. The results of the substrate preference test provided more evidence to support the endo-mode of CS10 during substrate hydrolysis, in that the enzyme did not have catalytic activity on ρ-nitrophenyl-β-D-cellobioside or cellobiose.
In summary, a positive clone with cellulase activity ( CS10 ) was screened from a metagenomic library of the gut microflora of Hermetia illucens (a black soldier fly) and the CS10 gene was functionally expressed in E. coli BL21 (DE3). The purified CS10 was a novel endo-β-1,4-glucanase, classified as glycosyl hydrolase family 5. The enzyme activity of CS10 was highly stable against various severe factors such as broad pH ranges, polar organic solvents, non-ionic detergents, and denaturants, under the extremely stressful conditions that occur frequently during industrial processes.
These results demonstrate that metagenomics, based on the immense amounts of the uncultured microorganism’s genetic diversity and activity screening, could develop as the more efficient technology to identify novel enzymes resistant against the various extreme environments encountered in industrial processes.
Acknowledgements
The authors declare that no competing interests exist. This work is supported by grants from the National Academy of Agricultural Science, Rural Development Administration (Project No. PJ00864902) and IPET project no. 110037-03-1-HD110.
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