Characterization of a Lichenase Isolated from Soil Metagenome

Abstract

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-${\beta}$-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley ${\beta}$-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at $50^{\circ}C$, with Azo-barley-glucan as the substrate. The metal ions $Mn^{2+}$, $Mg^{2+}$, $Ca^{2+}$, and $Fe^{2+}$ enhanced the enzymatic activity, whereas the $Cu^{2+}$ and $Zn^{2+}$ ions inhibited the enzymatic activity. The $K_m$ and $V_{max}$ values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

Introduction

Eukaryotic and prokaryotic microorganisms are the main sources of many useful genes for biocatalysts. However, because 99% of the microorganisms in the environment cannot be cultured by the use of traditional methods, the screening of unculturable microorganisms to identify novel genes related to the production of chemical compounds and enzymes is gaining interest for industrial applications [14]. In fact, culture-independent metagenomic approaches have been successfully employed for the identification of genes for a wide range of enzymes such as lipases, esterases, amylases, hydrolases, dehydrogenases, and oxidoreductases [12]. Biomass-hydrolyzing enzymes are specifically major targets for metagenomic screening for various industrial applications. Although many different types of such enzymes have already been reported from fungi and bacteria [4,15,21], biomass-hydrolyzing enzymes with novel or better properties are continuously being mined from unculturable microorganisms [16,17,28] to meet their growing demands in the biodegradation process to obtain biologically renewable resources from agricultural by-products.

The β-1,3-1,4-glucans are non-starch linear polysaccharides of cell walls in the endosperm of grains such as barley, oats, rye, rice, wheat, and sorghum [8]. Most β-1,3-1,4-glucans in the walls of the cereals are composed of about 30% β-(1,3)- and 70% β-(1,4)-glycosyl linkages [36]. The biodegradation of β-glucans is naturally catalyzed mainly by four types of β-glucanases [2]. β-1,3-D-glucan 3-glucanohydrolase (laminarinase, E.C. 3.2.1.39), β-1,4-D-glucan 4-glucanohydrolase (cellulase, E.C. 3.2.1.4), and endo-β-1,3-1,4-glucanase (lichenase, E.C. 3.2.1.73) are endotype enzymes hydrolyzing β-1,3-1,4-glucans. The β-1,3(4)-glucanases (E.C. 3.2.1.6) are able to hydrolyze both β-1,3-1,4-glucan and β-1,3-glucan. Among them, endo-β-1,3-1,4-glucanase (lichenase) is a member of the glycosyl hydrolase family 8 (also known as cellulase D) comprising enzymes such as cellulase (E.C. 3.2.1.4), and chitosanase (E.C. 3.2.1.132). Lichenases especially cleave β-1,4-glycosidic bonds in 3-O-substituted glucopyranose units of β-glucan, yielding mainly cellobiosyltriose and cellotriosyltetraose as the final products [3], whereas cellulase (E.C. 3.2.1.4), laminarinase (E.C. 3.2.1.39), and β-1,3(4)-glucanases (E.C. 3.2.1.6) catalyze the endohydrolysis of 1,4-linkages in β-D-glucans, 1,3-linkages in β-D-glucans, and 1,3- or 1,4-linkages in β- D -glucans, respectively. They also hydrolyze 1,4-linkages in β-D-glucans containing 1,3-linkages, although laminarinase (E.C. 3.2.1.39) shows limited action on mixedlink (1,3-1,4-)-β-D-glucans. Since β-1,3-1,4-glucans often increase the viscosity of the solution, lichenases are of great interest for the brewing and animal feed industries. Lichenase has been applied in brewing processes to reduce the viscosity and turbidity of a brewer mash containing high-molecular-weight β-glucans, and to increase the yield of the extract and filtration rate [30]. Lichenases have also been used in the animal feed industry to improve the digestibility of barley-based feed stuffs and the efficiency of feed conversion [5]. In addition, lichenase can be used in laundry detergents for the removal of stains containing βglucan [9]. It has been reported that various organisms, including bacteria, fungi, and plants, produce lichenase [15,25,34]. Several bacterial lichenase genes have been cloned and expressed from Bacillus and non-Bacillus species for biochemical characterization [15,18,32,34]. Moreover, metagenomic lichenase genes have been identified from the animal gut microbiota [11,35]. Walter et al. [35] isolated four clones showing lichenase activity from the largebowel microbiota of mice, and one of them was analyzed as a lichenase belonging to glycosyl hydrolase family 16. No further enzymatic characterization was reported, however [35]. Feng et al. [11] isolated four endo-β-1,4-glucanase genes from the cecum microbiota of rabbit. These enzymes were analyzed to belong to glycosyl hydrolase family 5 and one of them was enzymatically characterized [11]. However, there has not yet been any soil metagenomic lichenases reported.

In this study, we identified and biochemically characterized a lichenase gene from soil metagenomic DNA libraries, demonstrating the usefulness of the metagenomic approach to mine novel biocatalysts for potential industrial applications.

 

Materials and Methods

Construction and Screening of Soil Metagenomic Library

Metagenomic DNA was extracted from forest soil (Daejeon, Korea) by using the FastDNA Spin Kit for soil (MP Biomedicals, Solon, OH, USA) in accordance with the manufacturer’s instructions. Extracted total metagenomic DNA was partially digested with restriction enzyme Sau3AI and separated by agarose gel electrophoresis. DNA fragments between 1.0 and 5.0 kb were eluted from the gel and ligated with BamHI-digested pBluescript II SK+ (Stratagene, La Jolla, CA, USA). The ligation reaction mixture was transformed into E. coli DH5α, and plated onto Luria-Bertani (LB) plates (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, 1.5% (w/v) agar) containing 0.5% carboxymethylcellulose (CMC; Sigma-Aldrich, St. Louis, MO, USA) or lichenan (Megazyme, Ireland) as the substrate, 0.002% (w/v) trypan blue as the indicator, and 100 µg/ml ampicillin (Amp). The colonies were screened for β-glucanase activity, indicated by clear halos around the colonies. The putative clones isolated by primary screening were reconfirmed by Congo red screening [18]. Briefly, the colonies were streaked onto LB-AmpCMC plates and incubated at 37℃. After overnight growth, the colonies on the plates were washed off with deionized distilled water (ddH2O) to remove cells, stained with 0.1% (w/v) Congo red solution for 30 min, and destained three times for 10 min with 1 M NaCl to detect the presence of clear halos around colonies. The plasmid of the positive clone, named pBlue-mt-lic, was retransformed into E. coli DH5α to confirm the cellulolytic activity. The sequence of the metagenomic DNA insert in pBluemt-lic was then analyzed by DNA sequencing (Genotech Co. Ltd., Daejeon, Korea).

Sequence Analysis

Database analyses were performed by using the BLASTN and BLASTP programs of the National Center for Biotechnology Information at the National Institute of Health (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments and phylogenetic tree construction of the putative protein were performed using T-Coffee [26]. For domain detection, the amino acid sequence of the protein was submitted to Pfam (http://pfam.sanger.ac.uk/) [31].

Expression and Purification of Recombinant Protein

The identified gene encoding endo-β-1,3-1,4-glucanase (Mt-lic) was amplified by PCR with the use of primers, mt-lic-F (5’-GGAATTCCATATGAGAAAAAATAAGAGATTTTCGTTTAGC-3’, NdeI site underlined) and mt-lic-R (5’-CCGCTCGAGGTATGTCCACCAGTTGCCGG-3’, XhoI site underlined), employing the pBlue-mt-lic vector as a template. The PCR amplicon was purified and digested with NdeI and XhoI. The resulting 1.23 kb DNA fragment was inserted into NdeI-XhoI-digested pET21a(+) (Novagen, Madison, WI, USA), resulting in pET21-mt-lic. Restriction enzymes, DNA polymerase, T4 DNA ligase, and alkaline phosphatase were purchased from Takara Bio, Inc. (Otsu, Japan). Oligonucleotide primers were obtained from Bioneer Corp. (Daejeon, Korea).

The plasmid pET21-mt-lic was transformed into E. coli C43 (DE3) by electroporation. After growing overnight, the E. coli C43 (DE3) harboring the pET21-mt-lic expression vector was reinoculated into LB medium containing 100 µg/ml of ampicillin and grown at 37℃ until the optical cell density at 600 nm (OD600) reached 0.6. The cells were then induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) and cultured at 16℃ for an additional 24 h. The cultured cells were harvested and washed twice with ddH2O by centrifugation. The cell pellet was dissolved in binding buffer A (10 mM imidazole, 300 mM NaCl, and 50 mM NaH2PO4, pH 8.0), containing 1 mM phenylmethylsulfonyl fluoride and then lysed by sonication. Purification was performed with the His-tag/Ni-NTA method according to the manufacturer’s instruction (Qiagen, Valencia, CA, USA). The eluates were concentrated at 4℃ by the use of Amicon Ultracell 10 K membrane filters (Millipore, Billerica, MA, USA). The concentrated samples were then transferred to 20,000 MWC Slide-A-Lyzer dialysis cassettes (Thermo-Fisher, Rockford, IL, USA), and dialyzed against 1 L of 20 mM sodium phosphate buffer (pH 7.4) for 4 h, which was changed every 2 h. The amount of purified protein was determined using the Bradford protein assay kit (Bio-Rad Inc, Hercules, CA, USA) with bovine serum albumin as the standard [6]. Samples from each purification step, including the lysate, load-out, washout, and eluates were collected and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [18]. For the analysis by western blotting, eluates were subjected to 12% SDS-PAGE and the proteins were transferred onto a polyvinylidene fluoride membrane. The primary antibodies used were anti-His antibody (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed with Tris-buffered saline (TBS; 0.05 M Tris and 0.15 M NaCl, pH 7.6) containing 0.05% Tween-20 (TBS-T), the blots were probed with anti-mouse HRP-conjugated secondary antibody (1:10,000; Sigma-Aldrich, St. Louis, MO, USA). Bands were visualized by using the ECL detection reagent (GE Healthcare Bioscience, Buckinghamshire, UK).

Detection of Lichenase Activity by Zymography

The purified enzyme was separated by electrophoresis by using a 12% SDS polyacrylamide gel containing 0.2% (w/v) lichenan. The gel was washed twice with 0.05 M citrate buffer (pH 5.6) for 30 min to remove the SDS from the gel, soaked for 30 min in 2.5% (w/v) Triton X-100 solution, and stained with 0.2% (w/v) Congo red solution [18].

Enzyme Assay and Determination of Kinetic Parameters

The lichenase activity was assayed using Azo-barley glucan as the substrate (Megazyme) according to the manufacturer’s instructions. Briefly, 125 µl of pre-warmed Azo-barley glucan substrate solution (1% (w/v) dye-labeled barley glucan in 0.02% sodium azide) and 125 µl of pre-incubated lichenase in assay buffer (0.1 M sodium phosphate, pH 6.0) were mixed vigorously and incubated at 50℃ for 10 min. To terminate the enzyme reaction, 750 µl of precipitant solution A (4% (w/v) sodium acetate, 0.4% (w/v) zinc acetate in 80% (v/v) methoxyethanol solution, pH 5.0) was added. After centrifugation, the absorbance of the supernatant at 590 nm was measured. One unit (U) of enzyme activity was defined as the amount of enzyme that releases 1 µmol of glucose reducing sugar equivalent per minute under the specified assay conditions. The kinetic parameters (Km and Vmax) of lichenase were calculated with the use of Azo-barley glucan (0.1-1.7 mg/ml) from the Lineweaver-Burk plots, using the SigmaPlot program (Systat Software, Inc., San Jose, CA, USA).

Biochemical Characterization of Metagenomic Lichenase

To determine the optimal reaction temperature of the purified lichenase, the standard enzyme assay was performed at different temperatures between 20℃ and 90℃ using 100 mM sodium phosphate buffer (pH 6.5). The effect of pH on the activity of the purified enzyme was tested by measuring the enzymatic activity at different pH values (0.1 M sodium acetate buffer, pH 3.0–5.0; 0.1 M sodium phosphate buffer, pH 6.0–8.0; 0.1 M sodium borate buffer, pH 9.0) at 50℃. The effects of metal ions on the enzymatic activity were investigated by the addition of 1 mM of metal ions (MnCl2, MgCl2, CuSO4, ZnSO4, CaCl2, or FeCl3) or 1 mM of chelating agent ethylenediaminetetraacetic acid (EDTA) into the reaction mixture. The enzyme activities at each temperature and pH, and with different metal ions, were determined with the use of Azo-barley glucan as a substrate by employing the standard assay method described above. The substrate specificity of the purified enzyme was determined using the 3,5-dinitrosalicylic acid (DNS) method [22] after reaction at 50℃ for 10 min with CM-cellulose (Sigma-Aldrich), beechwood xylan (Sigma-Aldrich), barley glucan (Sigma-Aldrich), lichenan (Megazyme), medium molecular weight chitosan (Sigma-Aldrich), or soluble starch (Samchun Chemical, Korea), dissolved in 100 mM sodium phosphate buffer (pH 6.0) as substrates. The results are presented as an average of three trials.

 

Results and Discussion

Construction and Screening of the Soil Metagenomic DNA Plasmid Library

Small-insert metagenomic DNA plasmid libraries, which contain single genes or small operons encoding enzymes for the synthesis of novel biomolecules, are generally more effective than large-insert libraries for the isolation and characterization of functionally useful sections of environmentally derived DNA [10]. In this study, a plasmid library containing approximately 19,626 clones was constructed from metagenomic DNA isolated from a soil sample. When the plasmid DNA samples isolated from 10 randomly selected clones were analyzed by digestion with XbaI, the size of inserted metagenomic DNA fragments was found to be between 1.3 to 4.0 kb. The average size of the inserted DNA was approximately 1.9 kb, suggesting that the total library size was about 3.73 × 107 bp. The functionbased screening of this soil metagenomic DNA plasmid library identified one clone expressing CMCase activity upon primary screening on indicator plates and subsequent confirmation by Congo red staining (data not shown).

Sequence Analysis of the Metagenomic Lichenase

The CMCase-positive clone was sequenced, and an open reading frame of 1,230 base pairs was identified and named mt-lic. The nucleotide sequence of gene mt-lic was submitted to GenBank under Accession No. KM079629. The putative ribosome-binding site (5’-AAAGGAG-3’) was found 9 bp upstream of the initiation codon. An inverted repeated sequence that could form a stem-loop structure was found 29 bp downstream of the stop codon (Fig. 1A). The mt-lic gene encoded a protein with 409 amino acid residues with a molecular mass of 45.1 kDa. Analysis of the deduced amino acid sequence by SignalP [29] identified a signal peptide with a putative cleavage site after the first 31 residues. The BLASTX analysis showed that the amino acid sequence of Mt-lic exhibited 92% identity and 97% similarity with endo-β-1,3-1,4-glucanase having both lichenase and chitosanase activities, encoded by the bgc gene of Bacillus circulans WL-12 (GenBank Accession No. P19254) [7]. Mt-lic also showed a high degree of amino acid sequence homology with lichenases of Paenibacillus species, such as P. cookie (BAL46897.1) and P. lactis 154 (ZP_09002324.1), which belong to glycosyl hydrolase family 8 (data not shown). In addition, the conserved catalytic module with the amino acid sequence ATDGDLDIAYALLLASLQW, which is specific for glycosyl hydrolase family 8, was found in Mt-lic [33]. Furthermore, the potential catalytic amino acid residues Glu95 (proton donor), Asp156 (nucleophile), and Glu286 (proton acceptor) were identified, suggesting that Mt-lic belongs to subfamily b of glycosyl hydrolase family 8 [1]. Based on the amino acid sequences with the highest similarities, a phylogenetic tree of the deduced amino acid sequence of Mt-lic was constructed. The results suggested that Mt-lic was closely related to lichenases belonging to glycosyl hydrolase family 8. Interestingly, previously reported metagenomic lichenases from animal gut microbiota were shown to be distantly related to Mt-lic (Fig. 1B).

Fig. 1.Nucleotide and deduced amino acid sequences of the mt-lic gene. (A) The underlined amino acid sequence indicates a putative signal peptide. The arrow indicates the signal peptide cleavage position as determined by N-terminal sequencing of the purified protein. The conserved amino acid sequence of glycosyl hydrolase family 8 is boxed [33]. The residues Glu95 (proton donor), Asp156 (nucleophile), and Glu286 (proton acceptor) marked with triangles indicate the conserved catalytic residues of glycosyl hydrolase subfamily 8 [1, 13]. The underlined nucleotide sequence indicates a putative ribosome-binding sequence. The convergent arrows under the nucleotide sequence indicate a palindromic sequence, the putative transcription terminator. (B) Phylogenetic analysis of the metagenomic lichenase (Mt-lic) was performed by T-Coffee (http://www.tcoffee.org/). Protein sequences are labeled with gene names and GenBank accession numbers.

Expression and Purification of the Recombinant Metagenomic Lichenase from E. coli

To characterize the function of Mt-lic, the target gene was cloned into the pET21a(+) expression vector, which adds 6×His-tag to the C-terminus of the target protein, resulting in pET21-mt-lic. This vector was then transformed into several E. coli strains such as BL21 (DE3), Rosetta gami 2 (DE3), C41 (DE3), and C43 (DE3) [23]. However, except for the E. coli C43 (DE3)/pET21-mt-lic transformant, the expression of the 6×His-tagged Mt-lic was not detected with western blot analysis. Therefore, the E. coli C43 (DE3) strain was used for the remainder of the experiments. First, the expression of the lichenase was tested on LB-AmpIPTG agar plates containing 0.1% (w/v) lichenan. The lichenase activity was indicated by clear orange halos formed around the colonies of the E. coli C43 (DE3)/pET21-mt-lic transformant after Congo red staining (data not shown). The SDS-PAGE analysis of the crude extract showed that the target protein was being expressed (Fig. 2). After purification of the crude extract on a Ni-NTA column, a single band of the recombinant protein was observed on the SDS-PAGE gel, as described above (Fig. 2A). Furthermore, the Mt-lic activity was clearly detected by activity staining (Fig. 2B). However, the size of Mt-lic on the SDS-PAGE gel was smaller than expected based on the theoretical molecular mass of 45,133 Da, supporting the possibility of cleavage after the predicted signal peptide resulting in a protein with the expected molecular mass of 41.694 kDa. As expected, the N-terminal sequence of the purified mature protein was determined to be SPNKP (data not shown).

Fig. 2.Purification and activity staining of recombinant lichenase. (A) SDS-polyacrylamide gel electrophoresis of recombinant lichenase produced in E. coli C43 (DE3). Lanes: M, low-molecular-weight standard protein markers; 1, total cell lysate of the induced transformant harboring the pET21a(+) control plasmid; 2, total cell lysate of the induced transformant harboring the recombinant pET21-mt-lic plasmid; 3, purified recombinant lichenase. (B) Zymography analysis. Total cell lysates of control (1) and lichenase-producing (2) E. coli strains were separated on an SDS-PAGE gel containing 0.2% lichenan and stained with Congo red solution. The lichenase activity is indicated by a clear orange zone.

Characterization of the Recombinant Mt-lic Protein

When Azo-barley-glucan was used as the substrate, the optimal temperature for the purified metagenomic lichenase was 40–50℃, and its maximum activity was observed at 50℃ (Fig. 3A). This result is similar to those reported for the lichenases from Bacillus species [20,27]. The metagenomic lichenase retained more than 20% of its activity at temperatures higher than 60℃ (Fig. 3A). The optimum pH for metagenomic lichenase activity was pH 6.0, and we estimated that more than 80% of the activity was retained within the range of pH 5.0–6.5. However, when the pH was out of this range, the enzymatic activity decreased rapidly (Fig. 3B). In order to determine the effect of various metal ions on the hydrolytic properties of Mt-lic, the ability of this enzyme to hydrolyze the Azo-barley glucan was investigated with the addition of 1.0 mM of MnCl2, MgCl2, CaCl2, FeCl3, CuSO4, ZnSO4, or EDTA to the reaction mixture (Table 1). The enzymatic activity was slightly stimulated by Mg2+ and Ca2+ ions, whereas it was not affected by Mn2+ and Fe2+ ions. The chelating agent EDTA also slightly stimulated the enzymatic activity, suggesting the possibility of the inhibitory effects of some metal ions in the reaction mixture. In fact, in the presence of CuSO4 and ZnSO4, the Mt-lic activity was inhibited significantly and reduced to 74.85% and 23.42% of the initial activity, respectively. The activation and inactivation by these metal ions were similar to that shown by the purified lichenase EG1 from Bacillus licheniformis UEB CF [9].

Fig. 3.Effects of temperature (A) and pH (B) on lichenase activity. Buffers used were 0.1 M sodium acetate for pH 3.0–5.0, 0.1 M sodium phosphate for pH 6.0–8.0, and 0.1 M sodium borate for pH 9.0. The data represent the mean of three replications (± the standard deviation).

Table 1.aThe data represent the mean of three replications (± the standard deviation). bThe purified protein was preincubated with 10 mM EDTA for 12h and then dialyzed in high-pressure liquid chromatography-grade water overnight. The enzymatic activity in the absence of the metal ions was taken as 100%.

Substrate Specificity and Kinetic Parameters of the Recombinant Metagenomic Lichenase

In order to examine the substrate specificity, the enzymatic activity of the purified recombinant metagenomic lichenase on the substrates CM-cellulose, xylan, barley glucan, and lichenan was determined by DNS assay. The enzyme showed the highest hydrolytic activity on barley glucan (Table 2). When compared with the activity on barely glucan, the enzyme showed 70% of activity on lichenan. It is noteworthy that barley β-glucan and lichenan have different ratios of the β-1,3 and β-1,4 glycosidic bonds, with approximately 2:1 ratio for lichenan, and approximately 1:3 for barley glucan [2]. Therefore, the different hydrolytic activities of Mt-lic on barley β-glucan and lichenan could be due to the different ratios of the β-1,3 and β-1,4 glycosidic bonds in these substrates. The metag enomic lichenase showed significantly weak activity (<20%) on CM-cellulose, soluble starch, and xylan in comparison with barley glucan. Interestingly, Mt-lic showed only less than 10% hydrolytic activity toward chitosan compared with that toward lichenan under our experimental conditions (data not shown). However, it will require further investigation to determine whether Mt-lic cannot hydrolyze the linkages between GlcN-GlcN and GlcN-GlcAc in chitosan, unlike bgc-encoded endo-β-1,3-1,4-glucanase of B. circulans WL-12 [24], or whether a different assay condition is required to allow the hydrolysis of chitosan by Mt-lic. With Azo-barley glucan as the substrate, Lineweaver-Burk plots were used to determine the kinetic parameters of the purified metagenomic lichenase. The Km and Vmax values were determined to be 0.45 mg/ml and 24.83 U/min/mg protein, respectively (Fig. 4).

Table 2.aThe data represent the mean of three replications (± the standard deviation).

Fig. 4.The kinetic parameters of lichenase. The apparent Michaelis constants Km and Vmax were calculated using the Michaelis-Menten equation. The insert shows the resulting Lineweaver-Burk plot. The values are the means of three independent experiments (± the standard deviations).

In conclusion, a metagenomic lichenase gene (mt-lic) was identified through function-based screening of a soil metagenomic library, expressed in E. coli, and biochemically characterized. Its deduced amino acid sequence exhibited a greater similarity to the previously isolated B. circulans WL-12 endo-β-1,3-1,4-glucanase, having both lichenase and chitosanase activities. The purified recombinant enzyme was able to hydrolyze lichenan. The enzyme activity could be enhanced by the metal ions Mg2+ and Ca2+. This study demonstrates the possibility to employ a metagenomic approach to mine a novel lichenase that can be further improved through protein engineering for industrial applications.