Biotechnol Lett DOI 10.1007/s10529-015-1777-5
ORIGINAL RESEARCH PAPER
Direct detection, cloning and characterization of a glucoside hydrolase from forest soil Mei Hua • Shubo Zhao • Lili Zhang • Dongbo Liu • Hongmei Xia • Fan Li • Shan Chen
Received: 29 October 2014 / Accepted: 21 January 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract A glucoside hydrolase gene, egl01, was cloned from the soil DNA of Changbai Mountain forest by homologous PCR amplification. The deduced sequence of 517 amino acids included a catalytic domain of glycoside hydrolase family 5 and was homologous to a putative cellulase from Bacillus licheniformis. The recombinant enzyme, Egl01, was maximally active at pH 5 and 50 °C and it was stable at pH 3–9, 4–50 °C, and also stable in the presence of metal ions, organic solvents, surfactants and salt. Its activity was above 120 % in 2–3 M NaCl/KCl and over 70 % was retained in 1–4 M NaCl/KCl for 6d. Egl01 hydrolyzed carboxymethyl cellulose, beechwood xylan, crop stalk, laminarin, filter paper, and avicel but not
Electronic supplementary material The online version of this article (doi:10.1007/s10529-015-1777-5) contains supplementary material, which is available to authorized users. M. Hua S. Zhao L. Zhang D. Liu H. Xia F. Li (&) S. Chen (&) School of Life Science, Northeast Normal University, Changchun 130024, China e-mail: [email protected] S. Chen e-mail: [email protected] M. Hua e-mail: [email protected] D. Liu e-mail: [email protected] H. Xia e-mail: [email protected]
pNPG, indicating its broad substrate specificity. These properties make this recombinant enzyme a promising candidate for industrial applications. Keywords Changbai mountain forest soil Direct cloning Functional characterization Glucoside hydrolase
Introduction Cellulose, which consists of linear polymers of b-1,4linked glucoses, is the principal structural component of plant cell walls and is considered as the most abundant biomass in nature (Carroll and Somerville 2009). Degradation and conversion of cellulose are mostly dependent on cellulolytic enzymes from microorganisms. Cellulases have attracted increasing industrial interest and are widely used in various fields, such as textile, detergent, food processing, animal feed, biofuel, and biorefinery (Lange 2007). However, cellulases with high activities or with properties appropriate for industrial use are still lacking. PCR amplification technology has been frequently used for the direct isolation of functional genes from environmental DNA according to recent studies (Cucurachi et al. 2013; Itoh et al. 2014). Here, we have aimed to obtain cellulases from forest soil using the homologous PCR amplification approach. An new glucoside hydrolase, Egl01, was cloned, expressed,
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and purified. Recombinant Egl01 was characterized and was identified as a candidate for further research and possible industrial applications.
in NCBI (http://www.ncbi.nlm.nih.gov/Blast), and the signal peptide was predicted using the Signal P 4.0 server (http://www.cbs.dtu.dk/services/SignalP/). Expression and purification of recombinant Egl01
Materials and methods Strains, plasmids, and chemicals E. coli DH5a and pMD18-T from TaKaRa were used as the gene clone host and vector, respectively. E. coli BL21 (DE3) and pET-22b (?) (Novagen) were used for heterologous expression. DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from TaKaRa. The substrates carboxymethylcellulose (CMC), beechwood xylan, laminarin, Avicel, and filter paper (Whatman No.1) were purchased from Sigma. Crop stalks were dried at 80 °C for 24 h and ground into powder. All chemicals used were of analytical grade.
The recombinant was grown in LB medium containing 100 lg ampicillin/ml at 37 °C and 200 rpm up to an OD600 of 0.6–0.8, and then induced with 0.8 mM IPTG (final concentration) at 37 °C for 6 h. The cells were harvested and resuspended in lysis buffer (20 mM Tris/ HCl buffer, and 500 mM NaCl; pH 7.6) at 4 °C and lysed by sonication. After centrifugation at 14,0009g for 20 min, the supernatant was loaded onto the high-affinity Ni-NTA resin and purified by elution with a linear gradient of imidazole from 20 to 200 mM. Active fractions subjected to SDS-PAGE with single-band were pooled for future detection. Purified Egl01 was quantified by Bradford method using bovine serum albumin as the standard protein.
Sample collection and genomic DNA extraction Enzyme assay Soil samples were collected from Changbai Mountain forest (128°40 E, 42°50 N) in Jilin Province, China. Soils were collected from the surface stratum (3–15 cm deep), stored in ice box and immediately frozen at -80 °C before use. Soil genomic DNA was extracted using PowerMax Soil DNA isolation kit (MO BIO Laboratories, Inc., USA). The average concentration of purified DNA for downstream application was 89 ng ll-1.
Purified recombinant Egl01, 0.1 ml, was diluted to an appropriate concentration and mixed with 1.9 ml CMC (1 % w/v) dissolved in 200 mM Tris/HCl buffer (pH 7.6) to assay the activity. After incubation at 50 °C for 20 min, the reducing sugars from CMC were measured as D-glucose equivalents using the DNS method. One unit of activity was defined as the amount of enzyme to release 1 lmol in full reducing sugar per min.
Cloning of the gene from environment DNA Substrate specificity of Egl01 Degenerate primers (50 -GGAATTCCKTAYCAYTC HATWGAAA-30 and 50 - CATGCCTGVAWWCC RAACCA-30 ) (Javier et al. 2010) were used to detect and amplify the conserved regions of the genes. PCR was performed as follows: 5 min at 95 °C, followed by 15 cycles of 95 °C for 30 s, 65–50 °C (decreasing by 1 °C after each cycle) for 30 s, and 72 °C for 70 s, followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 70 s. Final extended at 72 °C for 10 min. The primer sets (forward: 50 -CGGGATCC GATGTCATATATGAAACGTTCC-30 and reverse: 50 -CCGCTCGAGTTTAGGTTCAGTGCCCC-30 ) with restricted enzyme sites for BamHI and XhoI were designed based on similar sequences of the conserved region and were used to clone the full-length gene egl01. Homology analysis was performed by BLAST
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The substrate specificity of recombinant Egl01 was determined in different substrates (1 % w/v) dissolved in 200 mM Na2HPO4/citric acid buffer (pH 5.0). The kinetic assays were performed under optimal temperature and pH conditions with CMC (1–10 mg ml-1) as the substrate. The Km and the Vmax were calculated according to the Lineweaver–Burk equation.
Results Cloning and expression of egl01 Partial gene fragments were amplified by PCR using degenerate primers and exhibited 71–99 % identity to
Biotechnol Lett
glucoside hydrolases from cultured or uncultured microbes in GenBank. Among the other genes, a partial gene fragment with 1,134 bp showed 99 % identity to a putative endo-b-1,4-glucanase from Bacillus licheniformis with genome sequenced (CAJ70710.1) (Waldeck et al. 2006). By using specific primers, full-length gene egl01 was directly cloned from the environmental genomic DNA of Changbai mountain forest soil. egl01 comprised an ORF of 1,554 bp encoded a 517-residue polypeptide with a theoretical molecular weight of 53.4 kDa and theoretical pI of 9.21, predicted with a 32-amino acid signal peptide at the N-terminal region. Sequence analysis revealed that the mature polypeptide was a modular enzyme with its catalytic domain of glucoside hydrolase family 5 (GH5) in the N-terminal portion and its cellulose binding domain 3 in the Cterminal portion. egl01 was expressed and purified as described in the Methods section. SDS-PAGE analysis revealed a single band with an MW of 53 kDa close to the theoretical value, and the activity of Egl01 was observed in zymogram (Fig. 1). The specific activity of the purified recombinant Egl01 on CMC was 152 U mg-1. Effects of pH and temperature on the activity of Egl01 The maximum activity of the enzyme with CMC was at pH 5, indicating that Egl01 was acidophilic (Fig. 2a). The maximum activity of Egl01 was at 50 °C measured over 20 min (Fig. 2b). Egl01 was stable over a wide pH range, with over 80 % activity from pH 4 to pH 8 and approx. 50 % activity at pH 3 and 11 (Fig. 2c). Egl01 was stable up to 50 °C for 3 h with 80 % activity remaining (Fig. 2d). Similar studies with other substrates with which Eg101 displayed relatively higher activity such as xylan and laminarin also gave the same results. Effects of metal ions and chemical reagents on the activity of Egl01 The effect of different metal ions and chemical reagents on the enzyme activity may result from the binding of substances to amino acid residues important for the catalysis or affect the charge distribution of enzyme molecules or active sites. These substances
Fig. 1 12 % SDS-PAGE and zymogram of Egl01. Lane 1 molecular mass markers, Lane 2 total protein of cells induced with IPTG, Lane 3 purified recombinant Egl01, Lane 4 zymogram of purified Egl01. In zymogram assay, Egl01 was resolved in SDS loading buffer and performed in SDS-PAGE with 0.1 % (w/v) CMC at 4 °C, then the PAGE was incubated at 50 °C for 30 min before stained by Congo Red
have thus been frequently used to probe into the response of enzymes’ catalytic site to inhibition or stimulation. The effects of various metal ions and chemical reagents on the activity of Egl01 were measured (Supplementary Table 1). No significant effects were found with metal ions (including Fe2?, Cu2? and Co2?) at 1 mM. Non-ionic surfactants (Triton X-100 and Tween 80), organic solvents, and SDS slightly decreased Egl01 activity. The greater effect was a 39 % inhibition by 2 % (v/v) SDS. Greater effects have been reported by Liu et al. 2011; Zhu et al. 2011. The zymogram of SDS-PAGE with 0.1 % CMC (2.5 % SDS totally) in Fig. 1 also suggested that Egl01 has outstanding stability. Effect of NaCl/KCl on the activity of Egl01 Figure 3 showed the effect of salt on Egl01 activity. The activity was stimulated with 1–4 M NaCl/KCl, and showed 139 and 135 % activity in 2 M NaCl and 3 M KCl, respectively (Fig. 3a). After pre-incubation for 6 d in 1 M–4 M KCl/NaCl, residual activities were over 76 % (4 M KCl) and 72 % (4 M NaCl) (Fig. 3b and c).
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Fig. 2 Effects of pH and temperature on the activity of Egl01. a and b The dependence of activity on pH and temperature. The optimal pH for Egl01 was tested in the range pH 3–12 at 50 °C. The optimal temperature for Egl01 was tested from 20–80 °C in the optimal pH buffer. c pH-stability and d thermostability of
Egl01. Egl01 was kept at pH 3–12 at 4 °C for 24 h or at 4–70 °C for 3 h. Residual activities were assayed under optimum conditions. 100 % activity was 118.5 Uml-1. The error bars represent the mean ± SD (n = 3)
Substrate specificity and hydrolysate of Egl01
Discussion
Table 1 shows the substrate specificity of the purified Egl01. Figure 4 shows hydrolysis of Egl01 on cellooligosaccharides examined with TLC. No hydrolysis was observed for G2 and G3. Partial products of G4 and G5 were detected to be G2 and G3; G4 was the smallest oligosaccharide that could be hydrolyzed. The Km and Vmax values were 3.4 mg/ml and 186 lmol/(minmg), respectively, similar to those of cellulases from Salinivibrio sp. NTU-05 [Km = 3 mg/ ml and Vmax = 143 mol/(minmg)] (Wang et al. 2009) and B. pumilus S124A [Km = 2.1 mg/ml and Vmax = 239 lmol/(minmg)] (Natesan and Nelson 2014).
In the present study, recombinant Egl01 showed some noticeable properties that were different to some endoglucanases already described. Firstly, Egl01 was acidophilic while many cellulases characterized from bacteria are alkalophilic (Trivedi et al. 2011; Annamalai et al. 2013). A short peptide ‘‘QGKLIWGTEPN’’ at the C-terminus of the endoglucanase from B. subtilis was considered as being probably related to this acidophilic property (Zhu et al. 2011). Interestingly, a similar short peptide ‘‘NGKLIWGTEPK’’ was also observed at the C-terminus of Egl01, thereby indicating that this peptide might actually be related to the acidophilic property of the glucoside hydrolase
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Biotechnol Lett Table 1 Substrate specificity of the recombinant enzymea Substrate
Special activity (U mg-1)
CMC
152 ± 2.4b
Crop stalk
12 ± 0.4
Filter paper Laminarin
8.1 ± 0.1 45 ± 0.6
Beechwood xylan
79 ± 0.4
Avicel
3.5 ± 0.1
pNPG
0
a
The activity was measured under optimum conditions, except for the filter paper and Avicel incubation at 50 °C for 120 min
b
Values represent the mean ± SD (n = 3) relative to control samples
Fig. 3 Effect of salts on the activity of Egl01. a activity and b, c halo-stability of Egl01 in NaCl/KCl. The reaction system contained 1–4 M NaCl/KCl as the final concentrations were tested under optimum conditions. For the halo-stability, Egl01 was incubated in 1–4 M NaCl/KCl as the final concentration for 1–6d at 4 °C. Reaction solution without any salt added was used as control with 100 % activity, 152.3 Uml-1. The error bars represent the mean ± SD (n = 3)
from Bacillus sp. Considering the typical acidophilia and stability over a broad pH range, Egl01 may be a candidate for biotechnological processes in acidic solutions or in combination with other acidophilic glucoside hydrolase. Secondly, Egl01 showed better activity and stability in the presence of high salt concentrations compared
Fig. 4 Hydrolysis of recombinant Egl01 on cello-oligosaccharides. TLC was performed with chloroform/methanol/acetic acid-water (50:50:15:5, by vol.). Sugars were visualized by incubating the TLC plate at 100 °C for 15 min after spraying with 5 % (v/v) sulfuric acid in ethanol. Lanes 1–4: G2-G5 treated with the recombinant Egl01 at 50 °C for 1 h; Lane 5, Standard sugars: glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4), and cellopentaose (G5)
with some cellulases from extremophiles (Liang et al. 2011; Li and Yu 2012). However, the recombinant Egl01with high halotolerance was directly derived from the forest soil, which is not an extreme environment. Similar properties were recently found in other enzymes (Xu et al. 2014; Nacke et al. 2012) thereby suggesting that some extreme enzyme properties may not only be due to the environment, but also to the specific structures formed during evolution. Further
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research may be helpful to assess the protein function change in extremophiles. Egl01 can hydrolyse a wide range of b-1,4-, b-1,3/ b-1,4-, or b-1,3/b-1,6-linked polysaccharides. Maximum activity of Egl01 was with CMC (152 U mg-1), and less activity on other related substrates. We speculate that Egl01 is probably an endoglucanase.
Conclusion A new glucoside hydrolase gene egl01 was directly cloned from forest soil using the homologous PCR amplification method. Recombinant Egl01 exhibited several special properties, such as acidophilia, halotolerance, organic reagent tolerance, and broad substrate specificity. All these properties show that Egl01 is a robust glucoside hydrolase that can maintain activity in tough environment and is a candidate for industrial applications. Acknowledgments This work was supported by National Natural Science Foundation of Jilin, China (Grant No. 20140101137JC) and State Key Laboratory Program of Microbial Technology of Shandong University. Supporting information Supplementary Table 1- Effect of metal ions and chemical reagents on Egl01.
References Annamalai N, Rajeswari MV, Elayaraja S, Balasubramanian T (2013) Thermostable, haloalkaline cellulase from Bacillus halodurans CAS1 by conversion of lignocellulosic wastes. Carbohydr Polym 94:409–415 Carroll A, Somerville C (2009) Cellulosic biofuels. Annu Rev Plant Biol 60:165–182 Cucurachi M, Busconi M, Marudelli M, Soffritti G, Fogher C (2013) Direct amplification of new cellulose genes from woodland soil purified DNA. Mol Biol Rep 40:4317–4325 Itoh N, Isotani K, Makino Y, Kato M, Kitayama K, Ishimota T (2014) PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes
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from the soil metagenome for biocatalysis. Enzyme Microb Technol 55:140–150 Javier Izquierdo A, Maria Sizova V, Lee Lynd R (2010) Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost. Appl Environ Microbiol 76:3545–3553 Lange JP (2007) Lignocellulose conversion: an introduction to chemistry, process and economics. Biofuel Bioprod Biorefin 1:39–48 Li X, Yu HY (2012) Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate, Bacillus sp. L1. J Ind Microbiol Biotechnol 39:1117–1124 Liang C, Xue Y, Fioroni M, Rodrı´guez-Ropero F, Zhou C, Schwaneberg U, Ma Y (2011) Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4. Appl Microbiol Biotechnol 89:315–326 Liu J, Liu WD, Zhao XL, Shen WJ, Cao H, Cui ZL (2011) Cloning and functional characterization of a novel endo-b1,4-glucanase gene from a soil-derived metagenomic library. Appl Microbiol Biotechnol 89:1083–1092 Nacke H, Engelhaupt M, Brady S, Fischer C, Tautzt J, Daniel R (2012) Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes. Biotechnol Lett 34:663–675 Natesan B, Nelson S (2014) Bacillus pumilus S124A carboxymethyl cellulase; a thermo stable enzyme with a wide substrate spectrum utility. Int J Biol Macromol 67:132–139 Trivedi N, Gupta V, Kumar M, Kumari P, Reddy CR (2011) An alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca. Carbohydr Polym 83:891–897 Waldeck J, Daum G, Bisping B, Meinhardt F (2006) Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. Appl Environ Microbiol 72:7879–7885 Wang CY, Hsieh YR, Ng CC, Chan H, Lin HT, Tzeng WS (2009) Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05. Enz Microb Technol 44:373–379 Xu JX, He BF, Wu B, Wang B, Wang C, Hu L (2014) An ionic liquid tolerant cellulase derived from chemically polluted microhabitats and its application in in situ saccharification of rice straw. Bioresour Technol 157:166–173 Zhu C, Xu Z, Song R (2011) The endoglucanase from Bacillus subtilis BEC-1 bears halo-tolerant, acidophilic and dithiothreitol-stimulated enzyme activity. World J Microb Biotechnol 27:2863–2871
ORIGINAL RESEARCH PAPER
Direct detection, cloning and characterization of a glucoside hydrolase from forest soil Mei Hua • Shubo Zhao • Lili Zhang • Dongbo Liu • Hongmei Xia • Fan Li • Shan Chen
Received: 29 October 2014 / Accepted: 21 January 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract A glucoside hydrolase gene, egl01, was cloned from the soil DNA of Changbai Mountain forest by homologous PCR amplification. The deduced sequence of 517 amino acids included a catalytic domain of glycoside hydrolase family 5 and was homologous to a putative cellulase from Bacillus licheniformis. The recombinant enzyme, Egl01, was maximally active at pH 5 and 50 °C and it was stable at pH 3–9, 4–50 °C, and also stable in the presence of metal ions, organic solvents, surfactants and salt. Its activity was above 120 % in 2–3 M NaCl/KCl and over 70 % was retained in 1–4 M NaCl/KCl for 6d. Egl01 hydrolyzed carboxymethyl cellulose, beechwood xylan, crop stalk, laminarin, filter paper, and avicel but not
Electronic supplementary material The online version of this article (doi:10.1007/s10529-015-1777-5) contains supplementary material, which is available to authorized users. M. Hua S. Zhao L. Zhang D. Liu H. Xia F. Li (&) S. Chen (&) School of Life Science, Northeast Normal University, Changchun 130024, China e-mail: [email protected] S. Chen e-mail: [email protected] M. Hua e-mail: [email protected] D. Liu e-mail: [email protected] H. Xia e-mail: [email protected]
pNPG, indicating its broad substrate specificity. These properties make this recombinant enzyme a promising candidate for industrial applications. Keywords Changbai mountain forest soil Direct cloning Functional characterization Glucoside hydrolase
Introduction Cellulose, which consists of linear polymers of b-1,4linked glucoses, is the principal structural component of plant cell walls and is considered as the most abundant biomass in nature (Carroll and Somerville 2009). Degradation and conversion of cellulose are mostly dependent on cellulolytic enzymes from microorganisms. Cellulases have attracted increasing industrial interest and are widely used in various fields, such as textile, detergent, food processing, animal feed, biofuel, and biorefinery (Lange 2007). However, cellulases with high activities or with properties appropriate for industrial use are still lacking. PCR amplification technology has been frequently used for the direct isolation of functional genes from environmental DNA according to recent studies (Cucurachi et al. 2013; Itoh et al. 2014). Here, we have aimed to obtain cellulases from forest soil using the homologous PCR amplification approach. An new glucoside hydrolase, Egl01, was cloned, expressed,
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and purified. Recombinant Egl01 was characterized and was identified as a candidate for further research and possible industrial applications.
in NCBI (http://www.ncbi.nlm.nih.gov/Blast), and the signal peptide was predicted using the Signal P 4.0 server (http://www.cbs.dtu.dk/services/SignalP/). Expression and purification of recombinant Egl01
Materials and methods Strains, plasmids, and chemicals E. coli DH5a and pMD18-T from TaKaRa were used as the gene clone host and vector, respectively. E. coli BL21 (DE3) and pET-22b (?) (Novagen) were used for heterologous expression. DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from TaKaRa. The substrates carboxymethylcellulose (CMC), beechwood xylan, laminarin, Avicel, and filter paper (Whatman No.1) were purchased from Sigma. Crop stalks were dried at 80 °C for 24 h and ground into powder. All chemicals used were of analytical grade.
The recombinant was grown in LB medium containing 100 lg ampicillin/ml at 37 °C and 200 rpm up to an OD600 of 0.6–0.8, and then induced with 0.8 mM IPTG (final concentration) at 37 °C for 6 h. The cells were harvested and resuspended in lysis buffer (20 mM Tris/ HCl buffer, and 500 mM NaCl; pH 7.6) at 4 °C and lysed by sonication. After centrifugation at 14,0009g for 20 min, the supernatant was loaded onto the high-affinity Ni-NTA resin and purified by elution with a linear gradient of imidazole from 20 to 200 mM. Active fractions subjected to SDS-PAGE with single-band were pooled for future detection. Purified Egl01 was quantified by Bradford method using bovine serum albumin as the standard protein.
Sample collection and genomic DNA extraction Enzyme assay Soil samples were collected from Changbai Mountain forest (128°40 E, 42°50 N) in Jilin Province, China. Soils were collected from the surface stratum (3–15 cm deep), stored in ice box and immediately frozen at -80 °C before use. Soil genomic DNA was extracted using PowerMax Soil DNA isolation kit (MO BIO Laboratories, Inc., USA). The average concentration of purified DNA for downstream application was 89 ng ll-1.
Purified recombinant Egl01, 0.1 ml, was diluted to an appropriate concentration and mixed with 1.9 ml CMC (1 % w/v) dissolved in 200 mM Tris/HCl buffer (pH 7.6) to assay the activity. After incubation at 50 °C for 20 min, the reducing sugars from CMC were measured as D-glucose equivalents using the DNS method. One unit of activity was defined as the amount of enzyme to release 1 lmol in full reducing sugar per min.
Cloning of the gene from environment DNA Substrate specificity of Egl01 Degenerate primers (50 -GGAATTCCKTAYCAYTC HATWGAAA-30 and 50 - CATGCCTGVAWWCC RAACCA-30 ) (Javier et al. 2010) were used to detect and amplify the conserved regions of the genes. PCR was performed as follows: 5 min at 95 °C, followed by 15 cycles of 95 °C for 30 s, 65–50 °C (decreasing by 1 °C after each cycle) for 30 s, and 72 °C for 70 s, followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 70 s. Final extended at 72 °C for 10 min. The primer sets (forward: 50 -CGGGATCC GATGTCATATATGAAACGTTCC-30 and reverse: 50 -CCGCTCGAGTTTAGGTTCAGTGCCCC-30 ) with restricted enzyme sites for BamHI and XhoI were designed based on similar sequences of the conserved region and were used to clone the full-length gene egl01. Homology analysis was performed by BLAST
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The substrate specificity of recombinant Egl01 was determined in different substrates (1 % w/v) dissolved in 200 mM Na2HPO4/citric acid buffer (pH 5.0). The kinetic assays were performed under optimal temperature and pH conditions with CMC (1–10 mg ml-1) as the substrate. The Km and the Vmax were calculated according to the Lineweaver–Burk equation.
Results Cloning and expression of egl01 Partial gene fragments were amplified by PCR using degenerate primers and exhibited 71–99 % identity to
Biotechnol Lett
glucoside hydrolases from cultured or uncultured microbes in GenBank. Among the other genes, a partial gene fragment with 1,134 bp showed 99 % identity to a putative endo-b-1,4-glucanase from Bacillus licheniformis with genome sequenced (CAJ70710.1) (Waldeck et al. 2006). By using specific primers, full-length gene egl01 was directly cloned from the environmental genomic DNA of Changbai mountain forest soil. egl01 comprised an ORF of 1,554 bp encoded a 517-residue polypeptide with a theoretical molecular weight of 53.4 kDa and theoretical pI of 9.21, predicted with a 32-amino acid signal peptide at the N-terminal region. Sequence analysis revealed that the mature polypeptide was a modular enzyme with its catalytic domain of glucoside hydrolase family 5 (GH5) in the N-terminal portion and its cellulose binding domain 3 in the Cterminal portion. egl01 was expressed and purified as described in the Methods section. SDS-PAGE analysis revealed a single band with an MW of 53 kDa close to the theoretical value, and the activity of Egl01 was observed in zymogram (Fig. 1). The specific activity of the purified recombinant Egl01 on CMC was 152 U mg-1. Effects of pH and temperature on the activity of Egl01 The maximum activity of the enzyme with CMC was at pH 5, indicating that Egl01 was acidophilic (Fig. 2a). The maximum activity of Egl01 was at 50 °C measured over 20 min (Fig. 2b). Egl01 was stable over a wide pH range, with over 80 % activity from pH 4 to pH 8 and approx. 50 % activity at pH 3 and 11 (Fig. 2c). Egl01 was stable up to 50 °C for 3 h with 80 % activity remaining (Fig. 2d). Similar studies with other substrates with which Eg101 displayed relatively higher activity such as xylan and laminarin also gave the same results. Effects of metal ions and chemical reagents on the activity of Egl01 The effect of different metal ions and chemical reagents on the enzyme activity may result from the binding of substances to amino acid residues important for the catalysis or affect the charge distribution of enzyme molecules or active sites. These substances
Fig. 1 12 % SDS-PAGE and zymogram of Egl01. Lane 1 molecular mass markers, Lane 2 total protein of cells induced with IPTG, Lane 3 purified recombinant Egl01, Lane 4 zymogram of purified Egl01. In zymogram assay, Egl01 was resolved in SDS loading buffer and performed in SDS-PAGE with 0.1 % (w/v) CMC at 4 °C, then the PAGE was incubated at 50 °C for 30 min before stained by Congo Red
have thus been frequently used to probe into the response of enzymes’ catalytic site to inhibition or stimulation. The effects of various metal ions and chemical reagents on the activity of Egl01 were measured (Supplementary Table 1). No significant effects were found with metal ions (including Fe2?, Cu2? and Co2?) at 1 mM. Non-ionic surfactants (Triton X-100 and Tween 80), organic solvents, and SDS slightly decreased Egl01 activity. The greater effect was a 39 % inhibition by 2 % (v/v) SDS. Greater effects have been reported by Liu et al. 2011; Zhu et al. 2011. The zymogram of SDS-PAGE with 0.1 % CMC (2.5 % SDS totally) in Fig. 1 also suggested that Egl01 has outstanding stability. Effect of NaCl/KCl on the activity of Egl01 Figure 3 showed the effect of salt on Egl01 activity. The activity was stimulated with 1–4 M NaCl/KCl, and showed 139 and 135 % activity in 2 M NaCl and 3 M KCl, respectively (Fig. 3a). After pre-incubation for 6 d in 1 M–4 M KCl/NaCl, residual activities were over 76 % (4 M KCl) and 72 % (4 M NaCl) (Fig. 3b and c).
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Fig. 2 Effects of pH and temperature on the activity of Egl01. a and b The dependence of activity on pH and temperature. The optimal pH for Egl01 was tested in the range pH 3–12 at 50 °C. The optimal temperature for Egl01 was tested from 20–80 °C in the optimal pH buffer. c pH-stability and d thermostability of
Egl01. Egl01 was kept at pH 3–12 at 4 °C for 24 h or at 4–70 °C for 3 h. Residual activities were assayed under optimum conditions. 100 % activity was 118.5 Uml-1. The error bars represent the mean ± SD (n = 3)
Substrate specificity and hydrolysate of Egl01
Discussion
Table 1 shows the substrate specificity of the purified Egl01. Figure 4 shows hydrolysis of Egl01 on cellooligosaccharides examined with TLC. No hydrolysis was observed for G2 and G3. Partial products of G4 and G5 were detected to be G2 and G3; G4 was the smallest oligosaccharide that could be hydrolyzed. The Km and Vmax values were 3.4 mg/ml and 186 lmol/(minmg), respectively, similar to those of cellulases from Salinivibrio sp. NTU-05 [Km = 3 mg/ ml and Vmax = 143 mol/(minmg)] (Wang et al. 2009) and B. pumilus S124A [Km = 2.1 mg/ml and Vmax = 239 lmol/(minmg)] (Natesan and Nelson 2014).
In the present study, recombinant Egl01 showed some noticeable properties that were different to some endoglucanases already described. Firstly, Egl01 was acidophilic while many cellulases characterized from bacteria are alkalophilic (Trivedi et al. 2011; Annamalai et al. 2013). A short peptide ‘‘QGKLIWGTEPN’’ at the C-terminus of the endoglucanase from B. subtilis was considered as being probably related to this acidophilic property (Zhu et al. 2011). Interestingly, a similar short peptide ‘‘NGKLIWGTEPK’’ was also observed at the C-terminus of Egl01, thereby indicating that this peptide might actually be related to the acidophilic property of the glucoside hydrolase
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Biotechnol Lett Table 1 Substrate specificity of the recombinant enzymea Substrate
Special activity (U mg-1)
CMC
152 ± 2.4b
Crop stalk
12 ± 0.4
Filter paper Laminarin
8.1 ± 0.1 45 ± 0.6
Beechwood xylan
79 ± 0.4
Avicel
3.5 ± 0.1
pNPG
0
a
The activity was measured under optimum conditions, except for the filter paper and Avicel incubation at 50 °C for 120 min
b
Values represent the mean ± SD (n = 3) relative to control samples
Fig. 3 Effect of salts on the activity of Egl01. a activity and b, c halo-stability of Egl01 in NaCl/KCl. The reaction system contained 1–4 M NaCl/KCl as the final concentrations were tested under optimum conditions. For the halo-stability, Egl01 was incubated in 1–4 M NaCl/KCl as the final concentration for 1–6d at 4 °C. Reaction solution without any salt added was used as control with 100 % activity, 152.3 Uml-1. The error bars represent the mean ± SD (n = 3)
from Bacillus sp. Considering the typical acidophilia and stability over a broad pH range, Egl01 may be a candidate for biotechnological processes in acidic solutions or in combination with other acidophilic glucoside hydrolase. Secondly, Egl01 showed better activity and stability in the presence of high salt concentrations compared
Fig. 4 Hydrolysis of recombinant Egl01 on cello-oligosaccharides. TLC was performed with chloroform/methanol/acetic acid-water (50:50:15:5, by vol.). Sugars were visualized by incubating the TLC plate at 100 °C for 15 min after spraying with 5 % (v/v) sulfuric acid in ethanol. Lanes 1–4: G2-G5 treated with the recombinant Egl01 at 50 °C for 1 h; Lane 5, Standard sugars: glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4), and cellopentaose (G5)
with some cellulases from extremophiles (Liang et al. 2011; Li and Yu 2012). However, the recombinant Egl01with high halotolerance was directly derived from the forest soil, which is not an extreme environment. Similar properties were recently found in other enzymes (Xu et al. 2014; Nacke et al. 2012) thereby suggesting that some extreme enzyme properties may not only be due to the environment, but also to the specific structures formed during evolution. Further
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research may be helpful to assess the protein function change in extremophiles. Egl01 can hydrolyse a wide range of b-1,4-, b-1,3/ b-1,4-, or b-1,3/b-1,6-linked polysaccharides. Maximum activity of Egl01 was with CMC (152 U mg-1), and less activity on other related substrates. We speculate that Egl01 is probably an endoglucanase.
Conclusion A new glucoside hydrolase gene egl01 was directly cloned from forest soil using the homologous PCR amplification method. Recombinant Egl01 exhibited several special properties, such as acidophilia, halotolerance, organic reagent tolerance, and broad substrate specificity. All these properties show that Egl01 is a robust glucoside hydrolase that can maintain activity in tough environment and is a candidate for industrial applications. Acknowledgments This work was supported by National Natural Science Foundation of Jilin, China (Grant No. 20140101137JC) and State Key Laboratory Program of Microbial Technology of Shandong University. Supporting information Supplementary Table 1- Effect of metal ions and chemical reagents on Egl01.
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