Biotechnol Lett DOI 10.1007/s10529-014-1742-8
ORIGINAL RESEARCH PAPER
Heterologous expression and characterization of a novel halotolerant, thermostable, and alkali-stable GH6 endoglucanase from Thermobifida halotolerans Yi-Rui Yin • Feng Zhang • Qing-Wen Hu • Wen-Dong Xian • Wael N. Hozzein • En-Min Zhou Hong Ming • Guo-Xing Nie • Wen-Jun Li
•
Received: 7 October 2014 / Accepted: 24 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 %
of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application. Keywords Alkali-stable endoglucanase Endoglucanase Glycoside hydrolase (GH 6) Halotolerant endoglucanase Thermobifida halotolerans Thermostable endoglucanase
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1742-8) contains supplementary material, which is available to authorized users. Y.-R. Yin Q.-W. Hu W.-D. Xian E.-M. Zhou H. Ming W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected]; [email protected] Y.-R. Yin E.-M. Zhou W.-J. Li State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and Conservation of Guangdong Higher Education Institutes, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China
W. N. Hozzein Bioproducts Research Chair, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia G.-X. Nie College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China W.-J. Li Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, ¨ ru¨mqi 830011, People’s Republic of China U
F. Zhang Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China
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Introduction
Construction of expression plasmid
Cellulose, a major component of the cell wall of plants and the most abundant renewable polysaccharide in nature, plays an important role in the circulation of materials and energy transfer. Endoglucanases (EC 3.2.1.4) are key enzymes in the initial stages of cellulose breakdown, which is an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol (Sun and Cheng 2002). Endoglucanases possess potential utility in textile, applies for biofuel, kraft pulp bleaching, and preparation of human and animal foods (Yennamalli et al. 2013). Although many microorganisms produce cellulases, extremophilic microorganisms offer an attractive opportunity for isolating new enzymes that can withstand harsh conditions, such as thermostability, acid-stability, alkaline-stability, and salt tolerance (Khandeparker et al. 2011). Searching for novel cellulases from extremophiles attracts increasing attention. Thermobifida spp. may become important industrial strains due to their thermostable and robust polysaccharide-degrading enzymes (Dhawan and Kaur 2007; Yang et al. 2007). Thermobifida halotolerans YIM 90462T is an aerobic, thermophilic, and halotolerant actinomycete isolated from a salt mine in Yunnan Province, south-west China (Yang et al. 2008). It contains an alkaline thermostable GH9 endoglucanase (Zhang et al. 2011) and a thermostable xylanase (Zhang et al. 2012). This paper reports a halotolerant and alkaline thermostable endoglucanase from T. halotolerans YIM 90462T, which belongs to the glycoside hydrolase family 6 (GH6) and is designated as Thcel6A. This study describes the cloning, heterologous expression, and characterization of Thcel6A. The salt-, alkali- and thermotolerance properties of this endoglucanase suggest that it may be useful for industrial applications.
Chromosomal DNA of T. halotolerans YIM 90462T was prepared as described previously (Li et al. 2007). Based on the partially sequenced genome sequence (unpublished data) of T. halotolerans YIM 90462T, the full sequence of Thcel6A was amplified by PCR with the forward primer, 50 -AGC GGA TCC ATG TCC CCC AAA CCC AT-30 and reverse primer 50 -GTC AAG CTT GCC GGC GGT GCA GGT GAG-30 , incorporated BamHI and HindIII restriction sites (underlined letters). The PCR product was digested with BamHI and HindIII and inserted into the vector pET28a with the same sites to yield the expression plasmid designated pET28a-Thcel6A.
Materials and methods Strain Thermobifida halotolerans YIM 90462T (=KCTC 16123T = DSM 44631T) was cultured on modified ISP medium 4 containing 10 % (w/v) NaCl at 45 °C (Yang et al. 2008).
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Expression of the recombinant Thcel6A The recombinant plasmid, pET28a-Thcel6A, was transformed into E. coli BL21 (DE3). The positive clones were isolated for Thcel6A expression. The transformants were cultured overnight at 37 °C in 5 ml LB broth containing 30 lg kanamycin/ml. 1 ml culture was inoculated into 100 ml Terrific broth and incubated at 37 °C and 220 rpm. IPTG was added at 1 mM at mid-growth point (OD600 & 0.6), and followed by further incubation 6 h at 28 °C and 220 rpm. Cells were harvested by centrifugation at 4,0009g and re-suspended in 50 mM Tris/HCl buffer (pH 8). After ultrasonic cell disintegration and centrifugation at 12,0009g for 30 min at 4 °C, cell-free extracts were applied to a Ni-chelating affinity column as the proteins possess a N- or C-terminal His-tag. The column was washed with five column volumes of buffer A (20 mM sodium phosphate, 0.5 mM NaCl, pH 8.0), followed by ten column volumes of buffer A with 20 mM imidazole, pH 8 and eluted with buffer A with 500 mM imidazole, pH 8. The eluted protein was used for enzyme characterization. The homogeneity of the purified enzyme was monitored by SDS-PAGE using 12 % (v/v) acrylamide gels. Proteins were visualized by Coomassie Brilliant Blue R-250 (Liu et al. 2010). Protein concentration was determined with protein assay kit (Sangon, China) using bovine serum albumin as standard. Enzymes assays The reaction mixture containing 0.5 % (w/v) carboxymethylcellulose (CMC) in Tris/HCl buffer (pH 8.5)
Biotechnol Lett
was pre-incubated at 55 °C before adding purified enzyme. Enzyme assays were measured at 55 °C for 30 min, the amount of reducing sugars released was determined by the 3, 5-dinitrosalicylic acid assay.
spraying of plates with freshly prepared 5 % (v/v) H2SO4 in ethanol.
Results and discussion Effects of temperature, pH, and salt Cloning, expression and purification of Thcel6A The effect of temperature on cellulase activity was examined in 50 mM Tris/HCl buffer (pH 8.5) from 5 to 85 °C. To evaluate the thermostability, cellulase solution (0.2 ml) was incubated at 70, 80 and 90 °C for 2 h, respectively, and residual activity was determined as described above. The effect of pH on Thcel6A was investigated using sodium citrate (pH 3–6), sodium phosphate (pH 6.5 and 7.5), Tris/HCl (pH 8–9); glycine/NaOH (pH 9–12). The pH stability test was performed by pre-incubating the enzyme in different buffer systems at 4 °C for 12 h, and then residual activity was determined under optimal assay conditions (55 °C, pH 8.5). The effect of high salinity on enzymatic activity was investigated using (0.5 %, w/v) CMC substrate containing NaCl up to 30 % (w/v) by the standard method. For salt-tolerant activity, Thcel6A was incubated in 1, 10, 20, 30 % NaCl in 37 °C and the residual activities were determined at intervals. Substrate specificity and kinetic parameter of cellulase Thcel6A
The cloned DNA fragment showed the highest amino acid sequence identity (81 %) to endoglucanase from Thermobifida fusca TM51 (Supplementary Fig. 1). The mature protein was predicted to consist of 443 amino acids with a molecular mass of 45.9 kDa and a calculated pI of 4.31. The nucleotide sequence of Thcel6A was deposited in the NCBI GenBank database under the accession number of KJ101553. SDSPAGE analysis indicated the molecular mass of the recombinant endoglucanase protein was good agreement with the theoretical one (Fig. 1). KDa
1
2
3
4
116.0 66.2
45.0
35.0
The substrate specificity was determined by comparing the activity on CMC with that on oats pelt xylan filter paper, b-glucan from barley and chitin (all from Sigma) at 0.5 % (w/v). Kinetic constants (Km and Vmax) were determined under optimal conditions,CMC was used as substrate from 2 to 18 mg/ ml. The initial velocity (V0) was determined by taking samples after 5 min of incubation at different substrate concentrations. The Lineweaver–Burk equation was used to calculate kinetic parameters Km and Vm, based on the enzymatic reactions.
25.0
18.4 14.4
TLC analysis of hydrolytic products Hydrolytic products from CMC were analyzed by TLC using silica gel 60 plates (Merck) developed with 1-butanol/acetic acid/water (2:1:1, by vol.). Sugars were detected by heating at 120 °C for 10 min after
Fig. 1 Analysis of the recombinant protein Thcelc6A on SDSPAGE. Lane 1, protein weight marker (116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4 kDa); lane 2, total protein in non-induced E. coli BL21/pET28a-Thcel6A; lane 3, total protein in IPTGiduced E. coli BL21/pET28a-Thcel6A; lane 4, purified ThCel6A
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Effects of temperature, pH and salt Maximal activity of the purified Thcel6A was at 55 °C. It showed high hydrolytic activities ([70 %) at 25–55 °C (Fig. 2a) and retained nearly 60 % of its
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initial activity after incubation for 1 h at 90 °C (Fig. 2b). The purified Thcel6A from BL21 (DE3) exhibited activity from pH 7.0 to 11.0, with the optimum being detected at pH 8.5 (55 °C) in a 30 min enzyme assay with 0.5 % CMC as substrate (Fig. 2c).
Biotechnol Lett b Fig. 2 Effects of temperature, pH and salt on the activity and
stability of the recombinant Thcel6A. a Temperature effect on the activity of Thcel6A. b The effect of temperature on stability at different temperatures (70, 80 and 90 °C) for 20, 40, 60, 80 and 120 min. c pH effect on the activity of Thcel6A. d The effect of pH on stability. The pH stability test was performed by pre-incubating the enzyme in different buffer systems at 4 °C for 12 h, and then residual activity was determined under optimal assay conditions (55 °C, pH 8.5). e Salt activation of Thcel6A. Activity was measured with CMC as substrate at 55 °C in 50 mM Tris/HCl buffer (pH 8.5) with different concentrations of NaCl (0 to 30 %). f salt-tolerance. The assay was carried out at 55 °C in 50 mM Tris/HCl buffer (pH 8.5) containing various concentrations of NaCl for 5, 10, 30, 60, 90 and 120 min. The primary activity was taken as 100 %. Each value in the figure represents the mean ± SE (n = 3). In all cases, 100 % activity = 34.5 U/mg protein. One unit of activity is defined as the amount of enzyme to release 1 lM glucoseequivalent reducing sugars per min
More than 80 % residual activity was detected after incubation 12 h at pH 4.0–12.0 (Fig. 2d). The conditions of its function were similar to the culture conditions of T. halotolerans YIM 90462T (20–50 °C, pH 6–9) (Yang et al. 2008). Maximal activity of the purified Thcel6A was in 5 % (w/v) NaCl, and showed high hydrolytic activities ([60 %) at 0–20 % NaCl (Fig. 2e). Enzyme activity increased to 252 % after incubation with 20 % NaCl for 10 min, and increased to 200 % after incubation with 10 or 30 % NaCl for 5 min (Fig. 2f). For individual species, environment stresses, for example extreme salinity (Goodarzi et al. 2008; Paul et al. 2008), can bias their amino acid composition due to their desired chemical characteristics. This adaptation is necessary to cope with the environmental stress. It can be quantified from the ratio of the acidic amino acids (Glu and Asp) to the basic amino acids (Lys, His and Arg) which is termed the AB ratio [AB = (Glu ? Asp)/(Lys ? His ? Arg)], and chosen AB Reads/AB Blast of greater than 1.2 are indicative of significant adaptation to salinity (Rhodes et al. 2010). The amino acids composition of the Thcel6A shows that its AB ratio is about 1.8 (Supplementary Table 1). The AB ratio of the endoglucanase (EOR71804.1) from Thermobifida fusca TM51 is 1.0 and shows the highest amino acid sequence identity with Thcel6A. Thus, the AB Thcel6A/AB Blast is much greater. Salt-tolerant cellulases derived from bacteria living in marine or saline environments have potential use in industrial applications (Gao et al. 2010) such as in the processing of sea food and food with a high salt content (Setati 2010).
1
2
3
4
Fig. 3 Thin-layer chromatography showing the hydrolysis products obtained by the action of ThCel6A on CMC. Lane 1, Glucose; lane 2, cellobiose; lane 3, contains the reaction mix with ThCel6A; lane 4, contains the reaction mix without ThCel6A. All reactions were performed under standard conditions for 24 h
Substrate specificity and kinetic constants of Thcel6A The enzyme showed the highest activities against substrates with b-1,4-glucan linkages, such as CMC (CMC activity as 100 %), and b-glucan from barley (402 %). Statistically significant but lower activities were found against microcrystalline cellulose (\11 %) and filter paper (36 %). The enzyme was unable to hydrolyze xylan from oat spelt, and chitin (Supplementary Fig. 2). Accordingly, Thcel6A represents a classical endoglucanase of GH6 of group A consistent with the prediction by Pfam. The Km value of Thcel6A for CMC was 80 mg/ml and the calculated Vm was 312 lM/mg. Compared with other endoglucanses, the Km and Vm values of Thcel9A from T. halotolerans YIM 90462T (Zhang et al. 2011) are higher than with a novel endoglucanase from Bacillus subtilis (Zafar et al. 2014). This indicates that Thcel6A has a lower affinity but higher catalytic efficiency for CMC.
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Analysis of hydrolytic products The hydrolytic products of CMC by Thcel6A were analyzed by TLC. After incubation at 55 °C for 24 h, the main enzymatic products from CMC were cellobiose and an oligosaccharide (Fig. 3). These results indicate that Thcel6A cuts randomly along the chains of cello-oligosaccharides, revealing both endo- and exo-glycolytic modes of action.
Conclusion We have cloned and characterized an endo-1,4-bglucanase from T. halotolerans YIM 90462T. The enzyme is thermotolerant, halotolerant and alkali/ Stable. It displays good activity in alkaline conditions and in present of NaCl. The enzyme activity was increased after pretreatment with high concentrations NaCl, which suggests that this novel endoglucanase has a potential for specific industrial applications and as a model protein for study about the relationship of protein function and osmotic pressure. Acknowledgments This work was supported by the Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), Yunnan Provincial Natural Science Foundation (2013FA004) and Scientific Research Fund of Xinxiang Medical University (2013QN126). W-J Li was also supported by the Hundred Talents Program of Chinese Academy of Sciences and Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014). Supporting information Predicted protein sequence PCR program Supplementary Fig. 1—Neighbor-joining tree of ThCe16A to other GH6 cellulases constructed using the amino acid sequences of them Supplementary Fig. 2—Substrate specificity of ThCe16A Supplementary Table 1—Composition of amino acids from amino acid sequence of ThCe16A Supplementary Table 2—Effect of metal ions and inhibitors on the ThCe16A activity
References Dhawan S, Kaur J (2007) Microbial mannanases: an overview of production and applications. Crit Rev Biotechnol 27:197–216
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Gao Z, Ruan L, Chen X et al (2010) A novel salt-tolerant endobeta-1,4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil. Appl Microbiol Biotechnol 87:1373–1382 Goodarzi H, Torabi N, Najafabadi HS, hetti M (2008) Amino acid and codon usage profiles: adaptive changes in the frequency of amino acids and codons. Gene 407:30–41 Khandeparker R, Verma P, Deobagkar D (2011) A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing. Nat Biotechnol 28:814–821 Li WJ, Xu P, Schumann P et al (2007) Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China), and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57:1424–1428 Liu SY, Shibu MA, Jhan HJ et al (2010) Purification and characterization of novel glucanases from Trichoderma harzianum ETS 323. J Agric Food Chem 58:10309–10314 Paul S, Bag SK, Das S et al (2008) Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes. Genome Biol 9:R70 Rhodes ME, Fitz-Gibbon ST, Oren A, House CH (2010) Amino acid signatures of salinity on an environmental scale with a focus on the Dead Sea. Environ Microbiol 12:2613–2623 Setati ME (2010) Diversity and industrial potential of hydrolase-producing halophilic/halotolerant eubacteria. Afr J Biotechnol 9:1555–1560 Shi R, Li Z, Ye Q et al (2013) Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresource Technol 142:338–344 Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83: 1–11 Yang CH, Yang SF, Liu WH (2007) Production of xylooligosaccharides from xylans by extracellular xylanases from Thermobifida fusca. J Agric Food Chem 55:3955–3959 Yang LL, Tang SK, Zhang YQ et al (2008) Thermobifida halotolerans sp. nov., isolated from a salt mine sample, and emended description of the genus Thermobifida. Int J Syst Evol Microbiol 58:1821–1825 Yennamalli RM, Rader AJ, Kenny AJ et al (2013) Endoglucanases: insights into thermostability for biofuel applications. Biotechnol Biofuels 6:136 Zafar M, Ahmed S, Khan MI, Jamil A (2014) expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli. Mol Biol Rep 41:3295–3302 Zhang F, Chen JJ, Ren WZ et al (2011) Cloning, expression and characterization of an alkaline thermostable GH9 endoglucanase from Thermobifida halotolerans YIM 90462T. Bioresour Technol 102:10143–10146 Zhang F, Chen JJ, Ren WZ et al (2012) Cloning, expression, and characterization of an alkaline thermostable GH11 xylanase from Thermobifida halotolerans YIM 90462T. J Ind Microbiol Biotechnol 39:1109–1116
ORIGINAL RESEARCH PAPER
Heterologous expression and characterization of a novel halotolerant, thermostable, and alkali-stable GH6 endoglucanase from Thermobifida halotolerans Yi-Rui Yin • Feng Zhang • Qing-Wen Hu • Wen-Dong Xian • Wael N. Hozzein • En-Min Zhou Hong Ming • Guo-Xing Nie • Wen-Jun Li
•
Received: 7 October 2014 / Accepted: 24 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 %
of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application. Keywords Alkali-stable endoglucanase Endoglucanase Glycoside hydrolase (GH 6) Halotolerant endoglucanase Thermobifida halotolerans Thermostable endoglucanase
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1742-8) contains supplementary material, which is available to authorized users. Y.-R. Yin Q.-W. Hu W.-D. Xian E.-M. Zhou H. Ming W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected]; [email protected] Y.-R. Yin E.-M. Zhou W.-J. Li State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and Conservation of Guangdong Higher Education Institutes, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China
W. N. Hozzein Bioproducts Research Chair, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia G.-X. Nie College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China W.-J. Li Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, ¨ ru¨mqi 830011, People’s Republic of China U
F. Zhang Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China
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Introduction
Construction of expression plasmid
Cellulose, a major component of the cell wall of plants and the most abundant renewable polysaccharide in nature, plays an important role in the circulation of materials and energy transfer. Endoglucanases (EC 3.2.1.4) are key enzymes in the initial stages of cellulose breakdown, which is an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol (Sun and Cheng 2002). Endoglucanases possess potential utility in textile, applies for biofuel, kraft pulp bleaching, and preparation of human and animal foods (Yennamalli et al. 2013). Although many microorganisms produce cellulases, extremophilic microorganisms offer an attractive opportunity for isolating new enzymes that can withstand harsh conditions, such as thermostability, acid-stability, alkaline-stability, and salt tolerance (Khandeparker et al. 2011). Searching for novel cellulases from extremophiles attracts increasing attention. Thermobifida spp. may become important industrial strains due to their thermostable and robust polysaccharide-degrading enzymes (Dhawan and Kaur 2007; Yang et al. 2007). Thermobifida halotolerans YIM 90462T is an aerobic, thermophilic, and halotolerant actinomycete isolated from a salt mine in Yunnan Province, south-west China (Yang et al. 2008). It contains an alkaline thermostable GH9 endoglucanase (Zhang et al. 2011) and a thermostable xylanase (Zhang et al. 2012). This paper reports a halotolerant and alkaline thermostable endoglucanase from T. halotolerans YIM 90462T, which belongs to the glycoside hydrolase family 6 (GH6) and is designated as Thcel6A. This study describes the cloning, heterologous expression, and characterization of Thcel6A. The salt-, alkali- and thermotolerance properties of this endoglucanase suggest that it may be useful for industrial applications.
Chromosomal DNA of T. halotolerans YIM 90462T was prepared as described previously (Li et al. 2007). Based on the partially sequenced genome sequence (unpublished data) of T. halotolerans YIM 90462T, the full sequence of Thcel6A was amplified by PCR with the forward primer, 50 -AGC GGA TCC ATG TCC CCC AAA CCC AT-30 and reverse primer 50 -GTC AAG CTT GCC GGC GGT GCA GGT GAG-30 , incorporated BamHI and HindIII restriction sites (underlined letters). The PCR product was digested with BamHI and HindIII and inserted into the vector pET28a with the same sites to yield the expression plasmid designated pET28a-Thcel6A.
Materials and methods Strain Thermobifida halotolerans YIM 90462T (=KCTC 16123T = DSM 44631T) was cultured on modified ISP medium 4 containing 10 % (w/v) NaCl at 45 °C (Yang et al. 2008).
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Expression of the recombinant Thcel6A The recombinant plasmid, pET28a-Thcel6A, was transformed into E. coli BL21 (DE3). The positive clones were isolated for Thcel6A expression. The transformants were cultured overnight at 37 °C in 5 ml LB broth containing 30 lg kanamycin/ml. 1 ml culture was inoculated into 100 ml Terrific broth and incubated at 37 °C and 220 rpm. IPTG was added at 1 mM at mid-growth point (OD600 & 0.6), and followed by further incubation 6 h at 28 °C and 220 rpm. Cells were harvested by centrifugation at 4,0009g and re-suspended in 50 mM Tris/HCl buffer (pH 8). After ultrasonic cell disintegration and centrifugation at 12,0009g for 30 min at 4 °C, cell-free extracts were applied to a Ni-chelating affinity column as the proteins possess a N- or C-terminal His-tag. The column was washed with five column volumes of buffer A (20 mM sodium phosphate, 0.5 mM NaCl, pH 8.0), followed by ten column volumes of buffer A with 20 mM imidazole, pH 8 and eluted with buffer A with 500 mM imidazole, pH 8. The eluted protein was used for enzyme characterization. The homogeneity of the purified enzyme was monitored by SDS-PAGE using 12 % (v/v) acrylamide gels. Proteins were visualized by Coomassie Brilliant Blue R-250 (Liu et al. 2010). Protein concentration was determined with protein assay kit (Sangon, China) using bovine serum albumin as standard. Enzymes assays The reaction mixture containing 0.5 % (w/v) carboxymethylcellulose (CMC) in Tris/HCl buffer (pH 8.5)
Biotechnol Lett
was pre-incubated at 55 °C before adding purified enzyme. Enzyme assays were measured at 55 °C for 30 min, the amount of reducing sugars released was determined by the 3, 5-dinitrosalicylic acid assay.
spraying of plates with freshly prepared 5 % (v/v) H2SO4 in ethanol.
Results and discussion Effects of temperature, pH, and salt Cloning, expression and purification of Thcel6A The effect of temperature on cellulase activity was examined in 50 mM Tris/HCl buffer (pH 8.5) from 5 to 85 °C. To evaluate the thermostability, cellulase solution (0.2 ml) was incubated at 70, 80 and 90 °C for 2 h, respectively, and residual activity was determined as described above. The effect of pH on Thcel6A was investigated using sodium citrate (pH 3–6), sodium phosphate (pH 6.5 and 7.5), Tris/HCl (pH 8–9); glycine/NaOH (pH 9–12). The pH stability test was performed by pre-incubating the enzyme in different buffer systems at 4 °C for 12 h, and then residual activity was determined under optimal assay conditions (55 °C, pH 8.5). The effect of high salinity on enzymatic activity was investigated using (0.5 %, w/v) CMC substrate containing NaCl up to 30 % (w/v) by the standard method. For salt-tolerant activity, Thcel6A was incubated in 1, 10, 20, 30 % NaCl in 37 °C and the residual activities were determined at intervals. Substrate specificity and kinetic parameter of cellulase Thcel6A
The cloned DNA fragment showed the highest amino acid sequence identity (81 %) to endoglucanase from Thermobifida fusca TM51 (Supplementary Fig. 1). The mature protein was predicted to consist of 443 amino acids with a molecular mass of 45.9 kDa and a calculated pI of 4.31. The nucleotide sequence of Thcel6A was deposited in the NCBI GenBank database under the accession number of KJ101553. SDSPAGE analysis indicated the molecular mass of the recombinant endoglucanase protein was good agreement with the theoretical one (Fig. 1). KDa
1
2
3
4
116.0 66.2
45.0
35.0
The substrate specificity was determined by comparing the activity on CMC with that on oats pelt xylan filter paper, b-glucan from barley and chitin (all from Sigma) at 0.5 % (w/v). Kinetic constants (Km and Vmax) were determined under optimal conditions,CMC was used as substrate from 2 to 18 mg/ ml. The initial velocity (V0) was determined by taking samples after 5 min of incubation at different substrate concentrations. The Lineweaver–Burk equation was used to calculate kinetic parameters Km and Vm, based on the enzymatic reactions.
25.0
18.4 14.4
TLC analysis of hydrolytic products Hydrolytic products from CMC were analyzed by TLC using silica gel 60 plates (Merck) developed with 1-butanol/acetic acid/water (2:1:1, by vol.). Sugars were detected by heating at 120 °C for 10 min after
Fig. 1 Analysis of the recombinant protein Thcelc6A on SDSPAGE. Lane 1, protein weight marker (116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4 kDa); lane 2, total protein in non-induced E. coli BL21/pET28a-Thcel6A; lane 3, total protein in IPTGiduced E. coli BL21/pET28a-Thcel6A; lane 4, purified ThCel6A
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Effects of temperature, pH and salt Maximal activity of the purified Thcel6A was at 55 °C. It showed high hydrolytic activities ([70 %) at 25–55 °C (Fig. 2a) and retained nearly 60 % of its
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initial activity after incubation for 1 h at 90 °C (Fig. 2b). The purified Thcel6A from BL21 (DE3) exhibited activity from pH 7.0 to 11.0, with the optimum being detected at pH 8.5 (55 °C) in a 30 min enzyme assay with 0.5 % CMC as substrate (Fig. 2c).
Biotechnol Lett b Fig. 2 Effects of temperature, pH and salt on the activity and
stability of the recombinant Thcel6A. a Temperature effect on the activity of Thcel6A. b The effect of temperature on stability at different temperatures (70, 80 and 90 °C) for 20, 40, 60, 80 and 120 min. c pH effect on the activity of Thcel6A. d The effect of pH on stability. The pH stability test was performed by pre-incubating the enzyme in different buffer systems at 4 °C for 12 h, and then residual activity was determined under optimal assay conditions (55 °C, pH 8.5). e Salt activation of Thcel6A. Activity was measured with CMC as substrate at 55 °C in 50 mM Tris/HCl buffer (pH 8.5) with different concentrations of NaCl (0 to 30 %). f salt-tolerance. The assay was carried out at 55 °C in 50 mM Tris/HCl buffer (pH 8.5) containing various concentrations of NaCl for 5, 10, 30, 60, 90 and 120 min. The primary activity was taken as 100 %. Each value in the figure represents the mean ± SE (n = 3). In all cases, 100 % activity = 34.5 U/mg protein. One unit of activity is defined as the amount of enzyme to release 1 lM glucoseequivalent reducing sugars per min
More than 80 % residual activity was detected after incubation 12 h at pH 4.0–12.0 (Fig. 2d). The conditions of its function were similar to the culture conditions of T. halotolerans YIM 90462T (20–50 °C, pH 6–9) (Yang et al. 2008). Maximal activity of the purified Thcel6A was in 5 % (w/v) NaCl, and showed high hydrolytic activities ([60 %) at 0–20 % NaCl (Fig. 2e). Enzyme activity increased to 252 % after incubation with 20 % NaCl for 10 min, and increased to 200 % after incubation with 10 or 30 % NaCl for 5 min (Fig. 2f). For individual species, environment stresses, for example extreme salinity (Goodarzi et al. 2008; Paul et al. 2008), can bias their amino acid composition due to their desired chemical characteristics. This adaptation is necessary to cope with the environmental stress. It can be quantified from the ratio of the acidic amino acids (Glu and Asp) to the basic amino acids (Lys, His and Arg) which is termed the AB ratio [AB = (Glu ? Asp)/(Lys ? His ? Arg)], and chosen AB Reads/AB Blast of greater than 1.2 are indicative of significant adaptation to salinity (Rhodes et al. 2010). The amino acids composition of the Thcel6A shows that its AB ratio is about 1.8 (Supplementary Table 1). The AB ratio of the endoglucanase (EOR71804.1) from Thermobifida fusca TM51 is 1.0 and shows the highest amino acid sequence identity with Thcel6A. Thus, the AB Thcel6A/AB Blast is much greater. Salt-tolerant cellulases derived from bacteria living in marine or saline environments have potential use in industrial applications (Gao et al. 2010) such as in the processing of sea food and food with a high salt content (Setati 2010).
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Fig. 3 Thin-layer chromatography showing the hydrolysis products obtained by the action of ThCel6A on CMC. Lane 1, Glucose; lane 2, cellobiose; lane 3, contains the reaction mix with ThCel6A; lane 4, contains the reaction mix without ThCel6A. All reactions were performed under standard conditions for 24 h
Substrate specificity and kinetic constants of Thcel6A The enzyme showed the highest activities against substrates with b-1,4-glucan linkages, such as CMC (CMC activity as 100 %), and b-glucan from barley (402 %). Statistically significant but lower activities were found against microcrystalline cellulose (\11 %) and filter paper (36 %). The enzyme was unable to hydrolyze xylan from oat spelt, and chitin (Supplementary Fig. 2). Accordingly, Thcel6A represents a classical endoglucanase of GH6 of group A consistent with the prediction by Pfam. The Km value of Thcel6A for CMC was 80 mg/ml and the calculated Vm was 312 lM/mg. Compared with other endoglucanses, the Km and Vm values of Thcel9A from T. halotolerans YIM 90462T (Zhang et al. 2011) are higher than with a novel endoglucanase from Bacillus subtilis (Zafar et al. 2014). This indicates that Thcel6A has a lower affinity but higher catalytic efficiency for CMC.
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Analysis of hydrolytic products The hydrolytic products of CMC by Thcel6A were analyzed by TLC. After incubation at 55 °C for 24 h, the main enzymatic products from CMC were cellobiose and an oligosaccharide (Fig. 3). These results indicate that Thcel6A cuts randomly along the chains of cello-oligosaccharides, revealing both endo- and exo-glycolytic modes of action.
Conclusion We have cloned and characterized an endo-1,4-bglucanase from T. halotolerans YIM 90462T. The enzyme is thermotolerant, halotolerant and alkali/ Stable. It displays good activity in alkaline conditions and in present of NaCl. The enzyme activity was increased after pretreatment with high concentrations NaCl, which suggests that this novel endoglucanase has a potential for specific industrial applications and as a model protein for study about the relationship of protein function and osmotic pressure. Acknowledgments This work was supported by the Key Project of International Cooperation of Ministry of Science & Technology (MOST) (No. 2013DFA31980), Yunnan Provincial Natural Science Foundation (2013FA004) and Scientific Research Fund of Xinxiang Medical University (2013QN126). W-J Li was also supported by the Hundred Talents Program of Chinese Academy of Sciences and Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014). Supporting information Predicted protein sequence PCR program Supplementary Fig. 1—Neighbor-joining tree of ThCe16A to other GH6 cellulases constructed using the amino acid sequences of them Supplementary Fig. 2—Substrate specificity of ThCe16A Supplementary Table 1—Composition of amino acids from amino acid sequence of ThCe16A Supplementary Table 2—Effect of metal ions and inhibitors on the ThCe16A activity
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