Biotechnol Lett DOI 10.1007/s10529-014-1531-4
ORIGINAL RESEARCH PAPER
Cloning and characterization of a novel GH44 family endoglucanase from mangrove soil metagenomic library Zhimao Mai • Hongfei Su • Jian Yang Sijun Huang • Si Zhang
•
Received: 27 February 2014 / Accepted: 31 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A novel endoglucanase gene, mgcel44, was isolated from a mangrove soil metagenomic library by functional-based screening. It encodes a 648-aa peptide with a catalytic domain of glycosyl hydrolase family 44. The deduced amino acid sequence of mgcel44 shares less than 50 % identity with endoglucanases in GenBank database. mgcel44 was cloned and overexpressed in Escherichia coli. The recombinant enzyme, MgCel44, has a molecular mass of 70.8 kDa as determined by SDS-PAGE. Its optimal
Z. Mai H. Su J. Yang S. Huang S. Zhang Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China e-mail: [email protected] H. Su e-mail: [email protected] J. Yang e-mail: [email protected] S. Huang e-mail: [email protected] Z. Mai H. Su S. Zhang Graduate University of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S. Zhang (&) South China Sea Institute of Ocean, Chinese Academy of Sciences, 164 Xingang Road, Guangzhou 530104, People’s Republic of China e-mail: [email protected]
pH and temperature for activity were 6 and 45 °C, respectively. It was highly active at 25–45 °C and pH 5–8. Its activity was enhanced in 0.5 M NaCl by [1.6fold and stable up to 1.5 M NaCl. MgCel44 was resistant to several organic solvents and had high activity at 15 % (v/v) solvent after incubating for 24 h at 25 °C. Keywords Cellulase Mangrove Metagenomic library Organic solvent-resistant Salt-tolerant
Introduction Cellulases have attracted enormous attention for their value in the conversion of renewable cellulosic biomass. They are classified into three types: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and b-glucosidases (EC 3.2.1.21) (Han et al. 1995; Cho et al. 2006; Lee et al. 2008). Based on sequence homology and hydrophobic clustering, the catalytic domain of endoglucanases have been assigned to glycosyl hydrolase families 5, 6,7,8, 9, 44, 45, 48, and 61 (Cantarel et al. 2009). Glycoside hydrolase family 44 (GH44) is primarily composed of endoglucanases that hydrolyze b-1,4-glycosidic bonds with an retained mechanism (Cantarel et al. 2009). Some tertiary structures of GH44 cellulases have been characterized (Kitago et al. 2007; Warner et al. 2010) but compared
123
Biotechnol Lett
to other glycoside hydrolase family endoglucanases, there are still only a few GH44 endoglucanases that have been kinetically described. During cellulose processing, lignocelluloses are usually pretreated with alkalis or acids to release cellulose (Klinke et al. 2004). The processed materials are then neutralized with acid or alkaline that, produce large amounts of salt (Zhang et al. 2012). Many enzymes rapidly lose their activity under such hypersaline conditions. Besides, organic solvents are often added to enzymatic reactions to solubilize the hydrophobic substrates and eliminate microbial contamination (Oikawa et al. 2001). Therefore, cellulases that remain stable in the presence of hypersaline and organic solvents might be useful in the enzymatic hydrolysis of cellulose. Microorganisms grown in pure culture serve as the starting point for enzyme exploration (Ogawa and Shimizu 1999). However, less than 1 % of the microorganisms are readily cultured with existing techniques (Amann et al. 1995). Due to the limitations in microbial cultivation, culture-independent metagenomic approach has been developed and is frequently used to identify novel cellulases (Voget et al. 2003; Liu et al. 2011). Mangrove environments, located in coastal intertidal wetlands, contain highly variable environmental conditions having both terrestrial and marine characteristics. Mangroves harbour highly diverse and unique microorganisms that may possess potentially novel cellulases capable of functioning under special conditions, such as hypersaline, anaerobic or organic-rich circumstances (Hyde and Lee 1995; Jiang et al. 2006). In this study, a novel GH44 endoglucanase gene was isolated from a mangrove soil metagenomic library. The gene, mgcel44, was overexpressed in Escherichia coli and then the recombinant enzyme MgCel44 was purified and characterized. The characteristics of organic solvent-resistant and salt-tolerant characteristics make it an ideal candidate for further research and industrial applications.
Materials and methods Bacterial strains, plasmids, and growth conditions Escherichia coli were grown at 37 °C on LB medium supplemented with appropriate antibiotics. His Binding
123
Purification Kit and gene expression plasmid pET22b(?) vector were from Novagen (USA). DNA isolation and purification kits were purchased from Sangon (China). E. coli DH5a, pMD19-T vector, restriction endonucleases, DNA polymerase, dNTPs, T4 DNA ligase and IPTG, were purchased from TaKaRa (Japan). All other chemicals were of analytical grade. Soil sampling, DNA isolation and metagenomic library construction Soil samples for metagenomic library construction were collected from Mangrove Reserve of Sanya City, Hainan, China. The topsoil samples were collected from depths of 0–10 cm and then stored at -20 °C until the DNA extraction was performed. The fosmid library was constructed follow by the instruction of CopyControlTM Fosmid Library Production Kit. Identification of cellulase-positive clones, subcloning and DNA sequence analysis For cellulase-positive clones screening of metagenomic library, recombinant E. coli strains were grown in LB medium with carboxymethylcellulose (CMC) to detect endo-b-1,4-glucanase activity. Clones showing endo-b-1,4-glucanase activity were surrounded by a yellow halo against a red background after incubation for 1–5 days at 37 °C. Only one clone was identified that showed endo-b-1,4-glucanase activity. The fosmid DNA of the clone was isolated and partially digested with Sau3AI. DNA fragments of 1–5 kb were recovered and ligated into pUC19 with BamHI. They were then transformed into E. coli DH5a. The subcloned library was rescreened of endo-b-1,4-glucanase activity in recombinant E. coli DH5a, and the plasmids from positive clone was sequenced. Nucleotide sequences were analyzed using the NCBI ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). The endo-b-1,4-glucanase gene, mgcel44, was assembled and translated to amino acid sequence by using the Vector NTI Suite 8 software (InforMax, Gaithersburg, MD, USA). Signal peptide was predicted using SignalP 4.0 Server (http://www.cbs.dtu. dk/services/SignalP/). Protein functional analysis was performed with InterProScan (http://www.ebi.ac.uk/ Tools/InterProScan/). Multiple sequence alignments were performed with Clustal X (Thompson et al.
Biotechnol Lett
1997). The sequence similarity search was performed with the BLAST program (http://www.ncbi.nlm.nih. gov/BLAST/). Expression and purification of cellulase MgCel44 The putative endo-b-1,4-glucanase gene mgcel44 was amplified from the positive clone fosSCSIO1 and ligated into the pET22b(?) vector (Novagen). The recombinant plasmid pET-mgcel44 was transformed into E. coli Rosetta (DE3) to express the target protein. The transformed cells carrying pET-mgcel44 were cultured in LB medium containing ampicillin (100 lg/ ml) and chloramphenicol (34 lg/ml) at 37 °C. When the OD600 of the bacterial culture reached 0.6, IPTG was added at 1 mM for further induction at 20 °C for 20 h. Induced cells were harvested, washed twice with Tris/HCl buffer (pH 7.6), and lysed by sonication (5 s, 150 W) on ice for several times. The lysate was centrifuged at 12,0009g for 20 min at 4 °C. MgCel44 was purified by using a Ni2?-NTA agarose gel column according to the manufacturer’s protocol (Novagen). The recombinant protein was eluted with column buffer (20 mM Tris/HCl, pH 8.0, 200 mM imidazole, and 300 mM NaCl) and then dialyzed three times in deionized double-distilled water at 4 °C. The purified protein was detected after SDS-PAGE. The protein concentration was determined using the Bradford method with bovine serum albumin as standard. Enzyme assay To measure the recombinant MgCel44 activity, a reaction mixture containing 100 ll diluted enzyme and 400 ll 1 % CMC in Na2HPO4/citric acid buffer (pH 6.0) was incubated at 45 °C for 30 min. The reaction was terminated with 500 ll 3,5-dinitrosalicylic (DNS). Reducing sugars were determined by the DNS method with D-glucose as standard. One unit (U) of MgCel44 activity was defined as the amount of enzyme releasing 1 lmol reducing sugar per min. Biochemical characterization of MgCel44 The characterization of the purified MgCel44 was detected using CMC as substrate. The effect of pH on MgCel44 activity was determined at 45 °C using 0.2 M McIlvaine buffer (pH 3–8), and 0.05 M
glycine/NaOH buffer (pH 8–11). pH stability studies were carried out by measuring the residual enzyme activity after incubating enzyme at different values pH as described above. The effect of temperature on MgCel44 activity was determined from 25 to 65 °C. The thermostability of the enzyme was determined by measuring the residual activity after incubating enzyme at different temperatures as described above for 1 h. 1 and 10 mM (final concentration) of metal ions, chelating agents (EDTA) and surfactants (SDS) were individually added to the reaction system to investigate their effects on the activity. MgCel44 activity was also measured in the presence of 0–3 M NaCl. The influence of NaCl on enzyme stability was tested by measuring the enzymatic activity after incubation with 0–3 M NaCl at 25 °C for 24 h. The effects of organic solvents on the enzyme were determined by measuring the enzymatic activity after incubation with 15 % (v/v) different log Pow organic solvents at 25 °C for 24 h. Activity was expressed as a percentage of the activity obtained in the absence of the metal ions, chemical agents and organic solvents. To investigate the substrate specificity, the enzyme activity was examined in different substrates. The Km and Vmax values for MgCel44 were determined in McIlvaine buffer (pH 6.0) containing 1 % (v/v) CMC as substrate at 45 °C by using the Lineweaver–Burk method.
Sequence accession number The nucleotide sequence for GH44 endoglucanase gene mgcel44 was deposited in GenBank database under the accession number KF424270.
Results Construction of the metagenomic fosmid library A metagenomic library containing 100,000 fosmid clones was constructed. BamHI restriction analysis of 18 randomly selected plasmids showed that the insert sizes ranged from 20 to 55 kb (data not shown). The average size of insert fragments was about 30 kb, and the total size of the library was estimated to be about 3 Gb. BamHI restriction analysis also showed that the
123
Biotechnol Lett
clones had different restriction patterns, indicating that the library was composed of random DNA clones.
Effect of metal ions and chemical agents on MgCel44 activity (Table 1)
Screening, cloning, and molecular analysis of the endo-b-1,4-glucanase gene
Activity of MgCel44 was only slightly affected by Na?, NH4?, and Ni2? at 1 mM whereas Zn2?, Mn2?, Cu2?, EDTA and SDS reduced enzyme activity. Other metal ions had no obvious effects.
By screening 10,000 fosmid clones on the CMC indicator plates, an independent clone with endo-b1,4-glucanase activity was isolated and confirmed to encode on endoglucanase gene by retransformation. A positive subclone of plasmid pUC-cel44 was sequenced. It contained a complete ORF coding for an endo-b-1,4glucanase. The overall G?C content of the ORF was 56 %, and the deduced product consists of 648 amino acids and contains a glycosyl hydrolase family 44 catalytic domain. The deduced protein sequence showed highest identity of 48 % with an endoglucanase from Micromonospora lupini str. Lupac 08 (WP_00 7457316.1), 48 % identity with endoglucanase from Streptomyces bingchenggensis BCW-1, 48 % identity with endoglucanase from S. himastatinicus ATCC 53653, 47 % identity with endoglucanase from Amycolatopsis mediterranei U32, 47 % identity with endoglucanase from A. mediterranei S699. All these five proteins shared high identities with MgCel44. Multiple sequence alignment showed that MgCel44 shared conserved residues with these proteins (Fig. 1). Expression and purification of the recombinant MgCel44 The gene mgcel44 was expressed in E. coli Rosetta (DE3) cells. The recombinant enzyme MgCel44 was purified with the Ni2?-NTA column. SDS-PAGE analysis showed that MgCel44 was purified to homogeneity, with a molecular mass approximately of 71 kDa which corresponds to the estimated molecular mass (Fig. 2). The effects of pH and temperature on MgCel44 (Fig. 3) The recombinant enzyme exhibited maximal activity at pH 6.0 and remained stable at pH 5.0–8.0 (maintaining over 80 % of activity). The optimal temperature of MgCel44 was 45 °C (Fig. 4). MgCel44 activity was stable below 40 °C (maintaining more than 85 % activity) but decreased greatly above 40 °C.
123
Effect of NaCl on the activity of MgCel44 (Fig. 5) MgCel44 displayed typical characteristics of salt tolerance. Its activity was enhanced I [ 1.6-fold in 0.5 M NaCl and it remained more than 50 % activity in 1.5 M NaCl. MgCel44. It was stable for 24 h in NaCl below 1.5 M (maintained over 50 % of activity). Effect of organic solvents on MgCel44 activity (Table 2) More than 80 % activity of MgCel44 was detected in 15 % (v/v) toluene, DMSO, methanol and acetone. It retained more than 50 % activity in acetonitrile and ethanol, but in ethyl acetate, 1-butanol and chloroform activity was greatly reduced. Substrate specificity of MgCel44 Substrate specificity of MgCel44 is shown in Table 3. Highest activities were with CMC and b-glucan from barley. The Km and Vmax of recombinant MgCel44 for CMC were 7.6 mg/ml and 16.3 lmol/min/mg, respectively.
Discussion Microorganisms and their related enzyme or gene resources have potential applications in industry. The mangrove environment possess highly diverse and unique microorganisms which represents an important biotechnological resources yet to be exploited (Sivaramakrishnan et al. 2006). The enzymes from culturable microorganisms from the mangrove environment have long been studied (Sudha 1981; Polizeli et al. 2005; Mishra 2010) but there are few reports on solvent-resistant or salt-tolerant cellulases. In this study, a metagenomic fosmid library was constructed from mangrove soil and a new endo-b-1,4-glucanase gene mgcel44 was identified. The predicted product of
Biotechnol Lett
Fig. 1 Multiple sequence alignment of MgCel44 with some similar endoglucanase MgCel44 was from mangrove soil metagenomic library in this study. The WP007457316.1 was from Micromonospora lupini str. Lupac 08. The YP004967448.1 was from Streptomyces bingchenggensis BCW-1. The WP0
09720086.1 was from Streptomyces himastatinicus ATCC 53653. The YP003769935.1 was from Amycolatopsis mediterranei U32. The YP005535968.1 was from Amycolatopsis mediterranei S699, respectively. Identical residues were shaded in black. The star symbol indicates the active site of enzyme
mgcel44 shares less than 50 % identity with other endob-1,4-glucanases, indicating it is a novel endoglucanase gene. The protein sequences similar to MgCel44 in the GenBank database were from genome sequencing which had not been characterized. So we cloned and expressed mgcel44 in E. coli. The recombinant MgCel44 was then purified and characterized. GH44 endoglucanases have a large range of optimal reaction pH and temperature. MgCel44 displays similar pH characteristics to a metagenomederived endoglucanase Cel5A (Voget et al. 2006). Its
activity was greatly inhibited by EDTA indicating that it was a metalloenzyme. 1 mM ions had no obvious effects on MgCel44 but 10 mM Zn2?, Mn2?, Cu2? and SDS were inhibition. This is similar to some endoglucanases from another metagenomic library (Feng et al. 2007; Liu et al. 2009). An interesting feature of MgCel44 was that its activity was significantly improved by NaCl and was stable at high salt concentrations. These results indicate that MgCel44 is a salt-tolerant enzyme. Halophilic cellulolytic enzymes had been found from
123
Biotechnol Lett
activity
100
Relative activity (%)
stability
80 60 40 20 0 3
4
5
6
7
8
9
10
11
pH
Fig. 2 10 % SDS-PAGE analysis of the purified MgCel44 1 protein marker; 2 purified MgCel44; 3 recombinant Escherichia coli harboring pET-mgcel44 induced with IPTG; 4 cell lysate of Escherichia coli harboring empty pET-22b(?) induced with IPTG
Fig. 3 Effect of pH on MgCel44 activity and stability. The activity of MgCel44 was determined at 45 °C from pH 3.0–11.0; pH stability of MgCel44 was measured by pre-incubating the enzyme at different buffers of pH 3.0–11.0 for 24 h at 4 °C. The error bars represent the mean ± SD (n = 3)
activity stability
microorganisms living in extreme environments and the high concentrations of salts are necessary for their stability at high temperatures (Bronnenmeier et al. 1995; Liebl et al. 1996; Voget et al. 2006). Mangrove soil is high salinity and the microorganisms and their enzymes usually were salt tolerant. Such as the activity of Cel5A rose to 1.6-fold in 0.5 M NaCl and remained elevated in 4 M NaCl (Gao et al. 2010) and r-BglNH remained elevated even in 5 M NaCl (Mai et al. 2013). This characteristic indicates MgCel44 has advantage in cellulose degradation which usually is hypersaline during the lignocelluloses processing. Organic solvent-resistant cellulases are useful in cellulose degradation industry but there are few reports on the halotolerant cellulases with organic solvent tolerance. MgCel44 displayed high activity in organic solvents. Cellulases with organic solvent tolerance have been isolated from different microorganisms. An endoglucanase from Haloarcula sp. G10 showed high activity in the presence of non-polar hydrophobic organic solvents with log Pow C 0.88 (Li and Yu 2013). An extracellular cellulase from Thalassobacillus sp. LY18 was highly active in non-ionic surfactants and was stable in water-insoluble organic solvents with log Pow C 2.13 (Li et al. 2012). Cellulases with organic solvent-resistance have also been isolated from uncultured microorganism from metagenomic libraries. An endoglucanase Cel5A from a soil metagenome was
123
Relative activity (%)
100 80 60 40 20 0 25
30
35
40
45
50
55
60
65
Temperature (°C) Fig. 4 Effect of temperature on MgCel44 activity and thermostability. The activity of MgCel44 was determined in McIlvaine buffer (pH 6.0) at different temperature; Thermostability assay was measured by incubating the enzyme at different temperature for 1 h. The error bars represent the mean ± SD (n = 3)
stable in 15 % (v/v) organic solvents (Voget et al. 2006). Three cellulases from metagenomic libraries were stable in 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfonate (Pottka¨mper et al. 2009). Compared to these cellulases, MgCel44 is a member of organic solvent-resistant and halotolerant endoglucanase from mangrove soil metagenomic library. Moreover, MgCel44 retained high activity in organic solvents with low log Pow after 24 h. Enzymes would, however, be less stable in lower log P values (Klibanov 1986) but MgCel44 did not follow this P trends. This is similar to
Biotechnol Lett Table 1 Effect of 1 or 10 mM metal ions and chemical reagents on MgCel44 activity Reagents
Relative activity (%)
(10 mM)
100 ± 2.2
?
Organic solvents
Log Pa
None
–
100 ± 3.6c
DMSO
-1.35
81 ± 5.3
Methanol Acetonitrile
-0.76 -0.34
87 ± 2.4 76 ± 5.4 78 ± 3.8
a
(1 mM) None
Table 2 Effect of organic solvents on MgCel44 activity
b
100 ± 3.2
ow
Relative activity (%)b
Na
105 ± 3.4
K?
85 ± 2.5
73 ± 3.2
Ethanol
-0.3
NH4?
102 ± 4.2
93 ± 6.8
Acetone
-0.24
88 ± 2.5
Mg2?
92 ± 3.1
78 ± 2.4
Isopropanol
0.14
67 ± 6.3
Zn2?
65 ± 2.1
53 ± 2.7
Ethyl acetate
0.68
15 ± 3.6
2?
Ca
92 ± 3.6
90 ± 3.4
1-Butanol
0.88
41 ± 6.7
Mn2?
67 ± 5.1
55 ± 6.5
Chloroform
1.97
39 ± 3.6
Cu2?
65 ± 2.1
43 ± 4.6
Toluene
2.73
102 ? 7.8
Ni2?
109 ± 3.6
95 ± 4.5
a
2?
Ba EDTA
89 ± 2.5 58 ± 5.7
65 ± 6.1 28 ± 6.1
SDS
88 ± 3.2
32 ± 5.2
109 ± 2.4
The Log Pow is the logarithm of the partition coefficient, P, of the solvent between n-octanol and water and is used as a quantitative measure of the solvent polarity b
Assay was performed under optimum conditions
c
a
Values represent the mean ± SD (n = 3) relative to the untreated control samples
Assay was performed under optimum conditions
b
Values represent the mean ± SD (n = 3) relative to the untreated control samples Table 3 Substrate specificity of MgCel44 Substrate
Specific activitya (U mg-1)
Carboxymethyl cellulose
7.6 ± 0.6b
120
b-Glucan from barley
5.5 ± 0.7
100
Avicel, microcrystalline cellulose
ND
pNPGlu Cellobiose
0.7 ± 0.3 ND
Relative activity (%)
180 160
activity
140
stability
80 60 40
Oat spelts xylan
0.8 ± 0.4
20
Laminarin
ND
Filter paper
ND
0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
NaCl (M) Fig. 5 Effect of NaCl on MgCel44 activity and stability. The activity of the enzyme was measured in the presence of 0–3 M NaCl. NaCl influence on enzyme stability was tested by measuring the residual activity of the enzyme after incubation with 0–3 M NaCl at 25 °C for 1 h
an organic solvents with solvent-tolerant lipase from Pseudomonas sp. strain S5 (Rahman et al. 2005). These characteristics indicate MgCel44 has potential applications for cellulose degradation under high organic solvent conditions and also is attractive use for transglycosylation in media containing organic solvent (Oikawa et al. 2001).
a
Assay was performed at the optimum condition
b
Standard deviations were shown behind the specific activities
MgCel44 had high specific activities against amorphous cellulose with a b-1,4-glucan linkage, such as CMC and b-glucan from barley but no activity, against cellobiose. Low activity was detected against xylan from oat spelts, indicating that MgCel44 can slowly hydrolyze b-1,4-xylose linkages. There were reports that GH44 family endoglucanases can hydrolyze xylan (Cho et al. 2006; Warner et al. 2011) but MgCel44 showed lower activity against xylan than other GH44 family endoglucanases. No obvious activity was detected against laminarin, indicating that MgCel44 could not hydrolyze b-1,3-glucan linkages. MgCel44
123
Biotechnol Lett
also cannot hydrolyze insoluble microcrystalline cellulose and filter paper that have high crystallinity cellulose. It is similar to some GH44 family endoglucanases (Rinco´n et al. 2001; Warner et al. 2011). MgCel44 includes a single domain but lacks the carbohydrate-binding domain that seems to be involved in the degradation of insoluble cellulose (Coutinho et al. 1993; Gill et al. 1999). The Vmax of MgCel44 for CMC was higher than the GH44 family endoglucanase from Ruminococcus flavefaciens FD-1(7.8 lmol/min/mg) (Warner et al. 2011) but lower than other family endoglucanases (Voget et al. 2006; Gao et al. 2010; Zhang et al. 2013). MgCel44 also had a lower Km value than some metagenome-derived endoglucanases, suggesting that it has a higher affinity for the CMC substrate (Liu et al. 2011; Zhang et al. 2013). In summary, we have constructed a large-size mangrove soil fosmid library and identified a novel endoglucanase gene mgcel44 by functional screening. MgCel44 is an organic solvent-resistant and salttolerant enzyme. These characteristics allow the potential application of MgCel44 in biomass conversion and food industry. This study also highlights the advantages of metagenomic libraries for cloning novel genes from mangrove soil through functional-based approaches. Acknowledgments This study was supported by the National Basic Research Program of China (973 Program, 2010CB 833801), National Natural Science Foundation of China (Grant No. 41230962).
References Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169 Bronnenmeier K, Kern A, Liebl W, Staudenbauer WL (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microbiol 61:1399–1407 Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acid Res 37:233–238 Cho KM, Hong SY, Lee SM, Kim YH, Kahng GG, Kim H, Yun HD (2006) A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains. Appl Microbiol Biotechnol 73: 618–630 Coutinho J, Gilkes N, Kilburn D, Warren R, Miller R Jr (1993) The nature of the cellulose-binding domain effects the
123
activities of a bacterial endoglucanase on different forms of cellulose. FEMS Microbiol Lett 113:211–217 Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, Hu YL, Wei J, Tang JL, Feng JX (2007) Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl Microbiol Biotechnol 75:319–328 Gao Z, Ruan L, Chen X, Zhang Y, Xu X (2010) A novel salttolerant endo-b-1, 4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil. Appl Microbiol Biotechnol 87:1373–1382 Gill J, Rixon J, Bolam D, Mcqueen-mason S, Simpson P, Williamson M, Hazlewood G, Gilbert H (1999) The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism. Biochem J 342:473–480 Han SJ, Yoo YJ, Kang HS (1995) Characterization of a bifunctional cellulase and its structural gene The cel gene of Bacillus sp. D04 has exo-and endoglucanase activity. J Biol Chem 270:26012–26019 Hyde K, Lee S (1995) Ecology of mangrove fungi and their role in nutrient cycling: what gaps occur in our knowledge? Asia-Pacific Symposium on Mangrove Ecosystems. Springer, pp 107–118 Jiang Y, Zheng T, Tian Y (2006) Research on mangrove soil microorganisms: past, present and future. Wei sheng wu xue bao = Acta microbiologica Sinica 46:848–851 Kitago Y, Karita S, Watanabe N, Kamiya M, Aizawa T, Sakka K, Tanaka I (2007) Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum. J Biol Chem 282:35703–35711 Klibanov AM (1986) Enzymes that work in organic solvents. ChemTech 16:354–359 Klinke HB, Thomsen A, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66:10–26 Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK, Chung CH, Lee YC, Lee JW (2008) Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour Technol 99:378–386 Li X, Yu HY (2013) Characterization of a halostable endoglucanase with organic solvent-tolerant property from Haloarcula sp. G10. Int J Biol Macromol 62:101–106 Li X, Wang HL, Li T, Yu HY (2012) Purification and characterization of an organic solvent-tolerant alkaline cellulase from a halophilic isolate of Thalassobacillus. Biotechnol Lett 34:1531–1536 Liebl W, Ruile P, Bronnenmeier K, Riedel K, Lottspeich F, Greif I (1996) Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes. Microbiology 142:2533–2542 Liu L, Feng Y, Duan CJ, Pang H, Tang JL, Feng JX (2009) Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World J Microbiol Biotechnol 25:1035–1042 Liu J, Liu W, Zhao X, Shen W, Cao H, Cui Z (2011) Cloning and functional characterization of a novel endo-b-1, 4-glucanase gene from a soil-derived metagenomic library. Appl Microbiol Biotechnol 89:1083–1092
Biotechnol Lett Mai Z, Yang J, Tian X, Li J, Zhang S (2013) Gene cloning and characterization of a novel salt-tolerant and glucoseenhanced b-glucosidase from a marine streptomycete. Appl Biochem Biotechnol 169(5):1512–1522 Mishra R (2010) Microbial biodiversity in mangroves of Bhitarakanika, Orissa-a study on genotypic, phenotypic and proteomic characterisation of some predominant bacteria. PhD Thesis submitted to North Orissa University, Orissa, India Ogawa J, Shimizu S (1999) Microbial enzymes: new industrial applications from traditional screening methods. Trends Biotechnol 17:13–20 Oikawa T, Tsukagawa Y, Chino M, Soda K (2001) Increased transglycosylation activity of Rhodotorula glutinis endo-bglucanase in media containing organic solvent. Biosci Biotechnol Biochem 65:1889–1892 Polizeli M, Rizzatti A, Monti R, Terenzi H, Jorge J, Amorim D (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591 Pottka¨mper J, Barthen P, Ilmberger N, Schwaneberg U, Schenk A, Schulte M, Ignatiev N, Streit WR (2009) Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids. Green Chem 11:957–965 Rahman RN, Baharum SN, Basri M, Salleh AB (2005) Highyield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5. Anal Biochem 341:267–274 Rinco´n MT, McCrae SI, Kirby J, Scott KP, Flint HJ (2001) EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain. Appl Environ Microbiol 67:4426–4431
Sivaramakrishnan S, Gangadharan D, Nampoothiri KM, Soccol CR, Pandey A (2006) a-Amylases from microbial sourcesan overview on recent developments. Food Technol Biotechnol 44:173–184 Sudha V (1981) Studies on Halophilic bacteria associated with mangrove sediment and a bivalve Mollusc Andara rhombea (Bora)(Arcidae) and (b) on L-asparaginase (antileukamic agent) from an extremely halophilic bacterium. Ph.D. Thesis, Annamalai University Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Voget S, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit W (2003) Prospecting for novel biocatalysts in a soil metagenome. Appl Environ Microbiol 69:6235–6242 Voget S, Steele H, Streit W (2006) Characterization of a metagenome-derived halotolerant cellulase. J Biotechnol 126:26–36 Warner CD, Hoy JA, Shilling TC, Linnen MJ, Ginder ND, Ford CF, Honzatko RB, Reilly PJ (2010) Tertiary structure and characterization of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum. Appl Environ Microbiol 76:338–346 Warner CD, Go RM, Garcı´a-Salinas C, Ford C, Reilly PJ (2011) Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1. Enzym Microb Technol 48:27–32 Zhang G, Li S, Xue Y, Mao L, Ma Y (2012) Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10. Extremophiles 16:35–43
123
ORIGINAL RESEARCH PAPER
Cloning and characterization of a novel GH44 family endoglucanase from mangrove soil metagenomic library Zhimao Mai • Hongfei Su • Jian Yang Sijun Huang • Si Zhang
•
Received: 27 February 2014 / Accepted: 31 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A novel endoglucanase gene, mgcel44, was isolated from a mangrove soil metagenomic library by functional-based screening. It encodes a 648-aa peptide with a catalytic domain of glycosyl hydrolase family 44. The deduced amino acid sequence of mgcel44 shares less than 50 % identity with endoglucanases in GenBank database. mgcel44 was cloned and overexpressed in Escherichia coli. The recombinant enzyme, MgCel44, has a molecular mass of 70.8 kDa as determined by SDS-PAGE. Its optimal
Z. Mai H. Su J. Yang S. Huang S. Zhang Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China e-mail: [email protected] H. Su e-mail: [email protected] J. Yang e-mail: [email protected] S. Huang e-mail: [email protected] Z. Mai H. Su S. Zhang Graduate University of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S. Zhang (&) South China Sea Institute of Ocean, Chinese Academy of Sciences, 164 Xingang Road, Guangzhou 530104, People’s Republic of China e-mail: [email protected]
pH and temperature for activity were 6 and 45 °C, respectively. It was highly active at 25–45 °C and pH 5–8. Its activity was enhanced in 0.5 M NaCl by [1.6fold and stable up to 1.5 M NaCl. MgCel44 was resistant to several organic solvents and had high activity at 15 % (v/v) solvent after incubating for 24 h at 25 °C. Keywords Cellulase Mangrove Metagenomic library Organic solvent-resistant Salt-tolerant
Introduction Cellulases have attracted enormous attention for their value in the conversion of renewable cellulosic biomass. They are classified into three types: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and b-glucosidases (EC 3.2.1.21) (Han et al. 1995; Cho et al. 2006; Lee et al. 2008). Based on sequence homology and hydrophobic clustering, the catalytic domain of endoglucanases have been assigned to glycosyl hydrolase families 5, 6,7,8, 9, 44, 45, 48, and 61 (Cantarel et al. 2009). Glycoside hydrolase family 44 (GH44) is primarily composed of endoglucanases that hydrolyze b-1,4-glycosidic bonds with an retained mechanism (Cantarel et al. 2009). Some tertiary structures of GH44 cellulases have been characterized (Kitago et al. 2007; Warner et al. 2010) but compared
123
Biotechnol Lett
to other glycoside hydrolase family endoglucanases, there are still only a few GH44 endoglucanases that have been kinetically described. During cellulose processing, lignocelluloses are usually pretreated with alkalis or acids to release cellulose (Klinke et al. 2004). The processed materials are then neutralized with acid or alkaline that, produce large amounts of salt (Zhang et al. 2012). Many enzymes rapidly lose their activity under such hypersaline conditions. Besides, organic solvents are often added to enzymatic reactions to solubilize the hydrophobic substrates and eliminate microbial contamination (Oikawa et al. 2001). Therefore, cellulases that remain stable in the presence of hypersaline and organic solvents might be useful in the enzymatic hydrolysis of cellulose. Microorganisms grown in pure culture serve as the starting point for enzyme exploration (Ogawa and Shimizu 1999). However, less than 1 % of the microorganisms are readily cultured with existing techniques (Amann et al. 1995). Due to the limitations in microbial cultivation, culture-independent metagenomic approach has been developed and is frequently used to identify novel cellulases (Voget et al. 2003; Liu et al. 2011). Mangrove environments, located in coastal intertidal wetlands, contain highly variable environmental conditions having both terrestrial and marine characteristics. Mangroves harbour highly diverse and unique microorganisms that may possess potentially novel cellulases capable of functioning under special conditions, such as hypersaline, anaerobic or organic-rich circumstances (Hyde and Lee 1995; Jiang et al. 2006). In this study, a novel GH44 endoglucanase gene was isolated from a mangrove soil metagenomic library. The gene, mgcel44, was overexpressed in Escherichia coli and then the recombinant enzyme MgCel44 was purified and characterized. The characteristics of organic solvent-resistant and salt-tolerant characteristics make it an ideal candidate for further research and industrial applications.
Materials and methods Bacterial strains, plasmids, and growth conditions Escherichia coli were grown at 37 °C on LB medium supplemented with appropriate antibiotics. His Binding
123
Purification Kit and gene expression plasmid pET22b(?) vector were from Novagen (USA). DNA isolation and purification kits were purchased from Sangon (China). E. coli DH5a, pMD19-T vector, restriction endonucleases, DNA polymerase, dNTPs, T4 DNA ligase and IPTG, were purchased from TaKaRa (Japan). All other chemicals were of analytical grade. Soil sampling, DNA isolation and metagenomic library construction Soil samples for metagenomic library construction were collected from Mangrove Reserve of Sanya City, Hainan, China. The topsoil samples were collected from depths of 0–10 cm and then stored at -20 °C until the DNA extraction was performed. The fosmid library was constructed follow by the instruction of CopyControlTM Fosmid Library Production Kit. Identification of cellulase-positive clones, subcloning and DNA sequence analysis For cellulase-positive clones screening of metagenomic library, recombinant E. coli strains were grown in LB medium with carboxymethylcellulose (CMC) to detect endo-b-1,4-glucanase activity. Clones showing endo-b-1,4-glucanase activity were surrounded by a yellow halo against a red background after incubation for 1–5 days at 37 °C. Only one clone was identified that showed endo-b-1,4-glucanase activity. The fosmid DNA of the clone was isolated and partially digested with Sau3AI. DNA fragments of 1–5 kb were recovered and ligated into pUC19 with BamHI. They were then transformed into E. coli DH5a. The subcloned library was rescreened of endo-b-1,4-glucanase activity in recombinant E. coli DH5a, and the plasmids from positive clone was sequenced. Nucleotide sequences were analyzed using the NCBI ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). The endo-b-1,4-glucanase gene, mgcel44, was assembled and translated to amino acid sequence by using the Vector NTI Suite 8 software (InforMax, Gaithersburg, MD, USA). Signal peptide was predicted using SignalP 4.0 Server (http://www.cbs.dtu. dk/services/SignalP/). Protein functional analysis was performed with InterProScan (http://www.ebi.ac.uk/ Tools/InterProScan/). Multiple sequence alignments were performed with Clustal X (Thompson et al.
Biotechnol Lett
1997). The sequence similarity search was performed with the BLAST program (http://www.ncbi.nlm.nih. gov/BLAST/). Expression and purification of cellulase MgCel44 The putative endo-b-1,4-glucanase gene mgcel44 was amplified from the positive clone fosSCSIO1 and ligated into the pET22b(?) vector (Novagen). The recombinant plasmid pET-mgcel44 was transformed into E. coli Rosetta (DE3) to express the target protein. The transformed cells carrying pET-mgcel44 were cultured in LB medium containing ampicillin (100 lg/ ml) and chloramphenicol (34 lg/ml) at 37 °C. When the OD600 of the bacterial culture reached 0.6, IPTG was added at 1 mM for further induction at 20 °C for 20 h. Induced cells were harvested, washed twice with Tris/HCl buffer (pH 7.6), and lysed by sonication (5 s, 150 W) on ice for several times. The lysate was centrifuged at 12,0009g for 20 min at 4 °C. MgCel44 was purified by using a Ni2?-NTA agarose gel column according to the manufacturer’s protocol (Novagen). The recombinant protein was eluted with column buffer (20 mM Tris/HCl, pH 8.0, 200 mM imidazole, and 300 mM NaCl) and then dialyzed three times in deionized double-distilled water at 4 °C. The purified protein was detected after SDS-PAGE. The protein concentration was determined using the Bradford method with bovine serum albumin as standard. Enzyme assay To measure the recombinant MgCel44 activity, a reaction mixture containing 100 ll diluted enzyme and 400 ll 1 % CMC in Na2HPO4/citric acid buffer (pH 6.0) was incubated at 45 °C for 30 min. The reaction was terminated with 500 ll 3,5-dinitrosalicylic (DNS). Reducing sugars were determined by the DNS method with D-glucose as standard. One unit (U) of MgCel44 activity was defined as the amount of enzyme releasing 1 lmol reducing sugar per min. Biochemical characterization of MgCel44 The characterization of the purified MgCel44 was detected using CMC as substrate. The effect of pH on MgCel44 activity was determined at 45 °C using 0.2 M McIlvaine buffer (pH 3–8), and 0.05 M
glycine/NaOH buffer (pH 8–11). pH stability studies were carried out by measuring the residual enzyme activity after incubating enzyme at different values pH as described above. The effect of temperature on MgCel44 activity was determined from 25 to 65 °C. The thermostability of the enzyme was determined by measuring the residual activity after incubating enzyme at different temperatures as described above for 1 h. 1 and 10 mM (final concentration) of metal ions, chelating agents (EDTA) and surfactants (SDS) were individually added to the reaction system to investigate their effects on the activity. MgCel44 activity was also measured in the presence of 0–3 M NaCl. The influence of NaCl on enzyme stability was tested by measuring the enzymatic activity after incubation with 0–3 M NaCl at 25 °C for 24 h. The effects of organic solvents on the enzyme were determined by measuring the enzymatic activity after incubation with 15 % (v/v) different log Pow organic solvents at 25 °C for 24 h. Activity was expressed as a percentage of the activity obtained in the absence of the metal ions, chemical agents and organic solvents. To investigate the substrate specificity, the enzyme activity was examined in different substrates. The Km and Vmax values for MgCel44 were determined in McIlvaine buffer (pH 6.0) containing 1 % (v/v) CMC as substrate at 45 °C by using the Lineweaver–Burk method.
Sequence accession number The nucleotide sequence for GH44 endoglucanase gene mgcel44 was deposited in GenBank database under the accession number KF424270.
Results Construction of the metagenomic fosmid library A metagenomic library containing 100,000 fosmid clones was constructed. BamHI restriction analysis of 18 randomly selected plasmids showed that the insert sizes ranged from 20 to 55 kb (data not shown). The average size of insert fragments was about 30 kb, and the total size of the library was estimated to be about 3 Gb. BamHI restriction analysis also showed that the
123
Biotechnol Lett
clones had different restriction patterns, indicating that the library was composed of random DNA clones.
Effect of metal ions and chemical agents on MgCel44 activity (Table 1)
Screening, cloning, and molecular analysis of the endo-b-1,4-glucanase gene
Activity of MgCel44 was only slightly affected by Na?, NH4?, and Ni2? at 1 mM whereas Zn2?, Mn2?, Cu2?, EDTA and SDS reduced enzyme activity. Other metal ions had no obvious effects.
By screening 10,000 fosmid clones on the CMC indicator plates, an independent clone with endo-b1,4-glucanase activity was isolated and confirmed to encode on endoglucanase gene by retransformation. A positive subclone of plasmid pUC-cel44 was sequenced. It contained a complete ORF coding for an endo-b-1,4glucanase. The overall G?C content of the ORF was 56 %, and the deduced product consists of 648 amino acids and contains a glycosyl hydrolase family 44 catalytic domain. The deduced protein sequence showed highest identity of 48 % with an endoglucanase from Micromonospora lupini str. Lupac 08 (WP_00 7457316.1), 48 % identity with endoglucanase from Streptomyces bingchenggensis BCW-1, 48 % identity with endoglucanase from S. himastatinicus ATCC 53653, 47 % identity with endoglucanase from Amycolatopsis mediterranei U32, 47 % identity with endoglucanase from A. mediterranei S699. All these five proteins shared high identities with MgCel44. Multiple sequence alignment showed that MgCel44 shared conserved residues with these proteins (Fig. 1). Expression and purification of the recombinant MgCel44 The gene mgcel44 was expressed in E. coli Rosetta (DE3) cells. The recombinant enzyme MgCel44 was purified with the Ni2?-NTA column. SDS-PAGE analysis showed that MgCel44 was purified to homogeneity, with a molecular mass approximately of 71 kDa which corresponds to the estimated molecular mass (Fig. 2). The effects of pH and temperature on MgCel44 (Fig. 3) The recombinant enzyme exhibited maximal activity at pH 6.0 and remained stable at pH 5.0–8.0 (maintaining over 80 % of activity). The optimal temperature of MgCel44 was 45 °C (Fig. 4). MgCel44 activity was stable below 40 °C (maintaining more than 85 % activity) but decreased greatly above 40 °C.
123
Effect of NaCl on the activity of MgCel44 (Fig. 5) MgCel44 displayed typical characteristics of salt tolerance. Its activity was enhanced I [ 1.6-fold in 0.5 M NaCl and it remained more than 50 % activity in 1.5 M NaCl. MgCel44. It was stable for 24 h in NaCl below 1.5 M (maintained over 50 % of activity). Effect of organic solvents on MgCel44 activity (Table 2) More than 80 % activity of MgCel44 was detected in 15 % (v/v) toluene, DMSO, methanol and acetone. It retained more than 50 % activity in acetonitrile and ethanol, but in ethyl acetate, 1-butanol and chloroform activity was greatly reduced. Substrate specificity of MgCel44 Substrate specificity of MgCel44 is shown in Table 3. Highest activities were with CMC and b-glucan from barley. The Km and Vmax of recombinant MgCel44 for CMC were 7.6 mg/ml and 16.3 lmol/min/mg, respectively.
Discussion Microorganisms and their related enzyme or gene resources have potential applications in industry. The mangrove environment possess highly diverse and unique microorganisms which represents an important biotechnological resources yet to be exploited (Sivaramakrishnan et al. 2006). The enzymes from culturable microorganisms from the mangrove environment have long been studied (Sudha 1981; Polizeli et al. 2005; Mishra 2010) but there are few reports on solvent-resistant or salt-tolerant cellulases. In this study, a metagenomic fosmid library was constructed from mangrove soil and a new endo-b-1,4-glucanase gene mgcel44 was identified. The predicted product of
Biotechnol Lett
Fig. 1 Multiple sequence alignment of MgCel44 with some similar endoglucanase MgCel44 was from mangrove soil metagenomic library in this study. The WP007457316.1 was from Micromonospora lupini str. Lupac 08. The YP004967448.1 was from Streptomyces bingchenggensis BCW-1. The WP0
09720086.1 was from Streptomyces himastatinicus ATCC 53653. The YP003769935.1 was from Amycolatopsis mediterranei U32. The YP005535968.1 was from Amycolatopsis mediterranei S699, respectively. Identical residues were shaded in black. The star symbol indicates the active site of enzyme
mgcel44 shares less than 50 % identity with other endob-1,4-glucanases, indicating it is a novel endoglucanase gene. The protein sequences similar to MgCel44 in the GenBank database were from genome sequencing which had not been characterized. So we cloned and expressed mgcel44 in E. coli. The recombinant MgCel44 was then purified and characterized. GH44 endoglucanases have a large range of optimal reaction pH and temperature. MgCel44 displays similar pH characteristics to a metagenomederived endoglucanase Cel5A (Voget et al. 2006). Its
activity was greatly inhibited by EDTA indicating that it was a metalloenzyme. 1 mM ions had no obvious effects on MgCel44 but 10 mM Zn2?, Mn2?, Cu2? and SDS were inhibition. This is similar to some endoglucanases from another metagenomic library (Feng et al. 2007; Liu et al. 2009). An interesting feature of MgCel44 was that its activity was significantly improved by NaCl and was stable at high salt concentrations. These results indicate that MgCel44 is a salt-tolerant enzyme. Halophilic cellulolytic enzymes had been found from
123
Biotechnol Lett
activity
100
Relative activity (%)
stability
80 60 40 20 0 3
4
5
6
7
8
9
10
11
pH
Fig. 2 10 % SDS-PAGE analysis of the purified MgCel44 1 protein marker; 2 purified MgCel44; 3 recombinant Escherichia coli harboring pET-mgcel44 induced with IPTG; 4 cell lysate of Escherichia coli harboring empty pET-22b(?) induced with IPTG
Fig. 3 Effect of pH on MgCel44 activity and stability. The activity of MgCel44 was determined at 45 °C from pH 3.0–11.0; pH stability of MgCel44 was measured by pre-incubating the enzyme at different buffers of pH 3.0–11.0 for 24 h at 4 °C. The error bars represent the mean ± SD (n = 3)
activity stability
microorganisms living in extreme environments and the high concentrations of salts are necessary for their stability at high temperatures (Bronnenmeier et al. 1995; Liebl et al. 1996; Voget et al. 2006). Mangrove soil is high salinity and the microorganisms and their enzymes usually were salt tolerant. Such as the activity of Cel5A rose to 1.6-fold in 0.5 M NaCl and remained elevated in 4 M NaCl (Gao et al. 2010) and r-BglNH remained elevated even in 5 M NaCl (Mai et al. 2013). This characteristic indicates MgCel44 has advantage in cellulose degradation which usually is hypersaline during the lignocelluloses processing. Organic solvent-resistant cellulases are useful in cellulose degradation industry but there are few reports on the halotolerant cellulases with organic solvent tolerance. MgCel44 displayed high activity in organic solvents. Cellulases with organic solvent tolerance have been isolated from different microorganisms. An endoglucanase from Haloarcula sp. G10 showed high activity in the presence of non-polar hydrophobic organic solvents with log Pow C 0.88 (Li and Yu 2013). An extracellular cellulase from Thalassobacillus sp. LY18 was highly active in non-ionic surfactants and was stable in water-insoluble organic solvents with log Pow C 2.13 (Li et al. 2012). Cellulases with organic solvent-resistance have also been isolated from uncultured microorganism from metagenomic libraries. An endoglucanase Cel5A from a soil metagenome was
123
Relative activity (%)
100 80 60 40 20 0 25
30
35
40
45
50
55
60
65
Temperature (°C) Fig. 4 Effect of temperature on MgCel44 activity and thermostability. The activity of MgCel44 was determined in McIlvaine buffer (pH 6.0) at different temperature; Thermostability assay was measured by incubating the enzyme at different temperature for 1 h. The error bars represent the mean ± SD (n = 3)
stable in 15 % (v/v) organic solvents (Voget et al. 2006). Three cellulases from metagenomic libraries were stable in 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfonate (Pottka¨mper et al. 2009). Compared to these cellulases, MgCel44 is a member of organic solvent-resistant and halotolerant endoglucanase from mangrove soil metagenomic library. Moreover, MgCel44 retained high activity in organic solvents with low log Pow after 24 h. Enzymes would, however, be less stable in lower log P values (Klibanov 1986) but MgCel44 did not follow this P trends. This is similar to
Biotechnol Lett Table 1 Effect of 1 or 10 mM metal ions and chemical reagents on MgCel44 activity Reagents
Relative activity (%)
(10 mM)
100 ± 2.2
?
Organic solvents
Log Pa
None
–
100 ± 3.6c
DMSO
-1.35
81 ± 5.3
Methanol Acetonitrile
-0.76 -0.34
87 ± 2.4 76 ± 5.4 78 ± 3.8
a
(1 mM) None
Table 2 Effect of organic solvents on MgCel44 activity
b
100 ± 3.2
ow
Relative activity (%)b
Na
105 ± 3.4
K?
85 ± 2.5
73 ± 3.2
Ethanol
-0.3
NH4?
102 ± 4.2
93 ± 6.8
Acetone
-0.24
88 ± 2.5
Mg2?
92 ± 3.1
78 ± 2.4
Isopropanol
0.14
67 ± 6.3
Zn2?
65 ± 2.1
53 ± 2.7
Ethyl acetate
0.68
15 ± 3.6
2?
Ca
92 ± 3.6
90 ± 3.4
1-Butanol
0.88
41 ± 6.7
Mn2?
67 ± 5.1
55 ± 6.5
Chloroform
1.97
39 ± 3.6
Cu2?
65 ± 2.1
43 ± 4.6
Toluene
2.73
102 ? 7.8
Ni2?
109 ± 3.6
95 ± 4.5
a
2?
Ba EDTA
89 ± 2.5 58 ± 5.7
65 ± 6.1 28 ± 6.1
SDS
88 ± 3.2
32 ± 5.2
109 ± 2.4
The Log Pow is the logarithm of the partition coefficient, P, of the solvent between n-octanol and water and is used as a quantitative measure of the solvent polarity b
Assay was performed under optimum conditions
c
a
Values represent the mean ± SD (n = 3) relative to the untreated control samples
Assay was performed under optimum conditions
b
Values represent the mean ± SD (n = 3) relative to the untreated control samples Table 3 Substrate specificity of MgCel44 Substrate
Specific activitya (U mg-1)
Carboxymethyl cellulose
7.6 ± 0.6b
120
b-Glucan from barley
5.5 ± 0.7
100
Avicel, microcrystalline cellulose
ND
pNPGlu Cellobiose
0.7 ± 0.3 ND
Relative activity (%)
180 160
activity
140
stability
80 60 40
Oat spelts xylan
0.8 ± 0.4
20
Laminarin
ND
Filter paper
ND
0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
NaCl (M) Fig. 5 Effect of NaCl on MgCel44 activity and stability. The activity of the enzyme was measured in the presence of 0–3 M NaCl. NaCl influence on enzyme stability was tested by measuring the residual activity of the enzyme after incubation with 0–3 M NaCl at 25 °C for 1 h
an organic solvents with solvent-tolerant lipase from Pseudomonas sp. strain S5 (Rahman et al. 2005). These characteristics indicate MgCel44 has potential applications for cellulose degradation under high organic solvent conditions and also is attractive use for transglycosylation in media containing organic solvent (Oikawa et al. 2001).
a
Assay was performed at the optimum condition
b
Standard deviations were shown behind the specific activities
MgCel44 had high specific activities against amorphous cellulose with a b-1,4-glucan linkage, such as CMC and b-glucan from barley but no activity, against cellobiose. Low activity was detected against xylan from oat spelts, indicating that MgCel44 can slowly hydrolyze b-1,4-xylose linkages. There were reports that GH44 family endoglucanases can hydrolyze xylan (Cho et al. 2006; Warner et al. 2011) but MgCel44 showed lower activity against xylan than other GH44 family endoglucanases. No obvious activity was detected against laminarin, indicating that MgCel44 could not hydrolyze b-1,3-glucan linkages. MgCel44
123
Biotechnol Lett
also cannot hydrolyze insoluble microcrystalline cellulose and filter paper that have high crystallinity cellulose. It is similar to some GH44 family endoglucanases (Rinco´n et al. 2001; Warner et al. 2011). MgCel44 includes a single domain but lacks the carbohydrate-binding domain that seems to be involved in the degradation of insoluble cellulose (Coutinho et al. 1993; Gill et al. 1999). The Vmax of MgCel44 for CMC was higher than the GH44 family endoglucanase from Ruminococcus flavefaciens FD-1(7.8 lmol/min/mg) (Warner et al. 2011) but lower than other family endoglucanases (Voget et al. 2006; Gao et al. 2010; Zhang et al. 2013). MgCel44 also had a lower Km value than some metagenome-derived endoglucanases, suggesting that it has a higher affinity for the CMC substrate (Liu et al. 2011; Zhang et al. 2013). In summary, we have constructed a large-size mangrove soil fosmid library and identified a novel endoglucanase gene mgcel44 by functional screening. MgCel44 is an organic solvent-resistant and salttolerant enzyme. These characteristics allow the potential application of MgCel44 in biomass conversion and food industry. This study also highlights the advantages of metagenomic libraries for cloning novel genes from mangrove soil through functional-based approaches. Acknowledgments This study was supported by the National Basic Research Program of China (973 Program, 2010CB 833801), National Natural Science Foundation of China (Grant No. 41230962).
References Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169 Bronnenmeier K, Kern A, Liebl W, Staudenbauer WL (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microbiol 61:1399–1407 Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acid Res 37:233–238 Cho KM, Hong SY, Lee SM, Kim YH, Kahng GG, Kim H, Yun HD (2006) A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains. Appl Microbiol Biotechnol 73: 618–630 Coutinho J, Gilkes N, Kilburn D, Warren R, Miller R Jr (1993) The nature of the cellulose-binding domain effects the
123
activities of a bacterial endoglucanase on different forms of cellulose. FEMS Microbiol Lett 113:211–217 Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, Hu YL, Wei J, Tang JL, Feng JX (2007) Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl Microbiol Biotechnol 75:319–328 Gao Z, Ruan L, Chen X, Zhang Y, Xu X (2010) A novel salttolerant endo-b-1, 4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil. Appl Microbiol Biotechnol 87:1373–1382 Gill J, Rixon J, Bolam D, Mcqueen-mason S, Simpson P, Williamson M, Hazlewood G, Gilbert H (1999) The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism. Biochem J 342:473–480 Han SJ, Yoo YJ, Kang HS (1995) Characterization of a bifunctional cellulase and its structural gene The cel gene of Bacillus sp. D04 has exo-and endoglucanase activity. J Biol Chem 270:26012–26019 Hyde K, Lee S (1995) Ecology of mangrove fungi and their role in nutrient cycling: what gaps occur in our knowledge? Asia-Pacific Symposium on Mangrove Ecosystems. Springer, pp 107–118 Jiang Y, Zheng T, Tian Y (2006) Research on mangrove soil microorganisms: past, present and future. Wei sheng wu xue bao = Acta microbiologica Sinica 46:848–851 Kitago Y, Karita S, Watanabe N, Kamiya M, Aizawa T, Sakka K, Tanaka I (2007) Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum. J Biol Chem 282:35703–35711 Klibanov AM (1986) Enzymes that work in organic solvents. ChemTech 16:354–359 Klinke HB, Thomsen A, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66:10–26 Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK, Chung CH, Lee YC, Lee JW (2008) Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour Technol 99:378–386 Li X, Yu HY (2013) Characterization of a halostable endoglucanase with organic solvent-tolerant property from Haloarcula sp. G10. Int J Biol Macromol 62:101–106 Li X, Wang HL, Li T, Yu HY (2012) Purification and characterization of an organic solvent-tolerant alkaline cellulase from a halophilic isolate of Thalassobacillus. Biotechnol Lett 34:1531–1536 Liebl W, Ruile P, Bronnenmeier K, Riedel K, Lottspeich F, Greif I (1996) Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes. Microbiology 142:2533–2542 Liu L, Feng Y, Duan CJ, Pang H, Tang JL, Feng JX (2009) Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World J Microbiol Biotechnol 25:1035–1042 Liu J, Liu W, Zhao X, Shen W, Cao H, Cui Z (2011) Cloning and functional characterization of a novel endo-b-1, 4-glucanase gene from a soil-derived metagenomic library. Appl Microbiol Biotechnol 89:1083–1092
Biotechnol Lett Mai Z, Yang J, Tian X, Li J, Zhang S (2013) Gene cloning and characterization of a novel salt-tolerant and glucoseenhanced b-glucosidase from a marine streptomycete. Appl Biochem Biotechnol 169(5):1512–1522 Mishra R (2010) Microbial biodiversity in mangroves of Bhitarakanika, Orissa-a study on genotypic, phenotypic and proteomic characterisation of some predominant bacteria. PhD Thesis submitted to North Orissa University, Orissa, India Ogawa J, Shimizu S (1999) Microbial enzymes: new industrial applications from traditional screening methods. Trends Biotechnol 17:13–20 Oikawa T, Tsukagawa Y, Chino M, Soda K (2001) Increased transglycosylation activity of Rhodotorula glutinis endo-bglucanase in media containing organic solvent. Biosci Biotechnol Biochem 65:1889–1892 Polizeli M, Rizzatti A, Monti R, Terenzi H, Jorge J, Amorim D (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591 Pottka¨mper J, Barthen P, Ilmberger N, Schwaneberg U, Schenk A, Schulte M, Ignatiev N, Streit WR (2009) Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids. Green Chem 11:957–965 Rahman RN, Baharum SN, Basri M, Salleh AB (2005) Highyield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5. Anal Biochem 341:267–274 Rinco´n MT, McCrae SI, Kirby J, Scott KP, Flint HJ (2001) EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain. Appl Environ Microbiol 67:4426–4431
Sivaramakrishnan S, Gangadharan D, Nampoothiri KM, Soccol CR, Pandey A (2006) a-Amylases from microbial sourcesan overview on recent developments. Food Technol Biotechnol 44:173–184 Sudha V (1981) Studies on Halophilic bacteria associated with mangrove sediment and a bivalve Mollusc Andara rhombea (Bora)(Arcidae) and (b) on L-asparaginase (antileukamic agent) from an extremely halophilic bacterium. Ph.D. Thesis, Annamalai University Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Voget S, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit W (2003) Prospecting for novel biocatalysts in a soil metagenome. Appl Environ Microbiol 69:6235–6242 Voget S, Steele H, Streit W (2006) Characterization of a metagenome-derived halotolerant cellulase. J Biotechnol 126:26–36 Warner CD, Hoy JA, Shilling TC, Linnen MJ, Ginder ND, Ford CF, Honzatko RB, Reilly PJ (2010) Tertiary structure and characterization of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum. Appl Environ Microbiol 76:338–346 Warner CD, Go RM, Garcı´a-Salinas C, Ford C, Reilly PJ (2011) Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1. Enzym Microb Technol 48:27–32 Zhang G, Li S, Xue Y, Mao L, Ma Y (2012) Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10. Extremophiles 16:35–43
123
