Gene cloning and characterization of a novel esterase from activated sludge metagenome
© Zhang et al; licensee BioMed Central Ltd. 2009
Received: 24 September 2009
Accepted: 22 December 2009
Published: 22 December 2009
A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216) and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤ C8). This EstAS had optimal temperature and pH at 35°C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications.
Lipolytic enzymes such as esterases (EC126.96.36.199) and lipases (EC188.8.131.52) catalyze both the fat hydrolysis and the synthesis of fatty acid esters including acylglycerides as biocatalysts . Lipolytic enzymes are ubiquitous α/β hydrolyzing enzymes existed in animals, plants, and microbes, including fungi and bacteria. Microbial esterases are showing considerable industrial potential where their regiospecificity and enantioselectivity are desired characteristics , such as production of fine chemicals, pharmaceuticals, in the food industry and are widely used in biotechnology [2–4].
Modern biotechnology has a steadily increasing demand for novel biocatalysts, thereby prompting the development of novel experimental approaches to find and identify novel biocatalyst-encoding genes. Metagenome is the total microbial genome directly isolated from natural environments, and the power of metagenomics is the access, without prior sequence information, to the so far uncultured majority, which is estimated to be more than 99% of the prokaryotic organisms [5–7]. In fact, the metagenomic approach was successful in searching for novel lipolytic enzymes in varied environments, and also, several genes encoding metagenomic esterases have been identified in metagenomic libraries prepared from varied environmental samples, including soils [6–9], marine sediment [10–12], pond and lake water [13–15], and tidal flat sediment .
Studies based on 16S rDNA library have extensively redefined and expanded our knowledge of microbial diversity in activated sludge from low-temperature aromatic wastewater treatment bioreactor, including members of various un-culturable groups (unpublished data). To the best of our knowledge, activated sludge microbial communities have not been exploited by culture-independent methods for isolation of lipolytic genes. Here, we report the isolation, sequence analysis, and enzymatic characterization of a novel esterase, EstAS, from an activated sludge derived metagenomic library. The discovery of EstAS led to the identification of a new family of bacterial lipolytic enzymes.
Materials and methods
Activated sludge was collected from a low temperature sequencing batch bioreactor (SBR) treating nitrogen-containing aromatic wastewater in our laboratory.
Bacterial strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study
Strain or plasmid
Source or reference
E. coli EPI300™-T1R
[F- e14-(McrA-) D(mcrC-mrr) (TetR) hsdR514 supE44 supF58 lacY1 or D(lacIZY)6 galK2 galT22 metB1 trpR55 l-]
E. coli TOP10
lac x74 recA1 deoR F - mcrA Δ (mrr-hsdRMS-mcrBC) φ80 lacZ ΔM15 Δ araD139 Δ (ara-leu)7697 galU galK
E. coli BL21(DE3)
F-, ompT, hsdSB (rB-, mB-), dcm, gal, λ(DE3), pLysS, Cmr
E. coli EPI300-FosB12L1
Positive clone from Fosmid genomic library, which carries the lipolytic gene
E. coli TOP10-EstAS
Positive clone from sublibrary, which carries the EstAS gene fragment
E. coli BL21(DE3)-EstAS
Positive clone, which carries the pEstAS-His expression vector
Cloning vector; Cmr
Cloning vector; Apr
Expression vector; Kmr
pCC2FOS, which carries the EstAS gene cluster (35 kb)
pUC118, which carries the complete lipolytic gene (EstAS)
pET28a carrying amplified Hin dIII -Nde I fragment containing lipolytic gene (EstAS)
DNA preparation and manipulation
E. coli cells were transformed by the calcium chloride procedure . Recombinant plasmid DNA was isolated by the method of Birnboim and Doly  or with a Tian-prep Mini kit (TianGen). Restriction enzymes, T4 DNA ligase and calf intestinal alkaline phosphatases were purchased from New England Biolabs (Ipswich, USA) or Takara (Tokyo, Japan) and used according to the manufacturers' instructions.
Construction of metagenomic DNA library and sublibrary
Activated sludge DNA extraction was carried out using SDS and proteinase K treatment , and the removal of humic acids (HAs) prior to DNA extraction was conducted by using HAs removing buffer . Approximately 100 μg of metagenomic DNA was run on a preparative pulsed-field gel (Bio-Rad CHEF DR®III; 0.1-40 s switch time, 6 V/cm, 0.5 × TBE buffer, 120° included angle, 16 h), and the appropriate size of DNA ranging from 30-50 kb was isolated, electroeluted, and dialyzed against 0.5 × TE buffer for further Fosmid library construction. The purified DNA fragments were end-repaired by End-repaired enzyme mix. After size fractionation and purification, the blunt-ended, 5'-phosphorylated DNA was ligated into the cloning-ready Copycontrol pCC2FOS vector, and the recombinant molecules were packaged in vitro with a MaxPlaxTM Lambda packaging kit (Epicentre Biotechnologies, Madison, Wisconsin, USA). The selected unique fosmid clone was named FosB12L1 (showing strong lipolytic activity on tributyrin plate), and purified, partially digested with Sau 3AI to obtain 3-5 kb size DNA, and ligated into a purified Bam HI/BAP pUC118 vector from Takara. Ligation products were transformed into E. coli TOP10 cells (Tiangen) and spread out on LB (ampicillin, 100 μg/ml) plates containing 1% (v/v) tributyrin as the indicator substrate .
Identification of lipolytic clones and DNA sequence analysis
The DNA fragment obtained was sequenced with primer walking method by SinoGenoMax Co. Ltd (Chinese National Human Genome Center, Beijing). The ORFs were analyzed using DNAstar (Lynnon Biosoft) and GeneTool software (Syngene), Database searches were performed with the BLAST program via GenomeNet World Wide Web server. Peptide sequences of various enzymes or subunits were extracted from National Center for Biotechnology Information (Washington, D.C).
Deduced amino acid sequences of 8 lipolytic enzymes were subjected to protein phylogenetic analysis. Sequence alignment was performed by using CLUSTAL_W program  and visually examined with BoxShade Server program. Phylogenetic tree was generated using the neighbor joining method of Saitou and Nei  with MEGA 4.0 software .
Protein expression and purification
Primers used in the study
Sequencing primer for pCC2FOS™
Sequencing primer for pCC2FOS™
M13 primer RV'
Sequencing primer for pUC118
M13 primer M2
Sequencing primer for pUC118
Genomic walking primer for EstAS gene
Genomic walking primer for EstAS gene
Genomic walking primer for EstAS gene
Genomic walking primer for EstAS gene
TCAGCCAT ATG TCTTACCCGATCGTCCTGG
Forward primer for EstAS gene
Reverse primer for EstAS gene
Characterization and biochemical properties of EstAS
The substrate specificity of the purified enzyme was analyzed using the following substrates of p-NP-fatty acyl esters [21, 25]: acetate (C2), butyrate (C4), hexanoate (C6), caprylate (C8), decanonate (C10), laurate (C12), myristate (C14) and palmitate (C16). The enzyme was incubated with the ester derivatives (0.5 mM) in 5 ml Tris-HCl buffer (50 mM, pH 8.0) at 40°C for 10 min. The reaction was quenched by adding 5 ml trichloroacetic acid (0.5 mM) and then recovered the original pH value with 5.15 ml NaOH (0.5 mM), and the amount of released p-NP was determined by an absorption increase at 405 nm against an enzyme-free blank on a Biospec-1601 spectrophotometer [26, 27]. One unit of esterase is defined as the amount needed to release 1 μmol p-NP per min under the above conditions. The highest enzyme activity on a substrate (i. e. p-NP-hexanoate) was defined as 100%. To determine the presence of esterase activity, the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) (Sigma Aldrich) was used as a chromogenic substrate, and the formation of methyresorufin was analyzed spectrophotometrically at 580 nm [1, 28, 29]. Candida rugosa lipase (Sigma Aldrich) was used as a positive control.
Using p-NP-hexanoate (0.5 mM) as substrate, the optimal temperature and pH of purified EstAS was determined, by measuring the enzyme activity after incubation at various temperatures (10-65°C) in 50 mM Tris-HCl buffer (pH 8.0) or after incubation at 35°C for 10 min in the following buffers: 50 mM phosphate buffer (pH 5.0-7.5), 50 mM Tris-HCl (pH 8.0-10.5).
Various metal ions (CoCl2, CaCl2, ZnCl2, MgCl2, K2SO4, FeSO4, CuSO4, Ni(NO3)2 and MnSO4), and chelating agent EDTA at final concentration of 1 mM were added to the enzyme in 50 mM Tris-HCl (pH 8.0), then assayed for esterase activity after preincubation at 35°C. Effect of detergents or reductors on esterase activity was determined by incubating the enzyme for 30 min at 35°C in 50 mM Tris-HCl (pH 8.0), containing (1%, v/v) Triton X-100, Tween 20 and 80, β-mercaptoethanol, 1, 4-dithiothreitol (DTT), sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), phenylmethanesulfonyl fluoride (PMSF) and diethypyrocarbonate (DEPC), respectively. The enzyme activity without metal ions and detergents was defined as 100%.
Nucleotide sequence accession number
The DNA sequence of EstAS was deposited in DDBJ/EMBL/GenBank under accession number of FJ386490.
Results and discussion
Construction of a metagenomic library and screening
About 100 μg DNA was extracted from 1 g activated sludge (wet-weight), and 1.5 μg of size-selected, pulse-field gel-purified high-molecular-weight (HMW) DNA suitable for fosmid cloning was obtained. 300 ng of 30-45 kb purified metagenomic DNA was ligated into the copy control pCC2FOS vector and transfected into E. coli EPI300-T1R, producing a metagenomic library of more than 100, 000 fosmids with insert sizes ranging from 28 kb to 40 kb (average size of 35 kb), covering approximately 3.0 Gbp of the total metagenomic DNA. The prokaryotic origin of the library was confirmed by end-sequencing of randomly selected fosmids and comparison with known ORFs in NCBI. Expression screening of the fosmid library based on the hydrolysis of emulsified tributyrin (1%) resulted in the detection of a recombinant clone, FosB12L1, forming a clear zone on the indicator plate.
Subcloning and identification of the esterase
Expression and purification of recombinant EstAS
Substrate specificity of EstAS
Effect of temperature and pH on EstAS
Effect of metal ions on esterase EstAS
Effect of metal ion on esterase activity
Relative activity (%)
100.0 ± 3.7
117.8 ± 2.1
100.5 ± 3.4
114.7 ± 1.3
81.7 ± 2.9
101 ± 4.1
103.8 ± 1.6
7.8 ± 2.3
192.9 ± 3.8
46.2 ± 5.2
121.7 ± 1.2
Effect of detergents and reductors on esterase EstAS
Effect of detergents and enzyme inhibitors on esterase activity
Relative activity (%)
100.0 ± 2.1
102.7 ± 2.7
101.9 ± 1.9
16.2 ± 9.3
119.6 ± 4.6
128.9 ± 0.8
135.8 ± 3.1
138.3 ± 2.1
100.3 ± 5.2
48.6 ± 0.7
In conclusion, we identified a new esterase EstAS belonging to family III lipases from SBR activated sludge metagenomic library. EstAS is a very interesting enzyme with high potential for downstream biotechnological applications. This was confirmed by extensive biochemical characterization, substrate specificity, stability towards addictives including metal ions and detergents, and also, wide pH and temperature spectra. This study also demonstrated that the metagenomic approach is very useful for expanding our knowledge of enzyme diversity, especially for bacterial esterases. Accessing the metagenomic pool of lipases and esterases can be an immediate source of novel biocatalysts, or yield enzymes that can be further specialized by directed evolution.
This work was supported by grants of Hi-Tech Research and Development Program of China ("863" program, No. 2006AA06Z316) and the Knowledge Innovation Program of the Chinese Academy of Sciences, No. KJCX2-YW-L08 and KSCS2-YW-G-055-01.
- Jaeger KE, Dijkstra BW, Reetz MT: Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol. 1999, 53: 315-351. 10.1146/annurev.micro.53.1.315.View ArticleGoogle Scholar
- Jaeger KE, Ransac S, Dijkstra BW, Colson C, Heuvel M van, Misset O: Bacterial lipases. FEMS Microbiol Rev. 1994, 15 (1): 29-63. 10.1111/j.1574-6976.1994.tb00121.x.View ArticleGoogle Scholar
- Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B: Industrial biocatalysis today and tomorrow. Nature. 2001, 409 (6817): 258-268. 10.1038/35051736.View ArticleGoogle Scholar
- Straathof AJ, Panke S, Schmid A: The production of fine chemicals by biotransformations. Curr Opin Biotechnol. 2002, 13 (6): 548-556. 10.1016/S0958-1669(02)00360-9.View ArticleGoogle Scholar
- Handelsman J: Metagenomics: Application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev. 2004, 68 (4): 669-685. 10.1128/MMBR.68.4.669-685.2004.View ArticleGoogle Scholar
- Lee SW, Won K, Lim HK, Kim JC, Choi GJ, Cho KY: Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl Microbiol Biotechnol. 2004, 65 (6): 720-726. 10.1007/s00253-004-1722-3.View ArticleGoogle Scholar
- Elend C, Schmeisser C, Leggewie C, Babiak P, Carballeira JD, Steele HL, Reymond JL, Jaeger KE, Streit WR: Isolation and biochemical characterization of two novel metagenome-derived esterases. Appl Environ Microbiol. 2006, 72 (5): 3637-3645. 10.1128/AEM.72.5.3637-3645.2006.View ArticleGoogle Scholar
- Henne A, Schmitz RA, Bomeke M, Gottschalk G, Daniel R: Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl Environ Microbiol. 2000, 66 (7): 3113-3116. 10.1128/AEM.66.7.3113-3116.2000.View ArticleGoogle Scholar
- Li G, Wang K, Liu YH: Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the metagenome. Microb Cell Fact. 2008, 7 (38):View ArticleGoogle Scholar
- Chu XM, He HZ, Guo CQ, Sun BL: Identification of two novel esterases from a marine metagenomic library derived from South China Sea. Appl Microbiol Biotechnol. 2008, 80 (4): 615-625. 10.1007/s00253-008-1566-3.View ArticleGoogle Scholar
- Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ, Lee JH: Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol. 2009, 81 (5): 865-874. 10.1007/s00253-008-1656-2.View ArticleGoogle Scholar
- Park HJ, Jeon JH, Kang SG, Lee JH, Lee SA, Kim HK: Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome. Prot Expr Purif. 2007, 52 (2): 340-347. 10.1016/j.pep.2006.10.010.View ArticleGoogle Scholar
- Ranjan R, Grover A, Kapardar RK, Sharma R: Isolation of novel lipolytic genes from uncultured bacteria of pond water. Biochem Biophys Res Commun. 2005, 335 (1): 57-65. 10.1016/j.bbrc.2005.07.046.View ArticleGoogle Scholar
- Rees HC, Grant S, Jones B, Grant WD, Heaphy S: Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries. Extremophiles. 2003, 7 (5): 415-421. 10.1007/s00792-003-0339-2.View ArticleGoogle Scholar
- Rhee JK, Ahn DG, Kim YG, Oh JW: New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol. 2005, 71 (2): 817-825. 10.1128/AEM.71.2.817-825.2005.View ArticleGoogle Scholar
- Wu C, Sun BL: Identification of novel esterase from metagenomic library of Yangtze River. J Microbiol Biotechnol. 2009, 19 (2): 187-193. 10.4014/jmb.0804.292.View ArticleGoogle Scholar
- Sambrook J, Russell DW: Molecular cloning: a laboratory manual. 2001, Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979, 7 (6): 1513-1523. 10.1093/nar/7.6.1513.View ArticleGoogle Scholar
- Zhou J, Bruns MA, Tiedje JM: DNA recovery from soils of diverse composition. Appl Environ Microbiol. 1996, 62 (2): 316-322.Google Scholar
- Xi F, Fu LY, Wang GZ, Zheng TL: A simple method for removing humic acids from marine sediment samples prior to DNA extraction. Chin High Technol Lett. 2006, 16 (5): 539-544.Google Scholar
- Roh C, Villatte F: Isolation of a low-temperature adapted lipolytic enzyme from uncultivated microorganism. J Appl Microbiol. 2008, 105 (1): 116-123. 10.1111/j.1365-2672.2007.03717.x.View ArticleGoogle Scholar
- Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987, 4 (4): 406-425.Google Scholar
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997, 25 (24): 4876-4882. 10.1093/nar/25.24.4876.View ArticleGoogle Scholar
- Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007, 24 (8): 1596-1599. 10.1093/molbev/msm092.View ArticleGoogle Scholar
- Hardeman F, Sjoling S: Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol. 2007, 59 (2): 524-534. 10.1111/j.1574-6941.2006.00206.x.View ArticleGoogle Scholar
- Jiang HF, Wang YQ, Liu CG: Comparison and improvement of three determination methods for lipase activity. Chem Eng. 2007, 24 (8): 72-75.Google Scholar
- Pignede G, Wang HJ, Fudalej F, Gaillardin C, Seman M, Nicaud JM: Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica. J Bacteriol. 2000, 182 (10): 2802-2810. 10.1128/JB.182.10.2802-2810.2000.View ArticleGoogle Scholar
- Panteghini M, Bonora R, Pagani F: Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate. Ann Clin Biochem. 2001, 38 (4): 365-370. 10.1258/0004563011900876.View ArticleGoogle Scholar
- Zandonella G, Haalck L, Spener F, Faber K, Paltauf F, Hermetter A: Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity. Chirality. 1996, 8 (7): 481-489. 10.1002/(SICI)1520-636X(1996)8:7<481::AID-CHIR4>3.0.CO;2-E.View ArticleGoogle Scholar
- Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J, Sussman JL, Verschueren KHG, Goldman A: The alpha/beta-hydrolase Fold. Protein Eng. 1992, 5 (3): 197-211. 10.1093/protein/5.3.197.View ArticleGoogle Scholar
- Bell PJL, Sunna A, Gibbs MD, Curach NC, Nevalainen H, Bergquist PL: Prospecting for novel lipase genes using PCR. Microbiology-Sgm. 2002, 148 (8): 2283-2291.View ArticleGoogle Scholar
- Verger R: Interfacial activation of lipases: Facts and artifacts. Trends Biotechnol. 1997, 15 (1): 32-38. 10.1016/S0167-7799(96)10064-0.View ArticleGoogle Scholar
- Jaeger KE, Eggert T: Lipases for biotechnology. Curr Opin Biotechnol. 2002, 13 (4): 390-397. 10.1016/S0958-1669(02)00341-5.View ArticleGoogle Scholar
- Yu MR, Qin SW, Tan TW: Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica. Process Biochem. 2007, 42 (3): 384-391. 10.1016/j.procbio.2006.09.019.View ArticleGoogle Scholar
- Lee MH, Lee CH, Oh TK, Song JK, Yoon JH: Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microbiol. 2006, 72 (11): 7406-7409. 10.1128/AEM.01157-06.View ArticleGoogle Scholar
- Nawani N, Dosanjh NS, Kaur J: A novel thermostable lipase from a thermophilic Bacillus sp.: Characterization and esterification studies. Biotechnol Lett. 1998, 20 (10): 997-1000. 10.1023/A:1005430215849. 10.1023/A:1005430215849.View ArticleGoogle Scholar
- De Simone G, Menchise V, Manco G, Mandrich L, Sorrentino N, Lang D, Rossi M, Pedone C: The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus. J Mol Biol. 2001, 314 (3): 507-518. 10.1006/jmbi.2001.5152.View ArticleGoogle Scholar
- De Simone G, Galdiero S, Manco G, Lang D, Rossi M, Pedone C: A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase. J Mol Biol. 2000, 303 (5): 385-10.1006/jmbi.2000.4195. 10.1006/jmbi.2000.4195.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.