- Open Access
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.
- Sodium Dodecyl Sulfate
- Activate Sludge
- Lipolytic Activity
Lipolytic enzymes such as esterases (EC188.8.131.52) and lipases (EC184.108.40.206) 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.
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.
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.
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