Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product
© JunGang et al; licensee BioMed Central Ltd. 2010
Received: 4 September 2010
Accepted: 7 November 2010
Published: 7 November 2010
In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications.
Lipolytic enzymes are ubiquitous α/β hydrolyzing enzymes existing in animals, plants, and microbes. The enzymes contain esterases (EC188.8.131.52) and lipases (EC184.108.40.206) which catalyze the hydrolysis and synthesis of fatty acid esters including acylglycerides . Due to some useful features such as broad substrate specificity, stability in organic solvents and regio-/enantioselectivity, lipolytic enzymes of microbial origin are widely used in industrial biotechnology, such as production of fine chemicals, pharmaceuticals, and fine chemicals synthesis [2–4].
Modern biotechnology has a steadily increasing demand for novel biocatalysts, thereby prompting the development of new experimental approaches to find and identify novel biocatalyst-encoding genes. Based on the direct cloning of the metagenome  for the construction of large clone libraries, metagenomics allows access to new sequences, genes, complete pathways and their products by multiple screening possibilities. With the advent of the metagenome approach, the so far uncultured microorganisms (estimated to more than 99%) [6–10] are now more readily accessible, resulting in an exponential increase in the number of potential biocatalysts. Indeed, the metagenomic approach was useful in mining novel lipolytic enzymes from environmental samples, and also, several genes encoding esterases have been isolated in metagenomic libraries prepared from highly diverse bacterial communities, including marine sediment [11–13], soils [8, 10, 14, 15], drinking water biofilm , pond and lake water [16, 17], and tidal flat sediment . Some of these enzymes display enhanced characteristics, therefore, searching for novel lipolytic enzymes still attracts considerable attention.
Pre-studies based on 16S rDNA library have extensively expanded our knowledge of microbial diversity in activated sludge from sewage treat plant, including members of varied un-culturable groups (unpublished data). Here, we report the cloning, sequence analysis, and biochemical enzymatic characterization of a novel esterase, EstMY, from an activated sludge derived metagenomic library. Our report demonstrates that metagenomics is a powerful approach in mining new industrial enzymes. The esterase EstMY constituted a new member of family IV of bacterial lipolytic enzymes.
Materials and methods
Activated sludge was collected from a sewage treatment plant treating nitrogen-containing aromatic wastewater on September 2008 in Mianyang City, SiChuan Province.
Bacterial strains, plasmids, and culture
Starting bacterial strains and plasmids used in this study
Strain or plasmid
Source or reference
E. coli TOP10
lac х74 recA1 deoR F - mcrA ∆ (mrr-hsdRMS-mcrBC) ϕ80 lacZ∆M15∆ araD139∆ (ara-leu)7697 galU galK
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 BL21(DE3)
F-, ompT, hsdSB (rB-, mB-), dcm, gal, λ(DE3), pLysS, Cmr
E. coli EPI300-FosD11L2
Positive clone from Fosmid genomic library, which carries the lipolytic gene
E. coli TOP10-EstMY
Positive clone from sublibrary, which carries the EstMY gene fragment
E. coli BL21(DE3)-EstMY
Positive clone, which carries the pEstMY-His expression vector
Cloning vector; Chlr
Cloning vector; Apr
Expression vector; Kmr
pCC1FOS, which carries the estMY gene cluster (31 kb)
pUC18, which carries the complete lipolytic gene (estMY)
pET28a carrying amplified Hin dIII -Nde I fragment containing lipolytic gene (estMY)
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 . For sequencing, this DNA was further purified by polyethylene glycol precipitation . 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. BugBuster Ni-NTA His. Bind Purification Kit was purchased from Novagen (Code No. NV70751-3, Novagen).
Construction of metagenomic DNA library and related sublibrary
Activated sludge DNA extraction was carried out as previously described using SDS and proteinase K treatment , and removing humic acids (HAs) prior to DNA extraction was conducted by removing HAs buffer, 100 mmol/L Tris-HCl pH 10.0, 100 mmol/L Na4P2O7 100 mM, Na2EDTA, 1.0%; PVP, 100mM NaCl, 0.05% Triton X-100 . Approximately 150 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 30-45 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 drop dialysis and concentration, the blunt-ended, 5'-phosphorylated DNA was ligated into the cloning-ready Copycontrol pCC1FOS vector, and the recombinant molecules were packaged into phage followed by phage transfection to E. coli EPI300 by using protocols described in MaxPlax™ Lambda packaging kit (Epicentre Biotechnologies, Madison, Wisconsin, USA). A fosmid clone showing strong lipolytic enzyme activity on a tributyrin agar plate was selected for further characterization and designated FosD11L2. The DNA was purified from the selected clone, partially digested with Sau 3AI in order to obtain 3-5 kb DNA fragments, ligated to the pUC18 vector and transformed into E. coli TOP10 cells (Transgen). Transformants were selected on LB (ampicillin, 100 μg/ml) plates containing 1% (v/v) tributyrin as the indicator substrate .
Genetic characterization and sequence analysis
The lipolytic DNA fragment obtained from positive clone E.coli TOP10-EstMY was sequenced with primer walking method by SinoGenoMax Co. Ltd (Chinese National Human Genome Center, Beijing). The ORFs were analyzed using DNASTAR (Lynnon Biosoft) software and ORF finder online analysis http://www.ncbi.nlm.nih.gov/projects/gorf/, Database searches for protein sequences was performed using BLAST and FASTA programs [24, 25]. Peptide sequences of various enzymes or subunits were extracted from National Center for Biotechnology Information (Washington, D.C).
Deduced amino acid sequences of 12 lipolytic enzymes were subjected to protein phylogenetic analysis. A phylogenetic tree was generated using the neighbor joining method of Saitou and Nei  with MEGA 4.0 software . A total of 6 sequences were aligned with the CLUSTAL_W program  and visually examined with BoxShade Server program. The length of each branch pair represents the evolutionary distance between the sequences.
Heterologous expression of gene estMY and purification of recombinant EstMY
Primers used in the study
Sequencing primer for pCC1FOS™
Sequencing primer for pCC1FOS™
M13 primer RV
Sequencing primer for pUC18
M13 primer M2
Sequencing primer for pUC18
Genomic walking primer for estMY gene
Genomic walking primer for estMY gene
Genomic walking primer for estMY gene
Genomic walking primer for estMY gene
GGCAT ATG GCCGCGCCCGTTCCGCCCATCAG Nde I
Forward primer for estMY gene
GGAAGCTT CTACGCTGCCGCCCTAGCGCCGATGHin dIII
Reverse primer for estMY gene
Polyacrylamide gel electrophoresis of enzyme in the presence of sodium dodecyl sulfate (SDS) was carried out by the method of Sambrook and Russell .
Characterization of recombinant EstMY and biochemical properties
The purified EstMY was subjected to a series of biochemical analysis, including determing the pH optimum, temperature optimum, substrate specificity, and effects of various detergents and metal ions. All measurements were carried out in triplicate. The values were the mean of the data. The substrate specificity of the purified EstMY protein was performed using the following substrates of p-NP-fatty acyl esters [23, 29]: 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 30°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). The enzymatic activity was measured by monitoring the p-nitrophenoxide production by absorbance at 405 nm against an enzyme-free blank, which was measured using a Ultraspec 3000 UV/vis spectrometer (Amersham Biosciences, Sweden) [30, 31]. One unit of enzyme activity was defined as the amount of activity required to release 1 μmol p-NP per minute under the above condition. The highest activities of enzyme assay using the substrate (i. e. p-NP-caprylate) was defined as the 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 methylresorufin was analyzed spectrophotometrically at 580 nm [32–34]. Candida rugosa lipase (Sigma Aldrich) was used as a positive control.
The optimum temperature of purified EstMY was determined by assaying lipolytic enzyme activities in a 50 mM Tris-HCl buffer (pH 8.0) for a temperature range of 20-65°C, in which p-NP-caprylate (0.5 mM) acted as substrate. Optimal pH was determined by examining the activity of the enzyme after incubation at 35°C for 10 min using p-NP-caprylate (0.5 mM) as substrate. The buffers used were: 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, CuCl2, Ni(NO3)2, and FeCl3), and chelating agent EDTA at final concentrations of 5 mM were added to the enzyme in 50 mM Tris-HCl (pH 8.0), whereafter it was assayed for esterase activity following 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 Triton X-100, Tween 20, Tween 80, β-mercaptoethanol, 1,4-dithiothreitol (DTT), sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), phenylmethanesulfonyl fluoride (PMSF), diethylpyrocarbonate (DEPC). The concentrations of metal ions, EDTA, detergents, and surfactants used were 5 mM, 3 mM, and 0.5% (v/v), respectively. The activity of the enzyme preparation in the absence of metal ions and detergents before incubation was defined as the 100% level.
Nucleotide sequence accession number
The DNA sequence of EstMY from activated sludge was deposited in GenBank under accession number of HM366454.
Results and discussion
Construction and screening of a metagenomic library
One hundred micrograms of prokaryotic DNA was extracted per gram of wet-weight activated sludge, and 1.5 μg of size-selected, pulsed field gel-purified high-molecular-weight (HMW) DNA suitable for fosmid library construction was obtained. Three hundred nanograms of 30-45 kb purified metagenomic DNA was ligated into the copy control pCC1FOS vector and then tranfected into E. coli EPI300-T1R, producing a metagenome library of more than 7,0000 fosmids with insert size ranging from 27 kb to 38 kb, with an average size of 32 kb, covering approximately 2.1 Gbp of the total metagenomic DNA. Given an average prokaryotic genome of approximately 5 Mbp, the metagenome library theoretically reached the size of over 400 prokaryotic genomes. 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 for hydrolytic activity based on the hydrolysis of emulsified tributyrin (1%) resulted in the finding of a recombinant clone, FosD11L2, forming a clear zone on the indicator plate. In order to identify the hydrolytic gene within a fragment of 31 kb, the insert was subject to further subcloning.
Subcloning and identification of the esterase gene
Amino acid sequence alignment indicated that this EstMY exhibited low identity with other esterase/lipases. EstMY shared the highest (71%) sequence identity with the ACL67845 esterase/lipase isolated from a marine metagenome library, 65% sequence identity to Est25 screened from a soil metagenomic library , followed by the putative lipase/esterase from other environmental samples (50-65% identity), the putative alpha/beta hydrolase from Burkholderia ubonensis Bu and Parvibaculum lavamentivorans DS-1 (ZP_02382719, 52% identity; and YP_001412150, 49% identity, respectively), members of the family IV hydrolases.
Expression and purification of recombinant EstMY
Substrate specificity of EstMY
Effect of temperature and pH on EstMY
Effect of metal ions on esterase
Effect of metal ions on esterase activity
Relative activity (%)
100.0 ± 2.9
126.4 ± 2.1
103.2 ± 3.6
100.9 ± 2.6
7.8 ± 2.7
36.2 ± 4.3
102.7 ± 3.2
10.9 ± 3.4
104.1 ± 3.7
23.7 ± 1.8
79.7 ± 2.6
Effect of detergents and reductors on esterase
Effect of detergents and enzyme inhibitors on esterase activity
Relative activity (%)
100.0 ± 2.1
101.7 ± 2.6
106.9 ± 4.9
129.7 ± 2.2
38.6 ± 2.7
101.3 ± 4.1
12.3 ± 2.9
129.6 ± 4.6
138.4 ± 2.1
156.7 ± 3.3
In conclusion, we identified a new esterase EstMY belonging to family IV lipases, whose encoding gene was isolated from activated sludge of a sewage treatment plant treating nitrogen-containing aromatic wastewater. EstMY is expected to show high potential for downstream biotechnological applications including synthetic organic chemistry. This was confirmed by its extensive biochemical characterization, which revealed the enzymes substrate specificity, wide pH and temperature spectra, and also, stability towards addictives including metal ions and detergents. Future work will establish the structure of this enzyme to gain more information about its catalytic mechanism. Our research also demonstrated the potential of metagenome strategy in bioprospecting novel genes and biocatalysts and expanded our knowledge of biocatalyst diversity, especially for bacterial esterases. Enlargement of the lipases/esterases pool can be an immediate source of genetic modification, or yield enzymes that can be further specialized by directed evolution, and also, this would optimize their industrial applications.
This work was supported by the grant No. 2005B049 from the Scientific Reserch Fund of Sichuan Provincial Education Department.
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