Isolation and Characterization of a Novel Lipase from a Metagenomic ...

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May 19, 2006 - quence (Arg97-Gly98 in LipG) that can serve as an oxyanion hole. All of the filamentous fungal lipases, including R. miehei lipase, which was ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 7406–7409 0099-2240/06/$08.00⫹0 doi:10.1128/AEM.01157-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 11

Isolation and Characterization of a Novel Lipase from a Metagenomic Library of Tidal Flat Sediments: Evidence for a New Family of Bacterial Lipases䌤 Mi-Hwa Lee,1 Choong-Hwan Lee,1 Tae-Kwang Oh,1 Jae Kwang Song,2* and Jung-Hoon Yoon1* Korea Research Institute of Bioscience and Biotechnology,1 and Korea Research Institute of Chemical Technology,2 Daejeon, Republic of Korea Received 19 May 2006/Accepted 26 August 2006

We cloned lipG, which encoded a lipolytic enzyme, from a Korean tidal flat metagenomic library. LipG was related to six putative lipases previously identified only in bacterial genome sequences. These enzymes comprise a new family. We partially characterized LipG, providing the first experimental data for a member of this family.

nant fosmids showed that the average insert size was approximately 35 kb (data not shown). The E. coli transformants were plated on Luria-Bertani agar plates containing emulsified tricaprylin. Four of the E. coli transformants exhibited lipolytic activity, as indicated by a transparent halo surrounding the colony. The plasmid designated pFosLip was finally isolated from the E. coli transformant showing the highest lipolytic activity and was used for further analysis of the tidal flat metagenomic lipase. An approximately 30-kb insert DNA from pFosLip was completely sequenced using random shotgun sequencing. The pFosLip plasmid was chimeric, containing two contigs with different G⫹C contents (GenBank accession no. DQ458963 and DQ478880). The open reading frame (ORF) finder (http: //www.ncbi.nlm.nih.gov/gorf/gorf.html) found a total of 32 ORFs, of which only 24 showed E values smaller than e⫺10 and database hits with moderate identity (32% to 79%) (Table 1). One of these ORFs, which we designated lipG, encoded a 300-amino-acid putative lipase. The predicted amino acid sequence for LipG included Ser169-Asp217-His285, a catalytic triad highly conserved in lipolytic enzymes of the ␣/␤ hydrolase superfamily (3). In addition, the sequence around Ser169 was Gly167-His168-Ser169-Leu170-Gly171, which matches the characteristic Gly-X-Ser-X-Gly motif (where X stands for any amino acid) found in lipolytic enzymes (24). A BLAST search of GenBank revealed that LipG is closely related to seven putative lipolytic enzymes, including six putative bacterial lipases known only from whole-genome sequences of bacteria (Table 2). Except for the lipase from the filamentous fungus Rhizomucor miehei (6), none of the bacterial proteins have been characterized. These putative lipases were generally from marine bacteria, which were not examined as a source for new enzymes until their genome sequences were determined. For example, Rhodopirellula baltica SH 1, a marine bacterium of the phylum Planctomycetes (9), Colwellia psychrerythraea, a strictly psychrophilic Arctic marine bacterium (18), and Idiomarina baltica, a marine bacterium with a high temperature optimum isolated from the surface water of the Central Baltic Sea (2), were recently isolated from rela-

Lipases (EC 3.1.1.3) are ubiquitous enzymes found in animals, plants, and microorganisms, including fungi and bacteria. Because microbial lipases have considerable industrial potential (10, 12, 14), additional microbial lipases with different characteristics are sought. Metagenomics, an approach to access global microbial genetic diversity, has been used to discover novel, potentially important enzymes, including lipases (5, 17, 23). Several genes encoding metagenomic lipases have been identified in metagenomic libraries prepared from various environmental samples, including soils (11, 16), pond and lake water (19, 20), and a solfataric field (21). Here, we describe the isolation, sequence analysis, and enzymatic characterization of a novel lipase-encoding gene, lipG, from a tidal flat-derived metagenomic library. The discovery of LipG led to the identification of a new family of bacterial lipolytic enzymes. We also partially characterize LipG, providing the first experimental data for a member of this new enzyme family. Isolation of a novel bacterial lipase gene and identification of a new family of bacterial lipases. Tidal flat sediments possess a unique microbial diversity including various unculturable microorganisms. For example, a previous report showed that 20% to 30% of the bacterial species isolated from tidal flat sediments are novel species (15). Therefore, we constructed a metagenomic library using genomic DNA isolated from sediments collected from tidal flats on the Korean west coast. This is the first reported library of this kind. Total DNA was isolated from the sediments as described previously (25, 26). Approximately 386,400 cells of Escherichia coli strain EPI300-T1 containing the pCC1FOS-based metagenomic DNA library (Epicentre) were prepared according to the manufacturer’s protocol. Restriction analysis of randomly selected recombi-

* Corresponding author. Mailing address for Jae Kwang Song: Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejeon 305-600, Korea. Phone: 82 42 860 7643. Fax: 82 42 860 7649. E-mail: [email protected] .re.kr. Mailing address for Jung-Hoon Yoon: Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon 305-333, Korea. Phone: 82 42 860 4276. Fax: 82 42 879 8595. E-mail: [email protected] 䌤 Published ahead of print on 1 September 2006. 7406

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TABLE 1. Annotation of ORFs identified on pFosLip ORF no.

Length (amino acids)

% G⫹C content

Most homologous protein

Putative source organism

% Identity/ similarity

E value

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

705 381 155 279 355 300 88 753 291 178 186 136 238 207 430 69 375 249 180 80 303 585 439 155

40.08 50.87 47.01 54.64 45.79 46.26 39.70 47.52 59.13 59.96 62.21 61.56 57.88 63.30 62.49 56.67 61.35 60.67 62.98 51.44 59.54 65.24 61.67 63.25

Ice nucleation protein homolog A540L Phosphoesterase Acetyltransferase Peptidase M20 Putative signal peptide protein Probable lipase Putative protease/amidase Cell surface protein Hypothetical protein RB2501_05900 Membrane protein, putative NnrU protein Conserved hypothetical protein Hypothetical protein MED297_12632 Hypothetical protein HCH_06703 Adenylosuccinate synthetase HflC protein HflK protein GTP-binding protein GTPase Hfq protein tRNA-adenosine transferase DNA mismatch repair protein mutL N-acetylmuramoyl-L-alanine amidase Hypothetical protein TIGR00150

Paramecium bursaria Chlorella virus Syntrophus aciditrophicus SB Syntrophobacter fumaroxidans MPOB Syntrophobacter fumaroxidans MPOB Marinomonas sp. strain MED121 Rhodopirellula baltica SH1 Bacteroides thetaiotaomicron VPI-5482 Methanosarcina acetivorans C2A Robiginitalea biformata HTCC2501 Pseudomonas syringae pv. phaseolicola Gammaproteobacterium KT 71 Shewanella sp. strain PV-4 Reinekea sp. strain MED297 Hahella chejuensis KCTC 2396 Pseudomonas fluorescens Pf-5 Uncultured bacterium 463 Thiomicrospira crunogena XCL-2 Marinobacter aquaeolei VT8 Hahella chejuensis KCTC 2396 Uncultured bacterium 463 Salmonella typhimurium LT2 Uncultured bacterium 463 Oceanospirillum sp. strain MED92 Shewanella oneidensis MR-1

34/50 50/67 51/73 44/65 32/50 38/54 49/62 32/48 54/68 32/48 39/63 50/68 37/59 48/63 67/79 57/85 46/63 50/69 67/79 79/91 53/68 47/62 47/62 53/72

2e⫺63 3e⫺74 5e⫺44 8e⫺67 4e⫺50 2e⫺27 2e⫺12 2e⫺22 6e⫺83 6e⫺10 3e⫺27 4e⫺11 6e⫺37 4e⫺40 3e⫺150 e⫺12 7e⫺75 e⫺63 e⫺62 6e⫺29 2e⫺167 e⫺168 8e⫺97 2e⫺31

tively untapped environmental regions and subjected to whole genome sequencing. We constructed a phylogenetic tree for LipG and other lipases. Jaeger and colleagues previously reported the extensive classification of bacterial lipolytic enzymes, mainly based on a comparison of their amino acid sequences (1, 13), which allowed us to classify LipG. For the phylogenetic analysis, we selected 38 bacterial lipolytic enzymes representing 8 different families. As shown in Fig. 1, LipG and the six putative bacterial lipases did not belong to any of the known lipase families. Therefore, we suggest that they comprise a new family of bacterial lipolytic enzymes. An important feature of the new family is an Arg-Gly sequence (Arg97-Gly98 in LipG) that can serve as an oxyanion hole. All of the filamentous fungal lipases, including R. miehei lipase, which was the most similar to LipG, have Arg-Gly oxyanion residues in the N-terminal part, whereas most bacterial and other fungal lipases have His-Gly residues (22). In fact, this Arg-Gly oxyanion hole sequence is known to be a unique signature sequence for filamentous fungal lipases. Unlike other

bacterial lipases, the oxyanion hole sequence of a lipase recently cloned from Photobacterium lipolyticum also contained the Arg-Gly sequence (22). Interestingly, P. lipolyticum has been isolated from tidal flat sediments. As suggested by the P. lipolyticum lipase, the new family of LipG-related lipases appears to be related to filamentous fungal lipases, including the lipase from R. miehei. Enzymatic characteristics of the tidal-flat-derived metagenomic lipase. We expressed LipG as a hexahistidine-tagged (His-tagged) protein and investigated its chain length specificity using p-nitrophenyl esters (Sigma), positional specificity toward triacylglycerols, and the additive effects of metal ions. The lipG gene from pFosLip was amplified by PCR and subcloned between the NdeI and XhoI sites in pET-22b(⫹) (Novagen). When E. coli strain BL21(DE3) (Novagen) harboring the resulting pET-LipG plasmid was grown at 37°C and induced at 18°C with 1 mM isopropyl-␤-D-thiogalactopyranoside, the Histagged LipG was expressed mostly in the soluble fraction. LipG was purified approximately 4.6-fold in a 61% yield from the soluble fraction by a single step of nickel-nitrilotriacetic

TABLE 2. Similarity in amino acid sequences for LipG and its closest homologuesa Protein

Source

GenBank accession no.

% Identity/similarity

E value

Reference

LipG Probable lipase Lipase family protein Lipase family protein Lipase Lipase Hypothetical protein Triacylglycerol hydrolaseb

Metagenome Rhodopirellula baltica SH 1 Idiomarina baltica OS145 Colwellia psychrerythraea 34H Desulfuromonas acetoxidans DSM684 Trichodesmium erythraeum IMS101 Photobacterium profundum SS9 Rhizomucor miehei

DQ458963 CAD74552 ZP_01041937 AAZ27340 ZP_00551941 ZP_00675753 CAG19796 3TGL

38/54 33/51 32/52 31/49 31/46 30/50 39/51

2e⫺27 7e⫺18 5e⫺20 3e⫺14 5e⫺22 e⫺2 e⫺7

This study 9 Unpublished 18 Unpublished Unpublished Unpublished 6

a The corresponding protein, source, and database accession number are given for the closest homologues to LipG identified by a BLAST search at the NCBI. The protein names and strain names of the microorganisms are given as originally designated by the authors. b Triacylglycerol hydrolase from R. miehei, which showed the highest identity to LipG, is the only fungal lipase among seven lipases similar to LipG.

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FIG. 1. Phylogenetic analysis of LipG and closely related proteins. Phylogenetic analysis was performed using the program MEGALIGN (DNASTAR, Madison, WI). Except for LipG, the protein sequences for previously identified families of bacterial lipolytic enzymes were retrieved from GenBank (http://www.ncbi.nlm.nih.gov). The units at the bottom of the tree indicate the number of substitution events.

acid affinity chromatography (QIAGEN) (data not shown). The lipase activity of the purified protein was quantitatively measured using a spectrophotometric method with p-nitrophenyl palmitate (pNPP) as a substrate (4). The production of p-nitrophenol was continuously monitored at 405 nm over a 20-min period at 37°C. One unit of lipase activity was defined as the amount of enzyme releasing 1 ␮mol of p-nitrophenol per min. The purified LipG was quantified using the commercial Bradford protein assay kit (Bio-Rad) with bovine serum albumin as a standard. The specific activity of the purified LipG was estimated to be 458.8 U mg⫺1, using pNPP as a substrate. We next examined the activity of the purified LipG using p-nitrophenyl esters with acyl chains of different lengths. We found that LipG had a high activity using relatively long-chain fatty acids as substrates (C14, C16, and C18) (Table 3). The catalytic efficiency toward pNPP, which was the best substrate for LipG, was approximately 30-fold higher than toward pnitrophenyl butyrate. To confirm the chain length specificity of LipG, we performed a pH-stat assay with mixed micelles of triacylglycerols (7), namely, tributyrin (C4), tricaprylin (C8), and triolein (C18:1 [cis-9]). In this assay, LipG showed the

highest activity toward triolein, followed in decreasing order by tricaprylin and tributyrin (94% and 60% of the activity with triolein, respectively). We further analyzed the positional specificity of LipG toward triacylglycerol using a simple continuous spectrophotometric method that employs 2,3-dimercapto-1-propanol tribu-

TABLE 3. Kinetic parameters for purified LipG using p-nitrophenyl esters Substrate (p-nitrophenyl ester)

Km (mM)

Kcat (min⫺1)

Kcat/Km (min⫺1 mM⫺1)

Acetate (C2) Propionate (C3) Butyrate (C4) Caproate (C6) Caprylate (C8) Caprate (C10) Laurate (C12) Myristate (C14) Palmitate (C16) Stearate (C18)

1.6 0.9 1.5 0.4 0.4 1.1 2.7 0.1 0.8 0.2

121 69 55 34 84 354 705 128 853 54

75 79 37 79 222 338 258 1,040 1,090 351

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tyrate (TBDMP; Sigma), a commercially available thioester analog of triacylglycerol (8). The optical density at 412 nm value for TBDMP hydrolysis was 1.124, which nearly matched the theoretical value (1.250) for the complete hydrolysis of TBDMP (data not shown), indicating that LipG hydrolyzed both of the TBDMP thioester groups. These results indicate that LipG is not specific for the position on triacylglycerol. We next examined the effect of divalent metal ions on the activity of LipG by adding 1, 5, or 10 mM CaCl2, CuCl2, MgCl2, FeSO4, ZnCl2, NiCl2, MnCl2, AgNO3, or CoCl2 to the assay solution. We found that 10 mM Ca2⫹ and 5 mM Mn2⫹ increased the lipase activity to more than 150% of that of the control. Moreover, the enzyme was strongly inhibited by chelation of divalent metal ions; the LipG activity was 54% and 10% of that of the control in the presence of 0.1 and 1 mM EDTA, respectively. These results indicate that divalent metal ions, especially Ca2⫹ or Mn2⫹, are necessary for the catalytic activity of LipG. In conclusion, we identified a new lipase family including LipG, which we isolated from a Korean tidal flat metagenomic library, and six putative lipases previously identified in bacterial genomes. LipG is related to lipases from marine bacterial sources and filamentous fungi and is the first experimentally characterized enzyme of this new family of bacterial lipolytic enzymes. This study also demonstrated that the metagenomic approach is very useful for expanding our knowledge of enzyme diversity, especially for bacterial lipases. This work was supported by the 21C Frontier program of Microbial Genomics and Applications (grants MG02-0401-001-1-0-0 and MG050103-3-0) from the Ministry of Science and Technology (MOST) of the Republic of Korea.

8.

9.

10. 11.

12.

13. 14. 15. 16.

17. 18.

19.

20.

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