Isolation and Characterization of a Novel Lipase from a Metagenomic Library of Tidal Flat Sediments: Evidence for a New Family of Bacterial Lipases

We cloned lipG, which encoded a lipolytic enzyme, from a Koreantidal flat metagenomic library. LipG was related to six putativelipases previously identified only in bacterial genome sequences.These enzymes comprise a new family. We partially characterizedLipG, providing the first experimental data for a member ofthis family.

INTRODUCTION

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Introduction

Lipases (EC 3.1.1.3) are ubiquitous enzymes found in animals,plants, and microorganisms, including fungi and bacteria. Becausemicrobial lipases have considerable industrial potential (10,12, 14), additional microbial lipases with different characteristicsare sought. Metagenomics, an approach to access global microbialgenetic diversity, has been used to discover novel, potentiallyimportant enzymes, including lipases (5, 17, 23). Several genesencoding metagenomic lipases have been identified in metagenomiclibraries prepared from various environmental samples, includingsoils (11, 16), pond and lake water (19, 20), and a solfataricfield (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 discoveryof LipG led to the identification of a new family of bacteriallipolytic enzymes. We also partially characterize LipG, providingthe first experimental data for a member of this new enzymefamily.

Isolation of a novel bacterial lipase gene and identification of a new family of bacterial lipases.

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Isolation of a novel...

Tidal flat sediments possess a unique microbial diversity includingvarious unculturable microorganisms. For example, a previousreport showed that 20% to 30% of the bacterial species isolatedfrom tidal flat sediments are novel species (15). Therefore,we constructed a metagenomic library using genomic DNA isolatedfrom sediments collected from tidal flats on the Korean westcoast. This is the first reported library of this kind. TotalDNA was isolated from the sediments as described previously(25, 26). Approximately 386,400 cells of Escherichia coli strainEPI300-T1 containing the pCC1FOS-based metagenomic DNA library(Epicentre) were prepared according to the manufacturer's protocol.Restriction analysis of randomly selected recombinant fosmidsshowed that the average insert size was approximately 35 kb(data not shown). The E. coli transformants were plated on Luria-Bertaniagar plates containing emulsified tricaprylin. Four of the E.coli transformants exhibited lipolytic activity, as indicatedby a transparent halo surrounding the colony. The plasmid designatedpFosLip was finally isolated from the E. coli transformant showingthe highest lipolytic activity and was used for further analysisof the tidal flat metagenomic lipase.

An approximately 30-kb insert DNA from pFosLip was completelysequenced using random shotgun sequencing. The pFosLip plasmidwas chimeric, containing two contigs with different G+C contents(GenBank accession no. DQ458963 and DQ478880). The open readingframe (ORF) finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)found a total of 32 ORFs, of which only 24 showed E values smallerthan 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 aminoacid sequence for LipG included Ser169-Asp217-His285, a catalytictriad highly conserved in lipolytic enzymes of the ${alpha}$ /ßhydrolase superfamily (3). In addition, the sequence aroundSer169 was Gly167-His168-Ser169-Leu170-Gly171, which matchesthe characteristic Gly-X-Ser-X-Gly motif (where X stands forany amino acid) found in lipolytic enzymes (24).

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TABLE 1. Annotation of ORFs identified on pFosLip

A BLAST search of GenBank revealed that LipG is closely relatedto seven putative lipolytic enzymes, including six putativebacterial lipases known only from whole-genome sequences ofbacteria (Table 2). Except for the lipase from the filamentousfungus Rhizomucor miehei (6), none of the bacterial proteinshave been characterized. These putative lipases were generallyfrom marine bacteria, which were not examined as a source fornew enzymes until their genome sequences were determined. Forexample, Rhodopirellula baltica SH 1, a marine bacterium ofthe phylum Planctomycetes (9), Colwellia psychrerythraea, astrictly psychrophilic Arctic marine bacterium (18), and Idiomarinabaltica, a marine bacterium with a high temperature optimumisolated from the surface water of the Central Baltic Sea (2),were recently isolated from relatively untapped environmentalregions and subjected to whole genome sequencing.

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TABLE 2. Similarity in amino acid sequences for LipG and its closest homologues^a

We constructed a phylogenetic tree for LipG and other lipases.Jaeger and colleagues previously reported the extensive classificationof bacterial lipolytic enzymes, mainly based on a comparisonof their amino acid sequences (1, 13), which allowed us to classifyLipG. For the phylogenetic analysis, we selected 38 bacteriallipolytic enzymes representing 8 different families. As shownin Fig. 1, LipG and the six putative bacterial lipases did notbelong to any of the known lipase families. Therefore, we suggestthat they comprise a new family of bacterial lipolytic enzymes.

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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.

An important feature of the new family is an Arg-Gly sequence(Arg97-Gly98 in LipG) that can serve as an oxyanion hole. Allof the filamentous fungal lipases, including R. miehei lipase,which was the most similar to LipG, have Arg-Gly oxyanion residuesin the N-terminal part, whereas most bacterial and other fungallipases have His-Gly residues (22). In fact, this Arg-Gly oxyanionhole sequence is known to be a unique signature sequence forfilamentous fungal lipases. Unlike other bacterial lipases,the oxyanion hole sequence of a lipase recently cloned fromPhotobacterium lipolyticum also contained the Arg-Gly sequence(22). Interestingly, P. lipolyticum has been isolated from tidalflat sediments. As suggested by the P. lipolyticum lipase, thenew family of LipG-related lipases appears to be related tofilamentous fungal lipases, including the lipase from R. miehei.

Enzymatic characteristics of the tidal-flat-derived metagenomic lipase.

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Enzymatic characteristics of the...

We expressed LipG as a hexahistidine-tagged (His-tagged) proteinand investigated its chain length specificity using p-nitrophenylesters (Sigma), positional specificity toward triacylglycerols,and the additive effects of metal ions. The lipG gene from pFosLipwas amplified by PCR and subcloned between the NdeI and XhoIsites in pET-22b(+) (Novagen). When E. coli strain BL21(DE3)(Novagen) harboring the resulting pET-LipG plasmid was grownat 37°C and induced at 18°C with 1 mM isopropyl-ß-D-thiogalactopyranoside,the His-tagged LipG was expressed mostly in the soluble fraction.LipG was purified approximately 4.6-fold in a 61% yield fromthe soluble fraction by a single step of nickel-nitrilotriaceticacid affinity chromatography (QIAGEN) (data not shown). Thelipase activity of the purified protein was quantitatively measuredusing a spectrophotometric method with p-nitrophenyl palmitate(pNPP) as a substrate (4). The production of p-nitrophenol wascontinuously monitored at 405 nm over a 20-min period at 37°C.One unit of lipase activity was defined as the amount of enzymereleasing 1 µmol of p-nitrophenol per min. The purifiedLipG was quantified using the commercial Bradford protein assaykit (Bio-Rad) with bovine serum albumin as a standard. The specificactivity 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-nitrophenylesters with acyl chains of different lengths. We found thatLipG had a high activity using relatively long-chain fatty acidsas substrates (C₁₄, C₁₆, and C₁₈) (Table 3). The catalytic efficiencytoward pNPP, which was the best substrate for LipG, was approximately30-fold higher than toward p-nitrophenyl butyrate. To confirmthe chain length specificity of LipG, we performed a pH-statassay with mixed micelles of triacylglycerols (7), namely, tributyrin(C₄), tricaprylin (C₈), and triolein (C_18:1 [cis-9]). In thisassay, LipG showed the highest activity toward triolein, followedin decreasing order by tricaprylin and tributyrin (94% and 60%of the activity with triolein, respectively).

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TABLE 3. Kinetic parameters for purified LipG using p-nitrophenyl esters

We further analyzed the positional specificity of LipG towardtriacylglycerol using a simple continuous spectrophotometricmethod that employs 2,3-dimercapto-1-propanol tributyrate (TBDMP;Sigma), a commercially available thioester analog of triacylglycerol(8). The optical density at 412 nm value for TBDMP hydrolysiswas 1.124, which nearly matched the theoretical value (1.250)for the complete hydrolysis of TBDMP (data not shown), indicatingthat LipG hydrolyzed both of the TBDMP thioester groups. Theseresults indicate that LipG is not specific for the positionon triacylglycerol.

We next examined the effect of divalent metal ions on the activityof 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 lipaseactivity to more than 150% of that of the control. Moreover,the enzyme was strongly inhibited by chelation of divalent metalions; the LipG activity was 54% and 10% of that of the controlin the presence of 0.1 and 1 mM EDTA, respectively. These resultsindicate 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 bacterialgenomes. LipG is related to lipases from marine bacterial sourcesand filamentous fungi and is the first experimentally characterizedenzyme of this new family of bacterial lipolytic enzymes. Thisstudy also demonstrated that the metagenomic approach is veryuseful for expanding our knowledge of enzyme diversity, especiallyfor bacterial lipases.

ACKNOWLEDGMENTS

This work was supported by the 21C Frontier program of MicrobialGenomics and Applications (grants MG02-0401-001-1-0-0 and MG05-0103-3-0)from the Ministry of Science and Technology (MOST) of the Republicof Korea.

FOOTNOTES

* 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: ajee{at}krict.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: jhyoon{at}kribb.re.kr.

Published ahead of print on 1 September 2006.

REFERENCES

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