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Journal of Biotechnology 133 (2008) 42–49

Novel thermophilic and thermostable lipolytic enzymesfrom a Thailand hot spring metagenomic library

Pacawadee Tirawongsaroj a,b,1, Rutchadaporn Sriprang a,1, Piyanun Harnpicharnchai a,Taksawan Thongaram a, Verawat Champreda a, Sutipa Tanapongpipat a,

Kusol Pootanakit b, Lily Eurwilaichitr a,∗a BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC),

113 National Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailandb Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand

Received 6 July 2007; received in revised form 24 August 2007; accepted 25 August 2007

bstract

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novelatatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipidcyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme.

Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities atlkaline pH, of 7–9 and 8–10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of0–75 ◦C, with optimal activity at 70 ◦C and were stable at 70 ◦C for at least 120 min.Both PLP and Est1 exhibited high V toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C and C ) and long

max 4 5

hain (C14 and C16) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases,hich usually show no activity for substrates longer than C10. We suggest that PLP and EstA enzymes are novel and have a; b potential use in

ndustrial applications.2007 Elsevier B.V. All rights reserved.

; Este

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eywords: Lipolytic enzyme; Metagenomic library; Patatin-like phospholipase

. Introduction

Lipolytic enzymes are important biocatalysts due to theirbility to utilize a wide range of substrates, and hence have aotentially broad spectrum of biotechnological uses. Lipoly-ic enzymes can be classified into eight families based onhe conserved sequences, motifs, and biological properties;amely: true lipase, GDSL, hormone-sensitive lipase (HSL)nd families III, V–VIII (Arpigny and Jaeger, 1999). In addi-

ion, the lipolytic enzymes can be classified into three mainroups based on the substrate specificity: lipases (EC 3.1.1.3),sterases/carboxylesterases (EC 3.1.1.1), and phospholipases.

∗ Corresponding author. Tel.: +66 2564 6700; fax: +66 2564 6707.E-mail address: lily@biotec.or.th (L. Eurwilaichitr).

1 These authors contributed equally to this work.

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168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2007.08.046

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ipases (EC 3.1.1.3) catalyze the hydrolysis of triglyceridesith long-chain fatty acids (≥10 carbon atoms). Most lipasesave activity at the aqueous/non-aqueous interface (referredo as interfacial activation), whose activity is associated withonformational changes of the lid domain in the protein reveal-ng the substrate-accessible site. However, some lipases contain

inor or no interfacial activation and thus display both lipasend esterase activity (Verger, 1997). Esterases (EC 3.1.1.1)refer water-soluble substrates and catalyze the hydrolysis oflycerolesters with short acyl chain (≤10 carbon atoms) toartial glycerides and fatty acids (Bornscheuer, 2002). Phospho-ipases catalyze the hydrolysis of various types of phospholipids.ipases are commonly used in the production of free fatty

cids, peptide synthesis and inter-esterification of oils and fats.oreover, these enzymes can be used as chiral catalysts for

ne chemical synthesis, or for stereo-selective conversion of aariety of amines and primary and secondary alcohols for pro-

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uction of cosmetics and pharmaceuticals (Jaeger and Eggert,002).

Some industrial processes in which lipolytic enzymes aremployed require harsh conditions, e.g. high temperature. Thenstability of well-characterized mesophilic enzymes in extremeonditions has prompted the search for new enzymes withetter-suited properties. The discovery that thermostability isorrelated with resistance to denaturation in organic solventsOwusu and Cowan, 1989) suggested that thermostable enzymesre likely to have properties apt for many industrial applications.

Molecular phylogenetic studies have shown that only a smallraction (<1%) of bacterial diversity present in an environmentalample can be cultured in the laboratory (Amann et al., 1995).he culture-independent, metagenomic approach (Rondon etl., 2000) can therefore be used to avoid the inherent loss ofiversity related to cultivation. Target enzymes including lipasesnd esterases (Robertson et al., 2004; Voget et al., 2003; Leet al., 2004; Rhee et al., 2005), amylases (Robertson et al.,004; Handelsman et al., 1998), chitinase (Cottrell et al., 1999),nd nitrilases (Robertson et al., 2004) have been successfullydentified using the metagenomic approach. Various types ofommercial products already available in the market have alsoeen found by this technique (Cowan et al., 2005).

Previously, our group reported the biodiversity and abun-ance of bacteria and archae from Bor Khleung and Jae Sawnot springs, Thailand (Kanokratana et al., 2004 and unpub-ished data). These studies demonstrated a complex communityf prokaryotes in Thailand’s hot springs, with over 80% of thoseicroorganisms representative of new strains not previously

ound elsewhere. It can be inferred from these data that novelnd interesting enzyme-producing microbes are present in thisnvironment. The hot spring sediment is therefore an attractiveesource for microorganisms producing thermotolerant lipolyticnzymes with potential use in many industries.

From functional screening of the metagenomic libraryerived from sediments of the Jae Sawn hot spring, we havesolated two genes encoding a novel esterase and a phospholi-ase. The esterase and phospholipase genes were heterologouslyxpressed in E. coli. The recombinant enzymes were purifiednd their biochemical properties were characterized. The iso-ated enzymes were found to function well at high temperature50–70 ◦C) and basic pH (8–9).

. Materials and methods

.1. Microorganisms and culture conditions

The bacterial strains used were: E. coli DH5�F′F′�(lacZYA-argF)U169 deoR endA1 hsdR17 supE44 thi-1ecA1 gyr96 relA1 (φ80dlacZ �M15)), E. coli BL21(DE3)LysS (F−ompT hsdSB (rB

−mB−) gal dcm (DE3) pLysS (CmR))

nd E. coli TOP10 (mcrA �(mrr-hsdRMS-mcrBC) �80�lacM15�lacX74 deoR recA1 araD139 � (ara leu) 7697 galU

alK λ− rpsL endA1 mupG). The cloning vector pTZ57R wasurchased from Fermentas (USA). The vector pZErO-2 was pur-hased from Invitrogen (Invitrogen, USA). An expression vectorET-28a (+) (Novagen, USA) was used for the expression of the

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otechnology 133 (2008) 42–49 43

ipase/esterase genes. E. coli was grown at 37 ◦C in LB brothSambrook and Russell, 2001) or on agar (1.5%, w/v) platesupplemented with appropriate antibiotics.

.2. DNA extraction from hot spring sediments

A soil sample from the Jae Sawn hot spring was taken at0–20 cm below the surface, with a temperature of 70 ◦C andH of 7. Approximately 4–10 �g of genomic DNA/g of wetoil was obtained following extraction method from Zhou et al.1996). DNA purification was performed by a modified DNAroughing method (Harnpicharnchai et al., 2007).

.3. Metagenomic library construction

The genomic DNA partially digested with Sau3AI was sub-ected to size fractionation using the above modified troughing

ethod. Fractions containing DNA fragments of approximately–10 kb were ligated with the zero-background cloning vector,ZErO-2 (Invitrogen, USA). To construct the library, approx-mately 1 �g of pZErO-2 was digested with BamHI. Theinearized plasmid was then purified by Wizard DNA cleanp kit (Promega, USA) and used to ligate with the partiallyigested genomic DNA. The ligated plasmids were transformednto E. coli TOP10 (Invitrogen) by electroporation and the trans-ormants were selected on LB supplemented with kanamycin50 �g/ml).

.4. Screening for lipase/esterase activity

Transformants from the library were screened forsterase/lipase activity by plating the transformed cellsnto Luria–Bertani (LB) agar plate containing 1% trioleoyl-lycerol (with an acyl chain length of 18 carbon atom, aommon substrate for true lipase screening) or 1% tributylglyc-rol (with an acyl chain length of four carbon atoms, a commonubstrate for esterase screening) plus 0.001% Rhodamine Bye and incubated at 37 ◦C for 2 days. Then, the plates wereverlaid with 0.7% agar containing d-cycloserine (60 �g/ml)nd further incubated at 37 ◦C for 2 days. The positive lipolyticnzyme candidates were selected from bacterial colonies pro-ucing clear halo zone, then their plasmids were isolated andnalyzed.

.5. DNA sequencing and sequence analysis

DNA was sent to Macrogen, Inc. (Seoul, South Korea)or sequencing. Sequences of bacterial lipase and esterasesed for comparative study were retrieved from pro-ein and nucleotide databases on the NCBI Entrez servert http://www.ncbi.nlm.nih.gov/Entrez/. Sequence similarityearches were performed by BLASTX analysis. Multiple align-

ents of enzymes with homologous proteins and phylogenetic

nalyses were performed with the Align X program, a compo-ent of the Vector NTI suite (InforMax, North Bethesda, MD).he nucleotide sequences of isolated genes have been deposited

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n GenBank under accession numbers EF413636 and EF413637or PLP and Est1, respectively.

.6. Expression and purification of recombinant lipolyticnzymes

The patatin-like phospholipase (PLP) and esterase (Est)enes were amplified by PCR with primer pairs PLPF (5′-ATATGCGCAGGATACATCTCG-3′)/PLPR (5′-AAGCTT-TACAACAGTTCCTTCAGC-3′) and ESTF (5′-CATATGCC-GCAGTAGATATCTTCC-3′)/ ESTR (5′-GGATCCTGAAC-TCCCCCTCAAAAATTC-3′), respectively. Restriction sites

or cloning are underlined. The 873 and 792 bp ampliconsor PLP and Est1, respectively, were cloned into pET-28a(+)Novagen), a vector designed for expression of N-terminalxHis tagged protein. The plasmids were introduced into E.oli strain BL21(DE3) (pLysS) and transformants were selectedn LB plates supplemented with kanamycin (50 �g/ml) andhloramphenicol (34 �g/ml).

.7. Expression and purification of lipase/esterase from E.oli

A culture of E. coli cells (grown to A600 0.2–0.4) was inducedy adding 1 mM IPTG (Promega, USA) and grown for an addi-ional 4 h. Cells were harvested by centrifugation and disruptedy BugBuster® Protein Extraction Reagent (Novagen, Ger-any). The supernatant was purified by using Hi-Trap affinity

olumn containing nickel-nitrilo-triacetic acid as described byhe manufacturer (Amersham Biosciences, UK). The eluted frac-ions with high lipolytic activity was concentrated and desaltedsing Amicon Ultrafiltration Unit (Millipore Co., USA). Therotein was subjected to SDS-PAGE analysis and visualized withoomassie Blue R-250.

.8. Enzyme assays

Lipolytic activities were determined by measuring themount of p-nitrophenol product released after lipolytic-atalyzed hydrolysis (Lee et al., 1993). One unit of enzymectivity was defined as the amount of enzyme required to release�mol of p-nitrophenol per minute from the p-nitrophenyl ester.

.9. Substrate specificity

Substrate specificities for p-nitrophenyl esters were deter-ined by incubation of purified enzymes in various con-

entrations (0.05–2 mM) of substrates; p-nitrophenyl acetate,

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ig. 1. Multiple sequences alignment of the primary structure of lipolytic enzymehospholipase gene from bacteria including Geobacter lovleyi SZ (ZP 01593172), Chlo8 (ZP 01182139), Carboxydothermus hydrogenoformans (ABB15198.1) and Aeromblock I–IV) of patatin-like phospholipase. Block I consists of a glycine-rich region wif the hydrolase motif, G-X-S-X-G, containing a serine residue located at the putatin other bacterial PLPs. Block IV contains the putative active-site aspartate. Black aespectively. (B) multiple alignment of Est1 and family V esterases from Sulfolobus ap. (X53869) and Psychrobacter immobilis. Open boxes indicate amino acid residuedentical or similar amino acid residues, respectively.

otechnology 133 (2008) 42–49

-nitrophenyl butyrate (Sigma), p-nitrophenyl valerate (Sigma,luka), p-nitrophenyl caprylate (Sigma), p-nitrophenyl laurate,-nitrophenyl myristate and p-nitrophenyl palmitate (Sigma) in0 mM Tris–HCl (pH 9.0) with 0.4 % Triton-X and 0.1% gumrabic (Rhee et al., 2005). The kinetic parameters were thenetermined by fitting the initial velocity data to the Michaelis-enten equation using the Kaleida Graph software package

Synergy Software, USA) (Chen et al., 2005).

.10. Enzyme characterizations

The optimal pH of the PLP and esterase enzyme activitiesere measured at pH ranging from 3.0 to 12.0 under stan-ard assay conditions at 70 ◦C. The buffers used were 50 mModium acetate (pH 3.0–5.0), 50 mM MES (pH 5.0–7.0), 50 mMEPES (pH 7.0–8.0), and 50 mM Tris–HCl (pH 8.0–12.0). Theroduction of p-nitrophenol from p-nitrophenyl butyrate wasonitored at 348 nm. The molar extinction coefficient (ε348 nm)

etermined at each pH tested were 24, 25 and 24 M−1 cm for pH.0, 4.0 and 5.0, respectively, in 50 mM sodium acetate; 25, 27nd 27 M−1 cm for pH 5.0, 6.0 and 7.0, respectively, in 50 mMES; 30 and 31 M−1 cm for pH 7.0 and 8.0, respectively, in

0 mM HEPES; and 31, 30, 25, 30, 27 and 31 M−1 cm for pH.0–12, respectively, in 50 mM Tris–HCl.

The optimal temperature for enzyme activity was determinedy subjecting reaction mixtures to different temperatures rang-ng from 30 to 80 ◦C in 50 mM Tris–HCl buffer (pH 9.0) for0 min. Thermostability of the enzymes was analyzed by mea-uring the residual activity after pre-incubating the enzyme at0, 60, 70, or 80 ◦C for 20–120 min in Eppendorf tubes withineral oil on top to prevent evaporation. Relative activity was

alculated as enzymatic activity at indicated temperature dividedy maximal activity at optimal temperature.

. Results and discussion

A metagenomic library was successfully constructed usingenomic DNA obtained from the sediments of the Jae Sawn hotpring. The library was in a plasmid vector (pZErO-2) under theontrol of a lacZ promoter (Plac). The Plac located upstreamf the multiple cloning site allows expression of promoterlessenes. The average library size obtained was 1 × 106 CFU/�gNA from the ligation reaction of metagenomic DNA andZErO-2. It was observed that more than 90% of the trans-

ormants contained genomic DNA fragments of the expectedize (1–10 kb). The remainders either lack or have undetectablymall inserts. From approximately 36,000 colonies screened,wo lipolytic-positive transformants, which formed clear halos

s. (A) Multisequence alignment comparing the isolated PLP to patatin-likeroflexus aurantiacus (EA058516.1), Bacillus cereus subsp. cytotoxis NVH 391-onas hydrophila (YP 857433). Open blocks indicate four conserved domains

th a conserved arginine residue. Block II is located next to block I and comprisesve active-site. Block III contains A-S-X-X-X-P sequence, which is conservednd grey boxes indicate regions with identical or similar amino acid residues,cidocaldarius (AF071233), Acetobacter pasteurianus (AB013096), Moraxellas belonging to the catalytic triad. Black and grey boxes indicate regions with

P. Tirawongsaroj et al. / Journal of Biotechnology 133 (2008) 42–49 45

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n tributylglycerol plates, were obtained. The plasmids were iso-ated from the two transformants and retransformed into new E.oli cells. The new transformants produced clear halos on trib-tylglycerol plates, suggesting that the plasmids contained genesesponsible for the esterase activity toward substrates with shortcyl chains. However, both plasmids failed to produce E. coliransformants with lipolytic activity on the agar plate contain-ng trioleoylglycerol, demonstrating that the enzymes producedy both plasmids have little or no activity toward triacylglycerolith acyl chain length of 18 carbon atoms.Sequence analysis showed that each transformant harbored

lasmids with inserts approximately 3 and 2 kb in size. Theselasmids were designated as pZErO-PLP and pZErO-Est1,espectively. The pZErO-PLP encodes a protein, designated asLP, of 291 amino acids with a theoretical molecular weight ofpproximately 32.3 kDa. Subsequent BLASTP analysis of PLPevealed a potential patatin-like phospholipase gene containinghe conserved motif of lipolytic enzymes (GXSXG). BLASTPnalysis revealed a moderate similarity (<50%) between PLPnd phospholipases from various bacteria, including the patatinEA058516.1) from Chloroflexus aurantiacus J-10-f1 (43%)Banerji and Flieger, 2004), the phospholipase family pro-ein (ABB15198.1) from Carboxydothermus hydrogenoformez39%), and the patatin-like phospholipase protein (EAV88283.1)rom Geobacter lovleyi SZ (41%). Further sequence analysisevealed a putative purine-rich ribosomal binding site of PLPocated 28 bp upstream of the ATG start codon. However, theotential −35 and −10 consensus promoter sequences were notound, suggesting that the isolated PLP gene was expressedsing the lacZ promoter. Multi-sequence alignment compar-son of the isolated PLP to patatin-like phospholipase genesrom bacteria such as G. lovleyi SZ (ZP 01593172), Clostrid-um cellulolyticum H10 (ZP 01593172), Bacillus cereus subsp.ytotoxis NVH 391-98 (ZP 01182139) Aeromonas hydrophilaYP 857433) and C. hydrogenoformans (YP 359744.1) wasurther analyzed. It revealed four conserved domains (blocks–IV), similar to those present in other patatin-like phospho-ipases from various bacteria (Fig. 1A) (Banerji and Flieger,004). From BLASTP analysis and multi-sequence alignment,he PLP gene could not be classified into any of the eight fam-lies of known bacterial lipases (Arpigny and Jaeger, 1999).herefore, it is inferred that PLP is not closely related to thestablished groups of lipolytic enzymes. Patatins are a groupf plant storage glycoproteins that show lipid acyl hydrolasectivity (Andrews et al., 1988) and catalyze the non-specificydrolysis of a wide range of acryl- and phospho-lipids (Sharmat al., 2004; Hirschberg et al., 2001). There are reports of patatinsresent in bacteria that are animal pathogens and plant pathogensuch as Bradyrhizobium japonicum, Leptospira interrogans andesorhizobium loti (Banerji and Flieger, 2004). Although PLP

xhibited less than 50% identity to patatin-associated lipolyticnzymes, it seemed most likely that a novel patatin-like phos-holipase homolog was obtained in this study.

BLASTP analysis of the ORF in pZErO-Est1 revealed autative esterase gene containing the conserved sequence motiff esterases/lipases (GXSXG). The ORF, designated as Est1,ncodes a protein of 264 amino acid residues with a theoretical

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otechnology 133 (2008) 42–49

olecular weight of approximately 29.3 kDa. BLASTP analysisevealed a moderate identity to a hydrolase in alpha/beta-foldamily from Pseudomonas mendocina ymp (ZP 01527185.1)39%), P. fluorescens PfO-1 (YP 258670.1) (33%) and also aoderate identity to an esterase from Rhodobacter sphaeroides

YP 345244.1) (31%). Sequence analysis of Est1 revealed autative ribosomal binding site located 5 bp upstream from theTG start codon and the potential −35 and −10 consensusromoter sequences were recognized (data not shown). FromSORT analysis, neither PLP nor Est1 showed potential secre-

ion signal sequences (Nakai and Horton, 1999).Multi-sequence alignment comparisons of Est1 with mem-

ers of each family of lipolytic enzymes as classified by Arpignynd Jaeger (1999) revealed that Est1 contains the serine residuehat forms a putative catalytic triad with histidine and aspar-ic residues, which are conserved among family V lipolyticnzymes (Fig. 1B). The 40% identity to known lipolytic enzymesuggests that Est1 is a novel enzyme from unculturable microbeshich maybe tentatively classified as a family V bacterial

ipolytic enzyme. The family V enzymes shown in Fig. 1Briginate from mesophilic bacteria (Acetobacter pasteurianusAB013096)) as well as from cold-adapted (Moraxella sp.X53869), Psychrobacter immobilis (X67712)) or heat-adaptedSulfolobus acidocaldarius) (AF071233) organisms. They shareome amino acid sequence similarity (20–25%) to variousacterial non-lipolytic enzymes, namely epoxide hydrolases,ehalogenases and haloperoxidases from Corynebacterium sp.nd Xanthobacter autotrophicus, which also possess the typical/�-hydrolase fold and a catalytic triad (Arpigny and Jaeger,999; Misawa et al., 1998; Verschueren et al., 1993) (Fig. 1B).

.1. Overexpression, purification and characterization ofecombinants PLP and Est1

In order to determine the biochemical properties of PLP andst1, the target enzymes of interest were expressed as N-terminalxHis tagged fusions in E. coli. PLP and Est1 were purifiedo 95% homogeneity by affinity chromatography with Ni-NTAesin. The purified proteins migrated as single bands on SDS-AGE with the expected molecular weights of approximately 32nd 29 kDa for PLP and Est1, respectively.

The optimal pH of PLP and Est1 activities were determined atarious pH values (pH 3.0–12.0). Using p-nitrophenyl butyrates the substrate, PLP exhibited more than 50% of its maximalctivity in the pH range of 7.0–9.0, with the highest activity atH 9.0 (Fig. 2A). Est1 exhibited high activity at a broader pHange, with more than 50% of its maximal activity occurring atH from 8.0 to 10.0 and the highest activity at pH 9.0 (Fig. 2B).

The effect of temperature on PLP and Est1 activities wasetermined by monitoring the hydrolysis of p-nitrophenylutyrate at various temperatures ranging from 45 to 80 ◦CFig. 2). Under the conditions used, both enzymes showed high-st activity at 70 ◦C. Fifty percent maximal activity was observed

etween 45 and 75 ◦C for PLP and 50 and 70 ◦C for Est1. Thehermostability of PLP and Est1 were determined by incubat-ng the enzymes for 20–120 min at 50–70 ◦C before measuringheir residual activities under standard assay conditions (Fig. 3).

P. Tirawongsaroj et al. / Journal of Biotechnology 133 (2008) 42–49 47

Fig. 2. Effect of pH on PLP (A) and Est1 (B). The enzymatic assay was performed at pHs ranging from 3.0 to 12.0 under standard assay conditions. The buffersused were 50 mM sodium acetate (pH 3.0–5.0) (�); 50 mM MES (pH 5.0–7.0) (�); 50 mM HEPES (pH 7.0–8.0) (�), and 50 mM Tris–HCl (pH 8.0–12.0) (×).The production of p-nitrophenoxide and p-nitrophenol products from p-nitrophenyl butyrate was monitored at 348 nm. Effect of temperature on PLP (C) and Est1( enzymT itropp

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D). The optimal temperature for enzyme activity was determined by assayingris–HCl buffer (pH 9.0) containing 0.4% Triton-X and 0.2% gum arabic. p-N-nitrophenyl butyrate was monitored at 405 nm.

oth PLP and Est1 displayed high thermal stability at temper-tures up to 70 ◦C in the absence of stabilizer. After incubationt 50–70 ◦C for 120 min, both enzymes retained approx. 80% ofaximal activity. Even after incubation at 80 ◦C, PLP retained

0% activity for 120 min and Est1 50% activity for 30 min.The optimal temperatures and thermostabilities of the puri-

ed enzymes are superior to those of other notable esterasesrom Bacillus stearothermophilus (Wood et al., 1995), Ther-oanaerobacter tengcogensis (Zhang et al., 2003) and Bacillus

p. (Burcu Bakir Ateslier and Metin, 2006). However, PLPnd Est1 have lower optimal activity and thermostability thansterases obtained from hyperthermophilic archaeon that growptimally at 100 ◦C, such as Pyrococcus furiosus (Almeida et

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ig. 3. Thermostability profiles of purified PLP (A) and Est1 (B). For determinationH 9.0 at 50 ◦C (�), 60 ◦C (�), 70 ◦C (©), or 80 ◦C (�) for 20–120 min. The residuapecified temperatures.

atic reactions at temperatures ranging from 30 to 80 ◦C for 10 min in 50 mMhenyl butyrate was used as a substrate. The production of p-nitrophenol from

l., 2006), Sulfolobus shibatae DAM5389 (Ejima et al., 2004).oth PLP and Est1 catalyzed the reactions with high stabil-

ty at high temperature even in the absence of any stabilizer.hese characteristics correspond well to the temperature of theot spring where the sediment was obtained (70 ◦C). Therefore,LP and Est1 could be used efficiently within a broad temper-ture range of 50–70 ◦C and are suitable for biotechnologicalpplications performed at high temperatures, pressure and in theresence of organic solvents. For example, PLP may be applied

n oil degumming (vegetable oil refining), which is conducted at0–80 ◦C (Clausen, 2001), while the Est1 may be used at tem-erature of exceeding 45 ◦C in various industrial processes, suchs removal of the pitch from pulp produced in paper industry and

of thermal stability, the purified enzymes were incubated in 20 mM Tris–HCl,l activity is defined as the ratio of the activity after and before incubation at the

48 P. Tirawongsaroj et al. / Journal of Biotechnology 133 (2008) 42–49

Table 1Kinetic parameters for recombinants PLP

Substrate (p-nitrophenyl ester) Vmax (�mol min−1 mg−) Km (mM) kcat (s−1) kcat/Km (s−1 mM−1)

Acetate (C2) 683 ± 5.0 0.59 ± 0.1 368.36 624.34Butyrate (C4) 1065 ± 79 0.14 ± 0.0 574.17 4101.2Valerate (C5) 818 ± 128 0.3 ± 0.04 440.80 1469.3Caprylate (C8) 177 ± 70 0.39 ± 0.05 240.77 617.36Laurate (C12) 178 ± 35 0.26 ± 0.0 95.9 368.85Myristate (C14) 194 ± 11 1.49 ± 0.5 104.57 70.18Palmitate (C16) 278 ± 13 0.39 ± 0.27 150.24 358.23

Table 2Kinetic parameters for recombinants Est1

Substrate (p-nitrophenyl ester) Vmax (�mol min−1mg−) Km (mM) kcat (s−1) kcat/Km (s−1 mM−1)

Acetate (C2) 181 ± 62 0.59 ± 0.4 90.55 153.47Butyrate (C4) 252 ± 44 0.15 ± 0.1 126.33 842.20Valerate (C5) 221 ± 14 0.12 ± 0.0 110.49 920.75Caprylate (C8) 63.5 ± 3.2 0.51 ± 0.4 31.78 62.31LMP

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almitate (C16) 28.0 ± 3.2

he removal of subcutaneous fat in the leather industry (Pandeyt al., 1999).

.2. Substrate specificity of PLP and Est1

To examine substrate specificity for each of the identifiednzymes, various p-nitrophenyl esters with different acyl chainsengths were used. Under our standard assay conditions ofH 9.0 and 70 ◦C, both PLP and Est1 showed hydrolyzingctivity toward short- and long- acyl chains of p-nitrophenylsters (C2–C16). The purified PLP showed the highest Vmaxnd kcat toward p-nitrophenyl butyrate (C4) with the value of065 ± 79 U mg−1 protein and 574.2 s−1 for Vmax and kcat,espectively, which was 1.3-fold higher than those valuesbtained when p-nitrophenyl valerate (C5) was used as the sub-trate (Table 1). The kcat/Km ratios indicated that p-nitrophenylutyrate (C4) was the best substrate among the p-nitrophenylsters tested.

The purified Est1 showed broad substrate specificity towardll acyl chain-length of p-nitrophenyl esters tested of varyingcyl-chain length (C2–C16). Est1 showed similar Vmax toward-nitrophenyl butyrate (C4) and p-nitrophenyl valerate (C5) withhe values of 252 ± 44 and 221 ± 14 U mg−1 protein, respec-ively. However, the kcat/Km ratios indicated that p-nitrophenylalerate was the best substrate among the p-nitrophenyl estersetermined (Table 2). The Vmax values of PLP toward vari-us substrates were higher than that of Est1. These activitiesre higher than the esterase activity exhibited by Sulfolobushibatae DSM5389 (Ejima et al., 2004) (Tables 1 and 2). In addi-ion, both PLP and Est1 exhibited broader substrate specificitieshan other microbial esterases, such as Burkholderia gladioli

TCC10248 (Reiter et al., 2000), Sulfolobus shibatae DSM5389

Ejima et al., 2004), and Bacillus sp. (Burcu Bakir Ateslier andetin, 2006) that hydrolyze p-nitophenyl esters ranging in chain

ength from C2 (acetate) to C6 (hexanoate). Unlike the esterases

sdp

0.81 ± 0.6 44.78 55.280.66 ± 0.0 23.01 34.860.05 ± 0.04 14.02 280.40

rom Aeropyrum pernix K1 (Wang et al., 2004) and other novelsterases obtained from metagenomic libraries (Kim et al., 2006;ark et al., 2007) having the ability to hydrolyze p-nitrophenyl

aurate, the substrate specificity assays show that both PLP andst1 possess good activity toward both short-chain (C4 and C5)nd long chain (C14 and C16) fatty acid esters. Further detectionf lipase activity on trioleoylglycerol revealed a lack of lipasectivity by both enzymes, which was unexpected given that thenzymes are able to hydrolyze esters with long acyl chain lengthC12–C16). Therefore, PLP and Est1 are novel lipolytic enzymesrom unculturable microbes displaying both esterase and lipasectivities, in which the latter acts only toward long-chain monoa-yl glycerol. In this respect, PLP and Est1 are similar to aipase from Sulfolobus acidocaldarius which hydrolyzes only-nitrophenyl palmitate but not trioleoylglycerol (Arpigny etl., 1998). In addition, based on functional characteristics, ournzymes might be similar to lipase B of Candida antarcticaCALB), which has neither a typical lid domain nor exhibitsnterfacial activation (Overbeeke et al., 2000; Rotticci et al.,000). The CALB active site has a characteristic narrow funnelhape, which is less accessible to triglycerides than carboxyliccid esters (Martinelle, 1995). Further study on functional andtructural relationships of both isolated enzymes will be per-ormed. In addition, application of the enzymes for asymmetricynthesis of optically pure compounds will be further inves-igated. Further studies will provide important data for futurepplication of the newly isolated thermophilic lipase/esteraseor promising biotechnological processes.

cknowledgements

We thank Dr. Kanyawim Kirtikara for her valuable discus-ion, Dr. Eric Dubreucq and Dr. Laurent Vaysse for criticaliscussion and Dr. Philip Shaw and Dr. Albert Ketterman forroof-reading of the manuscript. This work was supported by the

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