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Journal of Biotechnology 133 (2008) 4249

Novel thermophilic and thermostable lipolytic enzymes from 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,
BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 National Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand b 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

Abstract Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classied 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 at alkaline pH, of 79 and 810, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 5075 C, with optimal activity at 70 C and were stable at 70 C for at least 120 min. Both PLP and Est1 exhibited high Vmax toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C4 and C5 ) and long chain (C14 and C16 ) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which 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 industrial applications. 2007 Elsevier B.V. All rights reserved.
Keywords: Lipolytic enzyme; Metagenomic library; Patatin-like phospholipase; Esterase

1. Introduction Lipolytic enzymes are important biocatalysts due to their ability to utilize a wide range of substrates, and hence have a potentially broad spectrum of biotechnological uses. Lipolytic enzymes can be classied into eight families based on the conserved sequences, motifs, and biological properties; namely: true lipase, GDSL, hormone-sensitive lipase (HSL) and families III, VVIII (Arpigny and Jaeger, 1999). In addition, the lipolytic enzymes can be classied into three main groups based on the substrate specicity: lipases (EC, esterases/carboxylesterases (EC, and phospholipases.

Corresponding author. Tel.: +66 2564 6700; fax: +66 2564 6707. E-mail address: lily@biotec.or.th (L. Eurwilaichitr). These authors contributed equally to this work.

Lipases (EC catalyze the hydrolysis of triglycerides with long-chain fatty acids (10 carbon atoms). Most lipases have activity at the aqueous/non-aqueous interface (referred to as interfacial activation), whose activity is associated with conformational changes of the lid domain in the protein revealing the substrate-accessible site. However, some lipases contain minor or no interfacial activation and thus display both lipase and esterase activity (Verger, 1997). Esterases (EC prefer water-soluble substrates and catalyze the hydrolysis of glycerolesters with short acyl chain (10 carbon atoms) to partial glycerides and fatty acids (Bornscheuer, 2002). Phospholipases catalyze the hydrolysis of various types of phospholipids. Lipases are commonly used in the production of free fatty acids, peptide synthesis and inter-esterication of oils and fats. Moreover, these enzymes can be used as chiral catalysts for ne chemical synthesis, or for stereo-selective conversion of a variety of amines and primary and secondary alcohols for pro-

0168-1656/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.08.046

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duction of cosmetics and pharmaceuticals (Jaeger and Eggert, 2002). Some industrial processes in which lipolytic enzymes are employed require harsh conditions, e.g. high temperature. The instability of well-characterized mesophilic enzymes in extreme conditions has prompted the search for new enzymes with better-suited properties. The discovery that thermostability is correlated with resistance to denaturation in organic solvents (Owusu and Cowan, 1989) suggested that thermostable enzymes are likely to have properties apt for many industrial applications. Molecular phylogenetic studies have shown that only a small fraction (<1%) of bacterial diversity present in an environmental sample can be cultured in the laboratory (Amann et al., 1995). The culture-independent, metagenomic approach (Rondon et al., 2000) can therefore be used to avoid the inherent loss of diversity related to cultivation. Target enzymes including lipases and esterases (Robertson et al., 2004; Voget et al., 2003; Lee et al., 2004; Rhee et al., 2005), amylases (Robertson et al., 2004; Handelsman et al., 1998), chitinase (Cottrell et al., 1999), and nitrilases (Robertson et al., 2004) have been successfully identied using the metagenomic approach. Various types of commercial products already available in the market have also been found by this technique (Cowan et al., 2005). Previously, our group reported the biodiversity and abundance of bacteria and archae from Bor Khleung and Jae Sawn hot springs, Thailand (Kanokratana et al., 2004 and unpublished data). These studies demonstrated a complex community of prokaryotes in Thailands hot springs, with over 80% of those microorganisms representative of new strains not previously found elsewhere. It can be inferred from these data that novel and interesting enzyme-producing microbes are present in this environment. The hot spring sediment is therefore an attractive resource for microorganisms producing thermotolerant lipolytic enzymes with potential use in many industries. From functional screening of the metagenomic library derived from sediments of the Jae Sawn hot spring, we have isolated two genes encoding a novel esterase and a phospholipase. The esterase and phospholipase genes were heterologously expressed in E. coli. The recombinant enzymes were puried and their biochemical properties were characterized. The isolated enzymes were found to function well at high temperature (5070 C) and basic pH (89). 2. Materials and methods 2.1. Microorganisms and culture conditions The bacterial strains used were: E. coli DH5 F (F (lacZYA-argF)U169 deoR endA1 hsdR17 supE44 thi-1 recA1 gyr96 relA1 (80dlacZ M15)), E. coli BL21(DE3) pLysS (F ompT hsdSB (rB mB ) gal dcm (DE3) pLysS (CmR )) and E. coli TOP10 (mcrA (mrr-hsdRMS-mcrBC) 80 lac M15 lacX74 deoR recA1 araD139 (ara leu) 7697 galU galK rpsL endA1 mupG). The cloning vector pTZ57R was purchased from Fermentas (USA). The vector pZErO-2 was purchased from Invitrogen (Invitrogen, USA). An expression vector pET-28a (+) (Novagen, USA) was used for the expression of the

lipase/esterase genes. E. coli was grown at 37 C in LB broth (Sambrook and Russell, 2001) or on agar (1.5%, w/v) plates supplemented with appropriate antibiotics. 2.2. DNA extraction from hot spring sediments A soil sample from the Jae Sawn hot spring was taken at 1020 cm below the surface, with a temperature of 70 C and pH of 7. Approximately 410 g of genomic DNA/g of wet soil was obtained following extraction method from Zhou et al. (1996). DNA purication was performed by a modied DNA troughing method (Harnpicharnchai et al., 2007). 2.3. Metagenomic library construction The genomic DNA partially digested with Sau3AI was subjected to size fractionation using the above modied troughing method. Fractions containing DNA fragments of approximately 110 kb were ligated with the zero-background cloning vector, pZErO-2 (Invitrogen, USA). To construct the library, approximately 1 g of pZErO-2 was digested with BamHI. The linearized plasmid was then puried by Wizard DNA clean up kit (Promega, USA) and used to ligate with the partially digested genomic DNA. The ligated plasmids were transformed into E. coli TOP10 (Invitrogen) by electroporation and the transformants were selected on LB supplemented with kanamycin (50 g/ml). 2.4. Screening for lipase/esterase activity Transformants from the library were screened for esterase/lipase activity by plating the transformed cells onto LuriaBertani (LB) agar plate containing 1% trioleoylglycerol (with an acyl chain length of 18 carbon atom, a common substrate for true lipase screening) or 1% tributylglycerol (with an acyl chain length of four carbon atoms, a common substrate for esterase screening) plus 0.001% Rhodamine B dye and incubated at 37 C for 2 days. Then, the plates were overlaid with 0.7% agar containing d-cycloserine (60 g/ml) and further incubated at 37 C for 2 days. The positive lipolytic enzyme candidates were selected from bacterial colonies producing clear halo zone, then their plasmids were isolated and analyzed. 2.5. DNA sequencing and sequence analysis DNA was sent to Macrogen, Inc. (Seoul, South Korea) for sequencing. Sequences of bacterial lipase and esterase used for comparative study were retrieved from protein and nucleotide databases on the NCBI Entrez server at http://www.ncbi.nlm.nih.gov/Entrez/. Sequence similarity searches were performed by BLASTX analysis. Multiple alignments of enzymes with homologous proteins and phylogenetic analyses were performed with the Align X program, a component of the Vector NTI suite (InforMax, North Bethesda, MD). The nucleotide sequences of isolated genes have been deposited


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in GenBank under accession numbers EF413636 and EF413637 for PLP and Est1, respectively. 2.6. Expression and purication of recombinant lipolytic enzymes The patatin-like phospholipase (PLP) and esterase (Est) genes were amplied by PCR with primer pairs PLPF (5 CATATGCGCAGGATACATCTCG-3 )/PLPR (5 -AAGCTTCTACAACAGTTCCTTCAGC-3 ) and ESTF (5 -CATATGCCGGCAGTAGATATCTTCC-3 )/ ESTR (5 -GGATCCTGAACTTCCCCCTCAAAAATTC-3 ), respectively. Restriction sites for cloning are underlined. The 873 and 792 bp amplicons for PLP and Est1, respectively, were cloned into pET-28a(+) (Novagen), a vector designed for expression of N-terminal 6xHis tagged protein. The plasmids were introduced into E. coli strain BL21(DE3) (pLysS) and transformants were selected on LB plates supplemented with kanamycin (50 g/ml) and chloramphenicol (34 g/ml). 2.7. Expression and purication of lipase/esterase from E. coli A culture of E. coli cells (grown to A600 0.20.4) was induced by adding 1 mM IPTG (Promega, USA) and grown for an additional 4 h. Cells were harvested by centrifugation and disrupted by BugBuster Protein Extraction Reagent (Novagen, Germany). The supernatant was puried by using Hi-Trap afnity column containing nickel-nitrilo-triacetic acid as described by the manufacturer (Amersham Biosciences, UK). The eluted fractions with high lipolytic activity was concentrated and desalted using Amicon Ultraltration Unit (Millipore Co., USA). The protein was subjected to SDS-PAGE analysis and visualized with Coomassie Blue R-250. 2.8. Enzyme assays Lipolytic activities were determined by measuring the amount of p-nitrophenol product released after lipolyticcatalyzed hydrolysis (Lee et al., 1993). One unit of enzyme activity was dened as the amount of enzyme required to release 1 mol of p-nitrophenol per minute from the p-nitrophenyl ester. 2.9. Substrate specicity Substrate specicities for p-nitrophenyl esters were determined by incubation of puried enzymes in various concentrations (0.052 mM) of substrates; p-nitrophenyl acetate,

p-nitrophenyl butyrate (Sigma), p-nitrophenyl valerate (Sigma, Fluka), p-nitrophenyl caprylate (Sigma), p-nitrophenyl laurate, p-nitrophenyl myristate and p-nitrophenyl palmitate (Sigma) in 50 mM TrisHCl (pH 9.0) with 0.4 % Triton-X and 0.1% gum Arabic (Rhee et al., 2005). The kinetic parameters were then determined by tting the initial velocity data to the MichaelisMenten equation using the Kaleida Graph software package (Synergy Software, USA) (Chen et al., 2005). 2.10. Enzyme characterizations The optimal pH of the PLP and esterase enzyme activities were measured at pH ranging from 3.0 to 12.0 under standard assay conditions at 70 C. The buffers used were 50 mM sodium acetate (pH 3.05.0), 50 mM MES (pH 5.07.0), 50 mM HEPES (pH 7.08.0), and 50 mM TrisHCl (pH 8.012.0). The production of p-nitrophenol from p-nitrophenyl butyrate was monitored at 348 nm. The molar extinction coefcient (348 nm ) determined at each pH tested were 24, 25 and 24 M1 cm for pH 3.0, 4.0 and 5.0, respectively, in 50 mM sodium acetate; 25, 27 and 27 M1 cm for pH 5.0, 6.0 and 7.0, respectively, in 50 mM MES; 30 and 31 M1 cm for pH 7.0 and 8.0, respectively, in 50 mM HEPES; and 31, 30, 25, 30, 27 and 31 M1 cm for pH 8.012, respectively, in 50 mM TrisHCl. The optimal temperature for enzyme activity was determined by subjecting reaction mixtures to different temperatures ranging from 30 to 80 C in 50 mM TrisHCl buffer (pH 9.0) for 10 min. Thermostability of the enzymes was analyzed by measuring the residual activity after pre-incubating the enzyme at 50, 60, 70, or 80 C for 20120 min in Eppendorf tubes with mineral oil on top to prevent evaporation. Relative activity was calculated as enzymatic activity at indicated temperature divided by maximal activity at optimal temperature. 3. Results and discussion A metagenomic library was successfully constructed using genomic DNA obtained from the sediments of the Jae Sawn hot spring. The library was in a plasmid vector (pZErO-2) under the control of a lacZ promoter (Plac). The Plac located upstream of the multiple cloning site allows expression of promoterless genes. The average library size obtained was 1 106 CFU/ g DNA from the ligation reaction of metagenomic DNA and pZErO-2. It was observed that more than 90% of the transformants contained genomic DNA fragments of the expected size (110 kb). The remainders either lack or have undetectably small inserts. From approximately 36,000 colonies screened, two lipolytic-positive transformants, which formed clear halos

Fig. 1. Multiple sequences alignment of the primary structure of lipolytic enzymes. (A) Multisequence alignment comparing the isolated PLP to patatin-like phospholipase gene from bacteria including Geobacter lovleyi SZ (ZP 01593172), Chloroexus aurantiacus (EA058516.1), Bacillus cereus subsp. cytotoxis NVH 39198 (ZP 01182139), Carboxydothermus hydrogenoformans (ABB15198.1) and Aeromonas hydrophila (YP 857433). Open blocks indicate four conserved domains (block IIV) of patatin-like phospholipase. Block I consists of a glycine-rich region with a conserved arginine residue. Block II is located next to block I and comprises of the hydrolase motif, G-X-S-X-G, containing a serine residue located at the putative active-site. Block III contains A-S-X-X-X-P sequence, which is conserved in other bacterial PLPs. Block IV contains the putative active-site aspartate. Black and grey boxes indicate regions with identical or similar amino acid residues, respectively. (B) multiple alignment of Est1 and family V esterases from Sulfolobus acidocaldarius (AF071233), Acetobacter pasteurianus (AB013096), Moraxella sp. (X53869) and Psychrobacter immobilis. Open boxes indicate amino acid residues belonging to the catalytic triad. Black and grey boxes indicate regions with identical or similar amino acid residues, respectively.

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P. Tirawongsaroj et al. / Journal of Biotechnology 133 (2008) 4249

on tributylglycerol plates, were obtained. The plasmids were isolated from the two transformants and retransformed into new E. coli cells. The new transformants produced clear halos on tributylglycerol plates, suggesting that the plasmids contained genes responsible for the esterase activity toward substrates with short acyl chains. However, both plasmids failed to produce E. coli transformants with lipolytic activity on the agar plate containing trioleoylglycerol, demonstrating that the enzymes produced by both plasmids have little or no activity toward triacylglycerol with acyl chain length of 18 carbon atoms. Sequence analysis showed that each transformant harbored plasmids with inserts approximately 3 and 2 kb in size. These plasmids were designated as pZErO-PLP and pZErO-Est1, respectively. The pZErO-PLP encodes a protein, designated as PLP, of 291 amino acids with a theoretical molecular weight of approximately 32.3 kDa. Subsequent BLASTP analysis of PLP revealed a potential patatin-like phospholipase gene containing the conserved motif of lipolytic enzymes (GXSXG). BLASTP analysis revealed a moderate similarity (<50%) between PLP and phospholipases from various bacteria, including the patatin (EA058516.1) from Chloroexus aurantiacus J-10-f1 (43%) (Banerji and Flieger, 2004), the phospholipase family protein (ABB15198.1) from Carboxydothermus hydrogenoformez (39%), and the patatin-like phospholipase protein (EAV88283.1) from Geobacter lovleyi SZ (41%). Further sequence analysis revealed a putative purine-rich ribosomal binding site of PLP located 28 bp upstream of the ATG start codon. However, the potential 35 and 10 consensus promoter sequences were not found, suggesting that the isolated PLP gene was expressed using the lacZ promoter. Multi-sequence alignment comparison of the isolated PLP to patatin-like phospholipase genes from bacteria such as G. lovleyi SZ (ZP 01593172), Clostridium cellulolyticum H10 (ZP 01593172), Bacillus cereus subsp. cytotoxis NVH 391-98 (ZP 01182139) Aeromonas hydrophila (YP 857433) and C. hydrogenoformans (YP 359744.1) was further analyzed. It revealed four conserved domains (blocks IIV), similar to those present in other patatin-like phospholipases from various bacteria (Fig. 1A) (Banerji and Flieger, 2004). From BLASTP analysis and multi-sequence alignment, the PLP gene could not be classied into any of the eight families of known bacterial lipases (Arpigny and Jaeger, 1999). Therefore, it is inferred that PLP is not closely related to the established groups of lipolytic enzymes. Patatins are a group of plant storage glycoproteins that show lipid acyl hydrolase activity (Andrews et al., 1988) and catalyze the non-specic hydrolysis of a wide range of acryl- and phospho-lipids (Sharma et al., 2004; Hirschberg et al., 2001). There are reports of patatins present in bacteria that are animal pathogens and plant pathogens such as Bradyrhizobium japonicum, Leptospira interrogans and Mesorhizobium loti (Banerji and Flieger, 2004). Although PLP exhibited less than 50% identity to patatin-associated lipolytic enzymes, it seemed most likely that a novel patatin-like phospholipase homolog was obtained in this study. BLASTP analysis of the ORF in pZErO-Est1 revealed a putative esterase gene containing the conserved sequence motif of esterases/lipases (GXSXG). The ORF, designated as Est1, encodes a protein of 264 amino acid residues with a theoretical

molecular weight of approximately 29.3 kDa. BLASTP analysis revealed a moderate identity to a hydrolase in alpha/beta-fold family from Pseudomonas mendocina ymp (ZP 01527185.1) (39%), P. uorescens PfO-1 (YP 258670.1) (33%) and also a moderate identity to an esterase from Rhodobacter sphaeroides (YP 345244.1) (31%). Sequence analysis of Est1 revealed a putative ribosomal binding site located 5 bp upstream from the ATG start codon and the potential 35 and 10 consensus promoter sequences were recognized (data not shown). From PSORT analysis, neither PLP nor Est1 showed potential secretion signal sequences (Nakai and Horton, 1999). Multi-sequence alignment comparisons of Est1 with members of each family of lipolytic enzymes as classied by Arpigny and Jaeger (1999) revealed that Est1 contains the serine residue that forms a putative catalytic triad with histidine and aspartic residues, which are conserved among family V lipolytic enzymes (Fig. 1B). The 40% identity to known lipolytic enzymes suggests that Est1 is a novel enzyme from unculturable microbes which maybe tentatively classied as a family V bacterial lipolytic enzyme. The family V enzymes shown in Fig. 1B originate from mesophilic bacteria (Acetobacter pasteurianus (AB013096)) as well as from cold-adapted (Moraxella sp. (X53869), Psychrobacter immobilis (X67712)) or heat-adapted (Sulfolobus acidocaldarius) (AF071233) organisms. They share some amino acid sequence similarity (2025%) to various bacterial non-lipolytic enzymes, namely epoxide hydrolases, dehalogenases and haloperoxidases from Corynebacterium sp. and Xanthobacter autotrophicus, which also possess the typical / -hydrolase fold and a catalytic triad (Arpigny and Jaeger, 1999; Misawa et al., 1998; Verschueren et al., 1993) (Fig. 1B). 3.1. Overexpression, purication and characterization of recombinants PLP and Est1 In order to determine the biochemical properties of PLP and Est1, the target enzymes of interest were expressed as N-terminal 6xHis tagged fusions in E. coli. PLP and Est1 were puried to 95% homogeneity by afnity chromatography with Ni-NTA resin. The puried proteins migrated as single bands on SDSPAGE with the expected molecular weights of approximately 32 and 29 kDa for PLP and Est1, respectively. The optimal pH of PLP and Est1 activities were determined at various pH values (pH 3.012.0). Using p-nitrophenyl butyrate as the substrate, PLP exhibited more than 50% of its maximal activity in the pH range of 7.09.0, with the highest activity at pH 9.0 (Fig. 2A). Est1 exhibited high activity at a broader pH range, with more than 50% of its maximal activity occurring at pH 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 was determined by monitoring the hydrolysis of p-nitrophenyl butyrate at various temperatures ranging from 45 to 80 C (Fig. 2). Under the conditions used, both enzymes showed highest activity at 70 C. Fifty percent maximal activity was observed between 45 and 75 C for PLP and 50 and 70 C for Est1. The thermostability of PLP and Est1 were determined by incubating the enzymes for 20120 min at 5070 C before measuring their residual activities under standard assay conditions (Fig. 3).

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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 buffers used were 50 mM sodium acetate (pH 3.05.0) ( ); 50 mM MES (pH 5.07.0) ( ); 50 mM HEPES (pH 7.08.0) ( ), and 50 mM TrisHCl (pH 8.012.0) (). 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 (D). The optimal temperature for enzyme activity was determined by assaying enzymatic reactions at temperatures ranging from 30 to 80 C for 10 min in 50 mM TrisHCl buffer (pH 9.0) containing 0.4% Triton-X and 0.2% gum arabic. p-Nitrophenyl butyrate was used as a substrate. The production of p-nitrophenol from p-nitrophenyl butyrate was monitored at 405 nm.

Both PLP and Est1 displayed high thermal stability at temperatures up to 70 C in the absence of stabilizer. After incubation at 5070 C for 120 min, both enzymes retained approx. 80% of maximal activity. Even after incubation at 80 C, PLP retained 50% activity for 120 min and Est1 50% activity for 30 min. The optimal temperatures and thermostabilities of the puried enzymes are superior to those of other notable esterases from Bacillus stearothermophilus (Wood et al., 1995), Thermoanaerobacter tengcogensis (Zhang et al., 2003) and Bacillus sp. (Burcu Bakir Ateslier and Metin, 2006). However, PLP and Est1 have lower optimal activity and thermostability than esterases obtained from hyperthermophilic archaeon that grow optimally at 100 C, such as Pyrococcus furiosus (Almeida et

al., 2006), Sulfolobus shibatae DAM5389 (Ejima et al., 2004). Both PLP and Est1 catalyzed the reactions with high stability at high temperature even in the absence of any stabilizer. These characteristics correspond well to the temperature of the hot spring where the sediment was obtained (70 C). Therefore, PLP and Est1 could be used efciently within a broad temperature range of 5070 C and are suitable for biotechnological applications performed at high temperatures, pressure and in the presence of organic solvents. For example, PLP may be applied in oil degumming (vegetable oil rening), which is conducted at 5080 C (Clausen, 2001), while the Est1 may be used at temperature of exceeding 45 C in various industrial processes, such as removal of the pitch from pulp produced in paper industry and

Fig. 3. Thermostability proles of puried PLP (A) and Est1 (B). For determination of thermal stability, the puried enzymes were incubated in 20 mM TrisHCl, pH 9.0 at 50 C ( ), 60 C ( ), 70 C ( ), or 80 C ( ) for 20120 min. The residual activity is dened as the ratio of the activity after and before incubation at the specied temperatures.

48 Table 1 Kinetic parameters for recombinants PLP Substrate (p-nitrophenyl ester) Acetate (C2 ) Butyrate (C4 ) Valerate (C5 ) Caprylate (C8 ) Laurate (C12 ) Myristate (C14 ) Palmitate (C16 ) Table 2 Kinetic parameters for recombinants Est1 Substrate (p-nitrophenyl ester) Acetate (C2 ) Butyrate (C4 ) Valerate (C5 ) Caprylate (C8 ) Laurate (C12 ) Myristate (C14 ) Palmitate (C16 )

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Vmax ( mol min1 mg ) 683 1065 818 177 178 194 278 5.0 79 128 70 35 11 13

Km (mM) 0.59 0.14 0.3 0.39 0.26 1.49 0.39 0.1 0.0 0.04 0.05 0.0 0.5 0.27

kcat (s1 ) 368.36 574.17 440.80 240.77 95.9 104.57 150.24

kcat /Km (s1 mM1 ) 624.34 4101.2 1469.3 617.36 368.85 70.18 358.23

Vmax ( mol min1 mg ) 181 252 221 63.5 89.5 45.9 28.0 62 44 14 3.2 1.9 11 3.2

Km (mM) 0.59 0.15 0.12 0.51 0.81 0.66 0.05 0.4 0.1 0.0 0.4 0.6 0.0 0.04

kcat (s1 ) 90.55 126.33 110.49 31.78 44.78 23.01 14.02

kcat /Km (s1 mM1 ) 153.47 842.20 920.75 62.31 55.28 34.86 280.40

the removal of subcutaneous fat in the leather industry (Pandey et al., 1999). 3.2. Substrate specicity of PLP and Est1 To examine substrate specicity for each of the identied enzymes, various p-nitrophenyl esters with different acyl chains lengths were used. Under our standard assay conditions of pH 9.0 and 70 C, both PLP and Est1 showed hydrolyzing activity toward short- and long- acyl chains of p-nitrophenyl esters (C2 C16 ). The puried PLP showed the highest Vmax and kcat toward p-nitrophenyl butyrate (C4 ) with the value of 1065 79 U mg1 protein and 574.2 s1 for Vmax and kcat , respectively, which was 1.3-fold higher than those values obtained when p-nitrophenyl valerate (C5 ) was used as the substrate (Table 1). The kcat /Km ratios indicated that p-nitrophenyl butyrate (C4 ) was the best substrate among the p-nitrophenyl esters tested. The puried Est1 showed broad substrate specicity toward all acyl chain-length of p-nitrophenyl esters tested of varying acyl-chain length (C2 C16 ). Est1 showed similar Vmax toward p-nitrophenyl butyrate (C4 ) and p-nitrophenyl valerate (C5 ) with the values of 252 44 and 221 14 U mg1 protein, respectively. However, the kcat /Km ratios indicated that p-nitrophenyl valerate was the best substrate among the p-nitrophenyl esters determined (Table 2). The Vmax values of PLP toward various substrates were higher than that of Est1. These activities are higher than the esterase activity exhibited by Sulfolobus shibatae DSM5389 (Ejima et al., 2004) (Tables 1 and 2). In addition, both PLP and Est1 exhibited broader substrate specicities than other microbial esterases, such as Burkholderia gladioli ATCC10248 (Reiter et al., 2000), Sulfolobus shibatae DSM5389 (Ejima et al., 2004), and Bacillus sp. (Burcu Bakir Ateslier and Metin, 2006) that hydrolyze p-nitophenyl esters ranging in chain length from C2 (acetate) to C6 (hexanoate). Unlike the esterases

from Aeropyrum pernix K1 (Wang et al., 2004) and other novel esterases obtained from metagenomic libraries (Kim et al., 2006; Park et al., 2007) having the ability to hydrolyze p-nitrophenyl laurate, the substrate specicity assays show that both PLP and Est1 possess good activity toward both short-chain (C4 and C5 ) and long chain (C14 and C16 ) fatty acid esters. Further detection of lipase activity on trioleoylglycerol revealed a lack of lipase activity by both enzymes, which was unexpected given that the enzymes are able to hydrolyze esters with long acyl chain length (C12 C16 ). Therefore, PLP and Est1 are novel lipolytic enzymes from unculturable microbes displaying both esterase and lipase activities, in which the latter acts only toward long-chain monoacyl glycerol. In this respect, PLP and Est1 are similar to a lipase from Sulfolobus acidocaldarius which hydrolyzes only p-nitrophenyl palmitate but not trioleoylglycerol (Arpigny et al., 1998). In addition, based on functional characteristics, our enzymes might be similar to lipase B of Candida antarctica (CALB), which has neither a typical lid domain nor exhibits interfacial activation (Overbeeke et al., 2000; Rotticci et al., 2000). The CALB active site has a characteristic narrow funnel shape, which is less accessible to triglycerides than carboxylic acid esters (Martinelle, 1995). Further study on functional and structural relationships of both isolated enzymes will be performed. In addition, application of the enzymes for asymmetric synthesis of optically pure compounds will be further investigated. Further studies will provide important data for future application of the newly isolated thermophilic lipase/esterase for promising biotechnological processes. Acknowledgements We thank Dr. Kanyawim Kirtikara for her valuable discussion, Dr. Eric Dubreucq and Dr. Laurent Vaysse for critical discussion and Dr. Philip Shaw and Dr. Albert Ketterman for proof-reading of the manuscript. This work was supported by the

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grant (B21FJ0022) from the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand. References
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