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A Novel Esterase from Paenibacillus sp. PBS-2 Is a New Member of the β-Lactamase Belonging to the Family VIII Lipases/Esterases
A Novel Esterase from Paenibacillus sp. PBS-2 Is a New Member of the β-Lactamase Belonging to the Family VIII Lipases/Esterases
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1260-1268
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 20, 2014
  • Accepted : June 20, 2014
  • Published : September 30, 2014
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About the Authors
Young-Ok Kim
yobest12@korea.kr
In-Suk Park
Bo-Hye Nam
Dong-Gyun Kim
Young-Ju Jee
Sang-Jun Lee
Cheul-Min An

Abstract
Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative β-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Ser-x-x-Lys motif that is conserved among all β-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18℃. The enzyme is a serine protein and was active against p -nitrophenyl esters of C 2 , C 4 , C 8 , and C 10 . The optimum pH and temperature for enzyme activity were pH 9.0 and 30℃, respectively. Relative activity of 55% remained at up to 5℃ with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by Cd 2+ , Cu 2+ , and Hg 2+ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.
Keywords
Introduction
Esterases (E.C. 3.1.1.X) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds. Two major classes of hydrolase are of great importance: “true” esterases (E.C. 3.1.1.1, carboxylic-ester hydrolases) and lipases (E.C. 3.1.1.3, triacylglycerol hydrolases) [3] . According to their activity on substrates of different acyl chain lengths, carboxylester hydrolases are classified as esterases or lipases. Esterases act preferentially on water-soluble short-chain fatty acids, whereas lipases display maximal activity toward water-insoluble longchain triglycerides [35] . Most esterases and lipases harbor a minimally conserved consensus sequence around the active-site serine of G-x-G-x-G [27] or G-D-S-L [36] . More recently, esterases that contain a Gly-x-x-Leu motif [38] , as well as enzymes showing high homology to class C β-lactamases, which contain a Gly-x-Ser-x-Gly motif and a Ser-x-x-Lys motif, have been identified [1 , 28] .
Different sources yield different esterases. These enzymes are distributed widely in animals, plants, and microorganisms [23] . A wide variety of esterases exist that differ in their substrate specificity, protein structure, and biological function [24] . Many have a wide range of substrates, leading to the assumption that they evolved to enable access to carbon sources or to be involved in catabolic pathways. Moreover, esterases also show high region and stereo-specificity, which makes them attractive biocatalysts for the production of optically pure compounds in the synthesis of fine chemicals [2 , 7 , 26 , 39] . Lipases of microbial origin represent the most extensively used class and are receiving increasing attention owing to their relative ease of production and potential applications in biotechnology [10] . Cold-adapted microorganisms are a potential source of cold-active lipases/esterases and have been isolated from cold regions and evaluated. Compared with other lipases, smaller numbers of cold-active bacterial lipases/esterases have been investigated extensively. These include Moraxella sp. strain TA144 [9] , Aeromonas sp. LPB4 [21] , Pseudomonas sp. strain B11-1 [5] , Acinetobacter sp. No. 6 [33] , Psychrobacter sp. Ant300 [20] , Photobacterium lipolyticum sp. nov. [30] , Salinisphaera sp. P7-4 [19] , and Shewanella sp. ke75 [18] .
The strain Paenibacillus sp. PBS-2, previously isolated in our laboratory, exhibits polybutylene succinate-degrading activity [22] . After construction of a gene library from this strain in Escherichia coli XL1-Blue, we isolated a recombinant clone with lipolytic activity. This paper describes the cloning, sequencing, and biochemical characterization of the cloned enzyme to evaluate its potential applicability in biotechnological processes.
Materials and Methods
- Bacterial Strains, Plasmids, and Chemicals
Paenibacillus sp. PBS-2 was grown in Luria–Bertani (LB) broth at 25℃. E. coli XL1-Blue and BL21 (DE3) were used as the cloning hosts, and the pGEM-T easy vector (Promega, USA) and pET-22a(+) (Novagen, USA) were used as the cloning and expression vectors, respectively. E. coli XL1-Blue cells harboring the recombinant plasmids were grown in LB supplemented with 100 μg/ml ampicillin.
All other chemicals and solvents were of analytical grade and are available commercially. Tributyrin, p -nitrophenyl ( p -NP) acetate (C 2 ), butyrate (C 4 ), caprylate (C 8 ), caprate (C 10 ), palmitate (C 16 ), and stearate (C 18 ) were purchased from Sigma (USA), and p -NP myristate (C 14 ) was purchased from Fluka (USA).
- Screening the Genomic Library for Lipolytic Activity
The genomic DNA from Paenibacillus sp. PBS-2 was prepared for genomic library construction according to a method described previously [31] . Chromosomal DNA from Paenibacillus sp. PBS-2 was partially digested with Sau 3AI, ligated into the pUC118- Hin cII vector (TaKaRa, Japan), and used to transform E. coli XL1-Blue. A colony that formed a clear halo on a tributyrin (TBN)-LB plate containing ampicillin (100 μg/ml) was selected. TBN-LB plates were prepared as follows [16] : a TBN emulsion was prepared by emulsifying 5 ml of TBN in 45 ml of a gum arabic solution (200 mM NaCl, 10 mM CaCl 2 , and 5% gum arabic) for 2 min in a Waring blender. This TBN emulsion (50 ml) was then mixed with 450 ml of LB broth containing 100 μg/ml ampicillin and used to make TBN-LB plates for screening of colonies with lipolytic activity.
- Sequence Analysis of the Esterase Gene
The recombinant plasmid (pUCPBS-2) was purified from the transformant, and the insert was sequenced. Sequence analysis and database similarity searches were performed using the National Center for Biotechnology Information (NCBI) database ( http://www.ncbi.nlm.nih.gov/BLAST/ ). The SignalP ver. 3.0 software was used for the identification of potential signal peptides. Multiple sequence alignments were performed using ClustalW [34] . The presence of defined protein patterns was determined using the Prosite Database at ExPASY.
- Expression and Purification of the Esterase
The putative PBS-2 esterase gene was amplified from the pUCPBS2 plasmid using the primers 5’-TT GGATCC ATGGACTTTAAACCGGTT-3’ ( Bam HI adaptor restriction enzyme site underlined) and 5’-TT CTCGAG CAAACAAGAATAAACCGC ( Xho I). Primer pairs with restriction enzyme sites for Bam HI and Xho I were designed to add a His-tag to the C-terminal end of the protein. The esterase gene was cloned into an expression vector, pET28a(+) (Novagen), and the recombinant plasmid, pETPBS-2, was transformed into E. coli BL21 (DE3) cells. Transformed E. coli cells were cultivated in LB medium containing kanamycin (100 μg/ml) at 37℃. When the optical density (OD) reached 0.5–0.6 at 600 nm, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the cultures were further incubated at 18℃ overnight. The E. coli cells were then harvested and ruptured by sonication. The soluble proteins were recovered from the cell extract by centrifugation (10,000 × g , 20 min), and loaded onto a nickelnitrilotriacetic acid (Ni-NTA) column (Novagen). After washing with 60 mM imidazole, 500 mM NaCl, and 50 mM Tris-HCl buffer (pH 7.9), the bound esterase was eluted using 1,000 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl buffer (pH 7.9), and dialyzed against 50 mM Tris-HCl buffer (pH 8.0), followed by characterization of its biochemical properties. Protein concentration was determined using a bicinchoninic acid assay (BCA) method with bovine serum albumin as the standard (Sigma).
- β-Lactamase Activity Assay
To assay β-lactamase activity, pETPBS-2 vector, which has kanamycin resistance, was transformed into E. coli BL21(DE3), cultivated overnight, and plated onto LB plates containing 1 mM IPTG to verify gene expression. Small filter disks containing various concentrations of several β-lactam antibiotics (ampicillin, cefotaxime, ceftrazidime, ceftriaxone, mezlocillin, and penicillin) were placed onto the bacterial lawn, and the diameter of the clearance zone around the discs was recorded after incubation overnight at 37℃ [8] .
- Esterase Activity Assays
Esterase activity was measured using p -nitrophenyl ( p -NP) esters with fatty-acid chain lengths of C 2 –C 18 [30] . The s tandard assay mixture (1.0 μl) contained 10 mM p -nitrophenyl butyrate (pNPB) in ethanol, 50 mM Tris-HCl (pH 8.0), and an appropriate amount (10 μl) of the enzyme. Blank reactions were performed with compositions identical to the assay mixture but without the enzyme. The mixture was incubated at 30℃ for 5 min and the absorbance of p -NP liberated was then measured at 405 nm. For long-chain p -nitrophenyl esters (C 12 –C 18 ), 20 μl of esterase solution was added to 880 ml of reaction buffer containing 50 mM Tris-HCl (pH 8.0), 0.1% gum arabic, and 0.2% deoxycholate. After 5 min incubation at 37℃, the reaction was initiated by adding 100 μl of 8 mM substrate in isopropanol. The reaction was stopped by addition of 0.5 ml of 3 M HCl. After centrifugation, 333 μl of supernatant was mixed with 1 ml of 2 M NaOH, and the absorbance at 405 nm was measured. One unit of enzyme activity was defined as the release of 1 μmol of p -nitrophenol per minute from p -nitrophenyl ester.
- Biochemical Properties of the Esterase
The optimum temperature of the PBS-2 esterase was measured by assaying its hydrolytic activities toward p -nitrophenyl butyrate at temperatures of 5–80℃. To evaluate its thermostability, the enzyme was incubated at 5–80℃ for 30 min in 50 mM Tris-HCl buffer (pH 8 .0), a nd then the residual activity w as m easured under standard assay conditions. The optimum pH for the PBS-2 esterase was measured by assaying at 30℃ with various pH buffers ( i.e. , sodium acetate/acetic acid (pH 4–6), Tris/acetate (pH 6–7), Tris/HCl (pH 7–9), and sodium tetraborate/NaOH (pH 9–11) buffers). To evaluate its pH stability, the enzyme was incubated at various pH values (4–11) at room temperature for 30 min; residual activity was then measured under standard assay conditions. The effects of various metal ions, chemical reagents, detergents, and organic solvents on enzyme activity were assessed after preincubation in 50 mM Tris-HCl buffer (pH 8.0) at room temperature for 30 min.
- Nucleotide Sequence Accession Number
The DNA s equence of the cloned esterase gene has been submitted to the GenBank under the accession number KF972440.
Results and Discussion
- Cloning of a Gene Encoding Lipolytic Activity
A gene library from Paenibacillus sp. PBS-2 was screened for lipolytic activity on tributyrin agar plates to isolate esterases/lipases with biotechnological potential. To date, the only previous reports of esterases/lipases from Paenibacillus strains are EstA, a member of the type B carboxylesterases, from Paenibacillus sp. BP-23 [29] , and the lipolytic enzymes from the Paenibacillus polymyxa genome [14] . Of 3,500 recombinant clones screened, only one, E. coli XL1-Blue/pUCPBS-2, showed a clear zone on TBN-LB plates after 48 h of incubation, and was selected for further characterization. The recombinant plasmid (pUCPBS-2) was fully sequenced and found to contain a 3.1 kb DNA insert, based on restriction enzyme analysis (data not shown). Analysis of the insert nucleotide sequence revealed three major open reading frames (ORFs) corresponding to the cyclase superfamily, β-lactamase superfamily, and EamA-like transporter family, based on a homology search.
- Sequence Analysis of the Esterase Gene
The 3.1 kb DNA fragment contained a complete ORF of 1,134 nucleotides that encodes a predicted protein of 377 amino acids, with a deduced molecular mass of 42,003 Da and a pI of 5.45. The putative esterase gene was designated PBS-2 esterase ( Fig. 1 ). No signal sequence was found using the SignalP ver. 3.0 software. A putative ribosomal-binding site was identified 8 bp upstream of the start codon (AGGATG). No promoter-like motifs were identified in the sequence preceding the PBS2-esterase coding region. The predicted amino acid sequence of the cloned PBS-2 esterase was compared with other protein sequences in GenBank using the basic local alignment search tool (BLAST) software ( Fig. 2 A). The putative esterases were identified through whole-genome sequencing, but none have been biochemically characterized. The PBS-2 esterase exhibited the highest level of identity (69%) to a putative class C β-lactamase from Paenibacillus sp. JDR-2 (YP_003013228), followed by 41–42% identity to the putative β-lactamases of Clostridium sp. BNL1100 (YP_005146758), Clostridium papyrosolvens DSM 2782 (ZP_08192403), and Clostridium cellulolyticum H10 (YP_002506833), and 40% identity to the putative β-lactamases of Thermobacillus composti KWC4 (YP_007213950) and Paenibacillus mucilaginosus KNP414 (YP_004643483). The deduced amino acid sequence of PBS-2 esterase revealed a weak similarity at position 185–189 (GKRFG) to the classical lipase or esterase motif (G-x-S-x-G), and contained a β-lactamase class C motif (S-x-x-K). The consensus sequence of Ser-x-x-Lys, perfectly conserved among all penicillin-recognizing enzymes found to date [4 , 6 , 13] , surrounding the active-site serine residues (Ser residue in this motif) was found at position 58–61 in the PBS-2 esterase. However, although β-lactamases of classes A and C also contain a conserved triad of Lys-Ser/Thr-Gly (KTG-box) between the Ser-x-x-Lys motif and the C-terminus, no such signature was found in this sequence. Many conserved regions were found within the entire sequences of all seven protein sequences. Therefore, PBS-2 esterase most likely belongs to family VIII esterases/lipases, which contains primarily enzymes of approximately 380 residues that show sequence homology to class C β-lactamases and do not possess the mandatory lipase motif.
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Nucleotide sequence of the PBS-2 esterase gene and its deduced amino acid sequence. The conserved peptide sequence of the Ser-x-x-Lys motif is boxed. The sequence element (GKRFG)discussed in the text is underlined. The sequence has been submitted to GenBank under the accession number KF972440.
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Multiple protein sequence alignment (A) and phylogenetic analysis (B) of the PBS-2 esterase and six similar enzymes. (A) Deletions of amino acid residues are indicated by dashes (---) and amino acids conserved in all enzymes are marked by shadowing. Numbers refer to amino acid positions. The conserved motif involved in the catalytic triad is boxed. Sequences with the following accession numbers were obtained from EMBL/GenBank: Paenibacillus sp. PBS-2 (KF972440), Paenibacillus sp. JR-2 (YP_003013228), Clostridium sp. BNL1100 (YP_005146758), Clostridium papyrosolvens DSM 2782 (ZP_08192403), Clostridium cellulolyticum H10 (YP_002506833), Thermobacillus composti KWC4 (YP_007213950), and Paenibacillus mucilaginosus KNP414 (YP_004643483). (B) A phylogenetic tree of the aligned sequences was constructed using the neighborjoining algorithm in MEGA ver. 4.0. The degree of confidence for each branch point was determined by bootstrap analysis (1,000 replicates).
Fig. 2 B shows a phylogenetic tree, indicating the evolutionary relationship with other bacterial β-lactamases belonging to family VIII lipases/esterases, based on the amino acid sequence. The phylogram, generated using Phylip, showed that the Paenibacillus sp. PBS-2 esterase was more closely related to putative class C β-lactamases from Paenibacillus sp. JDR-2 than to other lipases and esterases.
- Expression and Purification of the Esterase
To investigate the biochemical properties of the esterase encoded by pUCPBS-2, the gene was placed under the control of the T7 promoter by cloning into the expression vector pET28a, resulting in the recombinant plasmid pETPBS-2. The plasmid was transformed into E. coli BL21 (DE3), and induced to express a recombinant protein using IPTG at various growth temperatures ( Fig. 3 ). When cultivated and incubated at 37℃, the resulting protein was insoluble; however, at 18℃, the resulting protein was both soluble and insoluble. The problem emanating from the abundant inclusion bodies that formed at 37℃ was resolved by reducing the temperature to 18℃; although some inclusion bodies remained, a large amount of soluble, active PBS-2 protein was produced. The recombinant enzyme was then purified to homogeneity by His-Bind resin affinity chromatography by means of the six-histidine tag at the C-terminus. SDS-PAGE analysis of the eluted fraction showed a distinct protein band at 48 kDa, which is close to the predicted mass of the deduced protein with the addition of a six-histidine tag. The purified enzyme had a specific activity of 6.7 U/mg toward p -NP butyrate at 30℃. Its activity was similar to those of EM2L8, PWTSB, and PWTSC, which had specific activities of 5.1, 5.3, and 6.9 U/mg toward PNPB, respectively [25 , 37] .
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Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of PBS-2 esterase. Lane M contains standard protein molecular-weight markers. Lanes 1 and 4 are non-induced cell lysate. Lanes 2 and 5 are the soluble fractions, and lanes 3 and 6 are the insoluble fractions after cell lysis. Lanes 1, 2, and 3 were obtained from cells cultured at 37℃ and lanes 4, 5, and 6 from cells cultured at 18℃. Lane 7 contains purified PBS-2 esterase. T, S, and P indicate total cell extracts, soluble protein, and insoluble protein, respectively.
- β-Lactamase Activity of PBS-2 Esterase
Since the PBS-2 amino acid sequence contained a possible β-lactamase consensus motif, we overexpressed the gene and investigated the activity of the enzyme toward β-lactam antibiotics. PBS-2 esterase could cleave the β-lactam rings of cefotaxime, ceftazidime, ceftriaxone, and penicillin ( Table 1 ). Metagenome-derived esterase ORF006, which contained a possible β-lactamase consensus motif, could also cleave the β-lactam rings of cefamandole, cefadroxil, and loracarbef [8] . This indicated a possible role for this gene in antibiotic resistance.
Activity of PBS-2 esterase against β-lactamase substrates.
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β-Lactamase activity was measured as the diameters of clear zones around the discs. All measurements were performed in triplicate. ND: not determined.
- Biochemical Characterization of the Esterase
The substrate specificity of PBS-2 esterase was determined using p -NP esters with C2 to C18 acyl chain lengths( Table 2 ). PBS-2 esterase showed a strong preference for short-chain (C 2 to C 8 ) fatty acid p -NP esters, and substrates with acyl groups of C 14 , C 16 , and C 18 were virtually inert as substrates for the enzyme. Carboxylesterases display maximum activities against substrates with acyl chain lengths of less than 10 carbon atoms, which are generally water-soluble [11 , 12] . Based on the substrate preference profile, PBS-2 esterase was classified as a carboxylesterase.
Specific activities of PBS-2 esterase againstp-nitrophenyl esters.
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All measurements were performed in triplicate. ND: not determined.
The effect of temperature on PBS-2 esterase activity was assessed using p -nitrophenyl butyrate as the substrate at temperatures of 5–80℃. Enzyme activity peaked at 30℃ ( Fig. 4 A). The activation energy for enzyme-catalyzed hydrolysis of p -NP butyrate was calculated to be 5.84 kcal/mol at 5–30℃ ( Fig. 4 B); this value was lower than those of other cold-adapted esterases: 11.2 kcal/mol for the esterase of Pseudomonas sp. B11-1 [32] , 9.0 kcal/mol for the esterase of Acinetobacter sp. No. 6 [33] , 11.25 kcal/mol for the esterase of Acinetobacter lwoffii 16C-1 [15] , 6.29 kcal/mol for the esterase of Shewanella sp. Ke75 [18] , and 7.69 kcal/mol for the esterase of Photobacterium sp. MA1-3 [17] . This suggests a high catalytic efficiency at this low temperature range. In fact, PBS-2 esterase showed as much as 55% of its maximum activity at 5℃. These results indicate that PBS-2 esterase is a cold-active enzyme. The enzyme was stable, with >80% residual activity after 30 min of incubation at 50℃. However, it was thermally unstable and lost its activity at temperatures greater than 60℃ ( Fig. 4 C).
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Effects of temperature and pH on the PBS-2 esterase. (A) Hydrolytic activity was measured at various temperatures at pH 8.0. (B) The logarithm of the enzyme turnover rate (k) (s-1) was plotted against the reciprocal of absolute temperature (T) (C) Residual hydrolytic activity was measured after exposure to various temperatures for 30 min. (D) Hydrolytic activity at various pH values was measured at 30℃. (E) Residual hydrolytic activity was measured after exposure to various pH values for 30 min. All measurements were performed in triplicate.
The activity of the recombinant PBS-2 esterase was evaluated under buffered conditions at pH values of 4 to 11. The enzyme exhibited at least 70% of its maximal activity at pH 8.0–10.0, with the highest activity at pH 9.0, indicating it to be an alkaline enzyme ( Fig. 4 D). Although its esterase activity differed somewhat depending on the incubation buffer used, the PBS-2 esterase was fairly stable at a pH range of 7.0 to 10.0, but its activity was markedly reduced below pH 5.0 when incubated for 1 h at room temperature ( Fig. 4 E).
- Effects of Metal Ions, Organic Solvents, and Detergents on Esterase Activity
The effects of metals and inhibitors have been investigated with respect to the suitability of lipases/esterases for industrial applications ( Table 3 ). PBS-2 esterase activity decreased by 50% in the presence of Cd 2+ . Moreover, compared with the control, esterase activity was severely inhibited by Cu 2+ and Hg 2+ , whereas ethylenediaminetetraacetic acid (EDTA) had no effect, suggesting that this esterase is not a metalloenzyme. The effect of phenylmethylsulfonyl fluoride (PMSF), a catalytic serine enzyme modifier that can irreversibly inhibit esterase activity, was also investigated. PBS-2 esterase was significantly (95%) inhibited by 5 mM PMSF. This result suggests that the serine residue in the catalytic triad of PBS-2 esterase was modified by PMSF, resulting in loss of its activity. Thereafter, the effects of various organic solvents and detergents were determined ( Tables 4 and 5 ). The PBS-2 esterase was highly stable in the presence of 30% methanol, ethanol, isopropanol, dimethyl sulfoxide (DMSO), acetone, and PEG-8000, but 30% acetonitrile inhibited the activity of PBS-2 esterase to 40% of the baseline value. Stability in the presence of organic solvents is a requisite property of enzymes used in organic synthesis in non-aqueous systems. With the exception of SDS, the tested detergents (1.0%) did not inhibit PBS-2 esterase activity.
Effects of metal ions and inhibitors on esterase activity.
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Activity in the absence of metal ions was set as 100%. All measurements were performed in triplicate.
Effects of organic solvents on enzyme activity.
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All measurements were performed in triplicate.
Effects of detergents on enzyme activity.
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All measurements were performed in triplicate.
In this study, a novel esterase produced by Paenibacillus sp. PBS-2, which showed similarity to β-lactamases, was characterized and found to exhibit high activity at low temperatures. In addition, it retained strong lipolytic activity in the presence of various organic solvents and detergents. These results suggest that this enzyme has considerable potential for application as a biocatalyst and detergent additive for use at low temperatures. Further work will establish the structure of this enzyme to gain more information about its catalytic mechanism.
Acknowledgements
This work was supported by a grant from the National Fisheries Research and Development Institute (NFRDI), Republic of Korea.
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