Staphylococcus haemolyticus lipase: biochemical properties, substrate speci city and gene cloning

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1 FEMS Microbiology Letters 179 (1999) 385^392 Staphylococcus haemolyticus lipase: biochemical properties, substrate speci city and gene cloning Byung-Chul Oh a, Hyung-Kwoun Kim a, Jung-Kee Lee a, Sun-Chul Kang b, Tae-Kwang Oh a; * a Microbial Enzyme Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon , South Korea b Department of Biotechnology, Taegu University, Taegu , South Korea Received 1 June 1999; received in revised form 23 August 1999; accepted 25 August 1999 Abstract Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kda by SDS-PAGE. Its optimum temperature and ph for the hydrolysis of olive oil was 28³C and ph 8.5, respectively. The enzyme was stable up to 50³C in the presence of Ca 2 and over the ph range 5^11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a preproenzyme of 711 amino acids. The preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49^67% amino acid sequence homology with other staphylococcal lipases. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Lipase; Staphylococcus haemolyticus; Triglyceride; Sequence homology 1. Introduction Lipases are a class of enzymes that catalyze the hydrolysis of long chain triacylglycerols. Many lipases have industrially important properties such as chain length selectivity, regiospeci city, and chiral selectivity. Therefore, the enzymes are often used for the production of free fatty acids, interesteri cation of fats and oils, and synthesis of useful esters and peptides. * Corresponding author. Tel.: +82 (42) ; Fax: +82 (42) ; Many staphylococci are able to produce extracellular lipases and some of them have been puri ed and their biochemical properties studied in detail [1,2]. In contrast to most other lipases, some staphylococcal lipases are distinguished by their extremely broad substrate speci city. Staphylococcus hyicus lipase (SHL) hydrolyzes neutral lipids almost irrespective of their chain length. In addition, it has a high phospholipase activity, which distinguishes the enzyme not only from other staphylococcal lipases but also from all bacterial lipases. Staphylococcal lipase genes have been identi ed from S. aureus [3,4], S. epidermidis [5,6], and S. hyicus [7]. These enzymes are produced as preproen / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (99)

2 386 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^392 zyme forms, which have molecular masses of approximately 80 kda. After secretion into the growth medium, proteolytic processing results in mature forms with molecular masses of 40^46 kda [8]. Many comparative studies of S. aureus lipase (SAL) and SHL have been done and in spite of high homology (50^51%) in amino acid sequences, they have found remarkable di erences in ph optimum, Ca 2 a nity, and substrate speci city [4,9]. In vivo chimeragenesis using SAL and SHL revealed that phospholipase activity in SHL was determined by three regions in its C-terminal domain and that chain length selectivity was de ned by elements within the region of residues 180^250 in SHL [10]. We isolated a S. haemolyticus strain producing an extracellular lipase from sewage treatment plants in Korea. In this paper the lipase enzyme was puri ed and its biochemical properties were studied. In addition, its corresponding gene was cloned and sequenced. We also compared its substrate speci city and amino acid sequence with other staphylococcal lipases. 2. Materials and methods 2.1. Puri cation of the lipase L62 S. haemolyticus L62 was cultivated in LB medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) at 37³C for 20 h. After ammonium sulfate was added to the culture supernatant to 80% saturation, the precipitate was collected by centrifugation at Ug for 30 min, dissolved in Tris-HCl bu er (20 mm, ph 8.0), and then dialyzed against the same bu er. The dialysate was applied to a DEAE-Sepharose CL-6B column (2.0U20 cm) pre-equilibrated with the same bu er. Although most proteins in the dialysate bound to the resin, lipase enzyme passed through the column. The active fractions were collected and re-loaded to a CM-Sepharose CL-6B column (1.0U20 cm) pre-equilibrated with the same Tris bu er. The bound proteins were eluted with a linear gradient of NaCl (0^0.5 M) in the same bu er. The active fractions were pooled, desalted, and then applied to a Resource S column (Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with 20 mm Tris bu er (ph 7.8). The lipase enzyme was eluted with a 0.3^0.4 M linear gradient of NaCl in the same bu er, concentrated, and stored at 320³C Lipase assay Lipase activity was measured by titrating free fatty acids released by hydrolysis of olive oil using the phstat method [11]. Olive oil emulsion was prepared by emulsifying 5 ml of olive oil in 495 ml of 20 mm NaCl, 1 mm CaCl 2, and 0.5% (w/v) gum arabic solution for 2 min at maximum speed in a Waring blender. After the ph of the substrate emulsion (20 ml) was adjusted to 8.5 by the addition of 10 mm NaOH solution, an appropriate amount (10^50 Wl) of the enzyme solution was added. The release rate of the fatty acid was measured with a ph titrator (718 Stat Titrino, Metrohm, Switzerland) for 5 min at 28³C. One lipase unit is de ned as the amount of enzyme liberating 1 Wmol of fatty acid per minute. Lipase activity for p-nitrophenyl esters was measured spectrophotometrically [12,13]. One milliliter of p-nitrophenyl esters (C 2^C 12, 10 mm in acetonitrile) was mixed with ethanol (4 ml) and 95 ml of 50 mm Tris-HCl (ph 8.5). An appropriate amount of the lipase was added to 1 ml of this freshly prepared substrate solution and the OD 405 was measured after 3 min incubation at 28³C. For long chain p-nitrophenyl esters (C 12^C 18 ), 20 Wl of lipase solution was added to 880 Wl of reaction bu er containing 50 mm Tris-HCl (ph 8.5), 0.1% gum arabic, and 0.2% deoxycholate. After 3 min incubation at 28³C, the reaction was initiated by adding 100 Wl of 8 mm substrate in isopropanol. The reaction was stopped by the addition of 0.5 ml of 3 M HCl. After centrifugation, 333 Wl of supernatant was mixed with 1 ml of 2 M NaOH and the OD 420 was measured. One unit of lipase activity was de ned as the amount of enzyme liberating 1 Wmol of p-nitrophenol per minute. Lipolytic activity of a protein on the SDS-PAGE gel was detected using tributyrin (TBN) agar plates. After gel running, the gel was washed sequentially with 50 mm Tris bu er (ph 8.0) containing 1% (v/v) TX-100 and washed twice with 50 mm Tris bu er. The gel was overlaid on TBN agar plate prepared with agar (1.5%) and tributyrin emulsion (1% tributyrin, 20 mm NaCl, 1 mm CaCl 2, and 0.5% (w/v) gum arabic).

3 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^ N-terminal amino acid analysis The puri ed lipase L62 was electrophoretically transferred to a polyvinylidene di uoride (PVDF) membrane (Bio-Rad Lab., Hercules, CA) from SDS-polyacrylamide gel [14]. The lipase band was cut out and its amino acid sequence was analyzed by Edman degradation using an Applied Biosystems model 476A protein/peptide sequencer (Applied Biosystems Inc.) Cloning of the lipase L62 gene To clone the lipase L62 gene, we made two degenerated primers: primer 1: 5P-CAA(G)TAT(C)AAA- (G)AAT(C)AAA(G)TAT(C)CC-3P; primer 2: 5P-G- TT(C)TG(ATGC)CC(ATGC)CCCAT(ATGC)CTA- (G)TG-3P. The nucleotide sequence of primer 1 was designed based on the N-terminal amino acid sequence (Q-Y- K-N-K-Y-P) of the puri ed mature lipase enzyme. In the case of primer 2, the sequence was designed based on the highly conserved sequence around the active site Ser residue. These two primer sets and S. haemolyticus genomic DNA made a 350-bp PCR product. This PCR product was used for Southern hybridization as a probe after labeling with the DIG DNA Labeling Kit (Boehringer Mannheim, Germany). The genomic DNA digested with ClaI and EcoRV showed a hybridization signal at around 4.2 kb. The corresponding DNA fragment was eluted from the gel and ligated with the plasmid pbluescript II SK(+) previously digested with the same enzymes. Escherichia coli XL1 Blue was transformed with this ligation mixture and plated on LB-TBN plates containing 100 Wg Wl 31 ampicillin. After overnight incubation at 37³C, one clear zone-forming colony was selected. S. haemolyticus L62 strain secreted as much as 9800 units l 31 of lipase to the culture medium. The lipase was puri ed from the culture medium by ammonium sulfate precipitation and three successive ion exchange column chromatographies. This enzyme's high pi value (9.7), which was obtained using the Novex Xcell II1 Mini-Cell kit with a pre-cast IEF gel (ph 3^10), was used e ectively in the enzyme puri cation process. The lipase passed through the DEAE column, while most other extracellular proteins including a 34-kDa protease protein bound tightly to the DEAE resin. Fig. 1 shows that a 45- kda protein was puri ed to homogeneity and after its renaturation process, it showed a lipolytic activity on the overlaid TBN agar plate. The speci c hydrolytic activity of the puri ed lipase was 596 U mg 31 and the puri cation yield was calculated to be about 42% of the total activity in the culture medium. The N-terminal 24-amino acid sequence of the 45- kda lipase enzyme was determined to be NH 2 -A-T- I-K-S-N-Q-Y-K-N-K-Y-P-V-V-L-V-H-G-F-L-G-L- V-. Seven amino acid sequences (Q-Y-K-N-K-Y-P) were used to make degenerated primers in our successive cloning experiment. 3. Results 3.1. Puri cation of the lipase L62 Fig. 1. SDS-PAGE (A) and zymogram (B) of S. haemolyticus L62 lipase. A: The enzyme was electrophoresed on a 12% SDS polyacrylamide gel. Lane M: the standard proteins; lane 1: culture supernatant; lane 2: ammonium sulfate precipitates; lane 3: sample after DEAE Sepharose CL-6B chromatography; lane 4: sample after CM Sepharose CL-6B chromatography; and lane 5: sample after Resource S chromatography. B: The 45-kDa protein showed lipolytic activity on TBN agar plate.

4 388 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^392 Fig. 2. E ects of temperature on enzyme activity and stability. A: The enzyme (1 mg ml 31 in 20 mm Tris-HCl, ph 7.8) was preincubated at various temperatures for 30 min in the presence of 10 mm Ca 2 (E) or 5 mm EDTA (F) and the residual activity was measured by the ph-stat method at 28³C and ph 8.5. The enzyme was assayed at various temperatures (b). B: The logarithm of the enzyme turnover rate (k) (s 31 ) was plotted against the reciprocal of absolute temperature (T). The values shown are activation energy calculated from the linear part of the plot Characterization of the lipase L62 The optimum temperature of the lipase L62 was 28³C and its activation energy for the hydrolysis of olive oil was calculated to be 8.63 kcal mol 31 in the range 4^28³C (Fig. 2). This value is much lower than those shown by enzymes from other sources: Antarctic bacteria, 12^kcal mol 31, and mesophilic Pseudomonas aeruginosa, 25 kcal mol 31 [15,16]. This implies that its catalytic e ciency is high in this Fig. 3. E ects of ph on enzyme activity and stability. A: The lipase activity was measured at various phs by the ph-stat method at 28³C. B: For ph stability test, the enzyme was preincubated at various ph bu ers for 24 h at 4³C and the residual activity was measured by the ph-stat method at 28³C and ph 8.5. E, 0.1 M sodium acetate (ph 4^6); a, 0.1 M potassium phosphate (ph 6.5^7.5); F, 0.1 M Tris-HCl (ph 7.5^9); O, 0.1 M KCl-glycine-KOH (ph 9^11); b, 0.1 M potassium phosphate (ph 11^12). temperature range. In fact, the lipase L62 showed at 4³C as much as 30% of the activity shown by it at 28³C. In the range over 28³C, its inactivation energy was determined to be kcal mol 31. The lipase L62 showed Ca 2 -dependent stability at elevated temperatures. In the presence of Ca 2,itis stable up to 50³C and a drastic decrease in stability occurred over 55³C, while its thermal stability was decreased by 10^15³ in the absence of Ca 2. The lipase L62 showed high activity at ph 8.5^9.5 when assayed at 28³C. Although its lipase activity is somewhat di erent depending on the various incubation bu ers used, the lipase L62 was fairly stable for 24 h from ph 5 to ph 11 (Fig. 3). As shown in Fig. 4A, the enzymatic hydrolysis of triacylglycerols by the lipase L62 depend on the acyl chain length in the substrate molecules. Tributyrin, tripropionin, and trimyristin are the preferred substrates whereas changing the length of the acyl chains dramatically reduces its activity. It showed about 2-fold higher activity against 1,3-rac-diolein and a little less activity against 1-rac-monoolein in comparison with triolein. On the other hand, the lipase L62 showed the highest activity toward p-nitrophenyl caprylate (C 8 ) among the synthetic substrates tested (C 2^C 18 ) (Fig. 4B). In addition, it showed no activity against phosphatidylcholine (data not shown), implying that it has no phospholipase activity.

5 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^ Fig. 4. Hydrolytic activity of the lipase L62 for various lipids. A: The hydrolytic activity of the lipase L62 for various chain lengths of triglycerides, 1,3-rac-diolein (1,3-DO), and 1-rac-monoolein (1-MO), was measured by the ph-stat method using each substrate emulsion (20 mm) prepared as described in Section 2 for olive oil emulsion. B: Its hydrolytic activity for p-nitrophenyl esters was measured by the spectrophotometric method. Two different assay methods were used for short chain (C 2^C 12, E) and long chain substrates (C 12^C 18, F) as described in Section Cloning and nucleotide sequence of the lipase L62 gene An E. coli transformant forming a clear halo on the TBN agar plate was selected as described previously. The recombinant plasmid (pshl) isolated from this transformant had about 4.2 kb insert DNA. Computer analysis found one major open reading frame of 2136 bp, which encodes a polypeptide of 711 amino acid residues (Fig. 5). The region upstream of the initiation codon ATG at position 1 contained a putative ribosome binding site and 335 and 310 promoter sequences. A 60-amino acid signal sequence and a cleavage site between Ala-60 and Ala-61 were con rmed by N-terminal amino acid analysis of the lipase puri ed from E. coli XL1 Blue (pshl). In addition, a 259-amino acid pro-region and a cleavage site between Glu-319 and Ala-320 were also determined by N-terminal amino acid analysis of the 45-kDa mature lipase puri ed from the S. haemolyticus strain. Therefore, the deduced sequence of the mature lipase contains 392 amino acids and corresponds to a molecular mass of Da. In our cloning experiment, E. coli XL1 Blue strain transformed with the plasmid (pshl) carrying the lipase L62 whole gene showed low lipase activity. This low lipase activity in the recombinant E. coli strain in comparison with the original staphylococcal strain (L62) was possibly caused by a low expression level of this staphylococcal gene in the E. coli system. In fact, we detected active prolipase protein from cell-free extract of the transformed E. coli (data not shown), implying that no processing of the proenzyme occurred in E. coli cells, although the N-terminal signal sequence of the expressed preproenzyme was correctly removed. The predicted amino acid sequence of S. haemolyticus lipase was compared with those of other staphylococcal lipases using the Clustal method. The mature part of the lipase L62 has 67%, 53%, 54%, 54%, and 49% homologies with S. aureus PS54, S. aureus NCTC8530, S. epidermidis RP62A, S. epidermidis 9 and S. hyicus lipases, respectively. In the case of preproenzyme, the total sequence of the lipase L62 was shown to have 45%, 37%, 38%, 38%, and 35% homologies with the same staphylococcal lipases, respectively. Sequence alignment of the lipase L62 with those staphylococcal lipases showed that Ser-119 in the mature enzyme is located in the conserved G-X- S-X-G sequence around the active site serine residue (Fig. 6). In addition, Asp-310 and His-352 are thought to be members of a catalytic triad of this enzyme together with Ser Discussion S. haemolyticus L62 strain produced and secreted as much as 9800 U l 31 of a 45-kDa lipase to the growth medium. The amount of the lipase L62 was calculated to be about 14% of the total extracellular proteins on the basis of the speci c activity (596 U mg 31 ). But from the protein band on the gel presented in Fig. 1, it looks as much as 40% of the total protein (Fig. 1A, lane 2). This discrepancy can be

6 390 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^392 explained from two postulations: that the lipase L62 was secreted as a complex with some compound, which complex has been already reported from several lipases [17] and that this `lipase complex' has much lower lipolytic activity in comparison with the puri ed enzyme. This lipase complex was evidenced from our two experimental results. First, the lipase L62 in culture supernatant was resistant to the attack of the endogenous 34-kDa protease, whereas the puri ed enzyme was rapidly degraded by it. Second, the total lipase activity was rather increased after ammonium sulfate precipitation in the puri cation process (data not shown). The cloned lipase gene suggests that the staphylococcal lipase is produced in an 80-kDa preproenzyme form, secreted, and processed rapidly to the 45-kDa Fig. 5. Nucleotide sequence of the L62 lipase gene and its deduced amino acid sequence. The numbering of nucleotides starts at the 5P end of the lipase gene and that of amino acids at the N-terminus of the preproenzyme. The putative 335, 310, ribosomal binding site (rbs), and stop codon (*) are shown. The N-terminal amino acid sequence from the puri ed proenzyme and mature enzyme are underlined. The sequence has been submitted to GenBank under accession number AF

7 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^ Fig. 6. Primary structure alignment of the mature lipases from S. haemolyticus (a), S. aureus PS54 (b), S. aureus NCTC8530 (c), S. epidermidis (d) and S. hyicus (e). In this alignment, the numbering starts at the N-terminal residue of the mature sequences. Amino acids conserved in all ve lipases (*) and the putative active site residues (2) are indicated. Arrows indicate the Ca 2 binding sites (u) and the Ser- 356 residue (v) in SHL. mature enzyme in this staphylococcal strain. In our experiment, neither prepro- nor proenzyme bands could be detected by Coomassie staining or activity staining of the SDS-PAGE gel. The optimum temperature for the lipase L62 was determined to be 28³C, however, it showed as much as 30% activity at 4³C (Fig. 2). This high activity at low temperature makes the enzyme applicable as an additive to detergents used at low temperatures and as a biocatalyst for biotransformation of labile compounds at cold temperatures. Sequence alignment using the Clustal method showed the lipase L62 has high homologies (49^ 67%) with other staphylococcal lipases, whereas it has less than 30% homology with all other bacterial lipases. This result shows that on the basis of sequence homology, the lipase L62 together with other staphylococcal lipases seems to make a separate lipase group. Although SAL and SHL have high homology (50^51%) in their amino acid sequences, their enzymatic properties such as substrate speci city are very di erent. This discrepancy in substrate speci city between these two homologous enzymes has not been explained clearly, for no X-ray crystal structure for the staphylococcal lipase has been elucidated yet. However, Nikoleit and co-workers showed that the C-terminal 146 amino acids of SHL are required for its phospholipase activity [4]. After that, Kampen and co-workers showed that three regions in the C-terminal domain of SHL were responsible for phospholipase activity and a region of residues 180^250 in SHL for its broad chain length selectivity [10]. Recently, they suggested that the Ser-356 residue plays an important role in determining phospholipase activity in SHL [18]. In the case of the lipase L62, it has no phospholipase

8 392 B.-C. Oh et al. / FEMS Microbiology Letters 179 (1999) 385^392 activity and shows relatively high hydrolytic activity for short chain length triglycerides such as tributyrin and tripropionin (Fig. 4) like SAL. This similarity in substrate speci city between the lipase L62 and SAL correlates well with their relatively high homology in total sequences (67%) or C-terminal part (146 amino acids) sequence (56%). In particular, the lipase L62 and SAL have Leu and Val residues, respectively, in the position matching the Ser-356 of SHL. On the other hand, Simons and co-authors showed that Asp-354 and 357 could be Ca 2 binding sites in SHL by site-directed mutagenesis in combination with isothermal titration calorimetry and they suggested that the calcium binding to the residues is important for its structural stabilization at elevated temperatures [19]. The lipase L62 was also stabilized at elevated temperatures by calcium binding (Fig. 2). Sequence alignment of the lipase L62 with SHL suggested that residues Asp-351 and Asp-354 are calcium binding sites in the lipase L62. But to determine de nitely the motifs involved in calcium binding or substrate binding in the lipase L62 enzyme, more detailed biochemical studies in combination with X-ray crystallography are needed. References [1] Go«tz, F., Verheij, H.M. and Rosenstein, R. (1998) Staphylococcal lipases: molecular characterisation, secretion, and processing. Chem. Phys. Lipids 93, 15^25. [2] Simons, J.-W.F.A., Go«tz, F., Egmond, M.R. and Verheij, H.M. (1998) Biochemical properties of staphylococcal (phospho)lipases. Chem. Phys. Lipids 93, 27^37. [3] Lee, C.Y. and Iandolo, J.J. (1986) Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. J. Bacteriol. 166, 385^391. [4] Nikoleit, K., Rosenstein, R., Verheij, H.M. and Go«tz, F. (1995) Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Eur. J. Biochem. 228, 732^738. [5] Farrell, A.M., Foster, T.J. and Holland, K.T. (1993) Molecular analysis and expression of the lipase of Staphylococcus epidermidis. J. Gen. Microbiol. 139, 267^277. [6] Simons, J.-W.F.A., Kampen, M.D., Riel, S., Go«tz, F., Egmond, M.R. and Verheij, H.M. (1998) Cloning, puri cation and characterisation of the lipase from Staphylococcus epidermidis. Eur. J. Biochem. 253, 675^683. [7] Go«tz, F., Popp, F., Korn, E. and Schleifer, K.H. (1985) Complete nucleotide sequence of the lipase gene from Staphylococcus hyicus cloned in Staphylococcus carnosus. Nucleic Acids Res. 13, 5895^5906. [8] Rollof, J. and Normark, S. (1992) In vivo processing of Staphylococcus aureus lipase. J. Bacteriol. 174, 1844^1847. [9] Simons, J.-W.F.A., Adams, H., Cox, R.C., Dekker, N., Go«tz, F., Slotboom, A.J. and Verheij, H.M. (1996) The lipase from Staphylococcus aureus. Eur. J. Biochem. 242, 760^769. [10] Kampen, M.D., Dekker, N., Egmond, M.R. and Verheij, H.M. (1998) Substrate speci city of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis. Biochemistry 37, 3459^3466. [11] Kim, H.-K., Park, S.-Y., Lee, J.-K. and Oh, T.-K. (1998) Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Biosci. Biotechnol. Biochem. 62, 66^71. [12] Lesuisse, E., Schanck, K. and Colson, C. (1993) Puri cation and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic ph-tolerant enzyme. Eur. J. Biochem. 216, 155^160. [13] Winkler, U.K. and Stuckmann, M. (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J. Bacteriol. 138, 663^670. [14] Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene di uoride membranes. J. Biol. Chem. 262, 10035^ [15] Feller, G., Thiry, M., Arpigny, J.L., Mergeay, M. and Gerday, C. (1990) Lipases from psychrotrophic antarctic bacteria. FEMS Microbiol. Lett. 66, 239^244. [16] Choo, D., Kurihara, T., Suzuki, T., Soda, K. and Esaki, N. (1998) A cold-adapted lipase of an alaskan psychrotroph, Pseudomonas sp. strain B11-1: Gene cloning and enzyme puri cation and characterization. Appl. Environ. Microbiol. 64, 486^491. [17] Jaeger, K.E., Adrian, F.J., Meyer, H.E., Hancock, R.E.W. and Winkler, U.K. (1992) Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein. Biochim. Biophys. Acta 1120, 315^321. [18] Kampen, M.D., Simons, J.-W.F.A., Dekker, N., Egmond, M.R. and Verheij, H.M. (1998) The phospholipase activity of Staphylococcus hyicus lipase strongly depends on a single Ser to Val mutation. Chem. Phys. Lipids 93, 39^45. [19] Simons, J.-W.F.A., Kampen, M.D., Ubarretxena-Belandia, I., Cox, R.C., Santos, C.M.A., Egmond, M.R. and Verheij, H.M. (1999) Identi cation of a calcium binding site in Staphylococcus hyicus lipase: Generation of calcium-independent variants. Biochemistry 38, 2^10.

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