Bacterial lipases for biotechnological applications

JOURNAL OF MOLECULAR CATALYSIS 6: ENZYMATIC zyxwvutsrqpon ELSEVIER Journal of Molecular Catalysis B: Enzymatic 3 (1997) 3-12 Bacterial lipases for biotechnological applications Karl-Erich Jaeger a**, Bernd Schneidinger a, Frank Rosenau a, Michael Werner a, Dietmar Lang b, Bauke W. Dijkstra b, Klaus Schimossek ‘, Albin Zonta ‘, Manfred T. Reetz ’ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP li Lehrstuhl Biologic der M ikroorganismen, Ruhr-Unirersitiit, D- 44780 Bochum, Germany h Laborator?j of Biophy sical Chemistm. 9747 AC Groningen, The Netherlands ’ Max-Planck Institutftir Kohlenforschung, D-45470 Miilheim, German! Received 15 July 1996; accepted I3 September 1996 Abstract Lipase genes originating from the Gram-negative bacteria Serrutiu marcescens and Pseudomonus urruginosa were lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and cloned. S. marcescens finally refolded to give enzymatically active lipase. The lipase operon of P. aeruginosa consisting of genes 1ipA and lip14 P. aeruginosa strain carrying a was cloned behind the T7 410 promoter and overexpressed in a lipase-negative chromosomal insertion of the gene encoding T7 RNA polymerase. A 3D structural model was built for P. aeruginosu lipase using the coordinates of the Burkholderiu cepacia lipase structure which has recently been solved in its open conformation by X-ray crystallography. Both lipases have been purified to homogeneity and were tested for their potential to catalyze biotechnologically important reactions. S. marcescens lipase stereoselectively hydrolyzed racemic isopropylideneglycerol acetate which is a basic building block in a variety of organic synthesis reactions. P. aeruginosa lipase was successfully used for kinetic resolution of chiral alcohols and amines giving enantiomeric excess values of 2 95% at reaction rates of 40-50%. Our results demonstrate that both lipases can be produced at levels of 100 mg/l for S. marcescens and 150 mg/l for P. arruginosa. The recombinant lipase proteins are promising candidates for biotechnological applications. Keywords: Lipase; Serratia marcescens; Pseudomonas aeruginosa; Overexpression; 3D structural model; Kinetic resolution 1. Introduction The estimated world-wide sales volume for industrial enzymes in 1995 is US$ 1 billion and this volume is foreseen to double until 2005 [l]. At least 75% of all these enzymes are hydrolases, and 90% of them are produced from microorganisms by fermentation. Following ’ Corresponding author. Phone: (49) 234 700 3101, fax: (49) 234 7094 425, e-mail: karl-erich.jaeger@rz.ruhr-uni-bochum.de. 138 I I 17/97/$17.00 Copyright P/I S 138 I-1 177(96)00039-2 proteases and carbohydrases, lipases are considered to be the third largest group based on total sales volume. Their biotechnological potential is relying on their ability to catalyze not only the hydrolysis of a given triglyceride, but also its synthesis from glycerol and fatty acids. The main application fields for lipases include detergents, dairy, diagnostics, oil processing, and biotransformations. Recently, special emphasis is lying on the production of chiral chemicals which serve as basic building blocks in the 0 1997 Elsevier Science B.V. All rights reserved 4 K.-E. Jaeger et al./ Joumal of M olecular Cataly sis B: Enzy matic 3 (1997) 3- 12 production of pharmaceuticals, agrochemicals, and pesticides or insecticides [2]. In a survey of enzyme supplier companies, 136 companies were listed which produced at least 23 different lipases, however, only 6 of them were of bacterial origin [I]. This underrepresentation stands in a sharp contrast to the actual knowledge in molecular biology and biochemistry of these lipases [3,4]. In this report, we present a summary of recent results obtained with extracellular lipases from the Gram-negaand Pseutive species Serratia marcescens prehensive studies on biotechnological applications of these two enzymes, we have constructed overexpression systems for both genes. The overexpressed proteins have been isolated and tested for their ability to catalyze the kinetic resolution of racemic substrates. domonas 2.1 .I. aeruginosa. lipase is a protein of M , 64800 which was purified to electrophoretic homogeneity. The structural gene was cloned and sequenced and the mechanism of secretion of this enzyme has been studied [5-71. However, overexpression of this lipase was not reported. P. aeruginosa lipase (M ,: 29000) is encoded by a structural gene ZipA which forms an operon with a second gene ZipH encoding a lipasespecific foldase which is involved in lipase folding and/or secretion. Both genes have been overexpressed and the corresponding proteins were purified and biochemically characterized [8]. Recently, experimental evidence was obtained for a cell-density dependent type of regulation called ‘quorum sensing’ [9] of lipase gene expression involving a regulatory protein LipR S. marcescens DOI. Only few biotechnological applications of these enzymes have been reported. The asymmetric hydrolysis of 3-phenylglycidic acid ester which is a key intermediate in the synthesis of Diltiazem hydrochloride, a widely used coronary vasodilator, was carried out with S. marcescens lipase [l I]. P. aeruginosa lipase was able to convert the diester dimethyl 5(3-(2(7-chlorochinolin-2-yl)ethyl)phenyl)4,6_dithianoanedioate to its (S&ester acid which is a chiral intermediate in the synthesis of the leukotriene receptor agonist Verlukast [12]. The production of this lipase has been scaled up to pilot plant bioreactors (volume: 1900 1) by Merck US [ 131. In order to enable more com- 2. Results and discussion 2.1. Lipase from Serratia O verexpression, marcescens purification and refold- ing t141 A fragment of chromosomal S. marcescens DNA carrying the lipase gene was cloned into the T7-expression vector pETlld and overexpressed in E. coli JM109/DE3. The vector contains a T7 promoter and the host strain a chromosomally encoded T7 RNA polymerase which are both inducible by addition of isopropyl-B-D-thiogalactopyranoside (IPTG) [ 151. Bacteria were grown until they reached the logarithmic growth phase (OD,,, = 0.6) and lipase gene expression was induced by addition of 0.4 mM IPTG. After further 3 h of growth lipase was produced at about 40% of total cellular protein. However, the overexpressed protein was not secreted but formed insoluble inclusion bodies inside the bacterial cytoplasm. These inclusion bodies were purified by centrifugation, denaturation in 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM Na,-EDTA, and 8 M urea, and subsequent gel filtration, and were finally refolded to enzymatic activity by rapid dilution in 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM Na,-EDTA, and 10 mM CaCl, [14]. Fig. 1 shows an SDS polyacrylamide gel electrophoresis analysis at various stages of the purification procedure. After gel filtration, the silver-stained gel (lanes 4-6) revealed only one major protein band at M , 64000 (theoretical M ,: 64800). Its identity was proven after blotting of this protein band by determination of the N-terminal amino acid sequence (amino acids no. l-20) showing 100% identity with the se- K.-E. Jaeger et al. / Journal of M olecular Cataly sis B: Enzy matic 3 11997) 3- 12 Fig. 1. Purification of S. marcescens lipase. Samples obtained at various stages of the purification procedure were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue R250 (lanes 1, 2, and 3 containing 10 kg of protein) and silver (lanes 4, 5, and 6 containing 5 (*g of protein). The band showing lipase (M ,: 64800) is marked with an arrow. Samples were obtained from: cell lysate obtained by ultrasonication (lane 1); soluble fraction (lane 2) and sediment (lane 3) after centrifugation (Sorvall RCSB, Rotor SS34, 6000g for 1.5 min at 4°C); gel filtration chromatography (Superdex TM200 column) (lane 4); ultracentrifugation (Kontron TGA 65. rotor SW 40. 100000~ for 60 min at 4°C) and refolding (lane 5); concentration of refolded lipase by ultrafiltration (lane 6). quence deduced from the DNA sequence of the cloned gene [5]. The ability to refold extracellular lipase to enzymatic activity enabled us to isolate significant amounts of protein (100 mg per liter of culture) which could be purified in a one-step purification giving electrophoretically homogeneous lipase protein with a yield of 30%. Recently, we have obtained a polyclonal antiserum against this lipase which will be used to study localization inside the bacterial cell. Furthermore, lipase has been subjected to crystallization experiments in order to determine its 3D structure by X-ray crystallography. 2. I .2. Secretion S. marcescens lipase does not possess an N-terminal signal sequence but instead contains multiple repeats of a glycine-rich consensus sequence (L-X-G-G-X-G-X-D) at the Cterminus. This sequence is indicative for a secretion system which consists of three different proteins, an ABC transporter (ABC), a membrane fusion protein (MFP), and an outer membrane component forming a pore-like structure (OMP) extending from the cytoplasm into the extracellular space. The corresponding genes are organized in an operon with the structural gene followed by the genes coding for ABC and MFP whereas the gene coding for OMP may be separated [ 161. We could demonstrate that S. marcescens lipase was secreted from E. coli if the strain contained a plasmid carrying either the prt D, E, and F genes for metalloprotease secretion in Envinia chrysanthemi or hlyB, C, and tolC genes for ol-hemolysin secretion in E. coli coding for the ABC, MFP, and OMP proteins [5]. More recently, an operon consisting of three genes named ZipB, C, and D was cloned from S. marcescens. Expression of these three genes in E. coli allowed secretion of both metalloprotease and lipase of S. marcescens [7]. Although these authors have studied secretion only in E. coli, genes lipB, C, and D presumably encode an ABC-secretion system used in S. marcescens. We have recently constructed a S. marcescens mutant strain carrying an insertion in the 1ipB gene coding for the ABC-transporter which exhibited a lipase- and proteasenegative phenotype [ 141. K.-E. Jaeger et al./ Journal of M olecular Cataly sis B: Enzy matic 3 (1997) 3- 12 6 2.1.3. Biotechnological applications Attempts to use S. marcescens lipase for synthesis reactions in organic solvent failed so far. At present, we assume that this lipase may need a water shell surrounding the molecule, and its replacement by organic solvent may cause irreversible denaturation. This view is supported by the finding that freeze-drying of S. marcescens lipase led to a decrease in enzyme activity by 75%. However, this lipase may be useful for kinetic resolution of chiral compounds. Racemic isopropylideneglycerol acetate (IPG) which is a basic building block in a variety of organic synthesis reactions was hydrolyzed at a reaction rate of about 25% giving a 35% enantiomeric excess of the D-enantiomer. Furthermore, we have observed the stereoselective esterification of the racemic alcohol ( R,S)-2-phenyl- 1-propan01 at a conversion rate of 3 1% and an enantiomeric excess of 20% of the ester isomer. At we are trying to immobilize S. present, marcescens lipase by using a newly developed method [17] in order to stabilize the enzyme thereby improving its biotechnological applications in organic chemistry. 2.2. Lipase from Pseudomonas 2.2.1. Cloning aeruginosa and overexpression Initially, we used a two-component T7 system developed by Darzins [ 181 to overexpress P. aeruginosa lipase. The gene coding for T7 RNA polymerase is stably integrated into the chromosome of P. aeruginosa strain ADD1976 and a 2.8 Kb XmnI/SmaI fragment of P. aeruginosa DNA containing genes ZipA and H necessary to obtain enzymatically active lipase were cloned into the SmaI-site of plasmid pEB 12 giving pELl1 with the lipase genes being expressed from the T7 gene10 promotor. Fig. 2B shows that we obtained an increase in extracellular lipase (mass of enzymatically active protein) of at least three orders of magnitude. In order to further improve this expression level and, in addition, to use this system for overex- pression of mutated lipase genes we constructed a novel host strain. A transposable element containing a ZacUV5 /ZacI q-regulated T7 RNA polymerase gene [ 183 was stably integrated into the chromosome of P. aeruginosa strain PABS which carries a deletion in the lipase structural P. aeruginosa gene ZipA to give strain PABST7.1 (Fig. 2A). In addition, a 2.8 Kb fragment containing genes ZipA and H was cloned into a pUC18-derived broad host range vector called pUCPKS which also contains the T7 gene10 promotor [19] giving pUCPL7. When P. aeruginosa strain PABST7.1 /pUCPL7 was grown in batch cultures (10 ml tryptone phosphate medium in Erlenmeyer flasks shaken at 180 rpm and 30°C) the yield of extracellular lipase was 0.15 g per 1 of culture supematant without any further optimization of media and growth conditions. At present, we are trying to set up a pilot-scale production protocol for extracellular P. aeruginosa lipase by high-cell density fermentation of this overexpressing strain. 2.2.2. 30 structural model A 3D structural model for P. aeruginosa lipase had previously been constructed [20,21]. However, this model was based on the X-ray structure of Burkholderia glumae (formerly P. glumae) lipase [22] which was solved in the closed conformation and could therefore not provide detailed information on the nature of the active site and substrate binding domains. This was the reason to build a new 3D structural model (Fig. 3) by using the X-ray coordinates P. of the lipase from B. cepacia (formerly cepacia) the structure of which was solved in its open conformation [24]. Equivalent amino acid residues in this structure have been replaced using the program 0 [25] and some of the side chain positions were fitted manually to prevent collisions between adjacent amino acid residues. Subsequently, 40 cycles of conjugate gradient minimisation were executed using the program XPLOR [26] and the final coordinates were analyzed with PROCHECK [27]. The resulting K.-E. Jaeger et al./Journal of Molecular Catal\sis B: Enzymatic 3 (1997) 3-12 attR lad Polymerase infection of Pseudomonas aeruginosa PABS t isolation of tet’ Cb” clones B / 1.000 3 5 z E x z 100 10 i E 1 : g ‘- 0.1 0,Ol Pseudomonas aeruginosa strains Fig. 2. Overexpression of lipase in P. aeruginosa. (A) Strategy to construct a host strain for overexpression of lipase by mserting the bacteriophage D3112-based transposable element mini-D31 12 (shown in large brackets, see also [ 181) containing the gene encoding RNA polymerase into the chromosome of the lipase-negative strain P. aeruginosa PABS giving expression strain P. aeruginosa PABST7.1. (B) Overexpression levels given in mg of enzymatically active lipase protein per I of P. aeruginosa culture supernatant obtained after cultivation of different strains as described in the text. Note that the ordinate is drawn to a logarithmic scale. 8 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA K.-E. Jaeger et al./Journal of M olecular Cataly sis B: Enzy matic 3 (1997) 3- 12 Fig. 3. 3D structural model of P. aeruginosa lipase shown as a ribbon plot drawn with MOLSCRIPT [23]. The catalyti c triad residues S82, D229, and H251 and residues Cl83 and C235 forming a disulfide bond are numbered and drawn in a ball-and-stic :k presentation. The Ca’+-ion is shown as a green ball. Amino-(N) and carboxyterminus of the lipase are indicated. Ramachandran plot showed a phi/psi distribution of conformational angles in the allowed regions with the exception of the active site serine (Ser-82): this residue is located at the so-called nucleophilic elbow which is a (3-eSerOLmotif placing the nucleophilic serine into the active site [28]. This motif has also been found in structures of hydrolytic enzymes sharing the characteristic cxy/p hydrolase fold [29]. The features of the actual model further include a core structure of six central P-strands with one of them divided into a parallel and an antiparallel part and a catalytic triad which is now completely surface-accessible and consists of amino acid residues Ser-82, Asp-229, and His-251. A Ca*+-ion (shown as a green ball in Fig. 3) is bound close to the active site and is stabilized by cooperative binding to residues Asp-209, Asp-253, and main chain oxygens O-257 and O-261. One disulfide bridge is formed between residues Cys-183 and Cys-235. A loop extending from Pro-122 to Leu-156 which contains an a-helix could serve as a possible lid covering the active site in the closed conformation. Based on this 3D structural model we have already introduced by site-directed mutagenesis ten different mutations into the lipase gene thereby exchanging amino acid residues involved in the K.-E. Jaeger et al. / Journal of M olecular Cataly sis B: Enzy matic 3 (1997) 3- 12 formation of the active site pocket in order to test their role in enzyme activity, substrate binding, and specificity of P. aemginosa lipase. Table I Enantioselective conversion 2.2.3. Biotechnological applications P. aeruginosa lipase hydrolyzed triglyceride substrates with a high stereoselectivity towards of amines by P. aeruginosa lipase ubstrate conversion rate0 [%I enantiomeric excess of amide PI -butylamine 55 32 47 96 28 >98 43 81 -pentylamine -methyl-Z-pentyhamine Ikctylamine N-amino-l-phenylbutane 93 -cyclohexylethylamine 38 *9b I-phenylethyhamine 30 98b 32 99 20 >99 b I~,3,4-tetmhydro-llaphtylamine I-naphtylethylamine NH2 63 9 \\ // a Values calculated from GC peaks represent area percent with the exception of N-acetyl-2-pentylamide b (RI-enantiomer determined by GC coinjection of amides obtained by amidation of the corresponding which is mol percent. optically pure amines. 10 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA K.-E. Jaeger et al./ Journal of M olecular Cataly sis B: Enzy matic 3 (1997) 3- 12 position sn-1 [30,31] which made it an interesting candidate for biotechnological applications. Recently, we have immobilized this lipase and used it for the kinetic resolution of two racemic model compounds, 1-phenylethanol and 2pentylamine, respectively. Both compounds were enantioselectively acylated with high efficiency giving enantiomeric excesses of > 99% ee for the alcohol and 96% ee for the amine with average conversion rates of 50% [32]. These results led us to further investigate the potential of P. aeruginosa lipase for stereoselective conversion of a variety of amines, as well as primary and secondary alcohols. Culture supernatants obtained from lipase-overexpressing and strains P. aeruginosa ADD1976/pELll PABST7.1 /pUCPL7 were sterile-filtered, concentrated by ultrafiltration, and freeze-dried giving crude lipase powder. This powder was suspended in tert-butyl methyl ether (for 2pentylamine in diethyl ether), and amines were Table 2 Enantioselective conversion acylated with ethyl acetate or alcohols with propionic anhydride by shaking at room temperature. Periodically, aliquots were withdrawn and analyzed by gas chromatography as described previously [32]. Table 1 shows that all amines tested were acylated to give the corresponding amides, although at differing conversion rates when compared after 48 h of reaction time. High enantiomeric excess rates exceeding 90% ee were usually obtained. Interestingly, those amines possessing bulky cyclohexyl-. or phenyl-groups ( 1-cyclohexylethylamine, lphenylethylamine, 1,2,3,4-tetrahydro1-naphthylamine, I-naphthylethylamine, and 1 aminoindane) were found to be efficiently acylated giving ee-values of 2 95%. An exception was 3-amino- 1-phenylbutane which reacted slowly (conversion rate: 26%) and with a lower ee (93%) than the other amines. It is to early to speculate about the detailed interactions between substrate molecules and selected amino of alcohols by P. aeruginosa lipase Substrate reaction time conversion rate [hl 1x1 alcohol recovered isomer’ ee[%] ester produced isomerb ee[%] (R+!+l-phenyl-lpropanol OH (R+S)-1-phenyl-2propanol 8r”” 6 49 s 73 6 53 S 22 (Rs)-2-phenyl-lpropanol a Determined by GC coinjection b Determined by GC coinjection n.d.: not determined. of the optically pure alcohol. of esters obtained by esterification of the optically pure alcohols. R 97 R 25 K.-E. Jaeger et al. / Journal II zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED of Molecular Catalysis 9: Enzymatic 3 (1997) 3-12 acid residues forming the active site pocket in P. aemginosa lipase, however, our results demonstrate that aromatic amines generally fit into the active site in P. aeruginosa lipase whereas the carbon side chain seems to be too short in butylamines and, on the contrary, too long in octylamines as reflected by low conversion rates and/or ee-values. Racemic primary and secondary alcohols were stereoselectively esterified with P. uerugimm lipase. The results presented in Table 2 demonstrate that these alcohols reacted much faster than did the amines (reaction time: 48 h); however, the overall stereoselectivity of the lipase as deduced from ee values was considerably lower towards alcohols as compared to amine substrates. It is interesting to compare the ee values obtained with racemic isomers (R,S)I -phenyl-2-propanol and ( R,S)-2-phenyl- 1-propanol. The latter compound representing the primary alcohol reacted much faster, however, with inverse stereoselectivity preferentially forming the (S&ester and with a somewhat lower stereoselectivity of 15% ee. Our results demonstrate that P. aemginosu lipase (i) accepts a wide variety of stereochemically different substrates and (ii) yields in reasonable ee-values at acceptable conversion rates. In conclusion, P. lipase proved to be a promising aeruginosu enzyme for biotechnological applications, particularly in the field of chiral resolution to produce enantiomerically pure compounds for synthetic organic chemistry. Acknowledgements KEJ wishes to thank Heinz-Jurgen Steinhoff, Lehrstuhl Biophysik, Ruhr-Universit’at Bochum, for his help and advice in preparing Fig. 3. References [I] T. Godfrey and S. West, Industrial Enzymology, Press, London, 1996, p. 3. [2] F. Theil, Chem. Rev.. 95 (1995) 2203-2227. Macmillan [3] K.-E. Jaeger, S. Ransac, B.W. Dijkstra, C. Colson. M. van Heuvel and 0. 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