Protein Engineering vol. 16 no. 12 pp. 1139-1145, 2003
© 2003 Oxford University Press
Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization
Institute of Chemistry and Biochemistry, Department of Technical Chemistry and Biotechnology, Greifswald University, Soldmannstrasse 16, D-17487 Greifswald, Germany 1Present address: Department of Biochemistry, Lund University, Getingevägen 60, 221 00 Lund, Sweden
2 To whom correspondence should be addressed. e-mail: uwe.bornscheuer{at}uni-greifswald.de
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It was shown recently that proline-ß-naphthylamidase from pig liver resembles the
-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and Vmax/Km values from 12 to 1000 l min–1 x 10–3 using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190–210 kDa. All enzymes exhibit the highest activity in the pH range 8–9 and between 60 and 70°C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-ß-naphthylamidase activity than rPLE.
Keywords: enzyme catalysis/gene technology/pig liver esterase/porcine intestinal carboxylesterase/site-directed mutagenesis
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Introduction |
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The enzymes of the carboxylesterase family (EC 3.1.1.1) are widely distributed and can be found in the tissues of all animals. They differ in biochemical, immunological and genetic properties and play different roles depending on which part of the organism they are expressed in (Satoh and Hosokawa, 1995
; Hosokawa, 1998). In general, these carb oxylesterases are responsible for detoxification, since they are able to degrade a number of exogenous compounds such as esters, amides and thioesters. Mammalian carboxylesterases are considered to constitute a family of isoenzymes (Schwer et al., 1997).
Pig liver esterase (PLE) has found numerous applications in organic synthesis and represents the most important carboxyl esterase for biocatalytic purposes (Adachi et al., 1986; Jones, 1990; Tamm, 1993). It is isolated from pig liver tissue and represents a heterogeneous enzyme consisting of three subunits, 
, ß and , which mostly exist as trimeric forms (Heymann and Junge, 1979; Junge and Heymann, 1979). Such heterogeneity makes the characterization of the enzyme difficult and irreproducible results can occur during its applications as the composition can change from batch to batch.
Recently, we were able to produce a recombinant -subunit of PLE by expression in Pichia pastoris. Key to successful expression was the removal of the C-terminal tetrapeptide HAEL (Lange et al., 2001). The amino acid sequence of PLE is identical with that of proline-ß-naphthylamidase from porcine liver (Matsushima et al., 1991). Owing to its purity and the absence of other isoenzymes, the recombinant PLE shows different properties to natural pig liver esterase (Musidlowska et al., 2001; Musidlowska-Persson and Bornscheuer, 2002).
The amino acid sequence of proline-ß-naphthylamidase shows 97% identity with porcine intestinal carboxylesterase (PICE) (David et al., 1998). PICE was first described by DiNella et al., who named the enzyme glycerol ester hydrolase and classified it as a lipase (DiNella et al., 1960). However, detailed investigations of its substrate specificity revealed that the enzyme has characteristic properties of a carboxylesterase (DiNella et al., 1960). It catalyses the hydrolysis of short- and medium-chain triglycerides, 1-monoglycerides and acyl-CoA derivatives.
In 1998 the group of Puigserver (David et al., 1998) purified and characterized the enzyme and determined its nucleotide sequence; however, the authors did not report functional expression of a recombinant version of PICE. Their studies revealed that significant differences in the properties of both PLE and PICE exist; despite the fact that they have very high sequence identity. In contrast to PLE, PICE is a homogeneous enzyme. The molecular mass of PICE monomer is 60 kDa and the predominant mature form is a tetramer. Monomers and trimers were also found, however. The formation of quaternary protein structure could be influenced by fatty acid modification of the monomers. The trimers consist of S-palmitoylated molecules (at Cys71) and monomers are both S-palmitoylated and N-myristoylated (at glycine residues located at the N-terminal glycine and at the G-XXX-S/T-consensus sequence). Tetramers are always a combination of one trimer and one monomer (Smialowski-Fleter et al., 2002). Also, differences in the substrate specificity of PICE and PLE isoenzymes, especially against methyl butyrate and proline-ß-naphthylamide, were reported (David et al., 1998).
In this paper, we describe the expression of recombinant porcine intestinal carboxylesterase by site-directed mutagenesis of recombinant pig liver esterase. The nucleotides encoding for the amino acids that differ between PICE and rPLE were exchanged stepwise and eight intermediate mutated genes were created. All gene products were extracellularly expressed in P.pastoris and characterized in detail. In this way, the influence of small changes in amino acid sequence on protein properties could be investigated.
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Materials
All chemicals were purchased from Fluka (Buchs, Switzerland), Sigma (Steinheim, Germany) and Merck (Darmstadt, Germany) at the highest purity available, unless stated otherwise. Oligonucleotides were obtained from Interactiva (Ulm, Germany) and MWG Biotech (Ebersberg, Germany).
Microorganisms, plasmids and growth conditions
Escherichia coli XL10-Gold Tetr
(mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F' proAB lacIqZ M15 Tn10 (Tetr) Amy Camr] was used for the cloning and mutation experiments. Cells were cultivated in low-salt Luria Bertani (LB) medium [yeast extract (10 g/l), peptone (10 g/l), NaCl (5 g/l)] supplemented with zeocin (25 g/l) (Invitrogen, Carlsbad, CA) at 37°C.
P.pastoris X33 (Invitrogen) was used for expression of recombinant PLE variants. The cultivation conditions were described previously (Lange et al., 2001).
The E.coli/P.pastoris shuttle vector pPICZA (Invitrogen) was used for mutation and cloning in E.coli and for expression of esterases under the control of the alcohol oxidase (AOX1) promoter in P.pastoris.
Recombinant DNA technologies
Unless stated otherwise, standard DNA technologies were used (Sambrook et al., 1989). A QIAprep Spin Miniprep kit, a Plasmid Midi kit and a PCR Purification kit (Qiagen, Hilden, Germany) were used for DNA purification. Restriction enzymes and other DNA-modifying enzymes were used as specified by the suppliers (New England BioLabs, Beverly, MA; Promega, Madison, WI).
DNA-sequencing reactions were carried out at MWG-Biotech (Ebersberg, Germany). Standard protocols were used for the preparation and transformation of competent E.coli cells (Chung et al., 1989). Linearized plasmids were transformed into P.pastoris by the lithium chloride method according to the supplier’s instructions.
Site-directed mutagenesis
This was carried out with a QuikChange Site-Directed Mutagenesis Kit (Stratagene). The PCR step was performed in a thermocycler (Thermocycler Progene, Techne, Cambridge). As template, the plasmid pPICZ-mPLE*-tag (5170 bp) harbouring the PLE gene without the natural signal sequence and without the terminal tetrapeptide HAEL but with myc-epitope and his-tag at the C-terminus was used. The PCRs were carried out according to the supplier’s instructions. The PCR reaction mixture was treated with DpnI (1 µl) for 60 min to digest template methylated non-mutated plasmid DNA. The mutated plasmids were transformed into E.coli XL10-Gold and after sequencing into P.pastoris X33.
Shake-flask cultivation of P.pastoris and secreted expression of the esterases
50 ml scale cultivation. Recombinant P.pastoris clones selected on zeocin plates were picked and grown in BMGY-Medium (10 ml) at 30°C and 200 r.p.m. until the OD600 value reached 2–6. The yeast cells were collected by centrifugation (5 min, 2000 g, room temperature) and resuspended to an OD600 of 1.0 with the BMMY induction medium (50 ml). Induction was performed by daily addition of methanol (0.5%, v/v). After a 48 h induction, cells were harvested by centrifugation. The esterase activity in the supernatants was determined with the pNPA assay.
250 ml scale cultivation.
Recombinant clones selected on zeocin plates were picked and grown in YPD medium (3 ml) at 30°C and 200 r.p.m. until the OD600 value was 
15. This preculture was used to inoculate BMGY medium (100 ml), which was then incubated overnight at 30°C until the OD600 was 2–6. The yeast cells were collected by centrifugation (5 min, 2000 g, room temperature) and resuspended to an OD600 of 1.0 with BMMY induction medium (250 ml). Induction was performed by daily addition of methanol (0.5%, v/v). After a 72 h induction, cells were harvested by centrifugation. Supernatants containing recombinant enzyme were concentrated using Centricons (20 ml, NMWL 30000, Ultracel-PL membrane, Millipore) for 15 min at 4000 g and 4°C. Activity during cultivation, after cell harvesting and in concentrated enzyme solution was determined by the pNPA assay (see below). Proteins were then analysed by gel electrophoresis (see below). Owing to the presence of disturbing peptides in the media (from yeast extract and peptone), protein concentrations were determined by densitometry using known concentrations of bovine serum albumin as a reference protein. For this, the National Institutes of Health (NIH) imager (available at http://rsb.info.nih.gov/nih-image/download.html) in combination with a special macro (Macintosh version, available from Dr T.Seebacher, E-mail thomas.seebacher{at}uni-tuebingen.de) for molecular mass and protein content determination was used.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
Concentrated P.pastoris culture supernatants (20 µl) were analysed by SDS–PAGE on polyacrylamide gels (12.5%) with a stacking gel (4%). The proteins in the low molecular weight standard mixture obtained from Sigma were used as reference. Gels were stained for protein detection with Coomassie Brilliant Blue. For esterase-activity staining, proteins were first renatured by incubation for 12 h in Triton X-100 solution [0.5% in 0.1 M tris(hydroxymethyl)aminomethane (TRIS)–HCl (pH 7.5)]. Next, the gel was incubated in a mixture of freshly prepared solutions of -naphthyl acetate and Fast Red. In the presence of hydrolytic (lipase or esterase) activity, released -naphthol forms a red complex with Fast Red (Krebsfänger et al., 1998).
Native polyacrylamide gel electrophoresis
Ferguson analysis. Concentrated P.pastoris culture supernatants (5–10 µl, 0.05–0.1 U) were mixed with sample buffer (10 µl). Samples were separated on polyacrylamide gels (4.5, 5, 5.5, 6, 7, 8, 9 and 10%) with a stacking gel (4.5%). The proteins in the high molecular weight standard mixture obtained from Sigma were used as reference. Gels were activity-stained as described above (without the renaturation step), followed by staining with Coomassie Brilliant Blue. The logarithm of relative mobility of proteins was plotted against the acrylamide concentration in the gel. From the calibration curve obtained from the slopes of standard proteins and their molecular masses, the molecular mass of the samples was calculated.
PhastSystem analysis. Concentrated P.pastoris culture supernatants were analysed by native PAGE using the PhastSystem (Pharmacia) on gels with a polyacrylamide gradient of 8–25%. The proteins in the high molecular weight standard mixture obtained from Pharmacia were used as reference. Gels were activity-stained as described above (without the renaturation step), followed by staining with Coomassie Brilliant Blue. The analysis on gradient native gels gives a linear relationship between relative mobility and the logarithm of molecular mass.
Isoelectric focusing
Concentrated P.pastoris culture supernatants were analysed by isoelectric focusing using the PhastSystem (Pharmacia) on gels with a pH gradient of 4–6.5. The proteins in the low pI calibration kit mixture obtained from Pharmacia were used as reference. Gels were activity-stained, then the bands were fixed with trichloroacetic acid solution (20%, w/v) for 5 min, followed by staining with Coomassie Brilliant Blue.
Glycoprotein analysis
The glycoprotein detection was performed with periodic acid–Schiff staining procedure for PhastSystem native gels (Van-Seuningen and Davril, 1992).
Esterase activity
Esterase activity was determined photometrically in sodium phosphate buffer (50 mM) with p-nitrophenyl acetate (10 mM dissolved in dimethyl sulfoxide) as the substrate. The amount of p-nitrophenol released was routinely determined at 410 nm (
= 15.6 x 103 [M–1*cm–1]) at room temperature and pH 7.5. In addition, activity measurements were performed at different pH values for pH profile determinations ( = 1.37 x 103 M–1 cm–1 for pH 5; 3.21 x 103 for pH 6.0; 11.68 x 103 for pH 7.0; 17.10 x 103 for pH 8.0; 18.00 x 103 for pH 8.5; 18.10 x 103 for pH 9.0; 18.11 x 103 for pH 9.5; and 18.16 x 103 for pH 10.0). One unit (U) of esterase activity was defined as the amount of enzyme releasing 1 µmol of p-nitrophenol per minute under assay conditions.
Methyl butyrate and tributyrin hydrolysis was measured by means of a pH-stat assay (Krebsfänger et al., 1998). A known amount of esterase (1 U, based on the pNPA assay) was used for each reaction. One unit of activity was defined as the amount of enzyme releasing 1 µmol of acid per minute under assay conditions.
Amidase activity
Proline-ß-naphthylamidase activity was determined photometrically with proline-ß-naphthylamide as described previously (Lange et al., 2001). A known amount of esterase (0.5 U, based on the pNPA assay) was used for each reaction. One unit (U) of amidase activity was defined as the amount of enzyme releasing 1 µmol of ß-naphthylamine per minute under the assay conditions.
Activity test on the agar plates
After replica-plating on to YPD agar plates, the colonies were grown for 24 h at 30°C and then every 24 h 100 µl of methanol were added to the lid of the Petri dish. After 2–3 days the plates were overlain with 10 ml of soft agar (0.5% agar in water) containing 100 µl of -naphthyl acetate solution (40 mg/ml in DMF) and 100 µl of Fast Red TR solution (100 mg/ml in DMSO). Esterase-positive colonies developed a red colour and the selection of the best transformants was made on the basis of the intensity.
Creation of homology models
The 3D structure of PLE and PICE (see Figure 7) were modelled based on the known structure of rabbit liver carboxylesterase [PDB entry: 1K4Y (Bencharit et al., 2002)] and human carboxylesterase 1 [hCE1, PDB entry 1MX1 (Bencharit et al., 2003)] using SWISS-MODEL, a fully automated protein structure homology-modelling server available at http://swissmodel.expasy.org/ (Peitsch, 1995, 1996; Guex and Peitsch, 1997).
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Results |
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Site-directed mutagenesis was performed using the QuikChange method. The plasmid pPICZ
-mPLE*-tag encoding for recombinant pig liver esterase was used as a template. The native N-terminal signal sequence of PLE was replaced here by the -factor signal sequence. At the C-terminus, the tetrapeptide HAEL was deleted followed by fusion of a myc-epitope and a his-tag (Lange et al., 2001). Starting from this gene of recombinant pig liver esterase, 22 nucleotides encoding for 17 amino acids were exchanged to obtain recombinant porcine intestinal carboxylesterase (Figure 1, Table I). The mutagenesis resulted in only nine and not 17 mutated genes as several nucleotides were very close to each other and thus were exchanged in one QuikChange reaction. All mutated genes were transformed to E.coli XL10-Gold, sequenced and transformed to P.pastoris X33 by genome integration.
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The transformants were first analysed by activity testing on agar plates followed by expression on the 50 ml scale to select the best clones for production of the enzymes on the 250 ml scale.
Expression of active enzyme during the cultivation was also monitored on a native gel (Figure 2). After 72 h, all secreted enzymes showed esterase activity (0.15–0.65 U/ml, pNPA-assay). Concentration of the supernatant resulted in enzyme preparations with 2–22 U/ml, corresponding to a specific activity of 4–377 U/mg protein (Table II).
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Kinetic studies were carried out by hydrolysis of p-nitrophenyl acetate in the concentration range 0.01–5 mM. The Vmax and Km values were calculated directly from Michaelis–Menten plots by fitting of the experimental points to a non-linear regression. The Vmax/Km values of the esterase variants differed by up to two orders of magnitude from 12 (PLE-PICEh) to 1000 l min–1 x 10–3 (PLE-PICEd) (Table II).
SDS–PAGE analysis (data not shown) showed a single esterase band for all recombinant enzymes with a molecular mass of 62–63 kDa. The slight difference from the calculated value was attributed to glycosylation, which was confirmed by periodic acid–Schiff reagent staining of a native gel (data not shown).
The molecular mass of the mature protein was analysed by native PAGE using a Ferguson plot and polyacrylamide gradient gels for the PhastSystem. Both methods confirmed that all enzymes, including recombinant porcine intestinal carboxylesterase, form a trimer as most active form (Figure 3, Table III). In addition, minor bands of monomers, tetramers and pentamers were detectable on the gels.
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Isoelectric focusing revealed that the activity-stained bands of all esterases were broad and not homogeneous, which is unexpected for single isoenzymes (Figure 4). This result could point to various post-translational modifications of enzyme molecules, which has been previously reported for Pichia-expressed proteins (Hirose et al., 2002
). However, the pI values of all enzymes were very similar, in the pH range 4.3–5.6, which closely matches the calculated value of 5.6. The pI value determined was also higher than the value for rPLE, which was expected after introducing mutation E77G.
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pH profiles were determined using the pNPA assay. All enzymes showed the highest activity in the pH range 8–9 (Table IV).
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Interesting results were obtained during the investigation of temperature profiles. The hydrolysis of tributyrin was performed using the pH-stat method in the temperature range 30–80°C. Figure 5 shows the relative activity of each enzyme at the highest temperatures (60–80°C). It was observed that with the number of introduced mutations the optimum in the profiles is shifted to the region of slightly higher temperatures. rPLE was most active at 60°C and completely inactive at 70°C. In contrast, rPICE showed the highest activity at 70°C and even at 80°C the enzyme still exhibited 80% of maximal activity.
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The substrate specificity was investigated towards two specific compounds, methyl butyrate and proline-ß-naphthyl amide, which according to literature (Heymann and Peter, 1993
), can be used to distinguish the - and -isoenzyme forms of pig liver esterase. However, there was no clear correlation between the number of mutations and the ratio of proline-ß-naphthylamidase activity to methyl butyrate hydrolase activity (Figure 6).
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Discussion |
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Site-directed mutagenesis of rPLE resulted in a group of recombinant enzymes, which can be considered as artificial isoenzymes of rPLE. The last product of the mutagenesis was rPICE. The characterization of the new esterases was considered to prove that small differences in the amino acid sequence could alter the properties of the mutated enzymes. First, considerably differences in the substrate specificity against p-nitrophenyl acetate could be observed. The highest Vmax/Km value was determined for PLE-PICEd (1000 l min–1 x 10–3), which is two orders of magnitudes higher than for PLE-PICEh. A similar pattern, but with smaller differences, was previously reported for different PLE isoenzymes. In the hydrolysis of methyl butyrate and proline-ß-naphthylamide, model substrates for the isoenzyme characterization (Junge and Heymann, 1979
; Heymann and Peter, 1993), there is no logical tendency for changes of proline-ß-naphthylamidase activity as compared with methyl butyrate hydrolase activity among the PLE mutants and rPICE. Eight of the enzymes show properties tending towards the -isoenzyme of PLE and PLE-PICEg rather behaves like the -isoenzyme. The stepwise introduction of mutations also resulted in considerable changes in the temperature profiles. The maximum of enzyme activity was shifted to higher temperatures with increasing number of mutations.
Significant differences were also found for the enantioselectivity of these new esterases in the hydrolysis of a range of acetates of secondary alcohols (Musidlowska-Persson and Bornscheuer, 2003). An up to 6-fold increase in enantioselectivity (E = 46) compared with rPLE (E = 8) was observed in the hydrolysis of (R,S)-1-phenylethyl acetate using a variant containing a single mutation (E77G). For other substrates, a switch in enantiopreference was observed with the introduction of certain mutations.
It is difficult to provide a clear explanation of how the introduction of the mutations can affect the catalytic properties, such as activity and enantioselectivity, as the 3D structures of PLE and PICE have not yet been elucidated. To try to understand this influence, homology modelling was performed. As templates for modelling, 3D structures of two carboxylesterases were used: rabbit liver carboxylesterase (RLCE, PDB entry 1K4Y) (Bencharit et al., 2002), with 75 and 76% identity with the PLE--isoenzyme and PICE, respectively, and human carboxylesterase 1 (hCE1, PDB entry 1MX1) (Bencharit et al., 2003), with 76 and 77% identity with the PLE--isoenzyme and PICE, respectively.
The homology models suggest that almost all mutated positions lie around the catalytic triad (Figure 7), with the exception of positions 112 and 195. Such localization of the mutation sites might explain the observed differences in activity of the enzyme variants created. The amino acids in positions 236 and 237 – responsible for the strongest change in activity towards pNPA – are located very close to the active site serine.
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Valine-236 is exchanged to alanine and alanine-237 to glycine and no changes in charge occur, but a reduced steric hindrance might take place, as the aliphatic side chain is shorter in alanine than valine and there is none in glycine. However, it remains difficult to provide a proper explanation for the observations without crystal structures of rPLE and the rPICE mutants.
As mentioned above, the major native form of mature PICE is a tetramer. However, for all new enzyme variants (including rPLE), trimers were detected as the most active form visible on the native gel after activity staining. Unfortunately, it was not possible to detect the proteins by Coomassie Brilliant Blue staining, hence it was difficult to confirm that the trimer was the main protein fraction. On the other hand, this seems to be very likely, since for the formation of a tetramer, myristoylation at the N-terminal glycine at the G-XXX-S/T-consensus sequence is probably required (Smialowski-Fleter et al., 2002). However, the glycine residue is not accessible for modification here, as the N-terminus was extended by two amino acids (glutamic acid and phenylalanine) for the introduction of specific restriction sites.
The functional expression of recombinant porcine intestine carboxylesterase has not been described previously in the literature. Because of the high identity with rPLE, it was possible to clone and express rPICE by performing site-directed mutagenesis of the rPLE-gene and avoid the approach using cDNA synthesis from pig intestinal mRNA. Some of the properties of the recombinant enzyme are similar to published data for the native form: pH optimum at 9.0 and a ratio of methyl butyrate hydrolase activity to proline-ß-naphthylamidase activity that is between the properties of the - and -isoenzymes of PLE.
The availability of recombinant porcine carboxylesterase, PICE, provides an easy access to this enzyme compared with extraction from pig intestine and thus makes further biochemical characterizations feasible.
Still, the question of whether PICE is identical with one of the PLE isoenzymes cannot be clearly answered. A range of properties of PICE closely match those of the ß-subunit of PLE (Heymann and Junge, 1979). The molecular mass and pI values are almost identical (60 and 59.7 kDa and pI 5.1 and 5.2 for PICE and the ß-subunit of PLE, respectively) and polyclonal antibodies against PICE cross-react with PLE (Smialowski-Fleter et al., 2002). However, the antibodies were directed against a peptide located between residues 281 and 296, where the -subunits of PLE and PICE show 87% sequence identity and a false-positive finding cannot be excluded. In addition, another enzyme from porcine intestine has been described, which also hydrolyses proline-ß-naphthylamide (Takahashi et al., 1989, 1991; Takahashi and Takahashi, 1990). This has a molecular mass of 58 kDa and shows 86% identity with the published N-terminal sequence of PICE, but apparently they are not identical. Hence additional experiments are required to clarify the relationship within this group of carboxylesterases.
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Acknowledgements |
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We thank the Konrad-Adenauer Foundation (St. Augustin, Germany) for a stipend to A.Musidlowska-Persson. We are also grateful to B.Lange (Greifswald University) for her support during her practical course and to Degussa (Project Houses Biotechnology and Catalysis) for financial support.
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Received May 12, 2003; revised October 10, 2003; accepted October 21, 2003
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