1Biotransformations Group and 3Structure Chemistry, Schering-Plough Research Institute, U-13-3000, 1011 Morris Avenue, Union, NJ 07083, USA
2 To whom correspondence should be addressed. e-mail: ningyan.zhang{at}spcorp.com; alex.zaks{at}spcorp.com
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Abstract |
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Keywords: directed evolution/irreversible inactivation/lipase/protein aggregation/stability
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Introduction |
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Even though the CALB gene has been sequenced and the crystal structure of the protein determined (Uppenberg et al., 1994a) neither sequence homology searches nor protein alignment analysis have revealed any clues for improving the thermal stability of the enzyme without negatively affecting its activity. Since rational engineering approaches have been only marginally successful in improving the lipase stability, we decided to turn to directed evolution methodology which has been quite successful in improving functional properties of many enzymes including lipases (Arnold and Moore, 1997
; Schmidt-Dannert and Arnold, 1999
; Jaeger and Reetz, 2000
; Petrounia and Arnold, 2000
; Arnold et al., 2001
; Farinas et al., 2001a
,b; Wintrode et al., 2001
; Jaeger and Eggert, 2002
; May et al., 2002
; Bulter et al., 2003
).
This paper reports the application of error-prone PCR and saturation mutagenesis combined with high-temperature screening to identify CALB mutants with improved resistance towards irreversible inactivation at elevated temperatures.
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Materials and methods |
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Glucose, glycerol, calcium chloride, sodium chloride, Tris-base, potassium phosphate, Tween-80, tributyrin and p-nitrophenyl butyrate (p-NB), were all from Fisher Scientific (Pittsburgh, PA). 6,8-Difluoro-4-methylumbelliferyl octanoate (DiFMU) was purchased from Molecular Probes, Inc. (Eugene, OR), SD-trp and other growth media were purchased from Bio101 (Carlsbad, CA) and Difco (Detroit, MI), respectively.
Cloning of CALB
The mature lipase B gene was cloned from C.antarctica ATCC 32657 by PCR. Forward (5'-AGTAGAATTCCTACCTTC CGGTTCGGAC-3') and reverse (5'-AGTACGTCGACTC AGGGGGTGAACGATGCC-3') primers were derived from the beginning and the end of lipase B gene from C.antarctica LF 058, respectively (Uppenberg et al., 1994a). Restriction enzyme recognition sites, EcoRI in forward primer and SalI in reverse primer, are shown as italics. Total DNA from C.antarctica ATCC 32657 was isolated by using the Qiagen DNeasy Tissue kit (Qiagen, Valencia, CA). PCR was conducted on a PE Applied Biosystems 9700 thermal cycler (Foster City, CA) under the following conditions: 95°C for 1 min followed by 25 cycles of (95°C for 45 s, 55°C for 45 s, 72°C for 120 s), and finally 72°C for 10 min. The reaction mixture (100 µl) contained 2 µl of Pfu polymerase (2.5 units/µl; Stratagene, La Jolla, CA), 250 ng of each primer, 348 ng of genomic DNA of C.antarctica ATCC 32657, 0.2 mM dNTP mix and 10 µl of 10x Pfu buffer. The PCR-amplified fragment was purified by a Qiagen QIAqucik PCR purification kit and was cloned into the EcoRI/SalI site of YEpFLAG-1 vector (Sigma, St Louis, MO). The recombinant plasmid was designated pWS52.
Gene mutagenesis and library construction
Error-prone PCR mutagenesis was carried out using a DiversifyTM PCR random mutagenesis kit from Clontech (Palo Alto, CA). The same set of PCR primers was used for CALB gene cloning and mutagenesis. Plasmid pWS52 and 23G5 mutant gene were used as templates for the first and second generation mutagenesis, respectively.
PCR-mutated genes and vector YEpFLAG-1 were digested with EcoRI and SalI and the fragments were separated on a 0.7% agarose gel. The desired DNA bands were excised and isolated from the gel using a Freeze-Squeeze kit from Bio-Rad (Hercules, CA). The digested vector backbone and PCR-mutated CALB genes were then ligated by T4 DNA ligase (New England Biolabs, Beverly, MA). The ligated genes were used to transform Escherichia coli ElectroMAX DH10B competent cells from Invitrogen Life Technology (Carlsbad, CA), and plated on LB agar plate containing 100 µg/ml ampicillin (Sigma). Transformants of E.coli were pooled and the plasmid DNA was isolated using a plasmid DNA Maxi preparation kit from Bio-Rad. The plasmid DNA library was then transformed into Saccharomyces cerevisiae BJ3505 according to protocols provided in the Yeast Maker Kit from Clontech. The BJ3505 yeast transformants were used for expressing CALB mutants.
Site-directed mutagenesis and saturation mutagenesis at amino acid positions 210, 221 and 281 of CALB were conducted by PCR with appropriate primers or by using the QuikChangeTM site-directed mutagenesis kit from Stratagene according to the manufacturers instructions.
Cell culture and enzyme expression
Saccharomyces cerevisiae transformants were inoculated into 96-well deep plates (Corning Inc., Acton, MA) containing 0.6 ml of SD-trp selective medium and grown at 30°C for 2 days on an orbital shaker (model ISF-4-V; Adolf Kuhner, Switzerland) at 200 r.p.m. The seed (0.1 ml) was transferred into 0.5 ml of fresh expression medium (1% yeast extract, 8% peptone, 1% glucose, 3% glycerol and 20 mM CaCl2) and grown at 20°C for 7 days at 200 r.p.m. The supernatant containing the enzyme was obtained by removing the cells by centrifugation at 2500 g (Eppendorf 5810R) for 10 min.
Enzyme screening
Culture supernatants (20 µl) containing the CALB enzyme were transferred from expression plates into 96-well assay plates (Corning Inc., Corning, NY), followed by the addition of 0.15 ml of 50 mM potassium phosphate (in the case of DiFMU) or TrisHCl (in the case of p-NB) buffer, pH 7.0 into each well. One set of plates was kept at room temperature while the duplicate set was sealed with an aluminum sealing tape (USA Scientific, Inc., Ocala, FL) and heated at 60 (for G1) or 70°C (for G2) for 1 h. The actual pH of the phosphate buffer at 60°C and Tris buffer at 70°C was 7.0 and 6.5, respectively. Following incubation, the heated plates were placed on ice for 15 min and equilibrated at room temperature for an additional 15 min. The first generation library was assayed with DiFMU as substrate. The reactions were initiated by introducing 30 µl of 0.8 M DiFMU solution in acetonitrile. The fluorescent response was measured every 20 s for a duration of 2 min at room temperature (Perkin-Elmer HTS7000 Bioassay plate reader; 360/465 nm excitation/emission). To maximize the signal-to-noise ratio the second generation mutant library was screened using a colorimetric assay with p-NB. The reaction was initiated by the addition of the enzyme supernatant (10 µl) into 50 mM TrisHCl buffer, pH 7.0 (160 µl) and 12 mM p-NB substrate in DMSO (30 µl). The residual activity (RA) was determined by measuring the change in absorbance at 405 nm for 1 min at room temperature with a 96-well plate reader (Model 384 plus; Molecular Devices, Sunnyvale, CA). The percentage of RA for mutants was calculated by dividing the activity after heat inactivation by the initial activity multiplied by 100. The standard deviation (SD) of RA calculated for every screen using 40 randomly picked WT-CALB clones and expressed as a percentage of the mean was 30%. The mutants with RAm > RAwt + 2.5 SD were selected for further confirmation. They were re-grown on a 50 ml scale and assayed at various dilutions in duplicate.
Nucleotide sequencing and analysis
Plasmid DNA from S.cerevisiae and E.coli were isolated using the Bio101 RPM yeast plasmid isolation kit and Bio-Rad Quantum isolation kit, respectively. DNA sequencing reactions were performed with the ABI Prism BigDyeTM v3.0 reaction kit according to the manufacturers instructions. Sequencing was conducted using an automatic capillary-sequencer (3700 DNA Analyzer; ABI Prism, Foster City, CA). Sequence analysis was conducted using Lasergene software (DNAStar Inc., Madison, WI).
Enzyme purification and protein assay
Lipase B was purified using agarose-bound Anti-FlagR M1 monoclonal antibodies (Sigma) on a BioCAD SPRINT perfusion chromatography system (Applied Biosystems). The enzyme solution was applied to a column containing 10 ml of Anti-FlagR M1 affinity resin equilibrated in 50 mM TrisHCl pH 7.4, 150 mM NaCl and 2 mM CaCl2. Enzyme fractions were eluted with 100 mM glycineHCl, pH 3.0 at 1 ml/min. Following the elution, the fractions containing CALB were adjusted to pH 7.0 with 1 M TrisHCl buffer, pH 8.0.
The enzyme concentration was determined using protein assay reagents from Bio-Rad with bovine serum albumin (BSA) as a standard.
Determination of kinetic constants
The initial rates of p-NB hydrolysis were determined at room temperature by following the formation of p-nitrophenol at A405 (18 200 M1 cm1) in a Shimadzu spectrophotometer (model 2401PC UV-VIS; Columbia, MA). The reaction was initiated by adding 50 µl of the purified CALB enzyme solution to the reaction mixture containing 950 µl of 50 mM TrisHCl, pH 7.0, 1% DMSO and varying the concentrations of p-NB. The initial rates of DiFMU hydrolysis at room temperature were determined by following the formation of fluorescent 6,8-difluorescent-7-hydroxy-4-methylcoumarin on a Perkin-Elmer plate reader (model HTS7000; Beaconsfield, UK). A CALB solution (20 µl) was added to the reaction mixture containing 180 µl of 50 mM potassium phosphate buffer, pH 7.0, 5% acetonitrile and varying concentrations of DiFMU. To determine kcat and KM, initial rates were determined at five different substrate concentrations over the range 0.11.6 mM for p-NB and 1.930 µM for DiFMU. For each initial rate, the average of three (for p-NB) and five (for DiFMU) measurements was used (SD was determined to be <10%). The kinetic constants were calculated by fitting the initial rate data into the MichaelisMenten equation using the GraFit software (version 5; Erithacus Software Ltd, Horley, UK).
Thermal denaturation of purified CALB
Approximately 0.8 ml of each purified enzyme preparation (0.06 mg/ml protein in 50 mM TrisHCl, pH 7.0) was placed into a 1.6 ml microcentrifuge tube and incubated at 70°C in a water bath. Periodically, 0.1 ml aliquots were removed, placed at room temperature for 1 h and then stored at 20°C for further analysis.
The protein aggregation at elevated temperature was determined by heating the purified enzyme preparations (0.1 mg/ml) under the above conditions followed by removal of protein aggregates by centrifugation at 14x103 g for 10 min. The concentration of the remaining soluble protein was determined with Bio-Rad protein assay reagent as described earlier in the text.
Circular dichroism studies
The far-UV circular dichroism (CD) spectra of WT-CALB and mutants were determined in a JASCO 810 spectropolarimeter with protein concentrations between 0.15 and 0.2 mg/ml in 50 mM TrisHCl, pH 7.0. Spectra were recorded using a 2 nm band width, 8 s response rate, scan rate of 50 nm/min and a 0.1 nm pitch. Four accumulations were recorded and averaged for each spectrum using a 0.1 cm path length cell. Temperature control was performed using a Jasco Peltier-type temperature controller (model PTC-424S/L).
The temperature-dependent far-UV CD spectra in Figure 2 were obtained using a heating rate of 1°C/min from 20 to 70°C with the spectrum at each temperature recorded during the increase in temperature. The temperature-dependent unfolding profiles in Figure 3 were obtained by heating at a rate of 1°C/min from 20 to 80°C and measuring the change in ellipticity at 226 nm. The spectra of refolded samples shown in Figure 4 were obtained by heating the sample from 25 to 70°C in the cuvette at a rate of 6°C/min. The sample was then equilibrated for 10 min at 70°C and then cooled to 25°C at a rate of 9°C/min. The spectrum of the refolded sample was recorded after a 10 min equilibration at 25°C.
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Results and discussion |
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Aspergillus and Pichia were successfully used for expressing active CALB (Hoegh et al., 1995; Rotticci-Mulder et al., 2001
). Both systems randomly integrated the lipase gene into the genomic DNA resulting in an undesirable clone to clone variation of protein expression. To circumvent this problem and to develop an expression system suitable for high-throughput screening we cloned CALB from strain ATCC 32657 into an S.cerevisiae Yeast FLAG expression system. The expression vector, YEpFLAG-1, contained a yeast alcohol dehydrogenase promoter, ADH2, regulated by glucose repression, and an
-factor leader sequence followed by an eight amino acid FLAG peptide. The presence of FLAG peptide had no effect on
-factor cleavage.
The amino acid sequence of the cloned CALB from ATCC 32657 was 99.4% identical to the lipase B from LF 058 (Uppenberg et al., 1994a) with only two differences, Thr57Ala and Ala89Thr. The N-terminus of CALB described in this study also contained an eight amino acid-long FLAG peptide followed by a GluPhe sequence which was derived from the EcoRI cloning site. The expression of active CALB was temperature dependent with maximum activity (
40 µmol of tributyrin hydrolyzed/min/ml of growth culture) obtained at 20°C. The activity level of WT-CALB expressed at 25 and 30°C was approximately half of that obtained at 20°C. Consequently, experiments described in this study were conducted using the lipase expressed at 20°C. No significant differences in the expression levels of the WT-CALB and the mutants were observed.
Screening of mutant libraries
Two rounds of error-prone PCR mutagenesis were used for generating libraries of CALB mutants. The first generation (G1) mutant library was constructed using the WT-CALB gene as template. The mutation conditions were selected to achieve a nucleotide mutational rate of 0.15% (determined by sequencing 12 randomly selected clones) resulting in a library consisting of
80% active mutants. The selection process consisted of incubating the mutant libraries for 1 h at an elevated temperature as described in Materials and methods, and then assaying for the RA at 20°C. It should be emphasized that this approach does not directly select for mutants that are active at elevated temperatures and consequently have higher Tm, but rather identifies variants with increased propensity for refolding (as compared with the wild-type enzyme) or/and those that are able to retain their native structure at the elevated temperature. After screening 10 000 colonies from the G1 library, only one mutant (23G5) was selected that satisfied both established selection criteria (RAmutant > RAparent + 2.5 SD and the activity prior to inactivation was at least 75% that of the parent). The sequence analysis of 23G5 revealed two amino acid substitutions, V210I and A281E. The gene encoding 23G5 was used as a template for generating the second generation (G2) error-prone PCR library. The nucleotide mutational rate of this library was made similar to that of the G1. Eight thousand four hundred colonies of the G2 library were screened, revealing a highly active mutant, 195F1, with RA exceeding that of the 23G5 by at least 2.5 SD. The new mutant acquired three mutations compared with the WT-CALB: it retained both amino acid substitutions of its parent, V210I and A281E, and acquired one additional mutation, V221D. In an attempt to improve the thermal stability even further, three additional saturation mutagenesis libraries (G3) were constructed by utilizing the 195F1 gene as a template and substituting each of the three mutated sites with each of the 19 remaining amino acids. The screening of 1800 colonies (600 from each of the three G3 libraries) revealed no new mutants that satisfied the selection criteria.
Thermal stability of 23G5 and 195F1 CALB mutants
Inactivation profiles. Irreversible thermal inactivation of WT-CALB and the two mutants was characterized by incubating each enzyme in an aqueous buffer at 70°C followed by cooling and assessing the RA at room temperature. The inactivation profiles of the WT-CALB, 23G5 and 195F1 mutants are presented in Figure 1. The inactivation of all three recombinant enzymes followed neither first- nor second-order kinetics indicating the contribution of multiple factors to the inactivation process. As illustrated in Figure 1, stability of both mutants against irreversible inactivation was significantly higher than that of the WT-CALB. The approximated T1/2 at 70°C of the WT-CALB, 23G5 and 195F1 mutants were 8, 211 and 232 min, respectively (Table I).
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Additional studies evaluating the temperature-dependent spectra for each protein at eight discrete temperatures provided an explanation for these findings (Figure 2). The spectra for WT-CALB recorded at several temperatures using a temperature ramp of 1°C/min (Figure 2A) indicated that the protein undergoes a large, 50%, spectral change by the time the temperature reaches 5256°C. This is consistent with the observed Tm of 57.7°C (Figure 3). Analysis of the spectrum in Figure 2A indicated that the ellipticity decreased along the entire spectrum and that there was no clear isosbestic point which is a signature for a two-state (folded versus unfolded) model (Kelly and Price, 2000). The decrease in ellipticity signal was consistent with a loss of protein due to temperature-induced protein precipitation. Inspection of the cuvette following heating to 70°C indicated that the sample contained a large amount of precipitate. The quantitative analysis of the soluble protein (see Materials and methods) indicated that <50% of WT-CALB remained in solution following a 10 min incubation at 70°C versus 70 and 95% remaining in solution for 23G5 and 195F1, respectively.
On the contrary, the temperature-dependent spectral profiles for the mutant proteins (Figure 2B and C) showed a classical isosbestic point near 211 nm which suggested protein unfolding of the mutants involved a two-state mechanism and was reversible. In fact, the samples were clear in the cuvette at 70°C, consistent with the fact that the samples did not undergo temperature-dependent precipitation like WT-CALB. Based on the temperature-dependent spectra (Figure 2B and C) mutants 23G5 and 195F1 appear to have Tm values between 48 and 52°C. These values correlate with that observed in Figure 3 (52.1 and 50.8°C, Table I). The observation that the mutant proteins unfolded reversibly suggested that the improved activity of the lipase mutants following heating to 70°C was mainly due to a higher recovery of active protein compared with the wild-type protein which underwent an irreversible denaturation at elevated temperatures to a much higher degree.
To further characterize the foldingrefolding profile for each enzyme the CD spectra were determined for samples equilibrated at 20°C (native) and compared with that of samples equilibrated at 70°C for 10 min (unfolded) followed by cooling to 20°C (refolded). The spectra for each protein at 20°C (native) and the ones following the refolding are shown in Figure 4. The refolded WT-CALB sample, cooled to 20°C after being heated to 70°C, had an ellipticity value that was only 40% at 226 nm compared with the 20°C native control. Conversely, the refolded ellipticity values for 23G5 and 195F1 were 61 and 55%, respectively, compared with their 20°C native controls. These results agree with the previously described temperature-dependent CD data which suggested that the mutant proteins retained a higher percentage of activity following heating to 70°C compared with the WT-CALB because the mutations introduced in 23G5 (V210I, A281E) and 195F1 (V210I, A281E, V221D) facilitated protein refolding perhaps by inhibiting protein aggregation in the unfolded state. These CD results also agree with the protein-recovery experiments where WT-CALB produced a larger amount of insoluble protein compared with the 23G5 mutant (>50 versus <20%).
So why do the amino acid substitutions in 23G5 and 195F1 mutants have such a profound effect on the protein aggregation? It is well accepted that protein aggregation at elevated temperatures is attributed to favorable interactions between hydrophobic regions of partly unfolded protein molecules (De Bernardez Clark, 1998; Broglia and Tiana, 2001
). The onset of aggregation occurs at prolonged sequences of hydrophobic amino acid residues, so-called nucleation sites. The amino acid sequence analysis revealed that the position 281 was in the middle of a long hydrophobic amino acid sequence (V272AAAALLAPAAAAIV286). The A281E substitution in the 23G5 mutant appears to disrupt the hydrophobic stretch which may in turn inhibit the nucleation site responsible for inducing protein precipitation. Similarly, the V221D substitution (195F1 mutant) decreases the hydrophobicity of the sequence P218LFVIDH224, although to a lesser extent than that seen at the 281 position. In contrast, position 210 is in the middle of a rather polar G207KNVQAQ213 sequence. Since its hydrophobicity is not altered significantly by the conservative V210I substitution, the effect of mutation on stability of the mutant was expected to be minimal.
To confirm the above hypothesis, the effect of a single amino acid substitution on thermal stability of CALB was investigated. The three CALB mutants with a single amino acid substitution were constructed using site-directed mutagenesis and their thermal inactivation at 70°C was analyzed (Table I). As expected, the A281E mutation had the most significant effect on stability increasing T1/2 22-fold compared with the WT-CALB. A single V221D mutation had a more moderate effect on stability (a 4-fold improvement). Consistent with these findings was the fact that the two single-site mutants showed a lower tendency to aggregate at elevated temperature than the WT-CALB (0, 32 and 52% of protein aggregated for A281E, V221D and WT-CALB, respectively, after incubation at 70°C for 10 min). As expected, the stability of the V210I mutant was similar to the wild-type enzyme.
Both the A281E and V221D mutations decreased the Tm value of the lipase. The decrease in stability may be due to mutation-induced structural perturbations. Analysis of a three-dimensional model of CALB (Figure 5A) based on the crystal structure (Uppenberg et al., 1994b) clearly shows that residue A281 is a part of an
-helix (
10) located at the top of the substrate-binding pocket in a highly hydrophobic environment (Figure 5B illustrates the residues within a 5 Å sphere with A281 at the center). The Glu side chain of A281E projects into the binding pocket. Due to a close contact with the neighboring hydrophobic residues, the carboxylate would have to be partially dehydrated which may destabilize the native conformation. Alternatively, the hydrophilic property of the side chain may induce a conformational change to facilitate solvent exposure of the carboxylic acid. This argument was supported experimentally when the Tm of the single A281E mutant was determined and found to be 7°C lower than that of the WT-CALB (Table I). Residue 221 is located on an extended strand on the surface of the protein with the Val side chain (or Asp of the mutant) exposed to the solvent (Figure 5C). Since the environment of the solvent-exposed residue is not likely to change upon unfolding, the substitution of Val for Asp should have only a small effect on Tm. This result is consistent with the experimental results (56°C for the mutant versus 58°C for the WT-CALB, Table I). Finally, if one assumes that the V210I mutant has the same side chain conformation as WT-CALB, i.e. projecting into the largely hydrophobic interior of the protein, the extra methyl group of Ile must contact the OH group of Y258 (Figure 5D). It would appear that the extra hydrophobicity of Ile over Val realized upon unfolding is counterbalanced by an unfavorable contact with the polar OH group of the Tyr (in the folded state) resulting in a minimal change in Tm which is also consistent with the experimental data.
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Acknowledgements |
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Received May 8, 2003; revised June 9, 2003; accepted June 23, 2003.