pHluorin-Based in Vivo Assay for Hydrolase Screening - Analytical

The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermop...
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Anal. Chem. 2005, 77, 2727-2732

pHluorin-Based in Vivo Assay for Hydrolase Screening Sascha Schuster,† Markus Enzelberger,†,‡ Harald Trauthwein,§ Rolf D. Schmid,† and Vlada B. Urlacher*,†

Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany, and Degussa AG, Rodenbacher Chaussee 4, D-63457 Hanau-Wolfgang, Germany

pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis. Over the past years, the importance of hydrolytical enzymes as catalysts for industrial applications has significantly increased due to their high chemo-, regio-, and stereoselectivity toward many different substrates under ambient temperature and neutral pH values.1 Modern chemical process technologies require biocatalysts with improved properties such as, e.g., higher activity, increased temperature stability, or selectivity. To create suitable catalysts, powerful methods such as protein design and directed evolution have been established.2-4 For mutant screening, simple and especially rapid assays are required. Most assays for hydrolytical activity are based either on absorbance or fluorescence measurements or on detection of pH changes via special indicators. Often, even both approaches have been combined.5-9 An assay for investigation of β-lactamase activity in vitro was reported, * Corresponding author. Fax: +49-711-685-3196. E-mail: itbvur@ itb.uni-stuttgart.de. † University of Stuttgart. ‡ Present address: MorphoSys AG, Lena-Christ-Str. 48, D-82152 Martinsried/ Planegg, Germany. § Degussa AG. (1) Petersen, M.; Kiener, A. Green Chem. 1999, 1, 99-106. (2) Pokala, N.; Handel, T. M. J. Struct. Biol. 2001, 134, 269-281. (3) Schmidt, M.; Baumann, M.; Henke, E.; Konarzycka-Bessler, M.; Bornscheuer, U. T. Methodol. Enzymol. 2004, 388, 199-207. (4) Reetz, M. T. P. Natl. Acad. Sci. U.S.A. 2004, 101, 5716-5722. (5) Kouker, G.; Jaeger, K. E. Appl. Environ. Microbiol. 1987, 53, 211-213. (6) Klein, G.; Reymond, J. L. Helv. Chim. Acta 1999, 82, 400-407. (7) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc. 1999, 121, 4306-4307. (8) Henke, E.; Bornscheuer, U. T. Anal. Chem. 2003, 75, 255-260. (9) Gupta, R.; Rathi, P.; Gupta, N.; Bradoo, S. Biotechnol. Appl. Biochem. 2003, 37, 63-71. 10.1021/ac0486692 CCC: $30.25 Published on Web 03/25/2005

© 2005 American Chemical Society

where enzyme-catalyzed cleavage of β-lactams leads to an alteration of the pH.10 For screening, enhanced green fluorescent protein (EGFP) acting as pH sensor, fused to the β-lactamase, was used. This assay is rather sensitive and can be applied for β-lactamase substrate evaluation but requires a number of handling steps such as cell disruption and centrifugation. The biggest challenge in assay development is, without any doubt, the improvement of the throughput. The use of capillary electrophoresis improved the sample throughput significantly.11 A state of the art flow cytometric device is capable of analyzing and sorting up to 50 000 cells/s. Originally being used as a valuable clinical tool for the analysis of cancer cells and the identification of bacteria with different fluorescent dyes, flow cytometry is now also frequently used for biotechnological applications such as expression analysis or on-line process monitoring of cell cultures.12-15 Combined with a sensitive assay, a cell sorter can be a very efficient tool for high-throughput screening of mutant libraries. A fluorescence activated cell sorting (FACS) assay for directed evolution of the native Escherichia coli (E. coli) surface protease ompT presented on the cell surface has been described that is based on the fluorescence resonance energy transfer (FRET) principle.16 Unfortunately, this assay is very laborious and only applicable for specific enzymes and/or substrates. Considering the limitations of the existing methods, we have developed an assay sensitive for screening of a wider range of enzymes and substrates, which can be combined with flow cytometric cell analysis and sorting. This novel in vivo assay is based on a change in pH during enzymatic hydrolysis. pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP) isolated from Aequorea victoria, was used as a sensor for determination of intracellular pH changes. (10) Puckett, L. G.; Lewis, J. C.; Bachas, L. G.; Daunert, S. Anal. Biochem. 2002, 309, 224-231. (11) Reetz, M. T.; Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew. Chem., Int. Ed. 2000, 39, 3891-3893. (12) Kenter, A. L.; Watson, J. V.; Azim, T.; Rabbitts, T. H. Exp. Cell Res. 1986, 167, 241-251. (13) Fuchs, B. M.; Wallner, G.; Beisker, W.; Schwippl, I.; Ludwig, W.; Amann, R. Appl. Environ. Microbiol. 1998, 64, 4973-4982. (14) Cormack, B. P.; Bertram, G.; Egerton, M.; Gow, N. A. R.; Falkow, S.; Brown, A. J. P. Microbiol-Uk 1997, 143, 303-311. (15) Harding, C. L.; Lloyd, D. R.; McFarlane, C. M.; Al-Rubeai, M. Biotechnol. Prog. 2000, 16, 800-802. (16) Olsen, M. J.; Gam, J.; Iverson, B. L.; Georgiou, G. Methods Mol. Biol. 2003, 230, 329-342.

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Several studies demonstrated the capability of pHluorin to detect dynamically the pH in various intracellular compartments, such as cytoplasm, trans-Golgi network, or peroxisomes.17-19 Starting with the mutation S202H, constructed for increasing the pH sensitivity of GFP, pHluorin was obtained by several rounds of directed mutagenesis.20 pHluorin has two pH-dependent excitation maxima, at 395 and 475 nm, thus facilitating easy monitoring of the intracellular pH value within the range of pH 5.5-8.0. The feasibility of the proposed assay was tested in microtiter-plate scale as well as in flow cytometric system for different hydrolylases, recombinantly expressed in E. coli. EXPERIMENTAL SECTION Chemicals and Reagents. If not stated otherwise, all chemicals were of analytical grade and purchased from Fluka (NeuUlm, Germany) or Sigma-Aldrich (Taufkirchen, Germany). [1-(4Methylphenyl)ethyl]-2-caprylamid was obtained from Bayer (Monheim, Germany). Isopropyl-β-D-thiogalactopyranoside (IPTG), restriction endonucleases, and T4 DNA ligase were purchased from MBI Fermentas (St. Leon-Rot, Germany), Taq DNA polymerase from Eppendorf (Cologne, Germany), and Pfu Turbo DNA polymerase from Stratagene (Amsterdam, The Netherlands). Escherichia coli Strains and Plasmids. The E. coli strain DH5R [supE44, lacU169 (80lacZ M15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1] was purchased from BD Biosciences Clontech (Heidelberg, Germany) and the Epicurian coli strain XL1-Blue [recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac (F¢proAB lacIqZDM15 Tn10 (Tetr))] from Stratagene (Amsterdam, The Netherlands). J. E. Rothman from the Memorial Sloan-Kettering Cancer Center (New York, USA) provided the gene encoding for ratiometric pHluorin in the bacterial expression vector pGEX_rat_pHluorin. J. Altenbuchner from the Institute of Industrial Genetics (Stuttgart, Germany) provided the expression vector pJOE2792.1. Site-Directed Mutagenesis. Site-directed mutagenesis was performed using the QuickChange Kit from Stratagene (Amsterdam, The Netherlands). Primers were purchased from Sigma Ark (Darmstadt, Germany) and Invitrogen (Karlsruhe, Germany). Instrumentation. The FLUOstar Model 403 absorption and fluorescence spectrophotometer from BMG LabTechnologies (Offenburg, Germany) was used for fluorescence measurements. FACS analysis and sorting were done on LSR II and FACSDiVa from Becton Dickinson Bioscience (Heidelberg, Germany). Cell disruption was carried out with the sonifier W250 (Branson, Dietzenbach, Germany). In vitro activity was measured with a pHstat device from Metrohm (Herisau, Switzerland) and the Ultrospec 3000 UV/visible spectrophotometer from Amersham Biosciences (Cambridge, England). Cloning Strategy. For the coexpression of ratiometric pHluorin and Pseudomonas fluorescens esterase (PFE), the plasmid pGEX_pH_PFE was engineered from pGEX_rat_pHluorin and pJOE2792.1. The vector pGEX_rat_pHluorin carries the pHluorin gene fused to the glutathion-S-transferase (GST) gene under the (17) Karagiannis, J.; Young, P. G. J. Cell Sci. 2001, 114, 2929-2941. (18) Machen, T. E.; Leigh, M. J.; Taylor, C.; Kimura, T.; Asano, S.; Moore, H. P. H. Am. J. Physiol.: Cell Physiol. 2003, 285, C205-C214. (19) Jankowski, A.; Kim, J. H.; Collins, R. F.; Daneman, R.; Walton, P.; Grinstein, S. J. Biol. Chem. 2001, 276, 48748-48753. (20) Miesenbock, G.; De Angelis, D. A.; Rothman, J. E. Nature 1998, 394, 192195.

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control of a tac-promotor. GST facilitates the easy and efficient purification of pHluorin if desired. A HindIII restriction site was inserted by site-directed mutagenesis downstream of the EcoRI restriction site located at the 3′ end of the GST-pHluorin fusion gene. Subsequently, the rhamnose inducible promotor (rha) and the PFE gene were excised from the pJOE2792.1 plasmid by the EcoRI and HindIII restriction endonucleases and cloned into the pGEX_rat_pHluorin plasmid. The new plasmid, pGEX_pH_PFE, carried the GST-pHluorin fusion gene and the PFE gene under the control of a tac and a rha promotor, respectively. To test other hydrolases, the PFE gene was replaced by other hydrolase genes of interest. The following plasmids were obtained: pGEX_pH_BSE (with a Geobacillus stearothermophilus esterase), pGEX_pH_ BsubpNBE (with a Bacillus subtilis p-nitrobenzyl esterase), and pGEX_pH_ACH (with an Arthrobacter crystallopoietes hydantoinase). Coexpression of pHluorin and Hydrolase. For the coexpression of pHluorin and hydrolase, 100 mL of LB medium supplemented with 100 µg/mL ampicillin (LB-Amp) was inoculated with a preculture of E. coli, harboring one of the plasmids listed above. It was then incubated at 37 °C with shaking (220 rpm) until OD578 ≈ 0.8-1.0. The coexpression of pHluorin and hydrolase was induced with 1.0 mM IPTG and 11.0 mM rhamnose. After 4 h incubation at 30 °C and 220 rpm, 10 mL aliquots were centrifuged at 4 °C and 4000 rpm for 15 min. The supernatant was discarded and the cell pellets stored at 4 °C. For the coexpression in microtiter-plate scale, bacterial colonies were cultivated in 200 µL LB-Amp medium per well for 24 h at 37 °C and 220 rpm (master plates). A 2 µL/well aliquot was then transferred into new microtiter plates (containing 200 µL LBAmp media per well) for subsequent cultivation and coexpression of pHluorin and hydrolase (production plates). The master plates were stored at -80 °C. The production plates were incubated at 37 °C under shaking (220 rpm) until OD578 ≈ 0.3. After induction with 1.0 mM IPTG and 11.0 mM rhamnose the plates were incubated for 24 h at 30 °C and 220 rpm. After centrifugation at 4 °C and 4000 rpm for 15 min, the supernatant was discarded and the production plates stored at 4 °C. Assay Setup. The cell pellets were resuspended in 50 mM potassium phosphate buffer (pH 8.0) supplemented with 3% (v/v) dimethyl sulfoxide (DMSO) as cosolvent and 2% (v/v) substrate. The biotransformation was performed for 45 min at 37 °C under shaking (220 rpm). A 250 µL aliquot of this cell solution was transferred into black microtiter plates. The fluorescence emission was measured at 508 nm upon excitation at 390 and 485 nm. Subsequently, the emission ratio (ratio 485/390) was calculated. FACS Analysis and Sorting. For FACS analysis and sorting cells were illuminated at 405 nm with a violet laser diode and at 488 nm with an argon ion laser. Forward scattering (FSC), side scattering (SSC), and the emission as well as the emission ratio of pHluorin upon excitation at 488 and 405 nm were measured by using a 530/30-nm band-pass filter. For each experiment, 106 cells were analyzed and sorted. Density plots were used in order to depict the cell population shifts more accurately. The level curves and color variations show differences of the event density in the same plot. The brighter the color in the plot the higher is the event density.

Figure 1. Emission ratios of pHluorin coexpressed with the Geobacillus stearothermophilus esterase (BSE) in the presence of different substrates. A significant change of the emission ratio compared to the reference (only expressing pHluorin) is visible only for tributyrin and methyl caproate.

pH-Stat Assay. In vitro hydrolase activity was determined using a pH-stat device. After cultivation, E. coli cells were resuspended in 5 mL of 50 mM potassium phosphate buffer (pH 8.0) and disrupted by sonication (three times for 1 min; output level, 80 W; duty cycle, 35%). After centrifugation for 15 min at 4000 rpm and 4 °C, 100 µL of the cell lysate was mixed with 50 mL of 50 mM potassium phosphate buffer (pH 8.0) supplemented with 3% (v/v) DMSO and 2% (v/v) substrate and placed into the reaction chamber. The released acid was titrated with 0.01 mM sodium hydroxide solution. pH-stat measurements were performed as described previously.21,22 One unit (U) hydrolase activity is defined as 1 µmol of released acid/min.

Table 1. In Vitro Activity of Geobacillus stearothermophilus Esterase (BSE) toward Different Substrates

RESULTS AND DISCUSSION Assay Development. The present in vivo assay for hydrolase screening is based on determination of intracellular changes in pH during hydrolysis of a substrate. The strength of this pH change depends on the hydrolase activity. Any intracellular pH change leads to alterations in fluorescence spectra of pHlourin, a mutant of GFP.20 pHluorin has two pH-dependent excitation maxima at 395 and 475 nm. Starting from pH 8.0, a pH decline results in a decrease of the peak at 395 nm and a simultaneous increase of the peak at 475 nm. Thus, the ratio of both maxima, the so-called fluorescence emission ratio, acts as an indicator for pH changes. The significant advantage of the emission ratio for detection of pH changes is its independence on the expression level of pHluorin and hydrolase, that is of course unconstant for different cells within a culture. To investigate the correlation between the pH and changes in the fluorescence emission ratio of pHluorin, pHluorin-producing E. coli DH5R cells were incubated for 45 min at 30 °C in potassium phosphate buffer at pH 6.0, 7.0, and 8.0, respectively, prior to emission measurements. The emission ratio increased significantly (almost by a factor of 2) with decreasing pH from 8.0 (0.98) over 7.0 (1.38) to 6.0 (1.95). These results indicate the strong pH dependency of pHluorin within this pH range. On this background all subsequent experiments were started at pH 8.0, if not stated otherwise.

Next the emission ratio during conversion of different substrates in E. coli cells producing pHluorin and the Geobacillus stearothermophilus (G. stearothermophilus) esterase was determined. The reference value was provided by the emission ratio of cells with pHluorin but without esterase activity. A significant increase in the emission ratio was only observed for tributyrin and methyl caproate (Figure 1). For these two substrates the emission ratios were almost twice as high as the reference value. Tricaprylin and butyl acetate, however, caused only very insufficient change in the emission ratio. The reasons for this were further examined by means of in vitro experiments on pH stat (Table 1). The G. stearothermophilus esterase showed the highest activity toward tributyrin. This is congruent with the result of the in vivo experiments (Figure 1). Tributyrin was therefore used as a standard for the comparison of the esterase activity toward the other substrates. Compared to tributyrin, the esterase activity toward methyl caproate was about 16% lower, also corresponding to the lower emission ratio observed in vivo (Figure 1). The esterase activity toward tricaprylin and butyl acetate, measured in vitro, was much lower. This explains such an insignificant change in emission ratio (Figure 1). Tributyrin and butyl acetate also show autohydrolysis under reaction conditions (Table 1), a phenomenon that might also affect the emission ratio of the reference (Figure 1). The correlation of the emission ratio and esterase activity was further investigated using inactivated esterase mutants from G. stearothermophilus and Pseudomonas fluorescence. The inactive

(21) Tietz, N. W.; Fiereck, E. A. Clin. Chim. Acta 1966, 13, 352-358. (22) Peled, N.; Krenz, M. C. Anal. Biochem. 1981, 112, 219-222.

substrate

rel activity (%)

autohydrolysis at 37 °Ca (µmol/min)

tributyrin methyl caproate tricaprylin butyl acetate phenyl acetate

100 84 10 4 107

5.6 nd nd 5.9 6.4

a

nd, not detected.

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Table 2. Emission Ratios of pHluorin for Different Hydrolase-Catalyzed Reactions emission ratio hydrolase

substrate [2% (v/v)]

ref

hydrolase activity

BSE inactivated mutant PFE inactivated mutant BSE ACH BsubpNBE (pH 8.0) BsubpNBE (pH 6.0)

tributyrin butyl acetate -caprolactone hydantoin N-(1-p-tolylethyl)octylamide N-(1-p-tolylethyl)octylamide

1.25 1.27 1.51 1.37 1.30 1.85

1.32 1.33 1.79 2.25 1.65 1.59

mutants were produced by site-directed mutagenesis replacing the active serine of the catalytic triad with alanine. The emission ratios of cells with the inactive esterase mutants were similar to the reference value (Table 2). These results confirm that the higher emission ratios of the wild-type esterase can be attributed to enzyme-mediated pH shifts through continuous substrate hydrolysis. Unfortunately, this in vivo assay is restricted to substrates that are not converted by the E. coli host strain. Conversion by the host is the most likely explanation for the discrepancy of the high emission ratio during -caprolactone hydrolysis (Table 2) and the reported low activity of the enzyme toward this substrate.23 In the metabolism of E. coli, a gluconolactonase of the pentosephosphate pathway converts 6-phosphogluconolactone into 6-phosphogluconate.24 It is likely that -caprolactone is also hydrolyzed by this gluconolactonase thus causing the strong pH shift detected by pHluorin. Permeability of the E. coli cell membrane for substrates is also an important issue for this assay. A pH shift due to substrate hydrolysis by a hydrolase can only occur when the substrate is able to diffuse in sufficient concentration through the cell membrane. The entry of any molecule is governed by its solubility in the cell’s boundary.25 All chosen substrates were able to penetrate the cell membrane. However, low permeability of more hydrophilic substances might prevent application of the assay. Cosolvents can be used not only to increase the solubility of hydrophobic substrates in buffer but also to support the penetration of the substrate into the cell.26 Several organic cosolvents were tested for the assay. The highest emission ratios after hydrolysis of tributyrin and methyl caproate were detected in the presence of gum arabic (2.42 and 1.98). Unfortunately, gum arabic has a decisive disadvantage: in a reaction emulsion of waterinsoluble substrate and phosphate buffer, E. coli cells stick to the dispersed gum arabic particles and are therefore unsuitable for flow cytometric analysis and cell sorting (data not shown). Much lower emission ratio values for both substrates were obtained when using Tween 20 (2.01 and 1.82). As a compromise DMSO (with values for both substrates 2.15 and 1.89) was considered best for the assay. In vitro measurements revealed a high activity of the G. stearothermophilus esterase toward phenyl acetate (Table 1). (23) Henke, E.; Bornscheuer, U. T. Appl. Microbiol. Biotechnol. 2002, 60, 320326. (24) Hucho, F.; Wallenfels, K. Biochim. Biophys. Acta 1972, 276, 176-179. (25) Al-Awqati, Q. Nat. Cell Biol. 1999, 1, 201-202. (26) Brayton, C. F. Cornell Vet. 1986, 76, 61-90.

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However, after 45 min hydrolysis of phenyl acetate, the fluorescence signal of E. coli cells producing pHluorin and BSE was comparable with that of the reference cells. Throughout the experiments, we noticed that the fluorescence intensity of pHluorin increased substantially with increasing buffer concentration (data not shown). For example, the fluorescence signal was up to four times stronger in a 500 mM buffer compared to a 50 mM buffer (data not shown). Further increase of buffer concentration, however, led to a slight reduction of the emission ratio from 2.24 (in 50 mM buffer) to 1.99 (in 1 M buffer). Such fluorescence quenching during hydrolysis may result from an in vivo pH shift to values below the stability limit of pHluorin (pH 5.0, data not shown). The results indicate that an increased buffer capacity prevents such strong pH shifts and subsequent quenching, thus retaining the fluorescence intensity at relative constant level. Extension of the Assay to Other Hydrolases. The assay was also tested on other hydrolase-catalyzed reactions. Two different model reactions were chosen: hydrolysis of hydantoin catalyzed by the Arthrobacter crystallopoietes hydantoinase (ACH) and carboxylic acid amide cleavage catalyzed by the Bacillus subtilis p-nitrobenzyl esterase (BsubpNBE). For the expression of ACH, 1 mM zinc sulfate was added after induction with rhamnose in order to support the enzyme’s correct folding and to serve as a catalytic factor for the hydrolysis reaction of hydantoin to N-carbamoyl glycine. The emission ratio of E. coli cells producing pHluorin and the recombinant hydantoinase was twice the value of the emission ratio of the reference (Table 2), i.e., of the same magnitude as the values obtained for tributyrin hydrolysis by esterase expressing cells (Figure 1). N-(1-p-Tolylethyl)octylamide was the substrate for the carboxylic acid amide cleavage reaction by BsubpNBE generating caprylic acid and 1-p-tolylethylamine. During this reaction both an acidic compound (caprylic acid) and a basic compound (1-ptolylethylamine) were released. The overall pH change and the resulting emission ratio thus reflects a sum signal derived from both compounds and is understandably not as high as that for hydantoin hydrolysis by ACH (Table 2). It is noteworthy that the pH shift can nevertheless be detected by pHluorin. To exemplify that the assay can also detect an increase in pH, amide cleavage was started at pH 6.0 instead of pH 8.0. In this case, cells without esterase activity, incubated at pH 6.0 were used as reference. The emission ratio of the cells with esterase activity was indeed lower (1.59) than that of the reference (1.85). This is in line with our expectation that an increase in pH can be registered by pHluorin as well (Table 2). Assay Establishment in Microtiter-Plate Scale. For downscaling of the assay eight single E. coli colonies producing the wild-yype esterase from G. stearothermophilus and eight colonies producing the corresponding inactivated mutant were picked and cultivated in microtiter plates. The assay was performed in 250 µL of potassium phosphate buffer containing 3% (v/v) DMSO as cosolvent and 2% (v/v) tributyrin as substrate. The emission ratios of eight E. coli cultures, which only expressed pHluorin, were used as reference. As indicated in Figure 2, esteraseexpressing cells showed higher emission ratios (2.0-2.3) after reaction for 45 min than the cells with inactivated esterase and controls (0.9-1.3). This meets the data obtained in the shake-

Figure 2. Emission ratios of E. coli cells expressing wild-type and inactivated mutants of Geobacillus stearothermophilus esterase (BSE). The hydrolysis of tributyrin is measured in microtiter-plate scale. Cells containing the active wild-type enzyme (squares) show approximately the 2-fold emission ratio than cells containing inactivated mutants (triangles).

Figure 3. FACS analysis of pHluorin-expressing E. coli cells in 50 mM potassium phosphate buffer at different pH values. A population shift is indicated by rearrangement of the lightly pink region (highest cell density).

flask experiments and demonstrates that pHluorin can be applied as a sensor of intracellular pH shifts in microtiter-plate scale. Combining the Assay and Flow Cytometry. The applicability of the assay for single cell sorting in flow-cytometric systems was also investigated. Measurements were carried out on LSR II and FACSDiVa from Becton Dickinson. In each experiment, 106 cells were analyzed. In the first set of experiments the behavior of E. coli cells with pHluorin in buffer at different pH was investigated. The obtained results have been presented graphically as emission ratio vs FSC density plot (Figure 3). As an indication of population shifts, quadrant gates were set as marker. The decrease of pH 8.0 to 6.0 was accompanied by a significant population shift toward higher emission ratios (Figure 3). Further E. coli cells, producing pHluorin and BSE esterase were analyzed during tributyrin hydrolysis. As reference cells without esterase activity were measured. Additionally, the impact of DMSO as cosolvent on the flow-cytometric assay was investigated in more detail. In the presence of DMSO, the shift of the

cell population with esterase activity toward higher emission ratios was stronger than in the absence of DMSO (data not shown), as this cosolvent increases the permeability of the cell membrane as described above. Finally, a mixed culture consisting of cells with and without esterase activity and pHluorin was analyzed during tributyrin hydrolysis. The cells, showing the maximum emission ratio, were sorted out of this culture and subsequently applied for fluorescence measurements. These fluorescence measurements confirmed the obtained results, indicating that only a strong pH shift can trigger a high emission ratio. The number of positive hits (cells with high hydrolytic activity) depends on the gate setting in FACS. A high emission ratio as indicator (narrow gate setting) may result in highly active enzymes. CONCLUSION A novel pHluorin-based assay was developed and successfully tested on different esterases as well as a hydantoinase. The assay is based on intracellular pH shifts and hence might be applied Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

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for monitoring of any hydrolytical reaction. This in vivo assay allows detection of pH changes in single E. coli cells, producing recombinant hydrolases. Therefore it can be used not only in microtiter-plate scale but also in combination with flow cytometric systems. For future studies, we are aiming to employ this assay for screening of hydrolase mutant libraries. ACKNOWLEDGMENT We would like to thank James E. Rothman from the Memorial Sloan-Kettering Cancer Center (New York, USA) for providing

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the plasmid pGEX_rat_pHluorin. We thank the Degussa AG for financial support of this project. Special thanks go to Jens Fleischer from BD Bioscience and Beate Roessle-Lorch for support with the FACS measurements and to Stefan Minning and Petra Traub for preliminary investigations.

Received for review February 5, 2005. AC0486692

September

8,

2004.

Accepted