- Email: [email protected]
β-Hydroxysteroid dehydrogenase: Activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion
Biochimie (1990) 72, 285-289 ~) Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier. Paris
285
/~-Hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion KM Lee, JF Biellmann* Laboratoire de Chimie Organique Biologique, UA CNRS 31, institut de Chimie. Universitd Louis Pasteur, I rue Blaise Pascal, 67008 Strasbourg, France
(Received 15 November 1989; accepted 22 March 1990)
Summary - The stability of purified/~-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions. The use of l-pentanol and l-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability. The NAD + Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion. Proteins, among them/3-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemul~ion, thus indicating that microemulsions may be utilized in protein release from cells. hydroxysteroid dehydrogenase/ Pseudomonas testosteroni/micelles / microemulsion/ stability / solubilization
Introduction
Microeraulsions and micelles present a n u m b e r of properties of interest to enzymologists: thermodynamic stability, ease of p r e p a r a t i o n and high exchange rates for the c o m p o n e n t s of the different phases. T h e transparency of these m e d i a renders possible the use of m a n y spectroscopic methods, especially for activity determination. T h e microemulsions generally consist of 4 c o m p o n e n t systems: water, organic solvent which is mostly h y d r o c a r b o n , surfactant and cosurfactant. Thus, the phase diagrams are 3-dimensional. Microemulsions and micelles have re,~ently generated much interest in the field of enzymology. T h e aspects studied thus far have included e n z y m e activity in media [1, 2], low w a t e r solubility proteins [3], enzyme acting on substrates with low water solubility [ 4 - 8 ] and biomimetic m e m b r a n e models [3]. ' r h e enzymic activity in microemulsion is a good model for the e n z y m e in heterogeneous conditions. In "he present study, we report a new use for microemulsions: the extraction of enzymes by treating whole cells with microemulsion. The usual m e t h o d s for cell disintegration tend to be vigourous. For P s e u d o m o n a s species, sonication seems to be most frequently used m e t h o d besides Manton-Gaulin homogenization [9]. A m e t h o d combining non-ionic detergent and osmotic pressure has b e e n described for cell disruption to
*Correspondence and reprints
release lysozyme [10]. We decided to work on /3-hydroxysteroid dehydrogenase [11, 12] extracted f r o m P s e u d o m o n a s testosteroni because the purified e n z y m e was available. T w o subsequent studies were therefore possible: first, a determination of the optimal conditions for observing enzymic activity and stability o v e r time of purified/3-hydroxysteroid dehydrogenase in microemulsion; then the extraction of the enzyme f r o m whole cells using microemulsion.
Materials and Methods
Sodium dodecyl sulfate (Fluka) and hexadecyltrimethyl ammonium bromide (Fluka) were recrystallized from ethanol, washed with pe,~ta~¢, and dried at 25°C under vacuum for 3 days. Triton X-100 (Sigma) was used directly as obtained. Cyclohexanefor UV spectroscopy (Fluka) was used without further purification, l-Butanol, l-pentanol and l-hexanol were purified by fractional distillation. The buffer used was 30 mM potassium phosphate pH 7.2 unless otherwise stated. Purified/~-hydroxystereid dehydrogenase [EC I. I. 1.5 1] from Pseudomonas testosteroni, crude dried cells of Pseudomonas testosteroni and testosterone were purchased from S~gma. The enzymes were dissolved in buffer immediately before use and protein concentration was estimated from the manufacturer's indication. Assays
The concentration of the total protein extracted from crude dried cells was estimated from the UV absorption intensity at 280 nm (1-
Abbreviations: NAD ÷, nicotinamide adenine dinucleotide; NADH, reduced nicotinamid adenine dinucleotide; CTAB, hexadecyltrimethyl-
ammonium bromide
286
KM Lee, JF Biellmann
cm cells). Actix~tyassays of/~-hydroxysteroid dehydrogenase were conducted in buffer and in microemulsion by following the absorption increase at 340 nm, due to the appearance of NADH. In buffer, the activi.*yof the enzyme was determined according to the published procedure [13]. The purified enzyme was dissolved in buffer (25 U / ml buffer). This enzyme solution (3 td) was added to the microemulsion(0.5 mi). At determined times (0. 1, 2, 4 and 8 h) at 20°C, a microemulsion containing NAD ÷ (5 raM; 0.1 ml) and another containing testosterone (50 raM; 0.1 ml) were added and enzymatic activity was determined by following the absorption increase at 340 am.
Kinetic constants of [3-hydroxysteroid dehydrogenase in buffer and in microemulsion A solution of testosterone in buffer was prepared: testosterone (8 rag) was dissolved in isopropanol (10 ml) and an aliquot of this solution (0.1 ml) was added to buffer (10 ml) containing Triton X100 (10 rag). To an aliquot of this homogeneous testosterone solution (27 ttM; 0.5 ml) was added a 10 mM solution of NAD + (10-70 ~tl) in buffer, buffer (0.19-0.13 ml) and enzyme solution(1/d; 25 U / ml). All solutions contained Triton X-100 at the same dilution (1 rag/ ml). The absorption increase was followed at 340 nm at 20°C. Two cationic Cb-2 microemuisions were prepared; one contained NAD + (5 raM) and the other testosterone (50 mM). To microemulsion Cb-2 (0.59-0.50 ml) was added the microemulsion conraining NAD ÷ (10-100 td) and the mieroemulsion with tezt,~sterone (100 td). To the resulting mieroemulsion system, a solution of purified enzyme in buffer (10 ~1; 25 U/ml) was added and the absorption increase at 340 nm was followed at 20oC.
Total protein extraction from Pseudomonas testosteroni cells with microemulsions Microemulsionswith the compositions indicated above were prepared. A suspension of Pseudomonas testosteroni cells (10-100 rag) in buffer (1 ml) was added at 20oC with magnetic stirring to each microemulsion (10 g). After 15 min, 1, 2, 4 and 8 h, the microemulsion was centrifuged for 3 rain at 10000 g and absorption at 280 nm was determined. The pH effect on the extraction of total protein v,as determined in 5 Cb-I microemulsions of varied pH, prepared with 30 mM phosphate buffer at pH 6, pH 7, pH 8 or with 30 mM glycine-sodium hydroxide buffer at pH 9 or pH 10. Cells of Pseudomonas testostetoni (10-100 rag) in buffer (1 ml) were added to these microemulsions (10 g) and after being magnetically stirred for 15 rain and then centrifuged for 3 min at 10000 g, absorption at 280 nm was determined. The calcium chloride effect on the total protein extraction was studied in the cationic microemulsion Cb-2 (10 g) which contained 0, 0.1 mM, 1 raM, 10 raM, 0.1 M or 1 M calcium chloride buffer. To each of these microemuisions, cells of Pseudomonas testosteroni (10 rag) in buffer of the same molarity were added with magnetic stirring. After 15 rain, each microemulsion was centrifuged for 3 rain at 10000 g and the absorption at 280 nm of the supematant was measured. Using microemulsions Cb-1 and Cb-3, a similar study was performed with 0.1 M calcium chloride.
Determination of the B-hydroxysteroid dehydrogenase extraction from Pseudomonas testosteroni cells with microemulsion A suspension of Pseudomonas testosteroni cells (10 rag) in buffer
Table l . Activity and stability of purified/~-hydroxysteroid dehydrogenase in buffer anti microemulsion; the composition of the microemulsions by weight. C, cationic; A, anionic; N, non ionic; b, 1-butanol; p, 1-pentanol; h, 1-hexanol.
Surfactant hexadecyltrimethyl ammonium bromide (CTAB)
Cosurfactant 1-butanol
Cb-1
1.5
Cb-2 Cb-3
Cyclohexane
Buffer
2.0
6.0
0.5
1.5
2.O
5.5
1.0
1.5
2.0
5.0
1.5
5.1
1.0
4.7
1.0
1-pentanol Cp-1
1.5
2.4 1-hexanol
Ch-i
Ab-1
1.5
2.8
Sodium dedecylsulfate (SDS)
1-butanol
1.2
2.0
5.8
1.0
2.0
4.8
1.0
Triton X- i00 Nb-1
2.7
Enzyme and microemu!sion (1 ml) was added to microemulsions (10 g) Cb-i, Cb-2 (containing 0; 0.1 M; 1.0 M calcium chloride) and Cb-3 with magnetic stirring. After 15 min, 1,2 and 4 h at 20°C, each microemulsion was centrifuged at 10000 g for 3 min and the supernatant was tested for dehydrogenase activity; a blank value was obtained in the following way: to the supernatant microemulsion (0.5 ml), a microemulsioa containing NAD ÷ (5 mM; 100/zl) was added, and the absorption increase at 340 nm determined. This blank value was compared with the value obtained with the mieroemulsion containing testosterone; to the supernatant microemnision (0.5 ml), a mieroemulsion containing N A D ÷ (5 mM; 100/zi) and a microemulsion containing testosterone (50 mM; 100 p,I) were added. The absorption increase at 340 nm was determined and the rates was corrected for the volume differences. The B-hydroxysteroid dehydrogenase activity was determined by the difference between rate of control and that of testosterone.
Results
and
Discussion
We thought that microemulsion might be an effective medium for disrupting the structure of the cell walls, and thus, for the release of the cytoplasmic enzymes. For this study, we chose ~3-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. Purified /~-hydroxysteroid dehydrogenase was quite stable in buffer (specific activity of the commercial preparation was 25 u n i t s / m g [13] and the activity decrease in 8 h was < 10%). In anionic (SDS) microemulsion, the dehydrogenase activity was very low and the activity loss quite rapid, in agreement with previous findings on other enzymes [4, 14]. In cationic microemulsions (CTAB), the activity at 5% water content was 40% of the activity at 15% water content, as found for other enzymes [14]. The activity loss was more rapid with lower water content (fig 1), and with higher water content, this loss was small enough justify further ( T i l t o n vA - I U1U, ~) , .~-~.... e . u u.~.. . y . ~. ~ t a l ; ~ J t i l u• l l | * . •, l l l l :~ I O l ~ l l l-U- .l.~1l-O. 'l -l tllU initial activity was higher than in the cationic microemulsion, and the enzyme was slightly more stable than in the cationic microemulsion. The surfactant Triton X-100, due to its aromatic ring, complicated protein determination by U V absorption at 280 nm and its study was discontinued. The nature of the cosurfactant in the cationic microemulsion had an influence on enzyme stability. With 1-pentanol and 1-hexanol, the activity was higher than in microemulsions with 1-butanol, and the activity showed greatest stability with 1-hexanol (fig 2). Microemulsions containing 1-pentanol or 1-hexanol, rather than l-butanol, seemed to be more favorable for maintaining enzyme activity. With 1-pentanol and 1-hexanol, the composition range for watero.in-oil microemulsions are much more restricted than with 1-butanol. So the effect of the water content of the microemulsion, with the higher alcohol content, can be determined within a small range of water concentration. For this reason, we focused our study on the microemulsions made with 1-butanol.
~"~-', /
|
1.0 *
t~l
°\
..
/
~~0~-~ O~ .
0
0
"[] Time (hr)
I
t
4
8
Fig 1. Activity and stability of purified//-hydroxysteroid dehydrogenase in cationic microemulsion (Cb-1; Cb-2; Cb-3) as a function of % H20: 5(o), 10 (F1), !5 (A). The kinetic constants were determined in buffer and in microemulsion Cb-2. The Michaelis constant of the coenzyme N A D + was found to be 0.22 mM in buffer, 0.35 mM expressed in overall concentration and 3.5 mM in watewool concentration * [ 15, 16]. A comparable observation of a higher Michaelis constant in microemulsion has also been reported for the Michaelis constant of N A D + with the horse liver alcohol dehydrogenase [14]. In contrast, the Michaelis constant of N A D ÷ with the alcohol dehydrogenase from Thermoanaerobium brockii [17] was the same in buffer and in mieroemulsion. The maximum velo~ties of the steroid dehydrogenase were 7.2 mmol-min -~" mgE -~ in buffer and 2.1 mmoi'min-~'mgE -~ in microemulsion. Microemulsions were used to study the release of total protein and of/3-hydroxysteroid dehydrogenase
*Overall concentration -- waterpool concentration x water volume fraction
2~S O~.
KM Lee, JF Biellmann
cationic microemulsions led to the same observed absorption intensity, and since the dehydrogenase was more stable in the cationic microemulsion, further studies were carried out with this system. The observed absorption at 280 nm increased with the quantity of cells. Presence of calcium chloride, effective on the partitioning of enzymes within micellar phases [1820], decreased absorptioa at 280 nm (table II). The pH of the microemulsion ha I a smaa effect. For example, for 10 mg of cells in cationic rmcroemulsion (1 ml) of otherwise constant composition, the absorption at 280 nm was 0.75 at pH 7.0, 0.65 at pH 6.0, and 0.5 at pH 10.0. Thus, the microemulsion effectively released protein from whole cells. When we turned our attention to the microemulsion extraction of fl-hydroxysteroid dehydrogenase from whole cells, we found that a second enzyme system was present which produced an absorption increase at 340 nm in the controls where no testosterone had been added. This alcohol dehTdrogenase likely acts on 1butanol of the microemulsion and is related to the enzymes previously i~olated from Pseudomonas species [21-23]. This activity (close to that o~ :he sterc~id dehydrogenase) interfered with the determination of /~-hydroxysteroid dehydrogenase activity. However, in the presence of testosterone, the absorption increase at 340 nm was significantly larger than in controls without the steroid. In contrast, the /~-hydroxysteroid dehydrogenase activity could be determined with sufficient precision (as shown in table II). At lower water content of the microemulsion, less activity was observed and added calcium chloride had a pronounced negative effect, e:;*.hibiting more than a 5-fold decrease in B-hydroxysteroid dehydrogenase activity in the
~ &
~-
t
< tt~ta__ --0
Time (hr) !
!
4
8
Fig 2. Activity and stability of purified fl-hydroxysteroid dehydrogenase in cationic microemulsions with the cosurfactants: 1-butanol Cb-2 (El), 1-pentanol Cp-1 (o), 1-hexanol Chd (A). from whole cells of Pseudomonas testosteroni. A suspension of cells was added to the microemulsion and the suspension was stirred and centrifuged. The absorption at 280 nm of the supernatant was determined as a measure of total protein extracted. Both anionic and
Table !I. Total protein extraction by microemulsion. *Same value with 0.1 mM; 1 mM and 10 mM CaCI2. **After 15 min-
8 h stirring.
Microemulsion (10 g)
SDS CTAB
Ab-1 Cb-1 Cb-2
Cb-3
CaCI2 M for l Og of microemulsion
Quantity of Pseudomonas cells (mg)
Absorption at 280 nra **
0 0 0.1 0*
10 10 10 10 40 100 10 40 !00 10 10
0.4 0.38 0.10 0.40* 1.22 2.43 0.12 0.40 0.80 0.43 0.12
0.1 0.1 0.1 0 0.1
289
Enzyme and microemulsion
Table I l L Extraction of steroid dehydrogenase activity from Pseudomonas testosteroni cells by microemulsions. /3-Hydroxysteroid dehydrogenase activity extracted from Pseudomonas testosteroni cells (10 mg in 1 ml buffer) by microemulsion (10 g) and expressed in/zmol.min-l-mg -~ (cell weight). Microemulsion
Cb-1 Cb-2 Cb-2 (0.1 and 1.0 M CaCI2) Cb-3
[~-Hydroxysteroid dehydrogenase activity ( i t m o l . m i n - l . m g -I ) extracted as a function o f time (h)
0.25
1
2
4
1,35 2.05 0.4 2.5
1.0 1.4 0.15 1.8
0.75 1.2 0.1 1.6
0.6 0.9 0.1 1.4
0.1 and 1.0 M calcium chloride. The time dependence of the activity was dose to that observed with the purified enzyme. In contrast, the activity of the interfering dehydrogenase was more pronounced in the presence of calcium chloride, exhibiting approximately 3-fold higher activity. Conclusion In conclusion, microemulsions are effective media for extraction of intercellular enzymes from whole cells [24]. The present results should prompt further investigation into the generality of this microcmulsion procedure for release of enzymes from whole cells. Acknowledgments The authors are grateful to Elf-Aquitaine for providing them with financial support.
References 1 Luisi PL (1985) Angew Chem lnt Ed Eng~ 24, 439-450 2 Martinek K, Levashov AV, Kiyachko NL, Khmelniski YL, Berezin IV (1986) Eur J Biochem 155,453-468 3 Waks M (1986) Proteins 1, 4-15 4 Lee KM, Biellmann JF (1986) Bioorg Chem 14, 262-273 5 Lee KM, Biellmann JF (1986) NewJ Chem 10, 675
6 7 8 9
Lee KM~ Biellmann .IF (1988) Tetrahedron 44, 1135-1140 Lee KM, Biellmann JF (1990) (in preparation) Hilhorst [~, Laane C, Weegcr C (1983) FEBS Lett 159,225-228 a, Maurer P, Lessmann D, Kurz G (1982) In: Methods in Enzymology (Colowick SP, Kaplan NO, eds) 261-270; b, Kohn LD, Utting JM (1982) In: Methods in Enzymology (Colowick SP. Kaplan NO, eds) 341-345; c, Vandecasteele JP, Guer~llot L (1982) In: Methods in Enzymology (Colowick SP, Kaplan NO, eds) 484-490 10 Schwinghamer EA (1980) FEMS Microb Left 7, 157-162 11 Battais E, Terouanne B, Nicholas JC, Descomps B, Crastes de Paulet A (1977) Biochimie 59, 909-917 12 Levy MA, Holt DA, Brandt M, Metcalf BW (1987) Biochemistry 26, 2270-2279 13 Schultz RM, Groman EV, Engel LL (1977) J Biol Chem 252, 3775-3790 14 a, Samama JP, Lee KM, Biellmann JF (1987) EurJ Biochem 163, 609-617; b, Larsson KM, Adlercretuz P, Mattiasson B (1987) Eur J Biochem 166~ 157-161 15 Martinck K, Levashov AV, Klyachko NL, Pantin VI, Berezin IV (1981) Biochim Biophys Acta 657,277-294 16 Bonner FJ, Wolf R, Luisi PL (1980) J Solid Phase Biochem 5, 255-268 17 Lee KM, Biellmann JF (!987) New J Chem 11,775-778 18 Luisi PL, Laane C (1986) Trends Biotechnol 153-161 19 Leser ME, Wei G, Luisi PL, Maestro M (1986) Biochem Biophys Res Commun 135,629-635 20 G6klcn KE, Hatton TA (1985) Biotechnology Progress, Vol 1, 1, 69-74 21 Groen BW, Vankleef MA, Duine JA (1986) Biochem J 234, 611-615 22 Ogushi S, Ando M, Tsuru D (1984) Agric Biol Chem 48, 597-601 23 Hou CT, Patel R, Barnabe N, Marczak I (1981) EurJ Biochem 119, 359-364 24 Giovenco S, Verheggen F, Laane C (1987) Enzyme Microbial Technol 9, 470-473
285
/~-Hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion KM Lee, JF Biellmann* Laboratoire de Chimie Organique Biologique, UA CNRS 31, institut de Chimie. Universitd Louis Pasteur, I rue Blaise Pascal, 67008 Strasbourg, France
(Received 15 November 1989; accepted 22 March 1990)
Summary - The stability of purified/~-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions. The use of l-pentanol and l-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability. The NAD + Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion. Proteins, among them/3-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemul~ion, thus indicating that microemulsions may be utilized in protein release from cells. hydroxysteroid dehydrogenase/ Pseudomonas testosteroni/micelles / microemulsion/ stability / solubilization
Introduction
Microeraulsions and micelles present a n u m b e r of properties of interest to enzymologists: thermodynamic stability, ease of p r e p a r a t i o n and high exchange rates for the c o m p o n e n t s of the different phases. T h e transparency of these m e d i a renders possible the use of m a n y spectroscopic methods, especially for activity determination. T h e microemulsions generally consist of 4 c o m p o n e n t systems: water, organic solvent which is mostly h y d r o c a r b o n , surfactant and cosurfactant. Thus, the phase diagrams are 3-dimensional. Microemulsions and micelles have re,~ently generated much interest in the field of enzymology. T h e aspects studied thus far have included e n z y m e activity in media [1, 2], low w a t e r solubility proteins [3], enzyme acting on substrates with low water solubility [ 4 - 8 ] and biomimetic m e m b r a n e models [3]. ' r h e enzymic activity in microemulsion is a good model for the e n z y m e in heterogeneous conditions. In "he present study, we report a new use for microemulsions: the extraction of enzymes by treating whole cells with microemulsion. The usual m e t h o d s for cell disintegration tend to be vigourous. For P s e u d o m o n a s species, sonication seems to be most frequently used m e t h o d besides Manton-Gaulin homogenization [9]. A m e t h o d combining non-ionic detergent and osmotic pressure has b e e n described for cell disruption to
*Correspondence and reprints
release lysozyme [10]. We decided to work on /3-hydroxysteroid dehydrogenase [11, 12] extracted f r o m P s e u d o m o n a s testosteroni because the purified e n z y m e was available. T w o subsequent studies were therefore possible: first, a determination of the optimal conditions for observing enzymic activity and stability o v e r time of purified/3-hydroxysteroid dehydrogenase in microemulsion; then the extraction of the enzyme f r o m whole cells using microemulsion.
Materials and Methods
Sodium dodecyl sulfate (Fluka) and hexadecyltrimethyl ammonium bromide (Fluka) were recrystallized from ethanol, washed with pe,~ta~¢, and dried at 25°C under vacuum for 3 days. Triton X-100 (Sigma) was used directly as obtained. Cyclohexanefor UV spectroscopy (Fluka) was used without further purification, l-Butanol, l-pentanol and l-hexanol were purified by fractional distillation. The buffer used was 30 mM potassium phosphate pH 7.2 unless otherwise stated. Purified/~-hydroxystereid dehydrogenase [EC I. I. 1.5 1] from Pseudomonas testosteroni, crude dried cells of Pseudomonas testosteroni and testosterone were purchased from S~gma. The enzymes were dissolved in buffer immediately before use and protein concentration was estimated from the manufacturer's indication. Assays
The concentration of the total protein extracted from crude dried cells was estimated from the UV absorption intensity at 280 nm (1-
Abbreviations: NAD ÷, nicotinamide adenine dinucleotide; NADH, reduced nicotinamid adenine dinucleotide; CTAB, hexadecyltrimethyl-
ammonium bromide
286
KM Lee, JF Biellmann
cm cells). Actix~tyassays of/~-hydroxysteroid dehydrogenase were conducted in buffer and in microemulsion by following the absorption increase at 340 nm, due to the appearance of NADH. In buffer, the activi.*yof the enzyme was determined according to the published procedure [13]. The purified enzyme was dissolved in buffer (25 U / ml buffer). This enzyme solution (3 td) was added to the microemulsion(0.5 mi). At determined times (0. 1, 2, 4 and 8 h) at 20°C, a microemulsion containing NAD ÷ (5 raM; 0.1 ml) and another containing testosterone (50 raM; 0.1 ml) were added and enzymatic activity was determined by following the absorption increase at 340 am.
Kinetic constants of [3-hydroxysteroid dehydrogenase in buffer and in microemulsion A solution of testosterone in buffer was prepared: testosterone (8 rag) was dissolved in isopropanol (10 ml) and an aliquot of this solution (0.1 ml) was added to buffer (10 ml) containing Triton X100 (10 rag). To an aliquot of this homogeneous testosterone solution (27 ttM; 0.5 ml) was added a 10 mM solution of NAD + (10-70 ~tl) in buffer, buffer (0.19-0.13 ml) and enzyme solution(1/d; 25 U / ml). All solutions contained Triton X-100 at the same dilution (1 rag/ ml). The absorption increase was followed at 340 nm at 20°C. Two cationic Cb-2 microemuisions were prepared; one contained NAD + (5 raM) and the other testosterone (50 mM). To microemulsion Cb-2 (0.59-0.50 ml) was added the microemulsion conraining NAD ÷ (10-100 td) and the mieroemulsion with tezt,~sterone (100 td). To the resulting mieroemulsion system, a solution of purified enzyme in buffer (10 ~1; 25 U/ml) was added and the absorption increase at 340 nm was followed at 20oC.
Total protein extraction from Pseudomonas testosteroni cells with microemulsions Microemulsionswith the compositions indicated above were prepared. A suspension of Pseudomonas testosteroni cells (10-100 rag) in buffer (1 ml) was added at 20oC with magnetic stirring to each microemulsion (10 g). After 15 min, 1, 2, 4 and 8 h, the microemulsion was centrifuged for 3 rain at 10000 g and absorption at 280 nm was determined. The pH effect on the extraction of total protein v,as determined in 5 Cb-I microemulsions of varied pH, prepared with 30 mM phosphate buffer at pH 6, pH 7, pH 8 or with 30 mM glycine-sodium hydroxide buffer at pH 9 or pH 10. Cells of Pseudomonas testostetoni (10-100 rag) in buffer (1 ml) were added to these microemulsions (10 g) and after being magnetically stirred for 15 rain and then centrifuged for 3 min at 10000 g, absorption at 280 nm was determined. The calcium chloride effect on the total protein extraction was studied in the cationic microemulsion Cb-2 (10 g) which contained 0, 0.1 mM, 1 raM, 10 raM, 0.1 M or 1 M calcium chloride buffer. To each of these microemuisions, cells of Pseudomonas testosteroni (10 rag) in buffer of the same molarity were added with magnetic stirring. After 15 rain, each microemulsion was centrifuged for 3 rain at 10000 g and the absorption at 280 nm of the supematant was measured. Using microemulsions Cb-1 and Cb-3, a similar study was performed with 0.1 M calcium chloride.
Determination of the B-hydroxysteroid dehydrogenase extraction from Pseudomonas testosteroni cells with microemulsion A suspension of Pseudomonas testosteroni cells (10 rag) in buffer
Table l . Activity and stability of purified/~-hydroxysteroid dehydrogenase in buffer anti microemulsion; the composition of the microemulsions by weight. C, cationic; A, anionic; N, non ionic; b, 1-butanol; p, 1-pentanol; h, 1-hexanol.
Surfactant hexadecyltrimethyl ammonium bromide (CTAB)
Cosurfactant 1-butanol
Cb-1
1.5
Cb-2 Cb-3
Cyclohexane
Buffer
2.0
6.0
0.5
1.5
2.O
5.5
1.0
1.5
2.0
5.0
1.5
5.1
1.0
4.7
1.0
1-pentanol Cp-1
1.5
2.4 1-hexanol
Ch-i
Ab-1
1.5
2.8
Sodium dedecylsulfate (SDS)
1-butanol
1.2
2.0
5.8
1.0
2.0
4.8
1.0
Triton X- i00 Nb-1
2.7
Enzyme and microemu!sion (1 ml) was added to microemulsions (10 g) Cb-i, Cb-2 (containing 0; 0.1 M; 1.0 M calcium chloride) and Cb-3 with magnetic stirring. After 15 min, 1,2 and 4 h at 20°C, each microemulsion was centrifuged at 10000 g for 3 min and the supernatant was tested for dehydrogenase activity; a blank value was obtained in the following way: to the supernatant microemulsion (0.5 ml), a microemulsioa containing NAD ÷ (5 mM; 100/zl) was added, and the absorption increase at 340 nm determined. This blank value was compared with the value obtained with the mieroemulsion containing testosterone; to the supernatant microemnision (0.5 ml), a mieroemulsion containing N A D ÷ (5 mM; 100/zi) and a microemulsion containing testosterone (50 mM; 100 p,I) were added. The absorption increase at 340 nm was determined and the rates was corrected for the volume differences. The B-hydroxysteroid dehydrogenase activity was determined by the difference between rate of control and that of testosterone.
Results
and
Discussion
We thought that microemulsion might be an effective medium for disrupting the structure of the cell walls, and thus, for the release of the cytoplasmic enzymes. For this study, we chose ~3-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. Purified /~-hydroxysteroid dehydrogenase was quite stable in buffer (specific activity of the commercial preparation was 25 u n i t s / m g [13] and the activity decrease in 8 h was < 10%). In anionic (SDS) microemulsion, the dehydrogenase activity was very low and the activity loss quite rapid, in agreement with previous findings on other enzymes [4, 14]. In cationic microemulsions (CTAB), the activity at 5% water content was 40% of the activity at 15% water content, as found for other enzymes [14]. The activity loss was more rapid with lower water content (fig 1), and with higher water content, this loss was small enough justify further ( T i l t o n vA - I U1U, ~) , .~-~.... e . u u.~.. . y . ~. ~ t a l ; ~ J t i l u• l l | * . •, l l l l :~ I O l ~ l l l-U- .l.~1l-O. 'l -l tllU initial activity was higher than in the cationic microemulsion, and the enzyme was slightly more stable than in the cationic microemulsion. The surfactant Triton X-100, due to its aromatic ring, complicated protein determination by U V absorption at 280 nm and its study was discontinued. The nature of the cosurfactant in the cationic microemulsion had an influence on enzyme stability. With 1-pentanol and 1-hexanol, the activity was higher than in microemulsions with 1-butanol, and the activity showed greatest stability with 1-hexanol (fig 2). Microemulsions containing 1-pentanol or 1-hexanol, rather than l-butanol, seemed to be more favorable for maintaining enzyme activity. With 1-pentanol and 1-hexanol, the composition range for watero.in-oil microemulsions are much more restricted than with 1-butanol. So the effect of the water content of the microemulsion, with the higher alcohol content, can be determined within a small range of water concentration. For this reason, we focused our study on the microemulsions made with 1-butanol.
~"~-', /
|
1.0 *
t~l
°\
..
/
~~0~-~ O~ .
0
0
"[] Time (hr)
I
t
4
8
Fig 1. Activity and stability of purified//-hydroxysteroid dehydrogenase in cationic microemulsion (Cb-1; Cb-2; Cb-3) as a function of % H20: 5(o), 10 (F1), !5 (A). The kinetic constants were determined in buffer and in microemulsion Cb-2. The Michaelis constant of the coenzyme N A D + was found to be 0.22 mM in buffer, 0.35 mM expressed in overall concentration and 3.5 mM in watewool concentration * [ 15, 16]. A comparable observation of a higher Michaelis constant in microemulsion has also been reported for the Michaelis constant of N A D + with the horse liver alcohol dehydrogenase [14]. In contrast, the Michaelis constant of N A D ÷ with the alcohol dehydrogenase from Thermoanaerobium brockii [17] was the same in buffer and in mieroemulsion. The maximum velo~ties of the steroid dehydrogenase were 7.2 mmol-min -~" mgE -~ in buffer and 2.1 mmoi'min-~'mgE -~ in microemulsion. Microemulsions were used to study the release of total protein and of/3-hydroxysteroid dehydrogenase
*Overall concentration -- waterpool concentration x water volume fraction
2~S O~.
KM Lee, JF Biellmann
cationic microemulsions led to the same observed absorption intensity, and since the dehydrogenase was more stable in the cationic microemulsion, further studies were carried out with this system. The observed absorption at 280 nm increased with the quantity of cells. Presence of calcium chloride, effective on the partitioning of enzymes within micellar phases [1820], decreased absorptioa at 280 nm (table II). The pH of the microemulsion ha I a smaa effect. For example, for 10 mg of cells in cationic rmcroemulsion (1 ml) of otherwise constant composition, the absorption at 280 nm was 0.75 at pH 7.0, 0.65 at pH 6.0, and 0.5 at pH 10.0. Thus, the microemulsion effectively released protein from whole cells. When we turned our attention to the microemulsion extraction of fl-hydroxysteroid dehydrogenase from whole cells, we found that a second enzyme system was present which produced an absorption increase at 340 nm in the controls where no testosterone had been added. This alcohol dehTdrogenase likely acts on 1butanol of the microemulsion and is related to the enzymes previously i~olated from Pseudomonas species [21-23]. This activity (close to that o~ :he sterc~id dehydrogenase) interfered with the determination of /~-hydroxysteroid dehydrogenase activity. However, in the presence of testosterone, the absorption increase at 340 nm was significantly larger than in controls without the steroid. In contrast, the /~-hydroxysteroid dehydrogenase activity could be determined with sufficient precision (as shown in table II). At lower water content of the microemulsion, less activity was observed and added calcium chloride had a pronounced negative effect, e:;*.hibiting more than a 5-fold decrease in B-hydroxysteroid dehydrogenase activity in the
~ &
~-
t
< tt~ta__ --0
Time (hr) !
!
4
8
Fig 2. Activity and stability of purified fl-hydroxysteroid dehydrogenase in cationic microemulsions with the cosurfactants: 1-butanol Cb-2 (El), 1-pentanol Cp-1 (o), 1-hexanol Chd (A). from whole cells of Pseudomonas testosteroni. A suspension of cells was added to the microemulsion and the suspension was stirred and centrifuged. The absorption at 280 nm of the supernatant was determined as a measure of total protein extracted. Both anionic and
Table !I. Total protein extraction by microemulsion. *Same value with 0.1 mM; 1 mM and 10 mM CaCI2. **After 15 min-
8 h stirring.
Microemulsion (10 g)
SDS CTAB
Ab-1 Cb-1 Cb-2
Cb-3
CaCI2 M for l Og of microemulsion
Quantity of Pseudomonas cells (mg)
Absorption at 280 nra **
0 0 0.1 0*
10 10 10 10 40 100 10 40 !00 10 10
0.4 0.38 0.10 0.40* 1.22 2.43 0.12 0.40 0.80 0.43 0.12
0.1 0.1 0.1 0 0.1
289
Enzyme and microemulsion
Table I l L Extraction of steroid dehydrogenase activity from Pseudomonas testosteroni cells by microemulsions. /3-Hydroxysteroid dehydrogenase activity extracted from Pseudomonas testosteroni cells (10 mg in 1 ml buffer) by microemulsion (10 g) and expressed in/zmol.min-l-mg -~ (cell weight). Microemulsion
Cb-1 Cb-2 Cb-2 (0.1 and 1.0 M CaCI2) Cb-3
[~-Hydroxysteroid dehydrogenase activity ( i t m o l . m i n - l . m g -I ) extracted as a function o f time (h)
0.25
1
2
4
1,35 2.05 0.4 2.5
1.0 1.4 0.15 1.8
0.75 1.2 0.1 1.6
0.6 0.9 0.1 1.4
0.1 and 1.0 M calcium chloride. The time dependence of the activity was dose to that observed with the purified enzyme. In contrast, the activity of the interfering dehydrogenase was more pronounced in the presence of calcium chloride, exhibiting approximately 3-fold higher activity. Conclusion In conclusion, microemulsions are effective media for extraction of intercellular enzymes from whole cells [24]. The present results should prompt further investigation into the generality of this microcmulsion procedure for release of enzymes from whole cells. Acknowledgments The authors are grateful to Elf-Aquitaine for providing them with financial support.
References 1 Luisi PL (1985) Angew Chem lnt Ed Eng~ 24, 439-450 2 Martinek K, Levashov AV, Kiyachko NL, Khmelniski YL, Berezin IV (1986) Eur J Biochem 155,453-468 3 Waks M (1986) Proteins 1, 4-15 4 Lee KM, Biellmann JF (1986) Bioorg Chem 14, 262-273 5 Lee KM, Biellmann JF (1986) NewJ Chem 10, 675
6 7 8 9
Lee KM~ Biellmann .IF (1988) Tetrahedron 44, 1135-1140 Lee KM, Biellmann JF (1990) (in preparation) Hilhorst [~, Laane C, Weegcr C (1983) FEBS Lett 159,225-228 a, Maurer P, Lessmann D, Kurz G (1982) In: Methods in Enzymology (Colowick SP, Kaplan NO, eds) 261-270; b, Kohn LD, Utting JM (1982) In: Methods in Enzymology (Colowick SP. Kaplan NO, eds) 341-345; c, Vandecasteele JP, Guer~llot L (1982) In: Methods in Enzymology (Colowick SP, Kaplan NO, eds) 484-490 10 Schwinghamer EA (1980) FEMS Microb Left 7, 157-162 11 Battais E, Terouanne B, Nicholas JC, Descomps B, Crastes de Paulet A (1977) Biochimie 59, 909-917 12 Levy MA, Holt DA, Brandt M, Metcalf BW (1987) Biochemistry 26, 2270-2279 13 Schultz RM, Groman EV, Engel LL (1977) J Biol Chem 252, 3775-3790 14 a, Samama JP, Lee KM, Biellmann JF (1987) EurJ Biochem 163, 609-617; b, Larsson KM, Adlercretuz P, Mattiasson B (1987) Eur J Biochem 166~ 157-161 15 Martinck K, Levashov AV, Klyachko NL, Pantin VI, Berezin IV (1981) Biochim Biophys Acta 657,277-294 16 Bonner FJ, Wolf R, Luisi PL (1980) J Solid Phase Biochem 5, 255-268 17 Lee KM, Biellmann JF (!987) New J Chem 11,775-778 18 Luisi PL, Laane C (1986) Trends Biotechnol 153-161 19 Leser ME, Wei G, Luisi PL, Maestro M (1986) Biochem Biophys Res Commun 135,629-635 20 G6klcn KE, Hatton TA (1985) Biotechnology Progress, Vol 1, 1, 69-74 21 Groen BW, Vankleef MA, Duine JA (1986) Biochem J 234, 611-615 22 Ogushi S, Ando M, Tsuru D (1984) Agric Biol Chem 48, 597-601 23 Hou CT, Patel R, Barnabe N, Marczak I (1981) EurJ Biochem 119, 359-364 24 Giovenco S, Verheggen F, Laane C (1987) Enzyme Microbial Technol 9, 470-473