Solubilization of bacterial cells in organic solvents via reverse micelles

Solubilization of bacterial cells in organic solvents via reverse micelles

Vol. 127, No. 3, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS larch 29, 1985 Pages 911-915 SOLUBILIZATION OF BACTERIAL CELLS IN ORGANI...

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Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

larch 29, 1985

Pages 911-915

SOLUBILIZATION OF BACTERIAL CELLS IN ORGANIC SOLVENTS VIA REVERSE MICELLES Gabriel HEring*, Pier Luigi L u i s i * , and Franz Meussdoerffer** * I n s t i t u t f~r Polymere and * * I n s t i t u t f~r Biotechnologie, Eigen~ssische Technische Hochschule, ZUrich, Switzerland Received February 12, 1985

Summary: A reverse micellar system containing Tween 85 and water in isopropylpalmitate was developed which permitted the s o l u b i l i z a t i o n of bacteria in the form of homogenous organic solutions. The presence of the bacteria in solution was demonstrated by l i g h t microscopy. Immediately after s o l u b i l i z a t i o n , isolated bacterial cells were observed, which by aging tend to form larger aggregates. Cells of Escherichia coli remained viable in this system for at least one day and retained B-galactosidase a c t i v i t y for an even longer period as indicated by the hydrolysis of x-gal. Cells of an alkane-degrading strain of Acinetobacter calcoaceticus remained viable in the system for several 'days. "~1985Acad~ieP . . . . . Inc.

I t is well established that many biological materials, among them enzymes, can be included into reverse micelles and maintained there even with preserved a c t i v i t y ( I - 4 ) . More recently the s o l u b i l i z a tion of nucleic acids, including a high molecular weight plasmid (2x 106 daltons) was reported (5). This finding was rather surprising since u n f i l l e d reverse micelles are not larger than 40-100 A° in diameter, depending on the water content of the system. Therefore i t seems that the micellar system is capable of restructuring itself

in a way that i t can include particles of much bigger size

than the original micelle. This observation raises the question of how far can the size l i m i t s of a system hosted into a reverse micelle be expanded, and whether one is s t i l l micelles a f t e r the s o l u b i l i z a t i o n still

dealing with reverse

(for the sake of s i m p l i c i t y , we

refer for the time being to our system as micellar system or

micellar solution). A p a r t i c u l a r l y challenging problem is the s o l u b i l i z a t i o n of entire cells.

In this communication we report the s o l u b i l i z a t i o n of two

types of bacteria, namely a strain of E. coli harbouring a recombinant plasmid and a strain of Acinetobacter calcoaceticus in a mi0006-291X/85 $1.50 911

Copyright © 1985 by Academic Press, Inc. All rights of reproductton m any form reserved.

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cellar system consisting of isopropylpalmitate, Tween 85 and water. Since the latter bacterium is known to degrade hydrocarbons, the bulk solvent of the micellar system might at the same time provide the carbon source for the bacteria, and therefore this micellar system may open interesting perspectives in biotechnology.

MATERIALS and METHODS Chemicals: Isopropylpalmitate and Tween 85 (Polyoxyethylen-Sorbitolt r i o l e a t e ) were from Fluka (Switzerland). 5-Bromo-4-chloro-3-indolylgalactopyranoside (x-gal) and Isopropyl-D-thiogalactoside (IPTG) were from Boehringer, Mannheim (Germany). Strains: Acinetobacter cribed by Kleber et al. ZUrich. E. coli K 12 and described by Dente Washington.

calcoaceticus, o r i g i n a l l y isolated and des(6) was a g i f t from Prof. Dr. A. Fiechter, 71/18 harbouring plasmid PEMBL 8, constructed et al. (7) was a g i f t from Dr. D. Clark,

S o l u b i l i z a t i o n : Acinetobacter calcoaceticus was grown in LB medium (8). E. coli 71/18 (PEMBL 8) was grown in LB supplemented with ampic i l l i n (60 ug/ml). Overnight cultures of bacteria were pelleted and m i c r o l i t e r aliquots of cell suspensions in water were added to the isopropylpalmitate solution of the surfactant, which already contained water. I f necessary, x-gal and IPTG or ethidiumbromide were added together with the c e l l s . Cells were kept in the organic solution in t i g h t l y closed vessels (in order to avoid any change in the composition of the organic solution by evaporation) at RT without shaking. For the determination of the concentration of viable c e l l s , aliquots of the micellar solution were diluted into l i q u i d LB medium and appropriate aliquots were plated on LB agar plates or LB plates containing a m p i c i l l i n (60 ug/ml). Light microscopy was performed with a Nikon Optiphot. RESULTS and DISCUSSION We have observed that several systems forming reverse micelles are capable to solubilize bacterial cells, i.e. providing clear organic solutions of the bacteria (e.g. Triton XlOO/hexanol/cyclohexane/ water). However, in these systems bacteria died quickly as indicated by the rapidly decreasing oxygen uptake. A system which permitted viability of cells for longer periods of time was Tween 85, isopropylpalmitate, water, with a surfactant content of I0% and a water content ranging from between 3-4%. Addition of glycerol (1%) to the system increased its capability to solubilized bacteria but reduced drastically the viability of the bacteria. With this system we have successfully solubilized two types of cells, namely Acinetobacter calcoaceticus and a strain of E. coli. 912

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The organic micellar system containing thebacteria at concentrations of up to lO8 cells per ml formed clear solutions while the same concentration of bacteria in water forms turbid suspensions. In the micellar organic solution the bacteria could be easily detected with a light microscope. When ethidiumbromide was added to the bacterial suspension prior to solubilization, a red fluorescence was observed in the micellar solution at the positions of the bacteria under ultraviolet light in the light microscope. Under the light microscope the mobility of the bacteria in the organic system appeared clearly reduced as compared to water. I n i t i a l l y , all cells appeared isolated from one another, but with time a tendency to aggregation was noticed. However no turbidity was observed and no precipitation occurred for several days. The rate of survival of Acinetobacter calcoaceticus (at about lO8 cells/ml) was tested by plating out (at 37°C) aliquots of the micellar system (kept at RT) after different time periods. As indicated in Table I, the number of viable cells did not decrease within 5 hours after solubilization, and even after 3 days in the organic system 2% of the cells were alive. Considering that no carbon or nitrogen source was provided and taking into account a severe oxygen limitation (no shaking), this number is f a i r l y high. The other type of solubilized cells is a strain of E. coli Kl2 harbouring plasmid PEMBL-8 which enables the strain to produce B-galac-

TABLE 1 Viability

of bacterial c e l l s in the m i c e l l a r system Tween/H20/ Isopropylpalmitate a

Time after solubilization (h) 0

Acinetobacter b E. coli

aTween 75 mM. ~o28.

60 (i00) 5

5

50 (16) 0.5

24

72

n.d.

13 (0)

0.05

n.d.

Cell concentration, 106 c e l l s / m l .

bData in parentheses are in the presence of I00 mM glycerol at mo 21

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Vol. 127, NO. 3, 1 9 8 5

1.5

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

B 4

3

0.2

1.0 r~ O

0.1

2

0.5

500

600

0 500

700

Figure ]

600

700

nm

nm

Absorption spectra of a micellar solution of E-Coli showing cleavage of x-gal by B.gakactosidase at different times. The substrate is added to the aqueous call suspension immediately prior to solubilization. A: Cells were induced with IPTG for 16 hours prior to solubilization. (l: t=O; 2: t= 5h; 3: t=2Oh; 4:t=48 h). B: IPT6 was added to the aqueous cell suspension immediately prior to solubilization. (l: t=O; 2: t=2Oh; 3: t=48h).

tosidase. The v i a b i l i t y of this strain in the reverse micelles was lower than for Acinetobacter (Table l ) . This might be due to the fact that the l a t t e r is able to degrade alkanes and therefore might be better adapted to survival in organic solutions. I t is noteworthy however, that no loss of plasmid as judged by the number of ampicillin resistent

clones occurred during 24 hours. Moreover, B-galacto-

sidase a c t i v i t y could be observed even after most of the cells were dead. When the synthesis of this enzyme was induced by IPTG prior to solubilization ( i . e . i f a large amount of galactosidase was present before the solubilization), enzyme a c t i v i t y in the reverse micellar solution kept increasing for more than 24 hours (Figure IA). Since i t was necessary to add the substrate (x-gal) to the bacteria immediately before solubilization, i t is not clear whether the eventual decrease of product formation is due to enzyme inactivation or exhaustion of the substrate. On the other hand, when IPTG was added together with the substrate to the celles immediately before the solubilization, only l i t t l e , however clearly increasing galactosidase a c t i v i t y was measured 914

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BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNIL;/~t Iur~o

(Figure IB).This might indicate that the cells in the reverse micellar solution are capable of protein biosynthesis, at least for some hours. The hydrolysis of x-gal in the presence of bacteria was compared with a control experiment consisting of a micellar solution under the same conditions but without c e l l s , which remained colourles ( Fig.l A,B ) during all experiments. Although preliminary, our data show that i t is possible to solubilize whole bacteria in reverse micellar solution with preserved v i a b i l i t y . Since both types of bacteria used in this study - the alkane degrading Acinetobacter and the E. coli harbouring a recombinat plasmid - are of biotechnological interest, our system might be of interest for cell bioconversions or degradations in organic systems. Further studies are under way to improve the described system and to elucidate in detail the structure of the cell-containing aggregates.

REFERENCES I) K. Martinek, A.V. Levashov, N.L. Klyachko, V.I. Pantin, I.V. Berezin, Biochem. Biophys. Acta 657, 277 (1981) 2) P. Douzou, E. Keh, C. Balny, Proc. Natl ' A c a d . Sci. USA 76, 681 (1979) 3) P.D.I. Fletcher, R.B. Freedman, J. Mead, C. Oldfield and B.H. Robinson, Colloid and Surfaces 10, 193 (1984) 4) S. Barbaric, P.L. L u i s i , J. Am. Chem. Soc. 103, 4239 (1981) 5) V.E. Imre and P.L. L u i s i , Biochem. and Biophys. Res. Com., Vol. 107, No. 2 538-545 (1982) 6) H.P. Kleber, W. Schoepp and H. Aurich, Z. Allg. Mikrobiol. 13, 445-467 (1973) 7) L.D. Dente, G. Cesareni and R. Cortese, Nucleic Acid Research 1__II, 1645-1655 (1983) 8) S.H. M i l l e r , "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory (1972)

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