Submitted to Enzyme and Microbial Technology, Revised 3/2/2000

CHIRAL EPOXIDE PRODUCTION USING

MYCOBACTERIUM SOLUBILISED IN A WATER-

IN-OIL MICROEMULSION

S. Prichanont

#, D. J. Leak

+ and D. C. Stuckey*

Key words: reverse micelles, cell encapsulation, biotransformation, chiral epoxide

RUNNING TITLE : EPOXIDE PRODUCTION IN MICROEMULSIONS

* to whom correspondence should be addressed. Phone 0171-594 5591, fax 0171-594

5629, e-mail d.stuckey@ic.ac.uk

Department of Chemical Engineering and Chemical Technology, and Department of

Biochemistry+ Imperial College of Science, Technology and Medicine, Prince Consort

Road, London SW7 2BY, UK. Department of Chemical Engineering, Chulalongkorn

University, Bangkok, Thailand#.

2

SUMMARY

The application of many biotransformation processes is limited because the

substrates/products are poorly water soluble, can be further metabolised, or are inhibitory.

Hence non-aqueous media (eg. two-phase systems, low water environments) are being

examined to determine whether they can be used to overcome these problems. One novel

approach is to encapsulate whole cells in water-in-oil (w/o) microemulsions (reverse

micelles). In this study we have investigated the influence of key system parameters on

system stability and epoxidation activity of Mycobacterium M156 cells in reverse micelles

comprised of a mixture of Tween 85 and Span 80 (10-20 w%, with an

hydrophilic/lipophilic balance [HLB] of 10 and a weight ratio of Tween 85 to Span 80 =

5.7) in n-hexadecane. It was found that the minimum allyl phenyl ether (APE)

concentration required in the bulk hexadecane solvent phase for epoxidation to occur was

15 mM, while the minimum molar ratio of water to surfactant (W0) was 35. The optimum

epoxidation rate achieved was 3.8 nmol/ mg dwt-min with an APE concentration of 50

mM, and a W0 of 50, with an enantiomeric excess (ee) of 86%. However, epoxidation was

found to terminate approximately 3 hours after initiation, and the causes for this were

postulated to be either: the deleterious effect of the solvent on the Mycobacteria;

inactivation of the energy generating system; an insufficient energy supply, or; the

instability of the monooxygenase enzyme. It was concluded that on balance emulsion

systems are not an economically viable system for producing phenyl glycidyl ether (PGE).

INTRODUCTION

Asymmetric synthesis by chemical or biocatalytic routes is currently of great interest due

to growing regulatory and public pressure over health concerns, since different

3

enantiomers can exhibit different modes of action and toxicities.1 S-Aaryl glycidyl ethers

are valuable intermediates in the production of -adrenergic receptor blocking agents

(blockers) and can be produced by the stereospecific epoxidation of allyl aryl ethers

catalysed by whole cells of alkene or alkane grown bacteria expressing hydrocarbon

monooxygenases. However, the large-scale biological production of optically active

epoxides presents a challenge since: most desirable substrates have a relatively low water-

solubility; the biocatalyst may be sensitive to product inhibition or toxicity, and; some

microorganisms contain enzymes which can further metabolise products to other undesired

compounds. A water-in-oil emulsion biotransformation system may provide solutions to

these problems due to its ability to encapsulate cells, and provide a non-polar environment

for both the substrate and product. Recently, work has been carried out in our laboratories

to define the influence of certain system parameters on cell encapsulation, and optimise

the stability of such a system.2

Most of the effort in the application of emulsion systems for biotechnological processes

has been devoted to enzyme encapsulation in reverse micelles (water-in-oil

microemulsions). Recently, however, it was found that large macromolecules, eg plasmids

with molecular weights of several million, can be solubilised in “reverse micelles”.3

Hence, there seems to be no constraint to the molecular weight of guest molecules, despite

the fact that unfilled reverse micelles are no larger than 4-10 nm,4 and Haering et al

5.

solubilised Escherichia coli in a system containing Tween 85, water, and

isopropylpalmitate. Early workers in this area4,5,6,7,8

all refer to these solutions as “reverse

micelles” or “o/w microemulsions”, nevertheless it is puzzling that with a bacterial cell

“solubilised” in the emulsion the droplet size must be around 1m, and hence by definition

4

they should be water-in-oil macroemulsions.9 However, since the cell suspensions do not

appear turbid, and they are stable for long periods of time, it appears that the cells are

solubilised and not suspended, and that the w/o emulsion system involved is a

microemulsion.7 Hence, in deference to past work, the emulsion used in this work will be

referred to as an oil-in-water microemulsion.

Despite recent advances in the chemical asymmetric synthesis of epoxides,10,11

there is still

no general method applicable to all substrates. Accordingly, biological processes have

attracted increasing interest from many researchers in recent years.12,13,14,15,16

These

include; direct conversion of alkenes to epoxides, two-step conversions of alkenes to

epoxides, and resolution of racemic mixtures.17

Of these, the direct conversion of olefins

using monooxygenases (see Equation 1) seems to be the most attractive route for the

production of optically active epoxides, particularly where the substrate itself is rather

valuable. This is due either to: the lack of stereoselectivity (e.g. haloperoxidase); added

complexity in the two-step conversion of alkenes, or; the loss of starting materials (50%)

from the chiral resolution of epoxides by selective ring opening of epoxide hydrolase or

other epoxide degrading enzymes in the absence of suitable methods for racemising the

unwanted enantiomer.

Equation 1 Direct conversion of alkenes to epoxides by monooxygenases.

R1CH

2O

2+NAD(P)H +CHR + H+ R

1CH CHR

2

O

+ H2O +NAD(P)

+

5

Monooxygenases from different types of organisms have different specificities. It was

found that monooxygenases in alkane-grown bacteria18

tend to produce racemic

epoxyalkanes,19,20

and are much more sensitive to product inhibition at low epoxide

concentrations than alkene-grown cells.21

Hence, alkene-specific monooxygenases are

more suitable for the production of epoxides on a large scale than alkane monooxygenases.

Monooxygenase catalysed epoxidation reactions are more attractive when catalysed by

whole cells rather than by an isolated enzyme for two reasons. Firstly, monooxygenase

enzymes are very unstable in vitro, and therefore good catalytic activity cannot be

maintained.18

Secondly, since monooxygenases are NAD(P)H-dependent, the regeneration

of the cofactor is easier to carry out in whole cells.22

By employing whole cells, however,

there is a possibility of further epoxide degradation by other enzymes resulting in a low

product yield. Resting rather than growing cells seem to be a more popular choice as: the

growth and bioconversion processes of the bacteria are separated, and therefore each step

can be optimised individually; the toxic effects of the substrate and the product on growth

can be avoided,23

and; the bioconversion reactions can be carried out under nonsterile

conditions.

Only two studies on the use of cells solubilised in microemulsions for biotransformations

have been carried out. Fadnavis and co-workers24

demonstrated that the hydrolysis of

methyl esters of racemic N-acetyl--amino acids by baker’s yeast was more efficient in a

microemulsion (AOT/(chloroform/isooctane)) than in water, which reflected the limitation

of substrate solubility in aqueous medium. Smolders et al.25

studied the 1,2

-

dehydrogenation of the steroid 16-methyl-Reichstein’s compound S-21-acetate (16MRSA)

6

by Arthrobacter simplex in a stirred flask containing a benzene derivative/phospholipids

microemulsion. They demonstrated that the water content of the system was the key

parameter affecting cell activity since the reaction rate was found to increase with

increasing water content. Hence, the objective of the current work was to utilise a water-

in-oil microemulsion system to carry out a whole cell chiral epoxidation, and to investigate

the influence of key system parameters on system stability and epoxidation activity.

MATERIALS AND METHODS

Microorganism

Mycobacterium sp. strain M156 was isolated at Imperial College, and was identified as

belonging to the genus Mycobacterium by N.C.I.M.B. (Aberdeen, U.K.). Its growth on a

large scale is detailed in a previous publication.26

Chemicals and Analytical Techniques

The chemicals used, and the analytical techniques employed have been detailed in a

previous publication.2

7

Experimental Procedures

Aqueous Solubilisation in a W/O Emulsion System in the Presence of APE: In order to

investigate the effect of APE on the solubilisation ability of the microemulsion solutions, a

series of solutions of (Tween 85 and Span 80)/n-hexadecane with a surfactant

concentration of 20 wt%, and an HLB of 10 were prepared as previously described,2 and

appropriate amounts of APE were added to each of these solutions to result in a final

concentration of 0, 5, 25, 100, and 150 mM, respectively. The solutions were titrated with

25 mM phosphate buffer solution (pH 7.5) in order to determine their aqueous capacities

using Karl Fischer titration.

The Effect of System Constituents and APE on System Stability: Four series of

solutions of (Tween 85 and Span 80)/n-hexadecane with a surfactant concentration of 20

wt%, and an HLB of 10 were prepared as previously described, however, 50 mM APE was

Gas chromatography

APE, PGE, and 2-pentanol were analysed by gas chromatography (Shimadzu 14A) using

an FID detector. The column used was 1.5 m long, with 3% SP2100 on Chromosorb WHP

80-100 mesh. Temperature programming consisted of a 120 oC initial temperature

followed by a rise of 15 oC/min to 200

oC where the temperature was held for 3 minutes

before being raised to 340 oC at the rate of 40

oC/min. The injector and detector were

maintained at 270 oC and 300

oC, respectively. The coefficient of variation of the

measurement was determined to be ±2.4% at the level of 0.3 mM PGE.

8

added to the second and fourth series. Buffer and cell suspension (heat-killed cells, 5 mg

dwt/mL) were added to the first and last two series of the prepared solution, respectively,

to a W0 of 50. The prepared solutions were then vortex mixed for 30 seconds, placed in a

37 o

C water bath, and monitored with a spectrophotometer at 540 nm every 30 minutes

until steady state was reached after 90 minutes. A similar experiment was carried out for

APE, however, in this case five series of solutions with 0, 25, 50, 100, and 150 mM APE

were prepared. Cell suspension (heat-killed cells, 5 mg dwt/mL) was added to all of the

prepared solutions, while other conditions were maintained as described above.

Epoxidation Assay in a W/O Emulsion System: Emulsion systems containing resting

cells of Mycobacterium sp. strain M156 were prepared by adding appropriate amounts of a

cell suspension (~6.5 mg dwt/mL in a pH 7.5, 25 mM phosphate buffer solution) to 4 mL

of Tween 85/Span 80 (20 wt%, HLB 10 - weight ratio of Tween 85 to Span 80 = 5.7) in air

saturated n-hexadecane contained in a number of 25 mL screw cap bottles. This resulted in

a final cell concentration of 0.25-0.5 mg dwt/mL. These were vortex mixed for 30

seconds, and then incubated in a 37 oC shaking water bath (280 rpm) for 2 minutes. To

initiate the reaction, appropriate amounts of APE were then added, and the samples

incubated again for appropriate time intervals. At set times an internal standard (2-

pentanol) was added to one bottle, and the cells were spun down immediately to stop the

reaction. However, the centrifuging time required before most of the cells were separated

was 20 minutes due to the high viscosity of the solutions. The supernatant liquid was

analysed by gas chromatography (Shimadzu 14A). Concentrations quoted in these studies

refer to the total solution volume in all cases. All samples were analysed in triplicate, and

9

the coefficient of variation of PGE measured from three repeated experiments was ±8.0%

at 0.3 mM.

System Stability During the Epoxidation: A ternary system (W0 = 50 and HLB = 10)

containing active Mycobacterium sp. strain M156 cells, and 50 mM APE was prepared

and the experiment was carried out following the procedures previously described.

However, instead of a gas chromatograph analysis for PGE, the optical density of the

samples was measured every 30 minutes at 540 nm using a Philips PU 8625 UV/VIS

spectrophotometer.

Determination of the Epoxide Enantiomeric Excess (ee): The PGE produced was

extracted from the cell suspension with ethyl ether in a volume ratio of 1:2 to the sample,

and the two phases were centrifuged. The PGE in the extract was then purified by thin-

layer liquid chromatography (TLC) using silica gel coated on glass as the stationary phase.

and the sample was analysed by HPLC (ATI Unicam Crystal 200). A sample solution (20

L) was injected into a Chiralcel OD-H column (0.46 x 25 cm-Daicel Chemical

Industries). The UV detector was set at 270 nm, and the mobile phase used was 30%

isopropanol/70% n-hexane. The coefficient of variation was 2.7%.

RESULTS AND DISCUSSION

In studying whole cell biotransformations in a w/o emulsion system, the cells are

presumed to be encapsulated inside a surfactant aggregate which protects them from

direct contact with the continuous organic phase. The location of the cells probably

10

depends on the size of the micelles; they are likely to partition more into the surfactant

layer when the water pools are small, or more into the bulk region of the water pool when

the pools are large. Thus, for a biotransformation to occur, nonpolar substrates have to

partition from the continuous phase, through the surfactant film, and/or an aqueous cell

environment until they reach the biocatalyst where the reaction takes place (see Figure 1).

Supply of the substrates, APE and oxygen, by a collision-fusion-fission process of the

emulsion droplets will not be significant since these substrates, especially APE, are

sparingly water soluble which will lead to a low probability of exchange between a globule

containing the substrate molecules and a cell-filled one. The product, on the other hand,

once produced would be stripped out of the cell environment owing to its preferential

solubility in the organic solvent.

The Effect of APE on the W/O Emulsion System

APE, when added to a w/o microemulsion, will distribute itself between the n-hexadecane

continuous phase, the interfacial phase of the surfactants, and the water pool based on its

respective partition coefficients. Thus APE is likely to play an influential role in

determining some system characteristics, i.e. system stability, and solubilisation capacity.

This effect of APE was investigated firstly by measuring the water content of the system

with varying APE concentrations. The results shown in Figure 2 indicate that the system

without APE could accommodate up to around 100% more water than those containing

APE, as the W0 value of the former was approximately twice as high as the latter.

According to Marszall27

, the effect of mixing oils (the continuous phase) on the phase

11

inversion temperature (PIT) of the emulsion, and hence its water solubilisation capacity is

given by28

:

PITmix = PITA A + PITB B

where A and B are the volume fractions of oils A and B, respectively, and PITmix, PITA,

and PITB are the respective PITs of emulsions in which the oil phase consists of a blend of

oil A and B, or A or B alone. Thus, APE should cause virtually no change in the

continuous phase characteristics since the amounts of APE added were exceedingly small

in comparison with n-hexadecane as it accounted for only 2 vol% in making up the final

concentration of 100 mM. As a result, it indicates that the marked effect of APE must have

been on the surfactant interfacial film.

Since APE is comprised of a very small hydrophilic part (an oxygen atom) in relation to

those of Tween 85 (a polyoxyethylene group) and Span 80 (a sorbitol ring), it is most

likely that APE would embed itself within the hydrophobic tails of the surfactants, and

thus increase the hydrophobicity of the surfactant mixture. The amounts of APE added (5-

150 mM) were probably enough to cause a significant effect on the interfacial

characteristics of the surfactant film since the concentration of the surfactant mixture in

the system was only around 127 mM. This increased hydrophobicity of the surfactant

mixture due to APE would inevitably reduce the critical temperature T (the highest

temperature at which the oil and non-ionic surfactant are immiscible), as well as the PIT.

Thus the experimental temperature, which was kept constant at 37 oC, was no longer close

to the PIT, resulting in the decreased solubilisation capacity of the system. The presence of

12

APE at different concentrations did not seem to significantly alter the apparent W0 values.

Emulsions containing 5, 25, 100, and 150 mM of APE showed W0 varying between 12-14,

and these were found not to be statistically different at the 95% confidence level on the t-

test. However, in calculating these W0 values, the amounts of APE present at the interface

acting as a surfactant were not taken into account, otherwise the values would appear

lower for the higher amounts of APE in the system. Following this line of argument, it can

be concluded that the higher the APE concentration, the lower the HLB temperature, and

the lower the solubilising ability of the system. The decrease in water content of the

system due to the increased APE concentration was not significant because the HLB

temperature was shifted away from the experimental temperature, making the system

insensitive to changes in the HLB of the surfactant mixtures.

Since APE was found to be surface active, its presence at the interface would unavoidably

affect the formation of the microemulsion droplets and their stability. The results shown in

Figure 3 illustrates the effect of APE on a variety of systems with a constant W0 of 50, at

an HLB = 10, and 20 wt% surfactant concentration. The APE molecules most likely

partitioned more into the hydrophobic rather than the hydrophilic part of the interface, thus

increasing the repulsive force due to the increased loss of the translational freedom of

motion of the hydrophobic tails. The buffer-containing system with APE was more stable

and resulted in lower light scattering than the one without APE (this difference could even

be noticed by eye). This might have resulted from smaller droplets being formed in the

former system thus lowering the repulsive forces due to the addition of APE, since lower

aggregation numbers would lead to a reduced loss of the translational freedom of motion.

On the other hand, when cells were also included in the system, the surfactants would be

13

arranged in such a way as to enclose the cells and their aqueous environment. The

increased repulsive forces due to the APE molecules at the interface could not then be

reduced by a reduction in emulsion size, and this resulted in the system with APE being

less stable than the one without. Moreover, it was found that the higher the APE

concentration the less stable the system, as shown in Figure 4. A higher APE concentration

in the system would enhance the repulsive forces, thus the aggregates formed would be

less stable than the system with less APE.

APE Distribution

In order to evaluate the distribution of APE between the continuous phase of n-hexadecane

and the interfacial film of Tween 85 and Span 80 mixture, log P values of these

compounds were verified. From the literature, and calculations using the hydrophobic

fragmental constants, the log P values of the following components were determined: n-

hexadecane = 8.829

, APE = 2.930

, and the mixture of Tween 85 and Span 80 (HLB = 10)

= 17.8. The smaller difference between the log P values of APE and n-hexadecane

compared to that of APE and the surfactant mixture implies that the APE molecules

should be preferentially distributed in the continuous phase rather than in the surfactant

interface, thus less APE is available for the reaction. Ideally, this problem could be tackled

by adjusting the hydrophobicity (log P) of the surfactant mixture to match that of APE so

that a heavy loading of the substrate in the organic continuous phase could be avoided.

However, this would dramatically reduce the solubilising ability of the system since it was

found earlier that the water content of the system (W0) with an HLB of 9 was only around

9, compared to 25 for the system with an HLB of 10. Thus the HLB of the system must be

14

kept at 10 in order to maintain a high solubilising ability, while a high loading of APE in

the continuous phase was required for the epoxidation reaction.

Parameters Affecting APE Epoxidation

The effect of different parameters such as surfactant concentration, substrate

concentration, and W0 on microbial epoxidation in the w/o microemulsion was evaluated,

and the results are summarised in Table 1. The cell suspension added to the reaction

medium was around 5.3 mg dwt/mL which resulted in a final cell concentration of 0.25-

0.5 mg dwt/mL for a W0 = 25-50 respectively with a 20 wt% surfactant concentration. The

Mycobacterium cell concentration employed was quite low, thus the reaction rates were

expected to be low. From the table, it can be seen that certain system conditions were

required for the biotransformation to occur.

First of all, the surfactant concentration when changed at a given water content, did not

physically affect the entrapped biocatalysts since the size of the emulsion droplets was

kept constant. However, an increased surfactant concentration at a given W0 resulted in an

increased water volume fraction in the system. PGE was not detected in any system with a

30 mM APE overall concentration, and W0 = 25 when the surfactant concentration varied.

As a consequence, it appears that the surfactant concentration is not a vital system

parameter controlling epoxidation.

The second parameter, the overall APE concentration, showed a marked influence on the

epoxidation, since PGE could not be detected when the substrate concentration was lower

15

than 15 mM at a W0 = 50. This indicates that the APE concentration was too low for the

reaction to occur since a large amount of it was distributed in the continuous phase where

the biocatalyst was not sited. Oxygen, on the other hand, was available for epoxidation

since the reaction could be detected at high APE concentrations. This was because oxygen

is more soluble in the more hydrophobic surfactant film than in the less hydrophobic n-

hexadecane continuous phase.

Finally, W0, a surrogate measure of emulsion size, appeared to be a vital parameter in

regulating the epoxidation. This is because the reaction did not seem to occur until the W0

value reached 35, corresponding to a 6.6% (v/v) water content. At a W0 value of 25,

epoxidation activity could not be observed under any conditions, either with increasing

APE concentrations up to 50 mM, or by varying the surfactant concentration. The reaction

rate was found to increase with W0, however, the numerical results are not shown here

since the epoxidation activity was too low to give any accurate numbers. Similar findings

were reported by Famiglietti et al. 7 for studies on the photosynthetic activity of A.

variabilis in an analogous system (Tween 85 and Span 80 / hexadecane, HLB = 10.33)

where no activity occurred up to a W0 value of 30. The lower W0 value used in our system

implies a closer contact between the cells and the surfactant film, as well as between the

cells and the substrates. This should favour the reaction, however, above the minimum

value of W0 = 35 required for activity the opposite was found. The explanation for this is

unclear at present, since the amount of water surrounding the cells should not have a

strong effect on the activity of the monooxygenase which was intact and well protected by

the cell membrane, unless the amount of water was not enough to constitute an “essential”

water layer around the cells. In addition, nonionic surfactants like Tween and Span are not

16

known for their ability to extract biomass. Cell viability, on the other hand, might be

reduced by encapsulation under these extreme conditions, however, enzyme activity

should still be present. Considering all the possibilities, this result might suggest that at

low W0 when the cells were in close contact with the substrates dissolved in the surfactant

film, APE inhibition occurred since the Ki for APE is only 5 mM (Caridis, unpublished

data). Moreover, the finding that the epoxidation rate increased with W0 may indicate that

the cells were solubilised in the bulk water pool according to the “water-shell” model.

Time-Course of the Reaction

Figure 5 shows PGE accumulation over time measured at W0 = 50 with 5, 50, and 75 mM

APE. PGE was found to slowly accumulate before reaching a maximum level in the latter

two cases, while in the case where 5 mM APE was used, no epoxidation could be detected.

Epoxidation started when 15 mM of APE was supplied; these results and the result when

100 mM APE was used are not shown here since the amounts of PGE detected in the first

90 minutes were too low to give any accurate numbers. However, the maximum amounts

found were measured at around 300 (0.16 mM) and 250 nmol/mg dwt (0.13 mM) for 15

and 100 mM APE, respectively, which were markedly less than the 640 and 488 nmol/mg

dwt found when 50 and 75 mM APE were supplied. From the different APE

concentrations mentioned above, the emulsion system supplied with 50 mM substrate

seemed to give the best epoxidation result, with an initial epoxidation rate of 3.8 nmol/mg

dwt-min. Similar amounts of PGE (around 180 nmol/mg dwt) were found at the initial

time point of every epoxidation which was some twenty minutes after APE addition. This

17

length of time, during which epoxidation might have occurred, was required in order to

spin down the cells from a viscous emulsion solution.

The lowest APE concentration required to initiate epoxidation was 15 mM, and this is the

reason why the epoxidation rate increased as the APE concentration increased. However,

50 mM APE was found to be the optimum concentration since the reaction rate reduced at

higher APE concentrations. The reason for this is still unclear, since the higher APE

concentration should not cause the lower epoxidation rate due to the higher aqueous phase

APE concentration. However, the reaction could have been suffering from substrate

inhibition. On the other hand, product inhibition is unlikely since PGE is preferentially

solubilised in the n-hexadecane continuous phase rather than in the interfacial film of the

surfactants since its log P is around 0.59, and the difference between this value and that of

the n-hexadecane is smaller than that with the surfactant mixture. Thus, PGE would be

effectively “pulled out” of the cell environment. Since the partition coefficient of PGE in

the two-liquid phase system of water-n-hexadecane was determined to be 10, the 0.3 mM

PGE (corresponding to 640 nmol/mg dwt) measured in the w/o microemulsion (W0 = 50,

9.5 vol% water content) would correspond to only approximately 0.03 mM PGE in the

water pool. This low concentration of PGE would not have a significant inhibitory effect

on the epoxidation, since Ki was found to be around 5 mM PGE (Caridis, unpublished

data). Thus, the degradation and inhibitory effects of PGE would be reduced dramatically

in this reaction medium.

Oxygen availability may have been limiting, however, its solubility in n-hexadecane is not

available. Nevertheless, it is expected to be more than its solubility in n-octane of around

18

24 mM31

, and since the mass of cells in the microemulsion was only 2 mg dwt, the

maximum uptake of O2 during the epoxidation would only be around 1.2 mM, well below

its solubility. From a theoretical standpoint, the accumulation of PGE was expected to

increase at a constant rate over the whole reaction period since there should have been no

substrate limitation, product inhibition, or product degradation, however, this did not

occur. Instead, after about 180 minutes from initiation PGE reached a steady

concentration, and the reaction ceased. In order to test whether this phenomenon was due

to the extreme conditions of the w/o microemulsion, the same experiment was carried out

in a two-liquid phase system without surfactant,32

but with a 9% (v/v) buffer solution

instead (corresponding to a W0 = 50 of the emulsion system). Surprisingly, it was found

that this phenomenon still occurred. In the two-liquid phase system the cell environment

was less hostile since the cells were surrounded by more water, and thus had less contact

with the surfactants or the organic solvent. As a consequence, the termination of reaction

at around 180 minutes after the start was not largely due to the w/o microemulsion system

itself, but common characteristics between the two systems.

Four hypotheses were put forward to explain this observation: firstly, n-hexadecane had

deleterious effects on M156; secondly, the energy-regeneration system of the bacteria was

inactivated, and this might have been caused by the damage done to the cell membrane by

n-hexadecane; thirdly, there was an insufficient energy supply; and lastly, the alkene

monooxygenase was not stable under maintenance conditions which might be due either to

heat, the buffer, and/or the organic solvent used since the time of the reaction termination

was similar regardless of the amounts of PGE produced. These possibilities have been

19

examined in a separate study,32

and the loss of activity has been shown to result from

inactivation of the oxygenase.

Stability of the System Under Epoxidation

The optical density of the w/o microemulsion system was measured at appropriate time

intervals during the APE epoxidation, and the results are shown in Figure 6. The system

was found to be optically stable for only 150 minutes after initiation, whether catalysing

epoxidation or not. This leads to two conclusions: firstly, the PGE produced did not play a

significant role at the surfactant-oil interface, hence, the change of the Gibbs free energy in

the formation of the aggregates was not altered. Secondly, water which is a by-product of

APE epoxidation was produced in such small amounts that the increased W0 value of the

system, and the system instability due to water production, was too low to exhibit any

notable effects.

Enantiomeric Excess (ee) of PGE Produced by M156

The optical purity of PGE produced by Mycobacterium sp. strain M156 was found to be

86% (the average of three analyses). Although the configuration of PGE produced was not

determined, it was expected to be in the S-form as obtained from other microorganisms.

Hence the biotransformation was capable of producing a relatively pure enantiomer.

20

CONCLUSIONS

In this paper, a w/o microemulsion system was employed as a medium for APE

epoxidation. It was discovered that certain conditions were required for APE epoxidation;

the minimum APE concentration for epoxidation was found to be 15 mM, while the

minimum W0 was 35. The optimum APE concentration was determined to be 50 mM;

while at higher concentrations substrate inhibition may have occurred. The observation

that a certain amount of water in the medium (W0) is required for the epoxidation was

unexpected, although it had been previously noted by another research group, because by

using whole cells the epoxidising enzyme should not be affected by the amount of water

surrounding the cells, unless there was not enough water to constitute the “essential” water

layer around the cells, and/or the function of the cell membrane was damaged under these

extreme conditions. However, this effect of W0 on the epoxidation may indicate that the

cell was located in the bulk water pool of the emulsion droplet according to the “water-

shell” model. The optimum reaction rate found at W0 = 50 and 50 mM APE was 3.8

nmol/mg dwt-min.

Under optimum conditions, the epoxidation was found to terminate approximately 180

minutes after initiation, and the same phenomenon was also found in a two-liquid phase

system of the same water content. Thus, the conclusion was drawn that the termination

was not caused by the surfactant film layer, or other factors that differentiated the

microemulsion from the two-liquid phase system. The deleterious effects of n-hexadecane

on M156, inactivation of the energy-generating system, an insufficient energy supply, and

the instability of the monooxygenase under maintaining conditions were all postulated

21

being responsible for this observation. Our recent work has shown that the loss of activity

was due to oxygenase inactivation.

The microemulsion epoxidation system was found to remain clear for only 150 minutes

after initiation, and APE was one of the factors contributing to this instability since it was

discovered to be surface active, and reduced the solubilising ability of the system. On

balance, it appears that significant improvements need to be made in this system before it

can become viable for the production of epoxides.

REFERENCES

1. Crossley, R. The relevance of chirality to the study of biological activity. Tetrahedron,

1992, 48(38), 8155-8178

2. Prichanont, S., Leak, D.J., and Stuckey, D.C. The solubilisation of Mycobacterium in a

water in oil microemulsion: system selection and characterisation. Colloids and Surfaces:

A., 2000, in press

3. Garcia-Carmona, F., Bru, R., and Sanchez-Ferrer, R. in Biomolecules in Organic

Solvents, A. Gomez-Puyou (Ed), CRC Press, Boca Raton, FL,.1992, 163

4. Haering, G., Luisi, P. L., and Meussdoerffer, F. Solubilisation of bacterial cells in

organic solvents via reverse micelles. Biochem. Biophys. Res. Comm.,1985, 127(3), 911-

915.

5. Haering, G., Pessina, A., Meussdoerffer, F., Hochkoeppler, S., and Luisi, P. L.

Solubilisation of bacterial cells in organic solvents via reverse micelles and

microemulsions Ann. NY. Acad. Sci., 1987, 506: 337-344

22

6. Hochkoeppler, A., and Luisi, P. L. Solubilisation of soybean protein mitochondria in

AOT/isooctane water in oil micremulsions. Biotechnol. Bioeng. 1989, 33, 148-153

7. Famiglietti, M., Hochkoppler, A., Wehrli, E., and Luisi, P. L. Photosynthetic activity of

Cyanobacteria in oil in water micoemulsions. Biotechnol. Bioeng. 1992, 40, 173-178

8. Pfammatter, N., Hochkoppler, A., and Luisi, P. L. Solubilisation and growth of Candida

pseudotropicales in water in oil microemulsions. Biotech. Bioeng, 1992, 40, 167-172

9. Binks, B.P. Surfactant monolayers at oil-water interfaces Chem. and Indust., 19th

July,

1993, 537-541

10. Katsuki, T., and Sharpless, K. B. The first practical method for asymmetric

epoxidation. J. Am. Chem. Soc., 1980, 102, 5974

11. Jacobsen, E. N. Asymmetric catalytic epoxidation of unfunctionalised olefins, in

Catalytic Asymmetrical Synthesis, Ojima I. (ed), VCH, New York, 1993, 159

12. Wingard, L. B., Jr., Roach, R. P., Miyawaki, O., Egler, K. A., and Klinzing, G.

Epoxidation of propylene utilising Nocardia corallina immobilised by gel entrapment or

adsorption. Enzyme Microb. Technol. 1985, 7, 503-509

13. Hamstra, R. S., Murris, M. R., and Tramper, J. Influence of immobilisation and

reduced water activity on gaseous-alkene oxidation by Mycobacteria PY1 and

Xanthobacter PY2 in a gas-solid reactor Biotech. Bioeng. 1987, 29, 884-891

14. Kovalenko, G. A., and Sokolovskii, V. D. Epoxidation of propene by microbial cells

immobilised on inorganic supports. Biotech. Bioeng. 1992, 39, 522-528

15. Frense, D., Miethe, P., Langwald, M., and Mohr, K. H. Epoxidation of lyotrophic

mesophases. Biotech. Letts., 1992, 14(2), 93-98

23

16. Wubbolts, M. G., Hoven, J., Melgert, B., and Witholt, B. . Efficient production of

optically active styrene epoxides in two-liquid phase cultures. Enzyme Microb. Technol.

1994, 16, 887-894

17. Leak, D. J., Aikens, P. J., and Mahmoudian, M. S. The microbial production of

epoxides. TIBTECH. 1992, 10, 256-261

18. Hartmans, S., de Bont, J. A. M., and Harder, W. Microbial metabolism of short-chain

unsaturated hydrocarbons. FEMS Microbiol. Rev. 1989, 63, 235-264

19. Subramanian, V. Oxidation of propene and 1-butene by Methylococcus ccapsulatus

and Methylsinus trichosporium. J. Ind. Microbiol., 1986, 1, 119-127

20. Weijers, C. A. G. M., van Ginkel, C. G., and de Bont, J. A. M. Enantiomeric

conversion of lower epoxyalkanes produced by methane-, alkane-, and alkene utilising

bacteria. Enzyme Microb. Technol. 1988, 10, 214-218

21. Habets-Crutzen, A. Q. H., and de Bont, J. A. M. Inactivation of alkene oxidation by

epoxides in alkene and alkane grown bacteria. Appl. Microbiol. Biotechnol. 1985, 22, 428-

433

22. Miyawaki, O., Wingard, L. B., Jr., Brackin, J. S., and Silver, R. S. Formation of

propylene oxide by Nocardia corallina immobilised in liquid parafin. Biotech. Bioeng.

1986, 28, 343-348

23. Onumonu, A. N. Improving the substrate range of alkene monooxygenases: directed

evolution vs isolation strategy. Ph.D Thesis, Imperial College of Science,Technology and

Medicine, London, UK, 1992

24. Fadnavis, N. W., Reddy, N. P., and Bhalerao, U. T. Reverse micelles, an alternative to

aqueous medium for microbial reactions: yeast-mediated resolution of -amino acids in

reverse micelles. J. Org. Chem., 1989, 54, 3218-3221

24

25. Smolders, A. J. J., Pinheiro, H. M., Noronha, P., and Cabral, J. M. S. Steroid

bioconversion in a micoemulsion system. Biotech. Bioeng.1991, 38, 1210-1217

26. Woodland, M. P., Matthews, C. S., and Leak, D. J. Properties of a soluble propene

monooxygenase from Mycobacterium sp. (strain M156). Arch. Microbiol., 1995, 163, 231-

234

27. Marszall, L., In Nonionic Surfactants: Physical Chemistry, Schick, M. J. (Ed), Marcell

Dekker, Inc., USA, 1987

28. Kunieda, K., and Shinoda, K. Evaluation of the Hydrophile-Lipophile Balance (HLB)

of nonionic surfactants. J. Coll. Interf. Sci. 1985, 107, 107

29. Vermue, M., Sikkema, J., Verheul, A., Bakker, R., and Tramper, J. Toxicity of

homologous series of solvents to the gram-positive bacteria Anthrobacter and Nocardia sp.

and the gram-negative bacteria Acinetobacter and Pseudomonas sp. Biotechnol. Bioeng.

1993, 42, 747-758

30. Hansch, C., and Leo, A., Pomona College Medicinal Chemistry Project, January, Issue

6, 1975

31. Gerrard, W. Solubility of Gases and Liquids, Plenum Press, New York, 1976

32. Prichanont, S., Leak, D.J., and Stuckey, D.C. Chiral epoxide production in a two

liquid phase system. Enzym. Microb. Technol. 1998, 22, 471-479

25

Table 1. Effects of Different Parameters on Microbial Epoxidation in W/O

Macroemulsions. Conditions: 37 oC, shaking speed 280 rpm.

[APE]

(mM)

W0 [Surfactants] (wt%) Epoxidationa

2

5

10

15

30

30

30

50

50

50

50

50

50

75

100

25

50

50

50

25

25

25

25

30

35

40

45

50

50

50

20

20

20

20

10

15

20

20

20

20

20

20

20

20

20

NO

NO

NO

YES

NO

NO

NO

NO

NO

YES

YES

YES

YES

YES

YES

a: The epoxidation was assumed not to occur if PGE was not detected by gas

chromatography (the detection limit was around 0.05 mM).

26

FIGURE CAPTIONS

Figure 1 Schematic representation of cell encapsulation in a surfactant aggregate:

(a) in a small aggregate where the cell is in close contact with a surfactant layer, and;

(b) in a large aggregate where the cell is surrounded by aqueous solution, an

additional phase that the substrates have to diffuse through before reaching the

biocatalyst.

Figure 2 The effect of APE concentration on the water content of the system.

Solutions of (Tween 85 and Span 80) in n-hexadecane with a 20 wt% surfactant

concentration at an HLB value of 10 with the addition of varying amounts of APE,

were tested for their water solubilisation capacity after 90 minutes. The experimental

temperature was controlled at 37 oC.

Figure 3 Effect of added components on the stability of the system. Different

components were added to solutions of 20 wt% (Tween 85 and Span 80) in n-

hexadecane: buffer solution, cell suspension (5 mg dwt/mL), and 50 mM APE. The

W0 and experimental temperature were kept constant at 50, and 37 oC, respectively.

Figure 4 The effect of APE concentration on the stability of the system. Various APE

concentrations were added to solutions of 20 wt% (Tween 85 & Span 80) in n-

hexadecane. The W0, cell concentration, and experimental temperature were kept

constant at 50, 0.5 mg dwt/mL (final concentration), and 37 oC, respectively.

Figure 5 APE epoxidation in the two-liquid phase and w/o emulsion systems at

various APE concentrations. The microemulsion used was a 20 wt% (Tween 85 and

Span 80) at an HLB of 10 in n-hexadecane. The water content, temperature, and

shaking speed of the systems were kept constant at W0=50 (9.5 vol%), 37 oC, and 280

rpm.

Figure 6 Stability of the system during the epoxidation reaction. The epoxidation

system was comprised of a w/o emulsion containing 0.5 mg dwt cells/mL, 20 wt%

(Tween 85 and Span 80) at an HLB of 10 in n-hexadecane, water 9.5 vol% (W0 = 50),

and 50 mM APE. The conditions were maintained at 37 oC, and a shaking speed of

280 rpm.

27

A P E P G E

O X Y G E N

(a)

A P E

O X Y G E N

P G E

(b)

surfactant

Mycobacterium sp. strain M156

28

0 5 25 100 150

0

5

10

15

20

25

Wa

ter

Co

nte

nt

(W0)

0 5 25 100 150

APE Concentration (mM)

29

0

0.5

1

1.5

2

2.5

3

0 30 60 90 120 150 180 210 240

Time (mins)

Op

tica

l D

en

sity

(5

40

nm

)

buffer

buffer & APE

cell suspension

cell suspension & APE

30

0

0.5

1

1.5

2

2.5

3

0 30 60 90 120 150 180 210

Time (mins)

Op

tica

l D

ensi

ty (

54

0 n

m)

no APE

25 mM APE

50 mM APE

100 mM APE

150 mM APE

31

0

100

200

300

400

500

600

700

800

0 30 60 90 120 150 180 210 240

Time (mins)

PG

E (

nm

ol/

mg

dw

t)

5mM APE

50 mM APE

75 mM APE

50 mM APE - no surfactants

32

0

0.5

1

1.5

2

2.5

0 30 60 90 120 150 180 210

Time (mins)

Op

tical

Den

sity

(540 n

m)