Enzyme-catalysed reactions in non-aqueous media

TIBTECH - JANUARY 1988 [Vol. 6]

--Abbreviations, AIST - Agencyfor I n d u s t r i a l Science and Technology

CIS

CST

- C o u n c i l f o r Industrial S t r u c t u re - C o u n c i l f o r Science

10 Nikkan Kogyo Shimbun (1986) 10 May 11 The Kagaku Shimbun (1987) 20 February 12 D. Swinbanks (1987) Nature 325,651 13 W. Lepkowski (1987) Chem. Eng. News 8 June, 13-14 14 D. Swinbanks (1986) Nature 323,284 References 15 B. Johnstone (1987) New Sci. April 2, 33-36 I The Sankei Shimbun (1985) June 26 2 Sci. Technol. Japan (1985) April/ 16 Jap. Soc. Promotion of Sci. Annu. Rep. 1983-84, Japanese Society of the June, 22-31 Promotion of Science, pp. 1-22 3 R. Schmid (1985) Naturwissenschaflen 17 Jap. Chem. Week (1985) October 31, 72, 534-538 4 Jap. Bioindustry Lett. (1986) 3, 1-9 4-8 5 Chem. Indust. Jap., 1985/86, Annual 18 Jap. Econ. J. (1985) September 21, 4-8 Report of the Japan Chemical Industry 19 R. D. Schmid (1985) Appl. Microbiol. Biotechnol. 22, 157-164 Association 20 Diet of the Federal Republic of 6 L. Lynn (1986) Science, 233,296-301 Germany, 10th term of election. 7 The Nikkan Kogyo Shimbun (1985) (1987) Report of the Commission of 13 December Inquiry on: 'Chancen und Risiken der 8 G. Gregory (1983) Chem. Econ. Eng. Gentechnologie' (Dated January 6th), Rev. 15, 18-22 pp. 273-275 9 Mainichi Daily News (1985) 24 May

invaluable help and for reviewing the manuscript, Mr A. H. Kirkwood for his excellent assistance with translation and Prof. J. N6esch for his support and advice.

Requirements for activity

Enzyme-catalysed reactions in non-aqueous media J. S. Deetz and J. David Rozzell The discovery that enzymes can function in apolar solvents has dramatically expanded the range of reactions which can be approached through biocatalysis. Key factors which influence both the activity and stability of enzymes in organic solvents include the ionic state of the enzyme, support characteristics, and the extent of hydration of biocatalyst and solvent. Industrial applications have begun to emerge in the areas of fat and oil processing. The long-standing belief that practical applications for enzymes are essentially limited to water-based chemistries has recently been challenged by several independent reports of enzyme catalysis in nonaqueous solutions ~ . By removing the constraints imposed by aqueous reaction conditions, many potential

J. S. Deetz and J. David Rozzell are at the Genetics Institute, Inc., 87 Cambridge Park Drive, Cambridge, MA 02140, USA.

products which are either insoluble or labile in water may now be produced with the use of enzymes 5'6. In addition, useful reactions which normally do not occur to an appreciable extent in water (e.g. transesterifications) may be catalysed efficiently by enzymes in organic solvents, and in the case of hydrolytic reactions where water is normally a reactant, the equilibrium may be shifted in non-aqueous media to favor condensation products (e.g. esterifications) 7-9.

~) 1988, Elsevier Publications, Cambridge 0166- 9430/88/$02.00

Strictly speaking, enzymes cannot function under totally anhydrous conditions; however, water can be a vanishingly small percentage of the overall reaction volume 1°'11. The a m o u n t of water required may be correlated in part with the solubility of water in the reaction solvent 11'12. Generally, the least polar solvents (most hydrophobic) require the least water for enzyme activity. For example, in our work with alcohol dehydrogenases, the requirement for water is satisfied at less than 0.1% (v/v) in hexane, whereas 5% water is needed for activity in acetonitrile. It is noteworthy that even water miscible solvents may be suitable for enzymatic reactions, provided sufficient water is present. Klibanov has suggested that the more hydrophilic solvents tend to strip the essential hydration layer from proteins~; thus, more water must be added to such solvents to prevent this desiccation and sustain enzyme activity. Klibanov has also shown that the enzyme must be presented to the organic solvent in a catalytically competent ionic state 2. The pH of the last aqueous solution from which the enzyme is recovered affects the pH of the micro-aqueous environment of the biocatalyst and hence, the charge balance of the protein.

TIBTECH - J A N U A R Y 1988 [Vol. 6] - Fig. 1

~~.~

Accordingly, we have found with lipases and proteases that optimal activity in organic solvents is generally achieved if the enzyme is first deposited on an inert support from an aqueous solution with a pH corresponding to the pH optimum in water. As a cautionary note, both the ionic character imparted by the support and the hydrogen-bonding capability of the organic solvent may shift the apparent 'pH optimum'; thus a pH-activity profile should be determined for each combination of support and solvent.

Relative enzyme activity How active are the enzymes in organic solvents? Often this question cannot be directly answered because comparable rate data for aqueous catalysis is not available. With lipases, for example, rate constants for esterification and transesterification reactions under aqueous conditions are difficult to determine since hydrolysis predominates. In the absence of such values, Klibanov has shown, however, that both yeast and porcine pancreatic lipase accelerate the rate of transesterification in hexadecane approximately 1.5-3 x 106-fold relative to the uncatalysed reaction 2. We have compared the rates of cinnamyl alcohol oxidation with horse liver alcohol dehydrogenase (HLADH) in both aqueous and organic media (Fig. 1). Cinnamyl alcohol is oxidized to cinnamaldehyde in one half-reaction with the concurrent reduction of cofactor; in the second half-reaction, octanal is reduced to octanol, and NAD+ is regenerated. Under optimal conditions in butyl ether, an initial rate of 23 ~mol (min. ~mol subunit) -1 HLADH was obtained for the overall conversion. This is approximately 15% of the rate of alcohol oxidation in aqueous buffer, pH 7.0, under saturating conditions of NAD + and cinnamyl alcohol. When this reaction was catalysed by yeast alcohol dehydrogenase (YADH) in butyl acetate containing 1% H20 (v/v), activities ranged from less than 1% to greater than 64% of that in aqueous buffer. This large variation was due to the influence of different supports. Yeast alcohol dehydrogenase was least active on unmodified glass,

~~~CHO

CH2OH

CinnamylAlcohol

Cinnamaldehyde HLADH,N A D

+

CH3(CH2)6CHO Octanal

+

CH3(CH2)6CH2OH Octanol

Reaction catalysed by horse liver alcohol dehydrogenase (HLADH) in organic solvents.

whereas on a glycophase glass its performance was superior. Clearly, if the amount of water, the ionic state of the enzyme, and the characteristics of the support are optimized for each system, high enzymic activity may be retained in organic reaction media.

Stability in organic solvents Perhaps the most neglected aspect of this emerging technology is enzyme stability in organic solvents. It is widely assumed that most solvents are e n z y m e denaturants; however, this tenet is now being aggressively challenged. Enzyme stability is greatly influenced by the amount of water in the organic solvent. In a groundbreaking paper 11, Zaks and Klibanov measured the half-life of porcine pancreatic lipase at 100°C as a function of the water content in tributyrin 11. (Tributyrin served as both a substrate and the reaction solvent for lipase-catalysed transesterification with heptanol.) At this extreme temperature the lipase was remarkably stable in dry tributyrin, with a half-life greater than 12 h. However, above a critical concentration of water (in this case 0.3% (w/w)) the stability of the lipase decreased precipitously. At water concentrations greater than 1% the lipase was instantly inactivated. Klibanov suggested that water was a necessary participant in the microscopic steps of thermoinactivation11; therefore, the loss of activity may be retarded under relatively anhydrous conditions. We have examined the stability of horse liver alcohol dehydrogenase in butyl acetate at 25°C as a function of water concentration. In dry butyl acetate, the half-life of HLADH was greater than 100 h; however, as the

concentration of water approached saturation in the solvent ( - 2 % v/v), the stability of the enzyme decreased. These results underscore the balance that must be maintained between activity and stability with respect to the water concentration. The window of optimum water content must be experimentally determined for each enzyme and solvent pair to achieve maximum productivity.

Supports One great advantage to nonaqueous catalysis is that the enzyme may be effectively immobilized by simple adsorption onto an inert support TM. As an integral component of the catalyst, the support may influence the microenvironment of the enzyme, and thus its activity and stability. Consequently, the choice of the support is of paramount importance in the development of an efficient and economical biocatalytic process. In the simplest case, enzymes may function in organic solvent as suspended powders 13. This strategy has been particularly useful for crude commercial preparations of lipases, which can catalyse esterification and transesterification reactions when added directly to the organic reaction milieu as a solid. In HLADH-catalysed reactions, the support may dramatically alter the local concentrations of substrates and products in the microenvironment of the enzyme through partitioning effects. For example, HLADH is severely inhibited by its substrate, cinnamyl alcohol, at concentrations above 0.1 mM in aqueous solution. This inhibition can be effectively eliminated (Fig. 2) at overall concentrations up to 50 mM in the organic solvent butyl acetate w h e n the hydrophilic carrier, glycophase

TIBTECH - JANUARY 1988 [Vol. 6]

--Fig. 2 16

12

\ ,_'~'> 4

0 0

I

I

I

I

I

0.1

0.2

0.3

0.4

0.5

1/rnM-' Lineweaver-Burk plot for the horse liver alcohol dehydrogenase-catalysed reaction of cinnamyl alcohol to cinnamaldehyde in butyl acetate. The dotted line shows that the reaction is still inhibited by the substrate. However, in butyl acetate, inhibition occurs only at substrate concentrations above 50 mM. In water, the enzyme is inhibited by concentrations of cinnamyl alcohol above 0.1 raM.

controlled pore glass, is used. The partition coefficient for cinnamyl alcohol between water and butyl acetate cannot account for this large effect. In an elegant study of biphasic catalysis, Tanaka and Fukui have demonstrated with polymerentrapped enzymes that the hydrophobicity of the support influences the partitioning of substrates into the immediate aqueous environment of the enzymes 14-16. Increasing the content of hydrophobic residues systematically increased the activity of the catalyst toward hydrophobic substrates. Hence, the choice of support may be an effective means to control the local concentrations of substrates and products for catalysis. The support need not be solid and insoluble in the organic solvent. If enzyme is added to solvent containing a critical ratio of water to surfactant, it may be incorporated into a reverse micelle 17-21. The micelle is essentially a water droplet entrapped in an ionic cage of surfactant. Enzymes may be very active in reverse micelles, with kca t values approaching those observed in water; however, the micellar structure may easily be disrupted, and the amount of soluble enzyme may be limiting. Thus, the practical application of such systems remains to be proven.

In an ingenious example of what may be a reverse micelle, Inada and co-workers have prepared enzymes which are completely soluble in organic solvents such as benzene and chloroform, by covalently attaching polyethylene glycol (PEG) to accessible lysine residues on the surface of the protein 22. These PEGmodified enzymes, which include several lipases 23, chymotrypsin24, catalase 25 and horseradish peroxidase 26, are highly active in the organic milieu if the appropriate amount of water is present for protein hydration 27. In addition, the covalent character of the PEG-cage imparts greater stability to the PEG-enzyme than is generally observed for reverse micelles. All these studies indicate the importance of the support as well as the choice of solvent and water concentration, as critical factors influencing the activity and stability of enzymes in non-aqueous media.

Complex reactions Complex reactions involving cofactors or coupled enzymes can also be carried out in apolar solvents 28'29. In the horse liver alcohol dehydrogenase-catalysed conversion of cinnamyl alcohol to cinnamaldehyde NAD + is an absolute requirement and must be added to the enzyme during the preparation of the catalyst. Since NAD + is virtually insoluble in apolar

solvents, the effective concentration of cofactor in the aqueous microenvironment of the enzyme is high. Hence, the dehydrogenase can be saturated with near stoichiometric amount of coenzyme, which is an economic advantage over aqueous reactions involving NAD +. Although this reaction demonstrates that the cofactor can be efficiently recycled in organic systems, it does not discriminate between cofactor that is 'fixed' at the NAD-binding site and regenerated there, and NAD + that dissociates from one active site and is regenerated at another. To determine whether reactions catalysed by two separate enzymes can be 'coupled' in organic solvents at the lower critical limit of water, the reaction shown in Fig. 3 was examined both in butyl acetate (1% v/v H20) and in hexane (0.1% v/v H20). In this scheme, NAD + is first reduced to NADH in the YADH-mediated reaction with octanol as reductant. Reducing equivalents are then transferred from NADH to the dye 2,6dichlorophenolindophenol (DCIP) in the diaphorase-catalysed reaction. Since the substrate specificities of both enzymes exclude cross reactivity, a positive reaction, as shown by the bleaching of the oxidized form of DCIP, can occur only if the cofactor can shuttle between the enzymes. The reaction was initiated by the addition of catalyst to solvent containing substrates and the m i n i m u m amount of water required for yeast alcohol dehydrogenase to function, as determined previously. Since - Fig. 3 Octanol

Octanal

NAD÷

S Dye (Red)

NADH

Diaphorase Dye (Ox)

Coenzyme recycling scheme for yeast alcohol dehydrogenase (YADH) and diaphorase.

TIBTECH - JANUARY

1988 [Vol. 6]

-Fig. 4

i i !~ii i i i !i~i only the oxidized form of the cofactor was used in catalyst preparation, reducing equivalents could only originate from octanol via the YADH-dependent half-reaction. As shown in Fig. 4 we observed the bleaching of DCIP only in the complete reaction mixture that contained 'co-immobilized' catalyst, that is, catalyst with both YADH and diaphorase deposited on the same support. In contrast, no reaction was observed when octanol was omitted, or when the YADH and diaphorase were immobilized on separate support particles. For this reaction to proceed the nicotinamide cofactor had to diffuse through a 'fluid' microenvironment within the support from the active site of one enzyme to another, even at limiting water concentrations. This demonstration suggests that other multienzyme systems (even those involving water soluble intermediates) may be coupled in non-aqueous media.

i~'!i~~iiti!~:~ iili~

~

~i~i~ili i i ~i !i i i i~i

Demonstration of NAD + recycling between yeast alcohol dehydrogenase (YADH) and diaphorase in organic solvents. Conditions: butyl acetate (1% H20) or hexane (0.1% H20); octanol (YADH substrate) 10 mM; 2,6-dichlorophenolindophenol (DCIP) (diaphorase substrate) saturating; enzyme (10 mg g - 7) and NAD + (0. 6 mg g - 1). 0 = YADH, • = diaphorase, ~ = YADH and diaphorase on same support particles.

S u c c e s s stories

Although much remains to be discovered about the use of enzymes in non-aqueous media, industrial applications have already begun to emerge. One of the most important areas of application is in the processing of fats and oils since the substrates, triglyceride oils and fatty acid esters, are very insoluble in water. Unilever is commercializing a lipase-catalysed process for upgrading triglyceride oils by interesterification 3°. Macrae and coworkers have reported that lipase from R h i z o p u s d e l e m a r can catalyse this inter-esterification using fatty acids, such as stearate and stearate esters, either in neat triglyceride oils or in hexane solutions of oils 31. The tipase initially catalyses the hydrolysis of esters w.ith the trace quantities of water in the oil followed by re-esterification of the resultant acids and alcohols. In this manner, the stearate content of low grade oils, such as palm oil, is increased to produce a cocoa butter-like fat. Further advantage is conferred by the 1,3-positional selectivity of the ]ipase, since the stearoy] moieties are predominantly in these positions in cocoa butter. Commercial introduc-

tion of this process is expected in the very near future. A second example of a nonaqueous biocatalytic process of industrial interest is the resolution of chiral 2-halopropionic acids by stereoselective esterification. Kirchner and Klibanov have developed a process for the production of optically active 2-chloropropionic acid or 2-bromopropionic acid, useful intermediates for the production of herbicides, using the stereospecificity of the lipase from Candida cylindracQe 13'32. This lipase catalyses a stereoselective esterification of the R-2-halopropionic acid with primary alcohols, n-butanol, n-hexanol, or n-octanol in organic solvents such as hexane or chloroform. If a racemic mixture of the acid is used, an equimolar mixture of the R-ester and unreacted s-acid are produced. These products may be separated and recovered by simple extraction, providing both isomers in greater than 90% optical purity. The Candida lipase is exceedingly stable under operating conditions, allowing repeated use of the enzyme and thus making the economics of the process attractive.

Future d i r e c t i o n s

One of the most immediate areas for development is in the stereoselective use of hydrolases such as lipases and amidases. The advantages of chemo-, regio- and stereospecificity exhibited by enzymes may be exploited in the synthesis and resolution of a wide variety of optically active alcohols, carboxylic acids, esters and amides. Dipeptide formation in organic solvents has already been reported 33, and it is tempting to speculate that ultimately large-volume products such as aspartame will be produced by similar biocatalytic processes operating in non-aqueous media. Another area of current investigation with fascinating possibilities for the future involves the use of enzymes in supercritical fluids and gases. Recent work by Klibanov et a]. 34'35 and Blanch and co-workers 36 demonstrate the feasibility of such processes with possible applications for food and chemical processing. The use of enzymes in organic milieux marks a radical departure from traditional enzymology and has changed the way we think about the potential for industrial applications of enzymes. This development may dramatically transform the area of biocatalysis, and broaden the scope of those processes which could be catalysed by enzymes. As this technology is advanced and combined with recombinant-DNA technology, which allows low-cost production of virtually any enzyme, we can look forward to an exciting era of creative exploration in the development of novel products and processes. References

1 Klibanov, A. M. (1986) Chemtech 16, 354-359 2 Zaks, A. and Klibanov, A. M. (1985) Proc. Nat] Acad. Sci. USA 82, 31923196 3 Inada, Y., Takahashi, K., Yoshimoto, T., Ajima, A., Matsushima, A. and Saito, Y. (1986) Trends Biotechnol. 7, 190-194 4 Luisi, P. L. (1985) Angew. Chem. Int. Ed. 24, 439-450 5 Kazandjian, R. Z. and Klibanov, A. M. (1985) J. Am. Chem. Soc. 10, 54485450 6 Dordick, J. S., Marietta, M. A. and Klibanov, A.M. (1986) Proc. Nat]

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Acad. Sci. USA 83, 6255-6257 7 Martinek, K. and Semenov, A. N. (1981) J. Appl. Biochem. 3, 93-126 8 Martinek, K., Semenov, A. N. and Berezin, I.V. (1981) Biochim. Biophys. Acta 658, 76-89 9 Martinek, K. and Semenov, A. N. (1981) Biochim. Biophys. Acta 658, 90-101 10 Cambou, B. and Klibanov, A. M. (1984) J. Am. Chem. Soc. 106, 26872692 11 Zaks, A. and Klibanov, A. M. (1984) Science 224, 1249-1251 12 Deetz, ~. S. and Rozzell, J. D. (1986) The World Biotech Report 1986 (Vol. 2) part 3, pp. 25-33, Online 13 Kirchner, G., Scollar, M. P. and Klibanov, A. M. (1985) J. Am. Chem. Soc. 107, 7072-7076 14 Fukui, S. and Tanaka, A. (1984) Adv. Biochem. Eng. Biotechnol. 29, 1-33 15 Fukui, S. and Tanaka, A. (1982) E n z y m e Eng. 6, 191-200 16 Koshiro, S., Sonomoto, K., Tanaka, A. and Fukui, S. (1985) J. Biotechnol. 2, 47-57 17 Martinek, K., Levashov, A. V., Klyachko, N. L., Pantin, V.I. and Berezin, I.V. (1981) Biochim. Biophys. Acta 657, 277-294 18 Martinek, K., Levashov, A. V., Khmelnitsky, Y.L., Klyachko, N.L. and Berezin, I. V. (1982) Science 218, 889-891 19 Grandi, C., Smith, R. E. and Luisi, P. L. (1981) J. Biol. Chem. 256, 837-843 20 Larsson, K. M., Adlercreutz, P. and Mattiasson, B. (1987) Eur. J. Biochem. 864, 1-5 21 Klyachko, N. L., Levashov, A. V., Pshezhetsky, A. V., Bogdanova, N. G., Berezin, I. V. and Martinek, K. (1986) Eur. J. Biochem. 161,149-154 22 Yoshimoto, T., Takahashi, K., Nishimura, H., Ajima, A., Tamanra, Y. and Inada, Y. (1984) Biotechnol. Lett. 6, 337-340 23 Takahashi, K., Tamaura, Y., Kodera, Y., Mihama, T., Saito, Y. and Inada, Y. (1987) Biochem. Biophys. Res. Commun. 142,291-296 24 Matsnshima, A., Okada, M. and Inada, Y. (1984) FEBS Lett. 178, 275277 25 Takahashi, K., Ajima, A., Yoshimoto, T. and Inada, Y. (1984) Biochem. Biophys. Res. Commun. 125,761-766 26 Takahashi, K., Nishimura, H., Yoshimoto, T., Saito, Y. and Inada, Y. (1984) Biochem. Biophys. Res. Commun. 121,261-265 27 Takahashi, K., Nishimura, H., Toshimoto, T., Okada, M., Ajima, A., Matsushima, A., Tamaura, Y., Saito, Y. and Inada, Y. (1984) Biotechnol. Lett. 6, 765-770

28 Kazandjian, R. Z., Dordick, J. S. and Klibanov, A.M. (1986) Biotechnol. Bioeng. 28,417-421 29 Grunwald, J., Virz, B., Scollar, M. P. and Klibanov, A.M. (1986) J. Am. Chem. Soc. 108, 6732-6734 30 Halling, P. J. and Macrae, A. R. (1982) European Patent Application 0064855 31 Macrae, A.R. (1983)J. A m . Oil. Chem. Soc. 60, 291-294 32 Klibanov, A. M. and Kirchner, G. (1986) US Patent 4601987 []

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Gas hold-up measurements in bioreactors Joep J. M. Hofmeester The gas hold-up of a gas-liquid dispersion is an important parameter in the fermentation industry. If it is too low, or too high, productivity can be adversely affected. Gas hold-up in fermentors cannot be calculated from physico-chemical correlations and, therefore, must be measured accurately for each fermentation. This article surveys a number of methods for measuring the gas hold-up in gas-liquid dispersions, making particular note whether these methods can be applied aseptically. Gas h o l d - u p (also called the v o i d available for gas h o l d - u p in p u r e fraction) is the relative v o l u m e of gas liquid, there is h a r d l y a n y informathat is p r e s e n t in the gas-liquid tion o n gas h o l d - u p in f e r m e n t a t i o n d i s p e r s i o n in a f e r m e n t o r (Fig. 1). Gas broths. T h e m a i n r e a s o n is that in f o a m on top of a f e r m e n t a t i o n b r o t h the gas h o l d - u p d e p e n d s on the does not c o n t r i b u t e to the gas holdp r o p e r t i e s of the b r o t h (e.g., ionic up. O n l y the s u b m e r g e d air b u b b l e s strength, b i o m a s s c o n c e n t r a t i o n , visare c o n s i d e r e d in gas h o l d - u p . Gas cosity of the broth etc.). T h e s e h o l d - u p d e t e r m i n e s the transfer of p r o p e r t i e s v a r y not o n l y w i t h the o x y g e n f r o m the gas to the liquid c u l t u r e strain, but also d u r i n g ferphase. If it is too low, the p r o d u c t i o n m e n t a t i o n . A s e c o n d r e a s o n is that rate in m o s t aerobic f e r m e n t a t i o n s gas h o l d - u p is rarely i m p o r t a n t a n d will fall. O n the other h a n d , h i g h gas s e l d o m m e a s u r e d in laboratory-scale h o l d - u p s h o u l d also be avoided. T h e f e r m e n t a t i o n s w h e r e m o s t of the gas fraction d e t e r m i n e s the m a x i - r e s e a r c h is done. (Laboratory ferm u m b r o t h v o l u m e in a specific m e n t o r s are n o r m a l l y not c o m p l e t e l y f e r m e n t o r since it e x c l u d e s pro- filled, so the level of the aerated b r o t h d u c t i v e liquid. T h e gas h o l d - u p is, m a y vary.) therefore, not o n l y an i m p o r t a n t Therefore, this article will not give process design p a r a m e t e r but, since a n y data on gas h o l d - u p in f e r m e n t a the total v o l u m e of a f e r m e n t o r tion m e d i a . Rather, it will describe c a n n o t be c h a n g e d easily, it also has a h o w to m e a s u r e the gas fraction in a large i n f l u e n c e on p r o d u c t i v i t y . p a r t i c u l a r process. A l t h o u g h m a n y correlations are Joep J. M. Hofmeester is at Gist- Defining gas hold-up Gas h o l d - u p can be m e a s u r e d as brocades, PO Box I, 2600 MA Delft, the Netherlands. the ratio of gas v o l u m e to total O 1988, Elsevier Publications, Cambridge 0166- 9430/87/$02.00