Effect of Organic Solvents

Topics: Solvent, Enzyme, Acetic acid Pages: 15 (1298 words) Published: October 12, 2013
SYNTHETIC EXPLOITATION
OF ENZYMES:
BIOCATALYSIS IN ORGANIC
SOLVENTS: FUNDAMENTALS

ENZYMES IN ORGANIC SYNTHESIS
1. Enzymes catalyze a broad spectrum of reactions with high turnover numbers. Rate enhancements approach 1012 fold.
2. Enzymes may accept a wide range of substrates.
3. Enzymes are highly regio and stereoselective.
4. Enzyme reactions take place under mild conditions; this minimizes problems of isomerization and racemization.
5. Enzymatic processes are less hazardous and polluting than conventional chemistry-based methodologies.

Problems associated with enzyme use
1. Instability: immobilization, encapsulation, modification. 2. Cofactor requirements: regeneration of cofactor in situ.
3. Limited water solubility of many organic substrates.

Representations of some organic solvent systems

a)biphasic system b)reverse micellar
system

organic phase

aqueous phase

c) organic solvent
system

biocatalyst

Carrea, Trends in Biotechnology, 2, 102-106, 1984

Scheme of a biocatalytic biphasic system involving co-factor regeneration. Fernandes and Cabral in “Organic Synthesis with Enzymes in Non-Aqueous Media” Wiley-VCH, 2008, pp 191-210.

Advantages of using enzymes in water-organic solvent biphasic systems Increased solubility of hydrophobic substrates
Smaller reaction volumes and increased volumetric productivity Minimization of substrate/product inhibition of the biocatalyst, because of their low concentration in the aqueus phase where reaction takes place Facilitated product recovery

Solvent requisites for application in biphasic systems
a) Biocompatible toward biocatalyst activity and/or stability b) High capacity to dissolve substrates and/or products
c) Low cost
d) Non-biodegradable
e) Non-toxic
f) Relatively high boiling point

Solvent physical parameters that might be correlated
with biocatalyst activity and/or stability
a) Log P (logarithm of partition coefficient between n-octanol and H2O) b) Dielectric constant
c) Hildebrand solubility
d) Dimroth-Reichardt ET

Effect of different organic solvents on the oxidation of testosterone in biphasic systems catalyzed by β-hydroxysteroid dehydrogenase Carrea et al., Arch. Biochem. Biophys., 159, 7-10, 1973

Lineweaver-Burk plot of testosterone oxidation in water-butyl acetate biphasic system, catalyzed by β-hydroxysteroid dehydrogenase. Km referred to substrate concentration in the organic phase: 2.5 mM Km referred to substrate concentration in the aqueous phase: 4.4 µM Partition coefficient of testosterone Corg/Caq = 563

Effect of ethyl acetate and butyl acetate on the kinetic parameters (K m and V max) of some hydroxysteroid dehydrogenases with various steroid substrates

Dependence of testosterone oxidation, catalyzed by crude (triangles) or purified (circles) β-hydroxysteroid dehydrogenase, on the rate of shaking of a water-butyl acetate biphasic system . Reaction time 20 min.

Effect of shaking on the stability of 3α-hydroxysteroid dehydrogenase in α
water and in a water-ethyl acetate biphasic system.

Shaking: 100 strokes/min.

Bioconversions in biphasic systems (years 1973-1984)

Carrea, Trends in Biotechnology, 2, 102-106, 1984

More recent examples of application of biphasic systems in biocatalysis (2000-2007)

Fernandes and Cabral in “Organic Synthesis with Enzymes in Non-Aqueous Media” Wiley-VCH, 2008, pp 191-210.

Representations of some organic solvent systems

a)biphasic system b)reverse micellar
system

organic phase

aqueous phase

c) organic solvent
system

biocatalyst

100.000

Activity, mM min-1

10.000

1

100.000

5

a)
2 3 4

6

b)

10.000

1.000

1.000

0.100

0.100

0.010

0.010

0.001
0.1

0.001

1.0

10.0

100.0

Water content of solvent, % (v/v)

0

10

20

30

40

Water content of enzyme, % (w/w)

a) Dependence of alcohol oxidase activity on the concentration of water in various...
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