synthesis of sugar fatty
acids
34–100 0.5.10−^3 –
5.0.10−^3Cao et al. (1996)3 peptide synthesis 56–97 0.5–10–4–2.7 Eichhorn et al. (1995, 1997)
Erbeldinger et al. (1998b)
4 optically pure acids 85–100 6.3.10−^2 –0.4 Kitahara et al. (1960)
Van der Werf et al (1995)
Michielsen et al. (1999a)
besides the main application in peptide synthesis, solid-to-solid bioconversions are also
applicable to the synthesis of sugar fatty acids and optically pure acids.
KineticsBy definition, the overall rate of a multi-process conversion is determined by the slowest
process, also called the rate-limiting process. In solid-to-solid bioconversions, the rate-
limiting process can be either dissolution, bioconversion, or crystallisation, depending on
process conditions like temperature, biocatalyst concentration, solid substrate and product
amounts (per cubic meter of suspension), stirring speed, etc. Erbeldinger et al. (1998a)
reported one of the first kinetic studies of enzymatic solid-to-solid conversions. They
investigated the effect of water (from 0 to 600 ml per mol substrates) on the initial rate of
thermolysin-catalysed dipeptide synthesis with equimolar amounts of solid
carbobenzoxy-L-glutamine (Z-Gln-OH) and solid L-leucamide (H-Leu-NH 2 ) in a closed
system without mixing. It appeared that the initial rate per mass unit of enzyme increased
rapidly from almost zero until a maximum was reached at about 50 ml of water per mol
substrates. The authors explain this with the finding of Kuhl et al. (1995), that water is
necessary to maintain enzyme activity. However, Jakubke et al. (1996) reported that
biocatalytic rates may decrease with increasing medium viscosity due to reduced protein
mobility. Since high substrate concentrations are often accompanied by raised viscosity,
this means that the increase in initial rate per mass unit of enzyme with increasing water
amount could also be due to increased protein mobility. At higher amounts of water, the
initial rate per mass unit of enzyme first decreased rapidly (between about 50 and 100 ml
of water per mol substrates), and then levelled at higher amounts of water per mol
substrates. This effect was explained by mass-transfer limitation, and emphasises that,
besides the amount of water, mixing is also a key parameter. The maximum rate per mass
unit of enzyme was attained at 20 mol of substrate per kg of water. The latter is
promising in terms of industrial application, as in combination with a high conversion
this can result in high overall product concentrations.
Improved solids mixing in solid-to-solid bioconversions can be obtained by rotary
homogenisation (Čeřovský, 1992), (ultra)sonication (Kuhl et al., 1992, 1995), and stirring
(Kuhl et al., 1992; Eichhorn et al., 1997). Kuhl et al. (1995) have reported on the
application of two different types of fluidized-bed batch reactors for the chymotrypsin-
catalysed synthesis of Z-Phe-Leu-NH 2 and for the thermolysin-catalysed synthesis of Z-
Ala-Phe-Leu-NH2 and Boc-Ala-Phe-Leu-NH 2. The solid substrate and enzyme particles
were suspended and mixed by an upward moisturized air stream. However, low
conversions of 10–40% were achieved, probably due to sticking of enzyme and substrate
Solid-to-solid bioconversions 243