4.2 Production of PCL precursors
The biocatalytic production of PCL or its precursors has been heavily
investigated over the last years (Table 3). Approaches based on isolated
enzymes, [15, 40-44] as well as whole cells,[16, 45-47] have successfully been established.
However, most of these approaches relied on cyclohexanol as a substrate[41-47], which needs to be synthesized from
cyclohexane employing an ecologically critical process[48]. Additionally, inhibition of CHMO by
cyclohexanol or substrate inhibition necessitated the development of
suitable reaction concepts, e.g., two-liquid phase[40] or fed-batch systems[43]. The highest productivity of
1.87 g L-1 h-1 was obtained with
isolated enzymes by employing an appropriate feeding strategy for the
complete conversion of 283 mM cyclohexanol to 6HA[43] (Table 3). The CHMO from Acinetobacterheterologously expressed in E. coli showed the highest total
turnover number (TTN) with almost 70,000 molε-CLmolCHMO-1 [46].
In general, whole-cell approaches show lower yields on biocatalyst, as
target enzymes constitute only about 1-10% (w/w) of cells, but avoid
the enormous effort to purify the enzymes.
Compared to cyclohexanol, cyclohexane is an even more challenging
substrate due to its high volatility and toxicity. In comparison to
solvent-sensitive E. coli employed to convert cyclohexanol to 6HA[47], we obtained a 10-fold higher specific
whole-cell activity and a similar yield on biocatalyst (Table 3).P. taiwanensis VLB120 is known to tolerate low-logP solvents and
can, therefore, be considered suitable for the biotransformation of the
more toxic substrate cyclohexane [49, 50].
Possible prolongation of the reaction with an appropriate substrate
feeding and the application of a high-cell density setup hold big
potential to further improve the product titer and the volumetric
productivity.
This study, for the first time, demonstrates a whole-cell approach
directly converting cyclohexane to the PCL precursor 6HA. The
biotransformation to ε-CL presented by Karande et al.[16] could be optimized by enhancing the
conversion, yield on biocatalyst, TTN, and specific activity (Table 3).
The use of isolated enzymes to convert cyclohexane to ε-CL suffered from
low conversion and TTN, which can be attributed to mass transfer
limitations or inherent instability of P450 monooxygenases[7, 15]. The cellular environment allows for more
stable catalytic activities with superior productivities. Efficient
cyclohexane mass transfer without cell toxification will constitute a
major future challenge and may be solved by cyclohexane feeding
potentially via the gas phase. The achieved increase in whole-cell
activity and conversion is a huge step forward towards the establishment
of an economically viable process [51].