Biocatalyst Formulation And Tuning

To facilitate its application in organic synthesis, we developed a lyophilized cell powder of Sphingomonas sp. HXN-200 as a biohydroxylation catalyst.29 Hydro-xylation of N-benzyl-piperidine with such catalyst powder showed 85% of the activity of a similar hydroxylation with frozen/thawed cells, shown in Figure 15.6. The fact that rehydrated lyophilized cells are able to carry out such a reduced nicotinamide adenine dinucleotide (NADH)-dependent hydroxylation indicates that these cells are capable of retaining and regenerating NADH at rates equal to or exceeding the rate of hydroxylation. To our knowledge, this is the first example of the use of lyophilized cells for a cofactor-dependent hydroxylation.

For an industrial biotransformation, it is often necessary to further optimize an appropriate biocatalyst. This includes the elimination of the follow-up enzymes in the wild-type strain by mutations and the improvement of other characteristics by additional mutations and the selection of improved strains. Alternatively, the genetic information for a desired enzyme might be introduced in a host that has many of the preceding characteristics, and that has no enzymes that could modify or degrade the desired product.

Figure 15.6 Hydroxylation of N-benzylpiperidine (5 mM) with lyophilized cell powder and frozen/thawed cells of Sphingomonas sp. HXN-200 at cell concentration of 4.0 g cdw/L.

Sphingomonas sp.



P% frozen/thawed cells S% frozen/thawed cells P% Lyophilized cells S% Lyophilized cells


Figure 15.6 Hydroxylation of N-benzylpiperidine (5 mM) with lyophilized cell powder and frozen/thawed cells of Sphingomonas sp. HXN-200 at cell concentration of 4.0 g cdw/L.


Perhaps surprisingly, we have found in our work that E. coli is an excellent

23 30_32

whole-cell biocatalytic host. , It is relatively easy to introduce new desired enzymes into various E. coli strains. Generally, for many of the hydroxylation reactions that we have studied, degradative enzymes or downstream pathway enzymes that could modify or eliminate desired hydroxylation products, are not present in E. coli. In working with two liquid-phase systems, E. coli is more sensitive to apolar solvents than Pseudomonas strains. However, we have developed mixed apolar phase systems, based on highly apolar solvents such as hexadecane or substituted phthalates,19 which are highly compatible with E. coli. Thus, it is possible to use very toxic substrates, and produce equally toxic products, which dissolve in the hexadecane or phthalate phase and have very little effect on the host organism present in the aqueous phase.33

The beauty of working with only a few host strains is that these can be optimized for growth and growth medium, expression system for a wide range of bio-catalysts, behavior in the presence of solvents, regeneration of coenzymes, downstream processing, recovery of cells for further biocatalysis use, and waste treatment. Thus, function can be optimized generally, cost can be minimized generally, and different enzymes can be introduced from a wide range of sources. In addition, each enzyme to be used can be optimized further for top biocatalytic performance by mutagenesis, directed evolution,34 or gene shuffling.35

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