Whole-cell catalysts

An (5)-specific alcohol dehydrogenase gene from Rhodococcus erythropolis and GDH from Bacillus subtilis were ligated into one plasmid, which was expressed in Escherichia coli strain DSM14 459 to provide an (S)-selective whole-cell catalyst. 

Groeger, H., Rollmann, C., Chamouleau, F. et al. (2007) Enantioselective reduction of 4-fluoroacetophenone at high substrate concentration using a tailor-made recombinant whole-cell catalyst. Advanced Synthesis and Catalysis, 349 (4-5), 709-712. 

Yamashita, S., Satoi, M., Iwasa, Y.etal. (2007) Utilization of hydrophobic bacterium Rho do co ecus opacusB-4 as whole-cell catalyst in anhydrous organic solvents. Applied Microbiology and Biotechnology, 74, 761-767. 

For the synthesis of (7 )-alcohols by reduction of the corresponding ketones, an E. coli whole-cell catalyst was constructed based on an ADH from L. kefir. Some preparative examples using these designer cells are summarized in section 9.4. 

For Baeyer-Villiger oxidations of ketones into lactones, recombinant whole-cell catalysts were constructed using the CHMO from A. calcoaceticus and cells of S. cerevisiae [127-129] or E. coli [130-132] as host system. These designer cells were used for example for the biotransformation of bicyclo[3.2.0]-hept-2-ene-6-one [133-136], In order to avoid the substrate and product inhibition, an adsorber... 

Based on the (/ )-specific ADH from L. kefir, a recombinant E. coli strain was constructed as a whole-cell biocatalyst, and co-expressed GDH was used for regeneration of NADPH [157]. These designer cells were applied for the reduction of 4-fluoroacetophenone to the corresponding optically active (/ )-4-fluorophe-nylethan-l-ol at 0.5 M educt concentration [158]. After a reaction time of 23 h, a conversion of >95% has been achieved, and the purified isolated chiral alcohol showed an ee value of >99% (87% yield). (S)-p-Halohydrins were obtained with this whole-cell catalyst by means of an enantioselective reduction of the corresponding ketones with both high conversions of >95% and enantioselectivities of >99% (Fig. 40). Base-induced cyclization of the [S-halohydrin led to enantiomeri-cally pure (S)-epoxides in high yield and enantiomeric purity (>99% ee) [159]. 

Fig. 40 Concept for the two-step synthesis of enantiomerically pure (S)-epoxides out of aliphatic 1-halogenated 2-ketones. The ketone was reduced by a recombinant whole-cell catalyst bearing alcohol dehydrogenase from Lactobacillus kefir (LKADH) and glucose dehydrogenase (GDH) for regeneration of NADPH. Base-induced cyclization of the enantiomerically pure (5)-(3-halohydrin intermediate gave the desired (S)-epoxides in high yield and enantiomeric purity (>99% ee)...

Biotransformation can serve as an alternative route towards enantiopure aziridines. (1R,25)-1 -Benzyl- and l-arylaziridine-2-carboxamides were obtained in enantiomerically pure form via kinetic resolution of their racemates by Rhodococcus rhodochrous IFO 15564 catalyzed hydrolysis <07OF521>. Rhodococcus erythropolis AJ270 was reported as an efficient whole cell catalyst for the synthesis of highly enantiopure 5,-l-arylaziridine-2-carboxamides and A-l-arylaziridine-2-carboxylic acids <07JOC2040>. Enantiopure 2-... 

Whole cell catalysts do not need immobiUzation, especially when mycelial micro-organisms are involved, since their morphological structure allows for easy filtration and re-utihzation. Carboxylesterases bound to the mycelia of molds have been advantageously employed as biocatalysts in water and/or organic solvents the first report of the use of fungal myceha in organic solvent dates back to 1978... 

Fig. 10 Hydantoinase-based whole cell catalysts in L-amino acid synthesis.

A biocatalytic enantioselective addition of ammonia to a C=C bond of an afl-unsaturated compound, namely fumaric acid, makes the manufacture of L-aspar-tic acid, l-27, possible [30], This L-amino acid represents an important intermediate for the production of the artificial sweetener aspartame. The biocatalytic production process, which is applied on an industrial scale by, e.g., Kyowa Hakko Ko-gyo and Tanabe Seiyaku, is based on the use of an aspartate ammonia lyase [E.C.4.3.1.1] [31]. As a biocatalyst, an immobilized L-aspartate ammonia lyase from Escherichia coli [32, 33] as well as Brevibacterium flavum whole-cell catalysts [32 a, 34] have been applied successfully. 

In recent years, the enantioselective hydrolysis of nitriles has been studied in more detail. Whereas in the past only whole cell catalysts had been investigated, it is now possible to assign the activities to specific enzymes occurring in the cell. These enzymes are nitrilases, nitrile hydratases and/or amidases. 

The same whole cell catalyst can be used in the hydration of 3-cyanopyridine to nicotinamide (Scheme 12.1-17). This vitamin, broadly applied in animal feeding, is currently produced biocatalytically on an industrial scale (> 3000 t/a) by the Lonza AG. For this substrate Yamada and Kobayashi showed that the whole cell catalyst of Rhodococcus rhodocrous Jl, containing a nitrile hydratase induced with crotonamide, can even tolerate substrate concentrations up to 12 m 121 (see Fig. 12.1-3). 

Baeyer-Villiger monoxygenase or whole cell catalyst... 

As whole cell catalyst, Pseudomonas putida, which accepts a wide range of substrates, is applied. Subsequent to the biotransformation, benzaldehyde is added, resulting in precipitation of the D-amide Schiff base, which can be easily isolated by filtration. An acidification step leads to the D-amino acid. The L-amino acid can be reused after racemization so that a theoretical yield of 100% D-amino acid is possible. 

Verseck, S., Becker, U., Doderer, K., Ofiwald, S., and Wienand, W. (2009) Production of amino adds using wild type and recombinant whole cell catalysts using platform technologies for enhandng production effidency. ACS Symposium series Vol 1009, Asymmetric synthesis and application of a-amino acids. 375-393. 

Kleser M, Hannemann F, Hutter M, Zapp J, Bernhardt R (2012) CYP105A1 mediated 3-hydroxyl-ation of glimepiride and glibenclamide using a recombinant Bacillus megaterium whole-cell catalyst. J Biotechnol 157 405 12... 

Schrewe, M., Ladkau, N., Buehler, B., and Schmid, A. (2013) Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv. Synth. Catal., 355, 1693-1697. 

In a related approach, nonactivated terminal carbons were directly aminated by using a recombinant whole-cell catalyst [33]. In the key steps, an oxygenase and an m-TA were coupled in vivo within a single Escherichia coU host (BL21) (Scheme 4.8). For the oxidation of the alcohol to the respective aldehyde, the NADH-dependent oxygenase AUcBGT from Pseudomonas putida was used, which allowed the oxyfunctionalization of medium-chain-length alkanes, fatty acids [34], and selected fatty acid methyl esters [35]. Subsequent reductive amination was achieved... 

Subsequently, bienzymatic whole cell catalysts were constracted by coexpressing the (S)-HnL and nitrilase activities simultaneously in the yeast Pichia pastoris and the bacterium Escherichia coli. The recombinant E. coU cells exhibited much higher HnL and nitrilase activities compared to the P. pastoris catalysts and were therefore studied in greater detail [63, 64]. The recombinant E. coli cells were... 

The bienzymatic approach was also used for the synthesis of a-alkyl-a-hydroxycarboxylic acids from ketones and cyanide. The conversion of ketones by HnLs is problematic because the reaction equilibrium is mainly on the side of the ketones and therefore these substrates are generally not quantitatively converted by HnLs ]68, 69]. Therefore, the presence of a second enzyme, such as a nitrilase, results in the establishment of an efficient cascade reaction. The feasibility of this biotransformation was demonstrated for the conversion of acetophenone plus cyanide at acidic pH-values by the recombinant whole-cell catalysts which simultaneously produced the nitrilase from P.Jluorescens EBC191 and the MeHnL. These cells converted acetophenone plus cyanide almost quantitatively to (S)-atroIactate (and (S)-atrolactamide) [61]. 

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