Carboligase

A model developed by Leksawasdi et al. [11,12] for the enzymatic production of PAC (P) from benzaldehyde (B) and pyruvate (A) in an aqueous phase system is based on equations given in Figure 2. The model also includes the production of by-products acetaldehyde (Q) and acetoin (R). The rate of deactivation of PDC (E) was shown to exhibit a first order dependency on benzaldehyde concentration and exposure time as well as an initial time lag [8]. Following detailed kinetic studies, the model including the equation for enzyme deactivation was shown to provide acceptable fitting of the kinetic data for the ranges 50-150 mM benzaldehyde, 60-180 mM pyruvate and 1.1-3.4 U mf PDC carboligase activity [10]. 

The optimal feeding profile based on the model is shown in Figure 3 and the simulation profiles are shown in Figure 4 for initial substrate concentrations of 90 mM benzaldehyde and 108 mM sodium pyruvate, and initial PDC activity of 4.0 U ml carboligase. Feeding was programmed at hourly intervals and the initial reaction volume would increase by 50% by the end of the simulated biotransformation. 

Simulation profile of fed batch PAC biotransformation kinetics at 6°C with initial PDC activity of 4.0 U carboligase ml, 90 mM benzaldehyde and 108 mM sodium pyruvate. Feeding was performed hourly as illustrated in Fig. 3 and the initial reaction volume of 30 ml (which would be used experimentally) increased to 45 ml at the end of reaction. 

Fed- batch PAC biotransformation kinetics at 6°C with optimal feeding program. Initial and final reaction volumes after 81 h were 30 and 45 ml, respectively. The biotransformation was carried out in 2.5 M MOPS, 1 mM MgS04,1 mM TPP with initial pH of 6,5 and initial carboligase activity of 3.82 U mf. The pH adjustment was performed manually using 30% (v/v) H2SO4. 

Neuberg, C. and Hirsch, J. (1921) An enzyme which brings about union into carbon chains (Carboligase). Biochemische Zeitschrift, 115, 282-310. 

Neuberg, C. and Ohle, H. (1922) Carboligase. IV. Biosynthetic carbon chain union in fermentation processes. Biochemische Zeitschrift, 128, 610-618. 

The second method consists of the fermentation of glucose by yeast carboligase in the presence of benzaldehyde, which during the process turns into ( )-l-phenyl-2-keto-propanol (11.3.5). This is reduced by hydrogen in the presence of methylamine, to give the desired ephedrine (11.3.4) [54,55]. 

Development of a High-Throughput Screening Assay for Carboligase Activity... 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

Fig. 2.2.1.2 Colorimetric assay for carboligase activity. A) DMA-HPP 10 reduces 2,3,5-triphenyltetrazolium 13 chloride to the respective formazan 15, which has an intense red color. B) Formation of the red formazan 15 dye can be observed only in the presence of DMA-HPP 10 neither substrate dimethoxyacetaldehyde 8 nor benzaldehyde 4 causes any change in color.

In order to increase the understanding of ThDP-dependent enzymes, the identification of amino acid side chains important for the catalysis of the carboligase reaction in pyruvate decarboxylase from Zymomonas mohilis (E.C. 4.1.1.1) and benzoylformate decarboxylase from Pseudomonasputida (E.C. 4.1.1.7) was a major task. Using site-directed mutagenesis and directed evolution, various enzyme variants were obtained, differing in substrate specificity and enantioselectivity. 

The project encompassed the comparative characterization of pyruvate decarboxylase from Z. mohilis (PDC) and benzoylformate decarboxylase from P. putida (BED) as well as their optimization for bioorganic synthesis. Both enzymes require thiamine diphosphate (ThDP) and magnesium ions as cofactors. Apart from the decarboxylation of 2-ketoacids, which is the main physiological reaction of these 2-ketoacid decarboxylases, both enzymes show a carboligase site reaction leading to chiral 2-hydroxy ketones (Scheme 2.2.3.1). A well-known example is... 

With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD, while BVDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxy-propiophenone product [7, 9]. 

B. Lingen, J. Grotzinger, D. Kolter, M. R. Kula, M. Pohl, Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis. Protein Eng. 2002, 15, 585-593. 

Enzymatic processes also advance in the area of large-scale pharma intermediates /flactam antibiotics can now be produced in a fully biotechnological process, including the semi-synthesis from the /flactam core to the penicillin or cephalosporin. A precursor to ephedrine, long produced by a whole-cell process in yeast, can be obtained from benzaldehyde and acetaldehyde with the help of pyruvate dehydrogenase acting as a carboligase. 

Pyruvate decarboxylase (PDC, E.C. 4.1.1.1) accepts other substrates besides pyruvate, its natural reactant As early as 1921, Neubergand Hirsch described the reaction of yeast with benzaldehyde and pyruvate to phenylacetylcarbinol (2-keto-3-hydroxy-propylbenzene) (Neuberg, 1921) in a carboligase side reaction which yields ephedrine after reaction with methylamine and catalytic hydrogenation (Figure 7.37). 

H. Bruhn, M. Pohl, J. Groetzinger, and M.-R. Kuia, The replacement of Trp392 by alanine influences the decarboxylase/ carboligase activity and stability of pyruvate decarboxylase from Zymomonas mobilis,... 

As demonstrated in Scheme 3, the ThDP-bound active aldehyde 6 as an acyl-donor may be added to a second aldehyde cosubstrate (acyl-acceptor) in an acyloin condensation-type reaction. This carboligase reaction was intensively investigated with acetaldehyde as an acyl-donor, which may be either condensed to a further acetaldehyde molecule yielding acetoin [1,26,27,29,63,153,154] or to a wide range of various aliphatic, aromatic and heterocyclic aldehydes [5,14,118,151,154-157,161]. 

A detailed investigation of the carboligase reaction mediated by PDC from yeast, wheat germ and Z. mobilis revealed that the stereo-control of this reaction is only strict with aromatic aldehydes as acylanion-acceptors, while the formation of acetoin (3-hydroxybutan-2-one) resulted in mixtures of the (R)- and (S)-enantiomer. 

The improvement of the carboligase reaction of PDCZ.m. is a further example for the potency of the rational approach in designing enzymes with... 

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