Whole pseudomonas putida

A very efficient and universal method has been developed for the production of optically pue L- and D-amino adds. The prindple is based on the enantioselective hydrolysis of D,L-amino add amides. The stable D,L-amino add amides are effidently prepared under mild reaction conditions starting from simple raw materials (Figure A8.2). Thus reaction of an aldehyde with hydrogen cyanide in ammonia (Strecker reaction) gives rise to the formation of the amino nitrile. The aminonitrile is converted in a high yield to the D,L-amino add amide under alkaline conditions in the presence of a catalytic amount of acetone. The resolution step is accomplished with permeabilised whole cells of Pseudomonas putida ATCC 12633. A nearly 100% stereoselectivity in hydrolysing only the L-amino add amide is combined with a very broad substrate spedfidty. 

Wackett LP, DT Gibson (1988) Degradation of trichloroethylene by tolnene dioxygenase in whole-cell studies with Pseudomonas putida FI. Appl Environ Microbiol 54 1703-1708. 

The naphtho[2,3-d]-l,3-dioxole was oxidized using recombinant E. coli whole cells overexpressing the gene for the naphthalene dioxygenase from Pseudomonas putida G7. The linear carbohydrate fragment was enzymatically formed after ozon-ization of the diol, by chain extension with a dihydroxyacetone fragment in an... 

Tan HM, Cheong SP, Tan TC (1994) An amperometric benzene sensor using whole ceU Pseudomonas putida ML2. Biosens Bioelectron 9 1-8... 

Combining whole-cell biocatalysis and radical polymerization, researchers at Imperial Chemical Industries (ICI) published a chemoenzymatic route to high-molecular-weight poly(phenylene) [86], This polymer is used in the fibers and coatings industry. However, since it is practically insoluble, the challenge was to make a soluble polymer precursor that could first be coated or spun, and only then converted to poly(phenylene). The ICI process starts from benzene, which is oxidized by Pseudomonas putida cells to cyclohexa-3,5-diene-l,2-diol (see Figure 5.17). The... 

Mutants of Pseudomonas putida were found to exhibit an arene dioxygenase activity, which has been exploited in whole-cell reactions for the regio- and enantioselective preparation of cw-dihydrodiols starting from benzene, substituted benzenes, and polycyclic or heteroaromatic compounds [48], The products are invaluable precursors for natural product synthesis, as exemplified in Scheme 8 [49],... 

C-Cl hydrolysis for 2-chloropropionic acid with whole cells of Pseudomonas putida (dehalogenase) 2000 t/y Zeneca Life Science Molecules [3]... 

The aminopeptidase from Pseudomonas putida ATCC 12633 has also recently been cloned and overexpressed in E. coli resulting in a highly efficient whole-cell biocatalyst for industrial applications 1291. The specific activity of this new biocatalyst is substantially increased (25 times) compared with the specific activity of the P. putida wild type cells without changing the other positive characteristics of the aminopeptidase. Even though the aminopeptidase from Pseudomonas putida exhibits the relaxed substrate specificity described above, an a-hydrogen atom in the substrate is an essential structural feature for the enzymatic activity. Therefore this enzyme can not be used for the resolution of higher substituted amino acids. 

Whilst the whole-cell approach has proved invaluable, the associated problems of overmetabolism and side reactions can be encountered. Another way to counter the problems of high cost in using isolated BVMOs is to use an NADH dependent enzyme, as NADH retails at approximately one tenth of the cost of NADPH. The Type 2 DKCMOs from Pseudomonas putida ATCC 17453 (= NCIMB 10007) are NADH dependent, and Grogan et al. were successftd in applying a complement of these enzymes, termed MOl, to the transformation of bicyclo[3.2.0]hept-2-en-6-one, to yield another enantiodivergent mix of lactones enantiomeric to those obtained... 

Benzaldehyde can be produced from benzoyl formate with whole cells of Pseudomonas putida ATCC 12633 as biocatalyst119 201 (Fig. 16.6-5). Alternatively, but less effectively, mandelic acid can be used as starting material. A pH of 5.4 was found to be optimal for benzaldehyde accumulation. At this proton concentration, partial inactivation of the benzaldehyde dehydrogenase isoenzymes and activation of the benzoyl formate decarboxylase are reported. Fed-batch cultivation prevented substrate inhibition. In situ product removal is necessary to prevent product inhibition. 

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. 

E = aminopeptidase, whole cells from Pseudomonas putida... 

Production of (5)-2-chloropropanoic acid from racemic 2-chloropropanoic acid by enan-tioselective degradation of the R-enantiomer with whole cells of R-dehalogenase-contain-ing Pseudomonas putida. (5)-2-chloropropanoic acid is a building block for a wide range of herbicides and is produced by ICI (2000 tons per year). The side product (S)-lactic acid is also of commercial interest. 

The c/s-dihydroxylation reaction catalyzed by these dioxygenases is typically highly enantioselective (often >98% ee) and, as a result, has proven particularly useful as a source of chiral synthetic intermediates (2,4). Chiral cis-dihydrodiols have been made available commercially and a practical laboratory procedure for the oxidation of chlorobenzene to IS, 2S)-3-chlorocyclohexa-3,5-diene-l,2-c diol by a mutant strain of Pseudomonas putida has been published (6). Transformation with whole cells can be achieved either by mutant strains that lack the second enzyme in the aromatic catabolic pathway, cw-dihydrodiol dehydrogenase (E.C. 1.3.1.19), or by recombinant strains expressing the cloned dioxygenase. This biocatalytic process is scalable, and has been used to synthesize polymer precursors such as 3-hydroxyphenylacetylene, an intermediate in the production of acetylene-terminated resins (7). A synthesis of polyphenylene was developed by ICI whereby ftie product of enzymatic benzene dioxygenation, c/s-cyclohexa-3,5-diene-1,2-diol, was acetylated and polymerized as shown in Scheme 2 (8). 

The presence of a biocatalyst, either whole cells [ 126] or enzymes [ 157], or any other biological surface-active materials either produced or present as substrates in the bioconversion system, such as fatty acids or long chain alcohols [ 127,184], were expected to lower interfacial tension and hence breakthrough pressure [126, 157,184]. A threefold decrease in the interfacial tension was observed in an aque-ous-tetradecane system when either Pseudomonas putida or bakers yeast cells were used, as compared to the cell-free system [126]. A decrease in the breakthrough pressure due to the presence of a surface-active agent, lauric acid, was also cited [184]. 

Field flow fractionation (FFF) can also be used for microbial cell separation. In the FFF technique, a field (may be gravitational, centrifugal, thermal-gradient, electrical, magnetic, etc.) is applied perpendicular to the fluid flow, causing particles to migrate with different velocities. Fields of sedimentation, diffusion, and electrical diffusion are manipulated to optimize the separations of microbes. Separation of Pseudomonas putida and E. coli has been achieved by hyperlayer FFF. Fractions of the whole cells were collected after the separation at different time intervals, dif-... 

Buhler, Schmid, and coworkers [36] described the development of a recombinant whole-cell biocatalyst for the direct terminal alkylamino-functionalization of fatty acid methyl esters (e.g., dodecanoic acid methyl ester). The model substrate was dodecanoic acid methyl ester, which was oxidized by an alkane monooxygenase (AlkBGT) from Pseudomonas putida GPol to the corresponding... 

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... 

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