Transaminases process development

Process Development A Fast Way to Identify Appropriate Transaminases... 

A new development is the industrial production of L-phenylalanine by converting phenylpyruvic add with pyridoxalphosphate-dependent phenylalanine transaminase (see Figure A8.16). The biotransformation step is complicated by an unfavourable equilibrium and the need for an amino-donor (aspartic add). For a complete conversion of phenylpyruvic add, oxaloacetic add (deamination product of aspartic add) is decarboxylated enzymatically or chemically to pyruvic add. The use of immobilised . coli (covalent attachment and entrapment of whole cells with polyazetidine) is preferred in this process (Figure A8.17). 

Celgene developed transaminase technology for the enantioselective conversion of chiral amines to ketones [25]. Low molecular weight aldehydes, such as propional dehyde, were used as the amine group acceptor. This process has been used on a... 

In the described examples, the pyridoxamine was covalently attached to the polymer while in most real transaminase enzymes the pyridoxamine coenzyme forms a noncovalent active holoenzyme with the protein (apoenzyme). A new artificial transaminase mimic was developed, in which the pyridoxamine binds noncovalently and reversibly to the polymer. The pyridoxamine attached, for example, to a steroid side chain 99 or 100, together with modified PEI 101 (molecular weight of 60000 and 8.7% dodecyl chains) forms the artificial holoenzyme (Figure 38a). The transamination of pyruvic acid was accelerated 28000-fold with 99 + 101 compared to 10 000 with the covalent pyridoxamine-polymer 98 enzyme mimic. This was due to the fact that the noncovalent system 99 - -101 is more dynamic and therefore can adopt a more suitable geometry for the reaction. The artificial transaminase shows effective rate enhancements in converting the ketoacid into the amino acid, but also the pyridoxamine is converted to pyridoxal. The conversion to pyridoxamine is a necessary step in the catalytic cycle to achieve high turnovers however, this was still not possible with the noncovalent model system. It was observed that the reverse process is very slow and actually in all artificial models so far thermodynamically unfavorable. However, it was possible to use sacrificial amino acids at elevated temperatures (60 °C) that were decarboxy-lated to recycle the pyridoxal 102 to pyridoxamine 100 with modest turnover numbers of 81 (Figure 38b). " ... 

Some of the typical hurdles to attain the performance requirements in the synthesis mode are equilibrium limitation, low chiral purity, enzyme inactivation/inhibition, and productivity. In a typical transaminase development work, first transaminase activity is demonstrated on a particular substrate. Once the activity is identified, process economy is calculated to identify bottlenecks in the process. In a majority of transaminase reaction cases, the reaction equilibrium favors the reaction in the reverse direction (i.e., toward the substrates) that reduces the overall yield and hence the cost of production. Ultimately, the cost of production drives the route selected and defines the requirements for enzyme modification in cases where these methods compare poorly to traditional methods. 

Several multienzymatic processes employing transaminases have been developed for the production of enantiopure amines and amino acids. 

An impressive example of a biotransformation process in aqueous media supplemented with a water-misdble cosolvent can be found in the transaminase-catalyzed synthesis of the sitagliptin key intermediate (S)-40 developed jointly by Codexis and Merck researchers (Scheme 2.16) [ 18,19]. As might be expeded, the ketone substrate was only soluble in water at less than 1 g/L. Various cosolvents were evaluated and both methanol and DMSO were suitable candidates with resped to both enzyme performance and increase of ketone solubility. The latter was ultimately chosen, and the final process operates at 50% (v/v) DMSO, which allows a remarkable substrate input of 200 g/L of ketone 39. This... 

To develop a biocatalytic process, screening of commercially available transaminases by Merck and Codexis provided no enz3une with detectable activity for amination of tile prositagliptin ketone 6 [28]. They therefore applied a combination of in silico design and directed evolution in an effort to confer such an enzyme. 

Transaminases are important enzymes in the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter the properties of transaminases, the reaction mechanisms, and their selectivity and substrate specificity are presented. The synthesis of chiral building blocks for pharmaceutically relevant substances and fine chemicals with transaminases as biocatalysts is discussed. Enzymatic asymmetric synthesis and dynamic resolution are discussed using transaminases. Protein engineering by directed evolution as well as rational design of transaminases under process condition is presented to develop efficient bioprocesses. 

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