Transaminases amino alcohols

Matosevic, S., Lye, G.J., and Baganz, F. (2011) Immobilised enzyme microreactor for screening of multi-step bioconversions characterisation of a de novo transketolase-(o-transaminase pathway to synthesise chiral amino alcohols. J. Biotechnol, 155, 320-329. doi 10.1016/j.jbiotec.2011.07.017... 

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. 

Given that transaminases are not able to aminate alcohols, one possibility for the synthesis of chiral amino alcohols is an enzyme cascade reaction carried out with whole cells [36]. Three enz3nnes cascaded in series were expressed in E. colt first the alcohol dehydrogenase oxidized tiie alcohol to the corresponding aldehyde, which is converted into the amine by the transaminase as shown in Scheme 29.15. The recycling of pyruvate and cofactor regeneration were achieved by the alanine dehydrogenase. 

For the in vitro screening of enzyme pairs and enzymatic pathways, immobilized enzyme microreactor systems (lEMR) were established. This includes the reversible immobilization of His -tagged enzymes via nickel-nitriloacetic acid (Ni-NTA) linkage on the surface-derivatized silica. First of all the kinetic parameters for a new enzymatic pathway have to be defined via a transketolase and a w-transaminase in a continuous flow system for a two-step asymmetric synthesis of chiral amino alcohols [147]. The apparenf value is found to be flow rate dependent with different flow rates ranging from 2 to 30 j1/min. The turnover rate was also found to be lower than that in solu-... 

Transaminases are most powerful tools for the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter several approaches for tiie preparation of fine chemicals or building blocks for pharmaceuticals were discussed, like asymmetric synthesis or kinetic resolution. The main limitations of transaminase-catalyzed reactions are the need to shift the equihbrium to the product side and substrate and product inhibition. Some solutions to overcome such inhibition were presented here for example, multienzyme cascades or biphasic extraction of the product. Protein engineering by directed evolution or rational enzyme design is a promising option to find transaminases with different substrate specificities and enantiopreferences. This is becoming more and more important for the pharmaceutical industry. Furthermore, it is a way to alter enzyme properties known so far, like thermostability and solvent and pH stability. Protein engineering has been assisted by the recently solved structures of certain transaminases. 

Vitamin B6 occurs naturally in three related forms pyridoxine (6.26 the alcohol form), pyridoxal (6.27 aldehyde) and pyridoxamine (6.28 amine). All are structurally related to pyridine. The active co-enzyme form of this vitamin is pyridoxal phosphate (PLP 6.29), which is a co-factor for transaminases which catalyse the transfer of amino groups (6.29). PLP is also important for amino acid decarboxylases and functions in the metabolism of glycogen and the synthesis of sphingolipids in the nervous system. In addition, PLP is involved in the formation of niacin from tryptophan (section 6.3.3) and in the initial synthesis of haem. 

A transaminase patented by Celgene Corporation (Warren, NJ), called an co-aminotransferase [(co-AT)E.C. 2.6.1.18] does not require an a-amino acid as amino donor instead it requires a primary amine and hence has the ability to produce chiral amines.125 126 A similar co-AT from Vibrio fluvialis has been described for the production of chiral amines along with chiral alcohols when coupled with AdH or chiral amino acids when coupled with an a-amino acid aminotransferase.127130 Another co-AT, ornithine (lysine) aminotransferase (E.C. 2.6.1.68), has been described for the preparation of a chiral pharmaceutical intermediate used in the synthesis of Omapatrilat, a vasopep-tidase inhibitor developed by Bristol-Myers Squibb, as well as the UAA A1 -piperidinc-6-carboxylic acid.131-132... 

Lflly, M., Bauer, F.F., Styger, G., Lambrechts, M.G., Pretorius, l.S. (2006b).The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates. FEMS Yeast Res., 6, 726-743. 

Vitamin B Three substances are classed under the term pyridoxine or adermine pyridoxol, pyridoxal and pyridoxamine. Pyridoxine was isolated by various study groups in 1938. Its structure was described by Folkers and Kuhn in 1939. Pyridoxal and pyridoxamine were discovered by Snell in 1942. Pyridoxal phosphate and pyridoxamine phosphate are biologically active substances. Intestinal absorption of Bg is dose-dependent and not limited. In alcoholism, a deficiency of vitamin Bg is encountered in 20—30% of cases, whereas the respective percentage is 50—70% in alcoholic cirrhosis. Vitamin Bg is an important coenzyme for transaminases, which transfer amino groups from amino adds to keto acids. In this way, biochemical pathways between the dtiic acid cycle and carbohydrate and amino acid metabolisms are created. (104)... 

FIGURE 12.1 The Ehrlich pathway exemplified for the conversion of the branched-chain amino acids L-valine, L-isoleucine, and L-leucine to the corresponding alcohols isobntanol, 2-methyl-1-bntanol, and 3-methyl-1-butanol. Adh, alcohol dehydrogenase KE)C, 2-ketoacid decarboxylase TA, transaminase. 

Tyrosine transaminase was purified about a hundredfold from acetone powder extracts of dog liver (the most potent source of the enzyme) by dialysis and alcohol fractionation. The presence of a phenolic hydroxyl, an alpha amino, and an alpha carboxyl group were required for a compound to be active as a substrate in this transamination system. Only the L-amino acid was attacked and the only effective keto acid is a-ketoglutaric acid. The enzyme, as would be anticipated, required pyridoxal pho hate as a coenz3rme. It contiuns copper but this apparently is unrelated to its... 

---