Racemases Achromobacter obae

For some years Foray s enzymatic process for L-lysine (L-Lys, 41) was competitive compared with fermentation. This chemoenzymatic L-Lys production was established with a capacity of 5000-10000 t/y. The key intermediate is a-amino-e-caprolactam (ACL), produced from cyclohexanone in a modified Beckmann rearrangement. The enantiospecific hydrolysis forming L-Lys is based on two enzymes L-ACL-hydrolase opens the ring of ACL to L-Lys and in the presence of the ACL-racemase the d-ACL is racemized. Incubating d,l-ACL with cells of Cryptococcus laurentii having l-ACL lactamase activity together with cells of Achromobacter obae with ACL-racemase activity, L-Lys could be obtained in a yield of nearly 100% (Scheme 24) [102]. 

Again a dynamic resolution is carried out, but this time the racemization is introduced by an enzyme, a racemase from Achromobacter obae (Fig. 19-30). The lactamase and racemase are applied as whole cells and are fortunately active at the same pH, so that they can be used in one reactor. Reaction conditions enabling... 

E1= L-aminolactam-hydrolase, whole cells from Cryptococcus laurentii E2= amino-lactam-racemase, whole cells from Achromobacter obae... 

L-Lysine, an essential amino acid, is used in very large quantities to supplement human foods and animal feeds. Traditionally, L-Lysine is produced by fermentation processes. An alternative route developed by Toray Ind. involves the chemical synthesis of D/L-a-amino-s-caprolactam followed by the selective hydrolysis of the L-a-amino-s-caprolactam catalyzed by intracellular lactamase in Cryptococcus laurentii, to give L-Lysine. The process can be improved by adding a second micro-organism, Achromobacter obae, containing a-amino-E-caprolactam racemase.Thus, quantitative yields of L-lysine are obtained. 

More elegant is selective hydrolysis of the L-enantiomer by an L-a-amino-8-caprolactamase which occurs in several yeasts, for example in Cryptococcus laurentii. The racemiza-tion of the remaining D-isomers is possible with a racemase of Achromobacter obae. The process can be performed as a one-step reaction the racemic aminocaprolactam is incubated with intact cells of C. laurentii and A. obae, producing almost 100% L-lysine. 

Synthesis of (S)- and (/ )-a-amino acids using nitrile hydratase (NHase) from Rhodococcus opacus, a-amino-e-caprolactam racemase (ACL) from Achromobacter obae, and different amidases (amidase 1, o-amino-peptidase from Ochrobactrum anthropi amidase 2, i-amino acid amidase from Brevundimonas diminuta) [111, 112]. / =CH3, CH CH, CH3CH(CH3), CH3CH(CH3)CH2. 

Based on the structural similarities of amino acid amides with ACL, we predicted that ACL racemase catalyzed the racemization of not only the cyclic amino lactam compounds but also amino acid amides. We soon discovered that ACL racemase from Achromobacter obae catalyzed the racemization of amino acid amides, such as 2-aminobutyramide and alanine amide [22], A previous study demonstrated that D-alanine was produced efficiently from L-alanine amide by a dynamic kinetic resolution reaction when the enzyme was used together with DAP from O. anthropi [23]. It had previously been unclear whether ACL racemase catalyzed the racemization of simple amino acid amides. 

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