Phenylalanine dehydrogenase catalyzed reductive amination of 6-(1 ',3 '-dioxolan-2 '-yl)-2-keto-hexanoic acid to 6-(1 ',3 '-dioxolan-2 '-yl)-2S-aminohexanoic acid with NADH regeneration and enzyme and cofactor retention

Two systems were analyzed that make it possible to reuse enzymes and cofact ors in the reductive amination of the lithium salt of 6-(1',3'-dioxolan-2'- yl)-2-keto-hexanoic acid (compound 1) to 6-(1',3'-dioxolan-2'-yl)-2S-aminoh exanoic acid, lithium salt (compound 2) catalyzed by phenylalanine dehydrog enase (PDH) with in situ NADH regeneration catalyzed by formate dehydrogena se (FDH). First, both enzymes and PEG-NADH, a mass-enlarged cofactor, were encapsulated within semipermeable microcapsules. These microcapsules cataly zed the biotransformation in the absence of external NAD. About 20 mg of co mpound 2 was formed per mt microcapsule per day. Substrate diffusion into t he microcapsules was not late-limiting. PEG(35,000)-NADH, but not PEG(20,00 0)-NADH, was retained within the microcapsules. The activity of the encapsu lated enzyme was about 15-25% of the activity of the free enzyme. If satura ting free NAD was added to microcapsules, the activity increased about thre efold. The microcapsules could be reused, and lost about half of their acti vity after eight days of continuous use at 40 degrees C. The stability of F DH limited the reusability of microcapsules. As an alternative system, enzy mes and PEG-NADH were used in a membrane reactor. Preliminary results showe d the production of about 1 g of product per day in a 10 mL reactor over a period of ten days at ambient temperature without loss of activity.