Reusability of ADH and GDH producing Arxula adeninivorans cells and cell extract for the production of 1-(S)-phenylethanol

An Arxula adeninivorans strain co-expressing ADH of Rhodococcus ruber and GDH of Bacillus megaterium, coding for (S)-specific alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) respectively, was used for the synthesis of enantiomerically pure 1-(S)-phenylethanol as permeabilized yeast cells (PC), permeabilized immobilized cells (PIC) or immobilized crude extract (IE). Permeabilization was achieved with Triton X-100 and resulted in cells that had the same activity as crude extract. m alginate immobilization allowed the entrapment of enzymes and PIC, which resulted in a gain of 60 and 82% activity respectively compared to crude extract. zyme preparations were used in successive reaction cycles. PC and IE lost activity after 10 reactions whereas PIC were active with over 80% activity for 28 cycles. After 5 PIC catalyzed reactions, 94.8% chemically pure 1-(S)-phenylethanol with ee of ≥99% was isolated, which is a conversion of 79.6% of the substrate, acetophenone. Furthermore the reaction medium containing BIS-TRIS buffer could be regenerated by the addition of Ca(OH)2, which precipitated calcium gluconate and restored full buffering capacity. ity is a key factor for the cost-effective use of an enzyme system in industry and these results indicate that A. adeninivorans PIC will be useful for the synthesis enantiomerically pure alcohols.