Preparative regio- and chemoselective functionalization of hydrocarbons catalyzed by cell free preparations of 2-hydroxybiphenyl 3-monooxygenase

Oxygenases are useful catalysts for the selective incorporation of molecula r oxygen into hydrocarbons. Here, we report on the application of isolated, cell free 2-hydroxybiphenyl 3-monooxygenase (HbpA) as catalyst for the reg io- and chemospecific hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybi phenyl. The catalyst was prepared from recombinant Escherichia coli using e xpanded bed adsorption chromatography and could be stored without significa nt loss of activity in lyophilized form. The reaction was performed in an a erated and thermostated simple stirred glass vessel in an aqueous (20% v/v) /organic (80% v/v) reaction medium. This allowed in situ product recovery p reventing substrate and product inhibition of the catalyst as well as decay of the labile product 2,3-dihydroxybiphenyl. Enzymatic regeneration of red uced nicotinamide cofactors was achieved using the formate/formate dehydrog enase system. We obtained volumetric productivities of up to 0.45 g l(-1) h (-1). No significant decrease of productivity was observed within 7 h and m ore. Product purification (purity 92%) was achieved using solid phase extra ction with aluminum oxide followed by crystallization as a polishing step ( purity > 99%). To our knowledge, these results show for the first time the perspectives of integrated enzyme and cofactor regeneration-based biocatalytic processes i n organic/aqueous emulsions, coupled with in situ product recovery. (C) 200 1 Elsevier Science B.V. All rights reserved.