Preparative application of 2-hydroxybiphenyl 3-monooxygenase with enzymatic cofactor regeneration in organic-aqueous reaction media

Abstract

2-Hydroxybiphenyl 3-monooxygenase (HbpA, E.C. 1.14.13.44) was partially purified on a gram scale from recombinant E. coli JM101 by expanded bed adsorption (EBA) chromatography. In order to develop an effective in vitro biotransformation system using isolated HbpA as catalyst, its stability and activity in the presence of different apolar and polar organic solvents was investigated. HbpA showed good stability in 80% (v/v) decanol and 10% (v/v) methanol. Since the reaction catalyzed by HbpA is cofactor dependent, enzymatic NADH cofactor regeneration by yeast alcohol dehydrogenase (YADH) was tested in the presence of 10% (v/v) methanol used both as a cosolvent and substrate for YADH. In an in vitro biotransformation on a 25ml scale 5.4mM of 2,2′-dihydroxybiphenyl was hydroxylated to 2,2′,3-trihydroxybiphenyl in 2.5h and 2,2′,3,3′-tetrahydroxybiphenyl in 23h at a mean volumetric productivity of 0.43g/(lh) and 0.05g/(lh), respectively. Additionally, HbpA and formate dehydrogenase (FDH) were applied in 80% (v/v) decanol to hydroxylate 2-hydroxybiphenyl with a productivity of 0.46g/(lh) and a total turnover number of 503. In 10% (v/v) aqueous methanol the total turnover number of 30 and enzyme stability for at least 60h were shown. This demonstrated the feasibility of using isolated HbpA for syntheses in the presence of polar as well as apolar solvents with enzymatic regeneration of reduced cofactors.