Hydroxylation is very important in the industrial production of fine chemicals. In spite of its ubiquity, however, hydroxylation has been one of the least-well understood of all enzymatic reactions, because there are many enzyme candidates which perform similar reactions and are often membrane proteins. We were interested in regioselective hydroxylation of chiral compounds with double chiral centers, terminal hydroxylation of long chain fatty acids and aromatic ortho-hydroxylation of daidzein(isoflavonoid), since chemical catalysis for the synthesis of these molecules require several steps of reaction and/or results very low yield, suggesting good advantages of using enzymes over chemical catalysts.

Chiral intermediates with double chiral centers are good candidates for dehydrogenase and carbonyl reductase. In combination with various enzymes such as transaminase and decarboxylase, we could generate valuable chiral amino alcohols for drug development in pharmaceutical industry.

In the case of ortho-specific hydroxylation of daidzein(isoflavonoid), P450 reaction was not fast enough to make an industrial scale biotransformation. Therefore, other means using tyrosinase was serendipitously found.  Tyrosinases (Ty) are type III copper containing protein catalyzing the two-step consecutive hydroxylation of monophenolic compounds (monophenols); ortho-hydroxylation of monophenols to arene cis-diols (catehols) and the subsequent oxidation to produce dioxygenated quinolic compounds (o-quinones, precursors for melanin formation).  

Here, increasing the ratio of monophenolase to catecholase activity (k1/k2) of Ty reaction on monophenols and efficient suppression of melanin formation are the key issues to achieve high yields of mono hydroxylation of monophenolic compounds. We could systematically control Ty reaction for selective production of 3′-ODI from daidzein, efficiently suppressing the melanin formation.

P450 is a very unique heme-containing mono-oxygenase enzyme showing diverse reaction mechanisms such as hydroxylation, desulfoxidation, dealkylation, deamination, etc.  Bacterial P450 reaction often requires redox partners and electron transfer donor cofactors such as NAD(P)H, resulting very low reaction rate and yield. Therefore, enhancement of specific activity, balance of electron acceptor (NAD(P)+) and donor (NAD(P)H), improvement of electron transfer efficiency, substrate/product transport are the key issues to make a successful biotransformation.

Using above three examples as model systems, we would like to explain how “enzyme screening, protein engineering and systems and synthetic biological approaches” work for this enzyme reaction, and what kinds of strategies are desirable to develop their industrial scale biotransformations.

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