Regioselective hydroxylation of isophorone

A biocatalytic process for the conversion of α-isophorone into ketoisophorone (KIP) comprising the enzymatic steps of:

• (a) converting α-isophorone into 4-hydroxy-α-isophorone (HIP) via catalytic action of a P450 monooxygenase wherein
• 1) at least 80% of α-isophorone is converted into HIP;
• 2) the P450 monooxygenase comprises
• i. a first polypeptide having at least 90% identity to SEQ ID NO:1 or
• ii. a second polypeptide having at least 62% identity to SEQ ID NO:3, said second polypeptide comprising an F at position 97 according to SEQ ID NO:3 and one or more substitutions at positions 88, 245, and 248 according to SEQ ID NO:3, wherein said one or more substitutions comprise F88W, L245X, and V248X, wherein “X” is any amino acid;
• and wherein the total turnover number of the second polypeptide is increased by at least 2-fold compared to a reference P450 monooxygenase according to SEQ ID NO:
• 3;
• and
• (b) converting the HIP from (a) into KIP via catalytic action of an enzyme having alcohol dehydrogenase or carbonyl reductase activity, wherein at least 40% of HIP is converted into KIP.