Mutant 3-hydroxybutyrate dehydrogenase from alcaligenes faecalis as well as methods and uses involving the same

A mutant 3-hydroxybutyrate dehydrogenase (3-HBDH) from Alcaligenes faecalis with improved performance relative to the wild-type 3-HBDH, wherein the mutant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:

• 1 (3-HBDH from Alcaligenes faecalis;
• wild-type 3-HBDH) or the amino acid sequence of SEQ ID NO:
• 2 (extended core sequence of wild-type 3-HBDH) and
• wherein the mutant has at least three amino acid substitutions relative to the wild-type 3-HBDH, wherein the amino acid at the position corresponding to
• position 253 of SEQ ID NO:
• 1 is substituted with Ile (253Ile), Ala (253Ala) or Cys (253Cys),
• position 233 of SEQ ID NO:
• 1 is substituted with Ser (233Ser), Ile (233Ile), Ala (233Ala), Pro (233Pro), Arg (233Arg), Thr (233Thr), Lys (233Lys), or Cys (233Cys), and
• position 234 of SEQ ID NO:
• 1 is substituted with Thr (234Thr), wherein improved performance relative to the wild-type 3-HBDH is
• increased thermal stability relative to the wild-type 3-HBDH;
• and/or
• increased substrate affinity relative to the wild-type 3-HBDH;
• and/or
• increased cofactor affinity.