Cyclohexylamine　oxidase　CHAOCCH12-C2　was　previously　discovered　and　utilized　in　the　chemo-enzymatic　synthesis　of　(S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline　((S)-1　a),　the　key　precursor　for　the　industrial　production　of　antitussive　dextromethorphan.　Herein,　structure-guided　semi-rational　engineering　and　random　mutagenesis　were　applied　to　CHAOCCH12-C2,　resulting　in　an　evolved　variant　WXF-FM　which　possessed　five　point　mutations:　H68Q/E198G/L200V/I201L/V209S　and　displayed　＞15-fold　higher　kcat　and　improved　stereoselectivity　towards　(R)-1　a　relative　to　the　WT　enzyme.　WXF-FM-catalyzed　deracemization　of　200　mM　of　rac-1　a　was　achieved　at　gram-scale　under　Turner＇s　deracemization　conditions,　affording　(S)-1　a　in　76　%　isolated　yield　with　97　%　ee,　demonstrating　the　effectiveness　and　great　potential　of　this　enzyme　in　the　practical,　green　synthesis　of　dextromethorphan.　Two　bulky　analogues　of　(S)-1　a　were　also　afforded　with　much　higher　optical　purities　in　WXF-FM-catalyzed　reactions　than　those　obtained　in　WT　enzyme-catalyzed　reactions.　Through　conducting　complete　deconvoluting　experiments,　presence　of　strong　cooperative　effect　was　revealed,　and　the　mutational　effect　of　H68Q　on　(R)-1　a　was　suggested　to　be　possibly　applicable　to　related　cyclohexylamine　oxidases.　Molecular　dynamics　(MD)　simulations　indicated　the　enlargement　of　binding　and　entrance　cavities,　and　the　formation　of　a　new　hydrogen-bonding　between　FAD　and　(R)-1　a　both　likely　contributed　to　the　enhanced　catalytic　activity.　©　2022　Wiley-VCH　GmbH.