4-Acetoxy-azacyclobutanone　(4AA)　is　a　highly　demanded　chemical　compound　used　in　the　production　of　Penem　and　Carbapenem　antibiotics.　However,　its　synthesis　is　constrained　by　the　preparation　of　methyl　(2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutanoate　[(2S,3R)-BHME].　In　light　of　stringent　environmental　regulations,　there　is　an　urgent　need　to　develop　an　effective　enzymatic　method　using　2-benzoylaminomethyl-3-oxy-butyrate　methyl　ester　(BOME)　as　the　substrate.　This　study　mined　a　carbonyl　reductase　AxSDR　from　Algoriella　xinjiangensis,　which　asymmetrically　reduces　BOME　to　(2S,3R)-BHME　using　isopropanol　(IPA)　as　a　cosubstrate.　The　mechanisms　underlying　the　high　stereoselectivity,　substrate　selectivity,　and　limited　activity　of　AxSDR　toward　BOME　were　analyzed　using　computer-aided　technology.　Based　on　these　analyses,　AxSDR　was　rationally　designed,　leading　to　the　identification　of　a　triple-point　variant,　G94T/H145Y/Y188L　(Mu3),　which　exhibited　a　2-fold　increase　in　catalytic　efficiency.　After　condition　optimization,　Mu3　cells　were　able　to　convert　300　mM　BOME,　achieving　a　space-time　yield　of　15.1　g/L/h.　The　sustainability　of　the　(2S,3R)-BHME　biosynthesis　method　was　further　enhanced　by　immobilizing　Mu3　on　IPA-tolerant　amino　resin.　The　space-time　yield　of　the　immobilized　enzyme　Mu3-imm　increased　to　75.3　g/(L·h)　and　was　maintained　at　50.2　g/(L·h)　after　100　uses.　These　results　demonstrate　the　significant　industrial　application　potential　of　Mu3-imm　in　reducing　the　costs　and　environmental　risks　associated　with　the　preparation　of　(2S,3R)-BHME　and　its　downstream　products　such　as　4-AA,　Penem,　and　Carbapenem　antibiotics.　©　2025　American　Chemical　Society.