Non-catalytic　sites　are　commonly　present　in　many　enzymes,　but　how　to　classify　and　modulate　such　sites　for　activity　improvement　is　not　well-explored.　Herein　a　non-catalytic　cavity　in　the　3-ketosteroid-Δ1-dehydrogenase　(Δ1-KstD)　from　Mycobacterium　smegmatis　(MsKstD1),　which　could　competitively　bind　to　substrate　to　form　non-catalytic　complex,　was　predicted　next　to　substrate　tunnel　by　the　CAVER/molecular　dynamics.　Based　on　that,　a　rational　design　was　performed　by　Focused　Site-directed　Iterative　Saturation　Mutagenesis　(FSISM)　for　blocking　the　non-catalytic　cavity　to　enhance　substrate　enter　active　site　to　improve　the　Δ1-dehydrogenation　activity　of　MsKstD1.　We　obtained　the　best　quadruple　mutant　(H132M\L113F\V419W\M51L)　with　10-fold　higher　specific　activity　toward　hydrocortisone　than　that　of　wild　type　enzyme.　The　Δ1-dehydrogenation　space　time　yield　toward　hydrocortisone　reached　36.0　g/L/h　to　produce　prednisolone.　This　work　displays　how　to　combine　focused　mutagenesis　with　computational　analysis　to　effectively　identify　and　modify　non-catalytic　cavity　to　enhance　enzyme　activity　for　efficient　production　of　Δ1-3-ketosteroid.　©　2022