L-ribulose,　a　kind　of　high-value　rare　sugar,　could　be　utilized　to　manufacture　L-form　sugars　and　antiviral　drugs,　generally　produced　from　L-arabinose　as　a　substrate.　However,　the　production　of　L-ribulose　from　L-arabinose　is　limited　by　the　equilibrium　ratio　of　the　catalytic　reaction,　hence,　it　is　necessary　to　explore　a　new　biological　enzymatic　method　to　produce　L-ribulose.　Ribose-5-phosphate　isomerase　(Rpi)　is　an　enzyme　that　can　catalyze　the　reversible　isomerization　between　L-ribose　and　L-ribulose,　which　is　of　great　significance　for　the　preparation　of　L-ribulose.　In　order　to　obtain　highly　active　ribose-5-phosphate　isomerase　to　manufacture　L-ribulose,　ribose-5-phosphate　isomerase　A　(OsRpiA)　from　Ochrobactrum　sp.　CSL1　was　engineered　based　on　structural　and　sequence　analyses.　Through　a　rational　design　strategy,　a　triple-mutant　strain　A10T/T32S/G101N　with　160%　activity　was　acquired.　The　enzymatic　properties　of　the　mutant　were　systematically　investigated,　and　the　optimum　conditions　were　characterized　to　achieve　the　maximum　yield　of　L-ribulose.　Kinetic　analysis　clarified　that　the　A10T/T32S/G101N　mutant　had　a　stronger　affinity　for　the　substrate　and　increased　catalytic　efficiency.　Furthermore,　molecular　dynamics　simulations　indicated　that　the　binding　of　the　substrate　to　A10T/T32S/G101N　was　more　stable　than　that　of　wild　type.　The　shorter　distance　between　the　catalytic　residues　of　A10T/T32S/G101N　and　L-ribose　illuminated　the　increased　activity.　Overall,　the　present　study　provided　a　solid　basis　for　demonstrating　the　complex　functions　of　crucial　residues　in　RpiAs　as　well　as　in　rare　sugar　preparation.　©　2022,　The　Author(s),　under　exclusive　licence　to　Springer　Science+Business　Media,　LLC,　part　of　Springer　Nature.