Ribose-5-phosphate　isomerase　A　(RpiA)　is　of　great　importance　in　biochemistry　research,　however　its　application　in　biotechnology　has　not　been　fully　explored.　In　this　study　the　activity　of　RpiA　from　Ochrobactrum　sp.　CSL1　(OsRpiA)　towards　D-allose　was　engineered　based　on　sequential　and　structural　analyses.　Strategies　of　alanine　scanning,　rational　design　and　saturated　mutagenesis　were　employed　to　create　three　mutant　libraries.　A　single　mutant　of　K124A　showed　a　45　%　activity　improvement　towards　D-allose.　The　reaction　properties　of　the　mutant　were　analyzed,　and　a　shift　of　optimal　pH　and　higher　thermal　stability　at　low　reaction　temperatures　were　identified.　The　conversion　of　D-allose　was　also　improved　by　40　%　using　K124A,　and　higher　activities　on　major　substrates　were　found　in　the　mutant＇s　substrate　scope,　implying　its　application　potential　in　rare　sugar　preparation.　Kinetics　analysis　revealed　that　Km　of　K124A　mutant　decreased　by　12　%　and　the　catalytic　efficiency　increased　by　65　%　towards　D-allose.　Moreover,　molecular　dynamics　simulation　illustrated　the　binding　of　substrate　and　K124A　was　more　stable　than　that　of　the　wild-type.　The　shorter　distance　and　more　relax　bond　angle　between　the　catalytic　residue　of　K124A　and　D-allose　explained　the　activity　improvement　in　detail.　This　study　highlights　the　potential　of　OsRpiA　as　a　biocatalyst　for　rare　sugar　preparation,　and　provides　distinct　evidences　for　its　catalytic　mechanism.