Ribose-5-phosphate　isomerase　B　is　of　great　importance　for　biocatalysis　and　biosynthesis,　but　the　multifunctional　residues　in　active　sites　hinder　the　research　efforts.　This　study　employed　rational　design　strategies　to　locate　the　key　residues　of　RpiB　from　Ochrobactrum　sp.　CSL1　(OsRpiB).　A　single-mutant　S9T　of　a　noncontact　residue　showed　80%　activity　improvement　toward　D-allose.　A　double-mutant　S98H/S134H　further　increased　the　activity　to　3.6-fold.　The　mutations　were　analyzed　by　kinetics　and　molecular　dynamics　analyses,　indicating　that　S9T　might　enhance　the　substrate　binding　and　catalysis　by　inducing　a　steric　effect,　and　S98H/S134H　could　strengthen　both　ring　opening　and　binding　of　D-allose.　Though　S98H/S134H　showed　low　temperature　stability,　its　potential　was　explored　by　isomerizing　D-allose　to　D-psicose　with　higher　conversion　and　in　less　reaction　time.　The　findings　of　this　study　were　beneficial　for　illustrating　the　complex　functions　of　key　residues　in　RpiBs　and　applying　OsRpiB　in　preparing　rare　sugars.