In　this　study,　we　attempted　to　find　new　and　efficient　microbial　enzymes　for　producing　rare　sugars.　A　ribose-5-phosphate　isomerase　B　(OsRpiB)　was　cloned,　overexpressed,　and　preliminarily　purified　successfully　from　a　newly　screened　Ochrobactrum　sp.　CSL1,　which　could　catalyze　the　isomerization　reaction　of　rare　sugars.　A　study　of　its　substrate　specificity　showed　that　the　cloned　isomerase　(OsRpiB)　could　effectively　catalyze　the　conversion　of　L-rhamnose　to　L-rhamnulose,　which　was　unconventional　for　RpiB.　The　optimal　reaction　conditions　(50°C,　pH　8.0,　and　1　mM　Ca2+)　were　obtained　to　maximize　the　potential　of　OsRpiB　in　preparing　L-rhamnulose.　The　catalytic　properties　of　OsRpiB,　including　Km,　kcat,　and　catalytic　efficiency　(kcat/Km),　were　determined　as　43.47　mM,　129.4　sec-1,　and　2.98　mM/sec.　The　highest　conversion　rate　of　L-rhamnose　under　the　optimized　conditions　by　OsRpiB　could　reach　26%　after　4.5　h.　To　the　best　of　our　knowledge,　this　is　the　first　successful　attempt　of　the　novel　biotransformation　of　L-rhamnose　to　L-rhamnulose　by　OsRpiB　biocatalysis.　©　2018　by　The　Korean　Society　for　Microbiology　and　Biotechnology.