Biocatalytic　technology　has　emerged　as　a　powerful　tool　for　synthesizing　functional　sugars　with　a　wide　range　of　physiological　functions.　D-lyxose　isomerase　is　an　important　aldose–ketose　isomerase　used　for　synthesizing　functional　sugars　such　as　L-ribose　and　D-mannose;　however,　at　present,　only　a　few　D-lyxose　isomerases　have　been　studied.　In　this　study,　a　metagenomic　approach　was　employed　to　mine　novel　D-lyxose　isomerases　from　the　goat　rumen　microbiome.　Eleven　full-length　D-lyxose　isomerase　genes　were　identified　using　sequence　alignment　and　phylogenetic　analysis.　All　11　genes　were　classified　as　Group　Ⅱ　D-lyxose　isomerases　and　display　low　similarity　to　previously　characterized　D-lyxose　isomerases.　Five　of　the　genes　were　selected　for　heterologous　expression　in　Escherichia　coli,　and　all　were　expressed　successfully　and　had　detectable　enzyme　activity.　Further　characterization　revealed　that　optimum　temperatures　for　the　five　enzymes　range　from　45　°C　to　60　°C,　and　the　optimum　pH　range　is　7.0–8.0,　with　high　relative　residual　activities　under　weakly　acidic　conditions.　The　recombinant　enzymes　have　a　broad　substrate　spectrum　and　are　active　against　L-ribose,　L-ribulose,　D-fructose,　and　D-mannose,　in　addition　to　exhibiting　the　highest　activity　against　D-lyxose.　Among　the　five　enzymes,　GR-LI2　displays　the　highest　activity　toward　L-ribulose,　and　GR-LI5　displays　the　highest　activity　against　fructose,　suggesting　that　these　two　enzymes　have　significant　potential　for　L-ribose　and　D-mannose　synthesis,　respectively.　©　2024　Elsevier　Ltd