Lactobacillus brevis protoplasts were fused by UV-heating inactivated parents,and the influencing factors of protoplast formation,regeneration and fusion were investigated.Further,the production capability of thymidine phosphorylase and hereditary stability of Lactobacillus brevis were characterized.The results indicated that Lactobacillus brevis was cultured in the fermentation medium containing 0.6% glycine and harvested at the logarithmic growth phase,which was hydrolyzed through adding 2 mg / mL lysozyme in 37℃ water bath for 2 h,protoplasts formation and regeneration rate reached individually 95% and 48%.The protoplasts were irradiated by 20 W ultraviolet light at distance 20 cm for 50 min,or heated at 60℃ for 60 min,the inactivation rates of which were almost 100%.Inactivated parental protoplasts were fused with 40% PEG6000 at 30℃ for 10 min,and thymidine phosphorylase productivity of fusant F15 after the 5 generation still could reach 1.500 U / mg wet bacterium,which was increased by 50% than that parents.