A strain producing β-galactosidase with transgalactosylation activity was screened from the soil.Mor-phological,physiological and biochemical properties as well as 16S rDNA sequence analysis were carried out.Based on taxonomy results,the strain was identified as Klebsiella pneumoniae 285.Fermentation conditions were optimized by single factor and orthogonal experiments.The enzyme activity reached 7.21 U/mL after cultured for 8 h at 30 ℃ when liquid volume in 250 mL flask was 40 mL,inoculation time was 6 h,initial pH was 7.0 and inoculation volume was 3%.When substrate concentrations were 40%(w/v) lactose and 20%(w/v) fructose(pH 7.0) at 3.5 U/mL enzyme activity,yield of lactulose reached approximately 20.8 g/L at 50 ℃ for 6 h with a productivity of 3.47 g/(L.h),as analyzed by HPLC.