借助形态观察、生理生化指标测定及16S rNDA序列分析,对从土壤中筛选得到的转半糖基β-半乳糖苷酶产生菌进行鉴定,确定其为肺炎克雷伯氏菌(Klebsiella pneumoniae)285。通过单因子试验和正交试验,对该菌株产转半乳糖基β-半乳糖苷酶的培养条件进行优化。优化的发酵条件为:种龄6 h,起始pH值7.0,接种量3%,装液量40 mL/250 mL,30℃下发酵8 h。该培养条件下产酶量达7.21 U/mL。以0.1 mol/L、pH值7.0 K3PO4缓冲溶液配制的40%乳糖、20%果糖(w/v)溶液为底物,调整酶活力至3.5 U/mL,50℃恒温水浴反应6 h,HPLC分析转半乳糖基产物,得乳果糖产量为20.8 g/L,即3.47 g/(L.h)。

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.