Abstract: Thymidine phosphorylase plays a critical role in synthesis of nucleosides. In this study，fermentation medium of Lactobacillus brevis for producing thymidine phosphorylase was optimized. Firstly，three factors of fermentation time (P=0.032)，inoculum size (P=0.037) and glucose concentrations (P=0.022) significantly affected thymidine phosphorylase were identified by Plackett-Burman experiment. Then，the three factors were further optimized through steepest ascent path approaching the maximum response region and response surface central composite design (CCD). The optimal fermentation medium were as follows ：initial pH 8.0，glucose 18 g/L，yeast extract 15 g/L，NaCl 7.5 g/L，peptone 10 g/L，thymidine 15mmol/L. Culture conditions were as follows ：rotating speed 110r/min，temperature 38 ℃，fermentation time 10.57 h，inoculum size 1.54%. Under optimum conditions，the capacity of Lactobacillus brevis to produce thymidine phosphorylase was greatly improved，thymidine phosphorylase activity reached 1.172 U/mg wet bacteria which was increased by 2.93 times more than that before optimization. Protein gel electrophoresis showed that thymidine phosphorylase content per gram wet bacteria with optimized fermentation medium was obviously higher than that before optimization.

Key words: Lactobacillus brevis, thymidine phosphorylase, response surface methodology, fermentation medium

摘要： 胸苷磷酸化酶在核苷类物质合成中具有重要作用，本研究以短乳杆菌为胸苷磷酸化酶生产菌种，对短乳杆菌产胸苷磷酸化酶发酵培养基进行优化。首先通过Plackett-Burman设计筛选出影响短乳杆菌产胸苷磷酸化酶的3个较为重要因素：发酵时间（P=0.030）、接种量（P=0.033）和葡萄糖浓度（P=0.019）。在此基础上采用最陡爬坡路径逼近最大响应区域，并利用响应面中心组合设计对影响显著因素进行优化，得到最适培养基组成成分和培养条件为：发酵初始pH 8.0，葡萄糖18 g/L，酵母膏15 g/L，NaCl 7.5 g/L，蛋白胨10 g/L，胸苷15 mmol/L，摇床转速110 r/min，发酵温度38 ℃，发酵时间10.57 h，接种量1.54%。在此优化条件下，短乳杆菌产胸苷磷酸化酶能力得到了很大提高，短乳杆菌胸苷磷酸化酶活从0.400 U/mg湿菌体提高到1.172 U/mg湿菌体，比优化前提高了2.93倍。蛋白质凝胶电泳分析显示经优化后每克湿菌体胸苷磷酸化酶的含量明显高于优化前。

关键词: 短乳杆菌, 胸苷磷酸化酶, 响应面法, 发酵培养基