产胸苷磷酸化酶短乳杆菌融合菌株的构建及其特性

Construction and characterization of Lactobacillus brevis fusion strains producing thymidine phosphorylase

利用紫外加热灭活双亲原生质体技术对短乳杆菌原生质体进行融合,考察短乳杆菌原生质体制备、再生及融合的影响因素,并对短乳杆菌融合子产酶能力和遗传稳定性进行了研究。结果表明:短乳杆菌在含0.6%甘氨酸发酵培养液培养至对数生长期,2 mg/mL溶菌酶于37℃恒温水浴酶解脱壁2 h,原生质体制备率和再生率可达95%和48%;20 W紫外灯距离20 cm照射50 min,60℃处理短乳杆菌原生质体60 min,原生质体的灭活率几乎为100%;将双亲灭活的原生质体用40%PEG6000,30℃恒温融合10 min,筛选融合子F15并进行5次传代测试其胸苷磷酸化酶活均在1.500 U/mg湿菌体,较亲本酶活提高了50%。

Lactobacillus brevis protoplasts were fused by UV-heating inactivated parents, and the influencing fac- tors 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 Lactobacil- lus 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 dis- tance 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.