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 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.