破壁方法对嗜热链球菌SP1.1胞内乳糖代谢关键酶活性的影响及其条件优化

Effect of Different Cell Wall Disruption Methods on Key Enzyme Activities Involved in Intracellular Lactose Metabolism in Streptococcus thermophilus SP1.1 and Optimization of Lysozyme Digestion Conditions

通过超声波破壁法、溶菌酶破壁法、反复冻融法和机械破壁法4种不同破壁方式对嗜热链球菌SP1.1进行破壁处理,对菌体的破壁率、提取的β-半乳糖苷酶和乳酸脱氢酶两种胞内酶的活力进行分析比较,以确定适合于嗜热链球菌细胞破壁方法及其最佳条件。结果表明:这4种菌体细胞破壁效果存在很大差异,其中经溶菌酶处理胞内酶提取效果最好,破壁率可达99.87%,β-半乳糖苷酶酶活力为0.387U,乳酸脱氢酶酶活力为2.375U,与其他3种方法的破壁效果差异极显著(P<0.01)。优化溶菌酶破壁条件,当处理8mL菌浓度为1.3×109CFU/mL的样品时,确定处理最佳温度为37℃,时间为30min,酶(20000U/mL)用量为1mL,此时破壁率可达99.99%,β-半乳糖苷酶酶活力为0.429U,乳酸脱氢酶酶活力为2.431U。

Four different cell wall disruption methods,such as ultrasonic treatment,lysozyme digestion,repeated freezing and thawing and mechanical disruption were respectively used to treat Streptococcus thermophilus SP1.1 cells,and the resultant cell wall disruption rates and the activities of the intracellular enzymesβ-galactosidase and lactate dehydrogenase in centrifuge supernatant obtained after cell wall disruption were compared with the aim of screening the most appropriate method for cell wall disruption of Streptococcus thermophilus SP1.1.These four methods exhibited a large difference in their effectiveness,and lysozyme digestion provided optimal extraction of β-galactosidase（0.387 U） and lactate dehydrogenase（2.375 U）,yielded a cell wall disruption rate of 99.87%,and was significantly different from three others（P 0.01）.The optimum cell wall disruption conditions for 8 mL of 1.3 × 109 CFU/mL cell suspension by lysozyme（20000 U/mL） digestion were temperature of 37 ℃ and treatment time of 30 min and enzyme loading of 1 mL.A cell wall disruption of 99.99%,a β-galactosidase activity of 0.429 U and a lactate dehydrogenase activity of 2.431 U were obtained under these conditions.