变溶菌素(Mutanolysin)酶解单核细胞增多性李斯特菌细胞壁条件的优化

Hydrolysis conditions optimization of Mutanolysin on Listeria monocytogenes

为了最大效率提取单核细胞增多性李斯特菌LB90SB2细胞壁表面蛋白,本研究对变溶菌素浓度和酶解时间进行了优化。选择20、60μg/m L的变溶菌素浓度进行30 min-4 h,37℃酶解以及37和4℃低温联合酶解,通过测定酶解前后OD600和CFU,判断细胞膜完整性和计算原生质体形成率,筛选最佳酶解浓度和酶解时间。在最佳酶浓度的条件下,测定不同酶解时间细胞壁蛋白提取物浓度。结果表明:在酶解30 min-4 h,原生质体形成率从0提高至91.00%,细胞壁蛋白浓度从0.272增加至1.735 mg/m L。△OD600在1.86%-14.50%变动,但△OD600的统计学处理差异不显著。60μg/m L变溶菌素作用4 h的原生质体形成率达到91.00%,远高于20μg/m L变溶菌素作用4 h的78%。最终确定变溶菌素浓度为60μg/m L,酶解时间4 h为酶解LM90SB2细胞壁的最佳条件。本研究为下一步研究细胞壁表面蛋白奠定基础。

In order to extract cell wall proteins of Listeria monocytogenes isolate LM90SB2, the concentration and hydrolysis time of mutanolysin were optimized. 20 μg/m L and 60 μg/m L mutanolysin was used to hydrolyse LM90SB2 at 37 ℃ or 37 ℃ and4 ℃ united action for 30 min-4 h, OD600 and CFU was measured before and after hydrolysis to judge membrane integrity and protoplast formation rate. Under optimal mutanolysin,concentration of cell wall proteins was measured. The results showed that the protoplast formation rate increased from 0 to 91.00% with hydrolysis time from 30 min to 4 h, proteins concentration were increased from 0.272 mg/m L to 1.735 mg/m L. △OD600was changed from 1.86% to 14.50% with hydrolysis time from 30 min to4 h, there was no significant difference by analysis of statistics. 91% of protoplast formation rate of LM90SB2 hydrolysed by 60μg/m L mutanolysin at 37 ℃ for 4 h was higher than 78% of protoplast formation rate hydrolysed by 20 μg/m L mutanolysin at the same condition. So 60 μg/m L mutanolysin at 37 ℃ for 4 h was the best hydrolysis condition for LM90SB2. This study provides basis for the research on cell wall surface proteins.