费氏丙酸杆菌谢氏亚种和植物乳杆菌亚油酸异构酶结构及酶学性质

Studies on the Characteristics of Linoleate Isomerase from P.freudenreichii ssp.shermanii and L.plantarum

【目的】对来源于植物乳杆菌(Lactobacillu plantarum)和费氏丙酸杆菌谢氏亚种(Propionibacteriu freudenreichii ssp.shermanii)亚油酸异构酶酶学特性开展研究,探讨不同来源的亚油酸异构酶酶学性质存在的差异。【方法】通过酶反应动力学方法及电泳技术测定了酶的最适反应条件、米氏常数、酶的分子量,并在此基础上,采用高效液相色谱(HPLC)与电喷雾离子化/质谱(ESI-MS)相结合的方法测定了L.plantarum和P.freudenreichii ssp.shermanii亚油酸异构酶的氨基酸序列。【结果】P.freudenreichii ssp.shermanii亚油酸异构酶的最适pH为8.0,最适反应温度是30℃,Km=20.53μmol·L-1,Vmax=0.44μg·ml-1·min-1;L.plantarum亚油酸异构酶的最适pH为7.5,最适反应温度是为50℃,Km=17.85μmol·L-1,Vmax=0.73μg.ml-1·min-1。【结论】来源于L.plantarum和P.freudenreichii ssp.shermanii亚油酸异构酶的全氨基酸序列不同,但两者具有一定的同源性,且两者酶学特性也有差异。

【Objective】 The attributes of linoleate isomerases from both Propionibacteriu freudenreichii ssp.shermanii and Lactobacillu plantarum were studied to understand the different characters of linoleate isomerase originated from different strains.【Method】 Dynamic and characteristic parameter such as Michaelis constant,molecular weight of linoleate isomerase with different origins were determined using the method of enzyme activity analysis and electrophoresis.The peptide mass fingerprints produced by P.freudenreichii ssp.shermanii and L.plantarum linoleate isomerase were studied by HPLC-ESI-MS system.【Result】 The optimum pH for P.freudenreichii ssp.shermanii linoleate isomerase was 8.0 and the optimum temperature was about 30℃.The Km and Vmax of linoleic acid was 20.53 μmol·L^-1 and 0.44 μg·ml-^1·min-1.The optimum pH for L.plantarum linoleate isomerase was 7.5 and the optimum temperature was about 50℃.The Km and Vmax of linoleic acid was 17.85 μmol·L^-1 and 0.73 μ·ml-^1·min-1,respectively.Molecular structure of linoleate isomerase produced by P.freudenreichii ssp.shermanii and L.plantarum showed some extent homogeny on sequences.【Conclusion】 The linoleate isomerase originated from the two strains has different attributes.The sequences of both linoleate isomerases have some extent homogeny and the characteristic parameters of experimental linoleate isomerase are different from those of reported.