A. Solikhin, A. Z. Mustopa, S. Suharsono, W. Putranto
{"title":"干酪乳杆菌胞外天冬氨酸蛋白酶的部分纯化、鉴定及在生产具有抗菌和抗氧化活性肽中的应用","authors":"A. Solikhin, A. Z. Mustopa, S. Suharsono, W. Putranto","doi":"10.14203/ANN.BOGOR.2018.V22.N2.47-56","DOIUrl":null,"url":null,"abstract":" Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.","PeriodicalId":41037,"journal":{"name":"Annales Bogorienses-Journal of Tropical General Botany","volume":"14 10 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity\",\"authors\":\"A. Solikhin, A. Z. Mustopa, S. Suharsono, W. Putranto\",\"doi\":\"10.14203/ANN.BOGOR.2018.V22.N2.47-56\",\"DOIUrl\":null,\"url\":null,\"abstract\":\" Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.\",\"PeriodicalId\":41037,\"journal\":{\"name\":\"Annales Bogorienses-Journal of Tropical General Botany\",\"volume\":\"14 10 1\",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2018-12-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Annales Bogorienses-Journal of Tropical General Botany\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.14203/ANN.BOGOR.2018.V22.N2.47-56\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Annales Bogorienses-Journal of Tropical General Botany","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.14203/ANN.BOGOR.2018.V22.N2.47-56","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 1
摘要
采用sephadex G-50层析凝胶过滤法对干酪乳杆菌wsp衍生的天冬氨酸蛋白酶进行了纯化。比活性提高22.81倍(51.5 U/mg),最终产率为1.9%。酶谱分析表明,纯化酶的分子量为37 kDa,具有溶胶活性。以酪蛋白为底物,在40℃和pH 6条件下酶活性最佳。酶活性被pepstatin A (0.5 mM和1 mM)显著抑制,证实该酶是一组天冬氨酸蛋白酶,而其他抑制剂如EDTA、PMSF和碘乙酸对酶活性无抑制作用。金属离子的加入降低了酶的活性,表明该蛋白水解酶是金属离子依赖性的。变性剂如滴滴涕倾向于增加溶酪蛋白活性。此外,该酶还能从脱脂牛奶中生成8kda、10kda和15kda的新肽。这些肽具有抗菌和抗氧化剂的潜力。
Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity
Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.