{"title":"利用肽聚糖结合基序LysM在乳杆菌细胞表面固定化β-半乳糖苷酶","authors":"Mai-Lan Pham, Anh-Minh Tran, Suwapat Kittibunchakul, Tien-Thanh Nguyen, G. Mathiesen, Thu-Ha Nguyen","doi":"10.3390/catal9050443","DOIUrl":null,"url":null,"abstract":"Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WCFS1 to display two different lactobacillal β-galactosidases, the heterodimeric LacLM-type from Lactobacillus reuteri and the homodimeric LacZ-type from Lactobacillus delbrueckii subsp. bulgaricus, on the cell surface of different Lactobacillus spp. The β-galactosidases were fused with the LysM domain and the fusion proteins, LysM-LacLMLreu and LysM-LacZLbul, were successfully expressed in Escherichia coli and subsequently displayed on the cell surface of L. plantarum WCFS1. β-Galactosidase activities obtained for L. plantarum displaying cells were 179 and 1153 U per g dry cell weight, or the amounts of active surface-anchored β-galactosidase were 0.99 and 4.61 mg per g dry cell weight for LysM-LacLMLreu and LysM-LacZLbul, respectively. LysM-LacZLbul was also displayed on the cell surface of other Lactobacillus spp. including L. delbrueckii subsp. bulgaricus, L. casei and L. helveticus, however L. plantarum is shown to be the best among Lactobacillus spp. tested for surface display of fusion LysM-LacZLbul, both with respect to the immobilization yield as well as the amount of active surface-anchored enzyme. The immobilized fusion LysM-β-galactosidases are catalytically efficient and can be reused for several repeated rounds of lactose conversion. This approach, with the β-galactosidases being displayed on the cell surface of non-genetically modified food-grade organisms, shows potential for applications of these immobilized enzymes in the synthesis of prebiotic galacto-oligosaccharides.","PeriodicalId":221236,"journal":{"name":"Catalysts (Basel, Switzerland)","volume":"8 1","pages":"0"},"PeriodicalIF":0.0000,"publicationDate":"2019-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"9","resultStr":"{\"title\":\"Immobilization of β-Galactosidases on the Lactobacillus Cell Surface Using the Peptidoglycan-Binding Motif LysM\",\"authors\":\"Mai-Lan Pham, Anh-Minh Tran, Suwapat Kittibunchakul, Tien-Thanh Nguyen, G. Mathiesen, Thu-Ha Nguyen\",\"doi\":\"10.3390/catal9050443\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WCFS1 to display two different lactobacillal β-galactosidases, the heterodimeric LacLM-type from Lactobacillus reuteri and the homodimeric LacZ-type from Lactobacillus delbrueckii subsp. bulgaricus, on the cell surface of different Lactobacillus spp. The β-galactosidases were fused with the LysM domain and the fusion proteins, LysM-LacLMLreu and LysM-LacZLbul, were successfully expressed in Escherichia coli and subsequently displayed on the cell surface of L. plantarum WCFS1. β-Galactosidase activities obtained for L. plantarum displaying cells were 179 and 1153 U per g dry cell weight, or the amounts of active surface-anchored β-galactosidase were 0.99 and 4.61 mg per g dry cell weight for LysM-LacLMLreu and LysM-LacZLbul, respectively. LysM-LacZLbul was also displayed on the cell surface of other Lactobacillus spp. including L. delbrueckii subsp. bulgaricus, L. casei and L. helveticus, however L. plantarum is shown to be the best among Lactobacillus spp. tested for surface display of fusion LysM-LacZLbul, both with respect to the immobilization yield as well as the amount of active surface-anchored enzyme. The immobilized fusion LysM-β-galactosidases are catalytically efficient and can be reused for several repeated rounds of lactose conversion. This approach, with the β-galactosidases being displayed on the cell surface of non-genetically modified food-grade organisms, shows potential for applications of these immobilized enzymes in the synthesis of prebiotic galacto-oligosaccharides.\",\"PeriodicalId\":221236,\"journal\":{\"name\":\"Catalysts (Basel, Switzerland)\",\"volume\":\"8 1\",\"pages\":\"0\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-05-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"9\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Catalysts (Basel, Switzerland)\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3390/catal9050443\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Catalysts (Basel, Switzerland)","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/catal9050443","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 9
摘要
Lysin motif (LysM)结构域存在于许多细菌肽聚糖水解酶中。它们可以与肽聚糖非共价结合,并被用于在细菌细胞表面显示异源蛋白。在这项研究中,我们旨在利用植物乳杆菌WCFS1的胞外转糖基酶Lp_3014衍生的LysM结构域来显示两种不同的乳酸杆菌β-半乳糖苷酶,即来自罗伊氏乳杆菌的异二聚体laclm型和来自德尔布鲁氏乳杆菌亚种的同二聚体lacz型。将β-半乳糖苷酶与LysM结构域融合,融合蛋白LysM- laclmlreu和LysM- laczlbul在大肠杆菌中成功表达,并在L. plantarum WCFS1细胞表面显示。结果表明,LysM-LacLMLreu和LysM-LacZLbul的β-半乳糖苷酶活性分别为179和1153 U / g干细胞重,表面锚定的β-半乳糖苷酶活性分别为0.99和4.61 mg / g干细胞重。LysM-LacZLbul也在其他乳杆菌属(包括L. delbrueckii亚种)的细胞表面显示。然而,在融合LysM-LacZLbul的表面展示试验中,无论是固定化产量还是活性表面锚定酶的数量,植物乳杆菌都被证明是最好的。固定化融合LysM-β-半乳糖苷酶催化效率高,可重复使用多次乳糖转化。随着β-半乳糖苷酶在非转基因食品级生物的细胞表面显示,这种方法显示了这些固定化酶在合成益生元半乳糖低聚糖方面的应用潜力。
Immobilization of β-Galactosidases on the Lactobacillus Cell Surface Using the Peptidoglycan-Binding Motif LysM
Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WCFS1 to display two different lactobacillal β-galactosidases, the heterodimeric LacLM-type from Lactobacillus reuteri and the homodimeric LacZ-type from Lactobacillus delbrueckii subsp. bulgaricus, on the cell surface of different Lactobacillus spp. The β-galactosidases were fused with the LysM domain and the fusion proteins, LysM-LacLMLreu and LysM-LacZLbul, were successfully expressed in Escherichia coli and subsequently displayed on the cell surface of L. plantarum WCFS1. β-Galactosidase activities obtained for L. plantarum displaying cells were 179 and 1153 U per g dry cell weight, or the amounts of active surface-anchored β-galactosidase were 0.99 and 4.61 mg per g dry cell weight for LysM-LacLMLreu and LysM-LacZLbul, respectively. LysM-LacZLbul was also displayed on the cell surface of other Lactobacillus spp. including L. delbrueckii subsp. bulgaricus, L. casei and L. helveticus, however L. plantarum is shown to be the best among Lactobacillus spp. tested for surface display of fusion LysM-LacZLbul, both with respect to the immobilization yield as well as the amount of active surface-anchored enzyme. The immobilized fusion LysM-β-galactosidases are catalytically efficient and can be reused for several repeated rounds of lactose conversion. This approach, with the β-galactosidases being displayed on the cell surface of non-genetically modified food-grade organisms, shows potential for applications of these immobilized enzymes in the synthesis of prebiotic galacto-oligosaccharides.