{"title":"乳双歧杆菌 JKL2022 的 CLA 转化亚油酸异构酶的异源表达和多相分析。","authors":"Arxel G Elnar, Yujin Jang, Geun-Bae Kim","doi":"10.1021/acs.jafc.4c05746","DOIUrl":null,"url":null,"abstract":"<p><p>The probiotic <i>Bifidobacterium breve</i> is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated <i>B. breve</i> JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar <i>B. breve</i> strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the <i>E. coli</i> BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of <i>lai</i> in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.</p>","PeriodicalId":41,"journal":{"name":"Journal of Agricultural and Food Chemistry","volume":" ","pages":"1425-1440"},"PeriodicalIF":5.7000,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Heterologous Expression and Polyphasic Analysis of CLA-Converting Linoleic Acid Isomerase from <i>Bifidobacterium breve</i> JKL2022.\",\"authors\":\"Arxel G Elnar, Yujin Jang, Geun-Bae Kim\",\"doi\":\"10.1021/acs.jafc.4c05746\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The probiotic <i>Bifidobacterium breve</i> is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated <i>B. breve</i> JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar <i>B. breve</i> strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the <i>E. coli</i> BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of <i>lai</i> in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.</p>\",\"PeriodicalId\":41,\"journal\":{\"name\":\"Journal of Agricultural and Food Chemistry\",\"volume\":\" \",\"pages\":\"1425-1440\"},\"PeriodicalIF\":5.7000,\"publicationDate\":\"2025-01-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Agricultural and Food Chemistry\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1021/acs.jafc.4c05746\",\"RegionNum\":1,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/11/25 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q1\",\"JCRName\":\"AGRICULTURE, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Agricultural and Food Chemistry","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1021/acs.jafc.4c05746","RegionNum":1,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/11/25 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"AGRICULTURE, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
摘要
益生菌布列夫双歧杆菌以其高效的共轭亚油酸（CLA）转化而闻名，但其CLA转化途径仍未得到充分探索。本研究调查了前列双歧杆菌 JKL2022 在活跃生长细胞、洗涤细胞状态和粗蛋白提取物中的 CLA 转化情况。此外，该研究还旨在确认 JKL2022 菌株中的 CLA 转化酶，并通过异源表达融合蛋白（LAI_sGFP 和 MBP_LAI）优化其纯化。与基因相似的 B. breve 菌株（JCM7017、JCM7019、JCM1192 和 JCM1273）相比，JKL2022 表现出更高的 CLA 转化率，尤其是在洗涤细胞（60.14 ± 7.60%）和粗蛋白组分（96.11 ± 6.63%）中观察到的 CLA 转化率。将多通道跨膜亚油酸异构酶（LAI）克隆到大肠杆菌 BL21(DE3)中，将其作为游离 LAI 或用超级夹子-GFP 或 MBP 标记修饰，并在 37 °C 下用 0.01 mM IPTG 表达，从而获得高活性蛋白质馏分。通过预测建模、分子对接和系统发育分析对 LAI 进行了表征。此外，反转录定量 PCR 分析表明，与 JCM 菌株相比，JKL2022 中 lai 的表达量上调（高出 20-140 倍）。然而，由于 LAI 的跨膜构型，为下游应用扩大其生产和纯化规模仍是一项挑战。
Heterologous Expression and Polyphasic Analysis of CLA-Converting Linoleic Acid Isomerase from Bifidobacterium breve JKL2022.
The probiotic Bifidobacterium breve is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated B. breve JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar B. breve strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the E. coli BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of lai in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.
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The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.
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