植物乳杆菌肌球交叉反应抗原MCRA的克隆表达及功能鉴定
详细信息Cloning, expression and enzymatic activity of the myosin cross reactive antigen gene from Lactobacillus plantarum ZS2058
-
摘要: 根据NCBI中已报道亚油酸异构酶基因的序列特征从植物乳杆菌ZS2058克隆获得了产CLA的关键酶基因肌球交叉反应抗原MCRA,构建表达载体后,在大肠杆菌中实现了表达,SDS-PAGE和Western Blot检测结果为胞内可溶表达,其大小约为67ku,将MCRA蛋白进行亲和层析后进行功能鉴定,GC-MS结果显示此蛋白能将亚油酸转化为羟基化衍生物10羟基-顺12-十八碳烯酸,将油酸转化为10-羟基-十八碳酸。
-
关键词:
- 肌球蛋白交叉反应抗原; /
- 亚油酸; /
- 共轭亚油酸; /
- 重组表达;
Abstract: Multiple enzymes were involved for CLA production in lactic acid bacteria (LAB) . Myosin cross reactive antigen (MCRA) gene was amplified with the specific primer from Lactobacillus plantarum ZS2058 according to reported putative linoleate isomerease in LAB in NCBI. MCRA gene was cloned and expressed successfully into Escherichia coli, the recombinant MCRA was analyzed by SDS-PAGE and Western Blot, and the results indicated the molecular weight of MCRA was 67ku. After purification with affinity chromatography, the activity of recombinant MCRA was analyzed. The GC-MS results showed that MCRA catalyzed linoleic acid into 10-hydroxy-cis-12-octadecenoic acid, and catalyzed oleic acid into 10-hydroxy-octadecanoic acid. -
[1] Belury M A.Dietary conjugated linoleic acid in health:physiological effects and mechanisms of action[J].Annual Review of Nutrition, 2002, 22:505-531.
[2] Jones E L, Shingfield K J, Kohen C, et al.Chemical, physical, and sensory properties of dairy products enriched with conjugated linoleic acid[J].Journal of Dairy Science, 2005, 88 (8) :2923-2937.
[3] Pariza M.Perspective on the safety and effectiveness of conjugated linoleic acid[J].American Journal of Clinical Nutrition, 2004, 79:1132S-1136S.
[4] Kepler C R, Hirons K P, McNeil J J, et al.Intermediates and products of the biohydrogenation of linoleic acid by Butyrivibrio fibrisolvens[J].Journal of Biological Chemistry, 1966, 241:1350-1354.
[5] Or-Rashid M M, Alzahal O, McBride B W.Comparative studies on the metabolism of linoleic acid by rumen bacteria, protozoa, and their mixture in vitro[J].Applied Microbiology and Biotechnology, 2011, 89 (2) :387-395.
[6] Liavonchanka A, Hornung E, Feussner I, et al.Structure and mechanism of the Propionibacterium acnes polyunsaturated fatty acid isomerase[J].Proceedings of the National Academy of Sciences of the United States of America, 2006, 103 (8) :2576-2581.
[7] Kishino S, Park S B, Takeuchi M, et al.Novel multi-component enzyme machinery in lactic acid bacteria catalyzing C=C double bond migration useful for conjugated fatty acid synthesis[J].Biochemical and Biophysical Research Communications, 2011, 416:188-193.
[8] Kil K S, Cunningham M W, Barnett L A.Cloning and sequence analysis of a gene encoding a67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with Class II major histocompatibility antigens[J].Infection and Immunity, 1994, 62:2440-2449.
[9] Rosson R A, Grund A D.Linoleate isomerase.World Patent:100846[P].2001.
[10] 钮晓燕, 陈卫, 田丰伟, 等.植物乳杆菌ZS2058在磷酸盐缓冲液体系中生物转化共轭亚油酸[J].微生物学报, 2007, 47 (2) :244-248. [11] Sambrook J, Fritsch E F, Maniatis T.Molecular Cloning:ALaboratory Manual.2nd Ed.New York:Cold Spring Harbor Laboratory Press, 1989.
[12] Volkov A, Liavonchanka A, Kamneva O, et al.Myosin cross-reactive antigen of Streptococcus pyogenes M49encodes a fatty acid double bond hydratase that plays a role in oleic acid detoxification and bacterial virulence[J].Journal of Biological Chemistry, 2010, 285 (14) :10353-10361.
[13] Rosberg-Cody E, Liavonchanka A, Gbel C, et al.Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection[J].BMC Biochemistry, 2011, 12:9.
[14] Bligh E G, Dyer W J.A rapid method of total lipid extraction and purification[J].Canadian Journal of Biochemistry and Physiology, 1959, 37:911-917.
计量
- 文章访问数:
- HTML全文浏览量:
- PDF下载量:
下载:
