Screening Lysophosphatidic-acid-producing Bacillus Using HPLC and Quadrupole Time-of-flight Mass Spectrometry
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摘要: 溶血磷脂酸(lysophosphatidic acid,LPA)是一种重要细胞外信号递质和细胞内第二信使。本研究采用高效液相色谱/四级杆飞行时间质谱(HPLC-QTOF-MS)技术对34株不同种类芽胞杆菌的代谢物进行测定。结果表明,供试的菌株中只有1株枯草芽胞杆菌Bacillus subtilis FJAT-8784的代谢物中检测出产溶血磷脂酸,与Metlin代谢物质谱数据库中溶血磷脂酸的匹配得分达96.73,该化合物的质子化分子离子[M+H]+质荷比为439.281 9,质量数为438.275 8,推断出其分子式可能为C21H43O7P,其相对含量占发酵上清液总代谢物的1.32%。
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关键词:
- 芽胞杆菌 /
- 溶血磷脂酸 /
- 高效液相色谱/四级杆飞行时间质谱
Abstract: Lysophosphatidic acid (LPA) is an important extracellular signal transmitter and intracellular secondary messenger. Metabolites in the supernatants of liquid culture media for 34 strains of Bacillus were determined by using HPLC and quadrupole time-of-flight mass spectrometry. It was found that the only B. subtilis strain that produced LPA was FJAT-8784. The obtained LPA showed a matching score of 96.73 according to the Metlin database. It had a molecular weight of 438.2758, and a mass to charge ratio of 439.2819. Consequently, the chemical formula for the isolated LPA was assumed to be C21H43O7P. In the supernatant, the total metabolites contained approximately 1.32% LPA. -
表 1 供试芽胞杆菌菌株
Table 1. Bacillus strains used
序号 菌株编号 地理来源 学名 中文名称 1 FJAT-8784 瑞典的标准菌株 Bacillus subtilis 枯草芽胞杆菌 2 FJAT-14227 德国的标准菌株 Bacillus novalis 休闲地芽胞杆菌 3 FJAT-14205 德国的标准菌株 Aneurinibacillusmigulanus 米氏解硫胺素芽胞杆菌 4 FJAT-14856 德国的标准菌株 Aneurinibacillusaneurinilyticus 解硫胺素硫胺素芽胞杆菌 5 FJAT-10042 德国的标准菌株 Fictibacillusbarbaricus 奇异虚构芽胞杆菌 6 FJAT-14221 德国的标准菌株 Bacillus acidiceler 酸快生芽胞杆菌 7 FJAT-10032 德国的标准菌株 Bacillus fordii 福氏芽胞杆菌 8 FJAT-10014 德国的标准菌株 Bacillus alcalophilus 嗜碱芽胞杆菌 9 FJAT-14272 德国的标准菌株 Bacillus cibi 食物芽胞杆菌 10 FJAT-14236 德国的标准菌株 Bacillus butanolivorans 食丁酸芽胞杆菌 11 FJAT-14270 德国的标准菌株 Bacillus ginsengihumi 人参土芽胞杆菌 12 FJAT-10033 德国的标准菌株 Bacillus fortis 强壮芽胞杆菌 13 FJAT-520 福建漳州 Bacillus coagulaus 凝结芽胞杆菌 14 FJAT-10010 德国的标准菌株 Bacillus endophyticus 内生芽胞杆菌 15 FJAT-14212 德国的标准菌株 Bacillus indicus 印度芽胞杆菌 16 FJAT-14252 德国的标准菌株 Bacillus isronensis 印空研芽胞杆菌 17 FJAT-8757 瑞典的标准菌株 Bacillus badius 栗褐芽胞杆菌 18 FJAT-16342 福建武夷山 Bacillus cereus 蜡样芽胞杆菌 19 FJAT-8762 瑞典的标准菌株 Bacillus clausii 克劳芽胞杆菌 20 FJAT-14215 德国的标准菌株 Bacillus cecembensis 科研中心芽胞杆菌 21 FJAT-16072 福建武夷山 Bacillus megaterium 巨大芽胞杆菌 22 FJAT-10015 德国的标准菌株 Alicyclobacilluscellulosilyticus 解纤维素脂环酸芽胞杆菌 23 FJAT-14213 德国的标准菌株 Bacillus lehensis 列城芽胞杆菌 24 FJAT-8754 瑞典的标准菌株 Bacillus amyloliquefaciens 解淀粉芽胞杆菌 25 FJAT-10037 德国的标准菌株 Bacillus hemicellulosilyticus 解半纤维素芽胞杆菌 26 FJAT-8764 瑞典的标准菌株 Bacillus firmus 坚强芽胞杆菌 27 FJAT-16041 福建武夷山 Bacillus methylotrophicus 甲基营养型芽胞杆菌 28 FJAT-8761 瑞典的标准菌株 Bacillus circulans 环状芽胞杆菌 29 FJAT-14210 德国的标准菌株 Bacillus koreensis 韩国芽胞杆菌 30 FJAT-16291 福建武夷山 Bacillus licheniformis 地衣芽胞杆菌 31 FJAT-8756 瑞典的标准菌株 Bacillus azotoformans 产氮芽胞杆菌 32 FJAT-8765 瑞典的标准菌株 Bacillus.flexus 弯曲芽胞杆菌 33 FJAT-14220 德国的标准菌株 Bacillus aryabhattai 阿氏芽胞杆菌 34 FJAT-14858 德国的标准菌株 Bacillus plakortidis 海绵芽胞杆菌 表 2 化合物鉴定结果
Table 2. Identification of related compound
菌株编号 菌株名称 化合物名称 分子式 质量数 得分 保留
时间高度 宽度 [M+H]+
质荷比m/zFJAT-8784 枯草芽胞杆菌
Bacillus subtilis溶血磷脂酸 C21H43O7P 438.2758 96.73 25.90 1216471.80 0.36 439.2819 -
[1] 何兰杰. 溶血磷脂酸--一种具有多种生物学功能的磷脂信号分子[J]. 细胞生物学杂志, 2000, 22(3):121-126. http://www.cnki.com.cn/Article/CJFDTOTAL-XBZZ200003004.htm [2] KROGFELT K A, UTLEY M, KRIVAN H C, et al. Specific phospholipids enhance the activity of β-lactamantibiotics against Pseudomonas aeruginosa[J]. Journal of Antimicrobial Chemotherapy, 2000, 46:377-384. doi: 10.1093/jac/46.3.377 [3] ZHANG H L, XU X Y, GAJEWIAK J, et al. Dual activity lysophosphatidic acid receptor Pan-Antagonist/Autotaxininhibitor reduces breast cancer cell migration in vitro and causes tumor regression in vivo[J]. Cancer Research, 2009, 69:5441. doi: 10.1158/0008-5472.CAN-09-0302 [4] SCHLEICHER S M, THOTALAL D K. Autotaxin and LPA receptors represent potential molecular targets for the radiosensitization of murine glioma through effects on tumor vasculature[J]. PLoS ONE, 2011, 6(7):e22182. doi: 10.1371/journal.pone.0022182 [5] NIKITOPOULOU I, KAFFE E, SEVASTOU I, et al. A metabolically-stabilized phosphonate analog of lysophosphatidic acid attenuates collagen-induced arthritis[J]. PLoS ONE, 2013, 8(7):e70941. doi: 10.1371/journal.pone.0070941 [6] JIANG G, XU Y, FUJIWARAY, et al. Alpha-substituted phosphonate analogues of lysophosphatidic acid (LPA) selectively inhibit production and action of LPA[J]. ChemMedChem, 2007, 2(5):679-690. doi: 10.1002/(ISSN)1860-7187 [7] 陈家华, 张延东. 酯键溶血磷脂酸和醚键溶血磷脂酸的全合成[J]. 北京大学学报:自然科学版, 2003, 39(2):151-157. http://www.cnki.com.cn/Article/CJFDTOTAL-BJDZ200302002.htm [8] HARA Y, SEKI M, MATSUOKA S, et al. Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis[J]. Genes Genet Syst, 2008, 83(6):433-442. doi: 10.1266/ggs.83.433 [9] LUY J, ZHANG Y M, GRIMES K D, et al. Acyl-phosphates initiate membrane phospholipid synthesis in gram-positive pathogens[J]. Molecular Cell, 2006, 23:765-772. doi: 10.1016/j.molcel.2006.06.030 [10] 孟兆玲, 齐元英, 柳仁民. 高效液相色谱-质谱联用技术的应用进展[J]. 化学分析计量, 2006, 15(6):99-102. http://www.cnki.com.cn/Article/CJFDTOTAL-HXFJ200606042.htm [11] TANGKIJVANICH P, MELTON A C, SANTISKULVONG C. Rho and p38 MAP kinase signaling pathways mediate LPA-stimulated hepatic myofibroblast migration[J]. Journal of Biomedical Science, 2003,(10):352-358. http://cn.bing.com/academic/profile?id=6abc3e48fdcafe86def475bc192354c5&encoded=0&v=paper_preview&mkt=zh-cn [12] DUNLOP M, MUGGLI E, CLARK E. Association of cyclin-dependent kinase-4 and cyclin D1 in neonatal cells after mitogenic stimulation by lysophosphatidic acid[J]. BiochemBiophys Res Commun, 1996, 218:132-136. doi: 10.1006/bbrc.1996.0023 [13] MURPH M, MILLS G B. Targeting the lipids LPA and SIP and their signaling pathways to inhibit tumour progression[J]. Expert Reviews in Molecular Medicine, 2007,(9):1-18. [14] 张兆辉, 卫涛涛, 余绍祖, 等. 溶血磷脂酸损伤小脑颗粒神经元并诱导细胞凋亡[J]. 卒中与神经疾病, 2004, 11(6):321-324. http://www.cnki.com.cn/Article/CJFDTOTAL-ZZYS200406000.htm [15] HIRAOKA H, ASAKA O, ANO T, et al. Characteristics of Bacillus subtilis RB14, coproducer of peptide antibiotics iturin A and surfactin[J]. Journal of General and Applied Microbiology, 1992, 38(6):635-640. doi: 10.2323/jgam.38.635 [16] SANAHUJA G, BANAKAR R, TWYMAN RM, et al. Bacillus thuringiensis:a century of research, development and commercial applications[J]. Plant Biotechnology Journal,2011, 9:283-300. doi: 10.1111/pbi.2011.9.issue-3