羟丙基-β-环糊精对新金色分枝杆菌合成ADD蛋白质组的影响

Proteomic differences in ADD synthesis by Mycobacterium neoaurum JC-12 with or without hydroxypropyl-β-cyclodextrins

新金色分枝杆菌(Mycobacterium neoaurum JC-12)可降解植物甾醇合成药物中间体雄甾-1,4-二烯-3,17-二酮(ADD),由于植物甾醇的溶解度较低,因此在转化体系中添加羟丙基-β-环糊精(HP-β-CD)作为助溶剂.本次研究在HP-β-CD存在与否条件下利用二维凝胶电泳(2-DE)和基质辅助激光解析电离飞行时间串联质谱技术(MALDI-TOFMS)分离和鉴定新金色分枝杆菌蛋白质表达量的差异;再使用反转录实时荧光定量PCR(RT-q PCR)对挑选出的5个与ADD合成相关的蛋白质编码基因进行转录水平分析.结果表明,2-DE图谱及质谱结果共鉴定到12个具有显著差异表达量的蛋白质点.其中,参与植物甾醇降解合成ADD途径的乙醇脱氢酶、烯酰-Co A水合酶、乙酰-Co A乙酰基转移酶、3α,7α,12α-三羟基-5β-胆甾-24-烯酰-Co A水合酶和4,5-9,10-双断裂-3-羟基-5,9,17三氧雄甾-1(10),2-二烯-4-酸酯水解酶的蛋白质表达量在羟丙基-β-环糊精存在条件下显著提高;参与糖、脂、氨基酸和核酸类物质代谢的乙二醛酶、3-氧代酰基-ACP合酶、3-酮脂酰-ACP还原酶、分支酸合酶、氮调节蛋白P-II和DNA结合蛋白的蛋白质表达量也有一定的提高,但磷酸甘油酸变位酶的蛋白质表达量有所下降.通过RT-q PCR分析的转录水平的变化情况与2-DE得到的蛋白水平的结果相符.综上所述,羟丙基-β-环糊精可促进降解植物甾醇合成ADD代谢途径及糖、脂、氨基酸和核酸类物质代谢途径相关酶的表达量.

This research aimed to explore the effect of hydroxypropyl-fl-cyclodextrin on the Androst-1,4-diene-3,17-dione （ADD） synthesis by Mycobacterium neoaurum JC-12. Differential proteins were separated and identified by two-dimensional gel electrophoresis （2-DE） and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry （MALDI-TOF- MS）. Furthermore, 5 proteins were analyzed by RT-qPCR at transcriptional level. All together 12 proteins were identified by 2-DE and MS. The expression level was improved for enoyl-CoA hydratase, alcohol dehydrogenase, 4,5-9,10-diseco-3- hydroxy-5,9,17-trioxoandrosta-1（10）,2-diene-4-oate hydrolase （DHDOH）, 3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholest- 24-enoyl-CoA hydratase （TCEH） and acetyl-CoA acetyltransferase, which participated in the synthesis of ADD. Moreover, the expression levels of glyoxalase, 3-oxoacyl-ACP synthase, chorismate synthase and 3-ketoacyl-ACP reductase involved in the metabolism of carbohydrate, lipid, nucleic acid and amino acid were also improved. However the expression level of phosphoglyceromutase was decreased. The RT-qPCR experiment was further applied to verify the results of 2-DE. The experiment showed that hydroxypropyl-fl-cyelodextrins can improve the expression of most enzymes that participate in the ADD synthesis and at the same time enhance the metabolism of carbohydrate, lipid, amino acid and nucleic acid.