【作者】 吴丹；

【导师】 饶志明；

【作者基本信息】 江南大学 ， 微生物学， 2016， 硕士

【摘要】 甾体激素类药物是指分子结构中含有甾体母核的一类化合物,由于其具有较强的抗过敏、抗感染、抗休克和抗病毒等作用,在医药行业中有着广泛的应用,因此也常被誉为“生命的钥匙”。目前甾体激素类药物合成方法主要为半合成法,也就是利用天然的甾体化合物作为骨架,以化学与生物转化相结合的方法对其进行改造。近年来,微生物转化因其绿色环保节能等优点,也越来越受到人们的关注。例如新金色分枝杆菌降解植物甾醇可合成较多的甾体激素类药物的中间体,如雄甾-1,4-二烯-3,17-二酮(Androst-1,4-diene-3,17-dione,ADD)、雄甾-4-烯-3,17-二酮(4-androstene-3,17-dione,AD)和9-OH AD,其中ADD是合成黄体酮,炔诺酮和雌酮的必要前体物质。作为助溶剂的羟丙基-β-环糊精(Hydroxypropyl-β-cyclodextrin,HP-β-CD)可有效增加转化体系中植物甾醇的溶解度并对植物甾醇的转化率有一定的提高。本论文以前期筛选的ADD高产菌株新金色分枝杆菌(Mycobacterium neoaurum)JC-12为研究模型来探究在HP-β-CD存在与否条件下,M.neoaurum JC-12合成ADD蛋白质组的差异。主要结论如下:(1)利用蛋白质二维凝胶电泳(2-DE)与基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术相结合的方法分离和鉴定M.neoaurum JC-12在HP-β-CD存在与否条件下具有表达差异的蛋白质,结果表明共鉴定出22个具有显著表达差异的蛋白质。其中,ADD合成途径中的乙醇脱氢酶、烯酰-CoA水合酶、乙酰-CoA乙酰基转移酶、3α,7α,12α-三羟基-5β-胆甾-24-烯酰-CoA水合酶、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、单加氧酶和短链脱氢酶在HP-β-CD存在与否条件下的蛋白质表达量都有显著的差异。参与糖,脂,氨基酸和核酸类物质代谢的β-羟-ACP脱氢酶、伸长因子Tu、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、甘氨酸氧化酶和磷酸核糖甲基甲酰甘氨脒合酶在HP-β-CD存在条件下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。参与转录、辅因子和能量相关蛋白吡哆胺5’-磷酸氧化酶、核黄素合酶的α亚基、核黄素合酶、F0F1 ATP合酶和DNA结合蛋白表达量均有一定的提高。以上结果说明HP-β-CD能提高M.neoaurum JC-12中大多数蛋白质的表达水平。(2)利用反转录实时荧光定量PCR(RT-qPCR)技术进一步分析2-DE中筛选出的差异表达蛋白质和ADD合成代谢途径中关键酶的基因转录水平。结果表明:RT-qPCR进一步验证了2-DE的结果且编码关键酶和差异蛋白质基因的转录水平也有显著的差异。(3)通过异源表达和Ni-NTA纯化获得重组酶3-甾酮-△1-脱氢酶(3-ketosteroid-Δ1-dehydrogenase,KSDD)并研究其纯酶的酶学性质。结果表明:KSDD在钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5中成功表达并被纯化。纯酶KSDD的最适反应温度为30℃,在30℃以下稳定性较好,温度越高其酶活下降越快,当温度达到60℃时,纯酶KSDD完全失活。纯酶KSDD的最适反应pH为7.0,在pH 3.0-pH 10.0之间纯酶KSDD都比较稳定。1 mmol·L-1 K+,Na+,Ca2+和EDTA对KSDD的酶活力有明显的提高,而1 mmol·L-1 Ag+、Mn2+、Cu2+和Fe3+对其酶活有不同程度的抑制作用。以不同浓度的AD为底物,纯酶KSDD的Km值为8.91μmol·L-1,Vmax值为6.43μmol·L-1·min-1。(4)利用重组菌C.crenatum SYPA 5-5/pXMJ19-ksdd进行全细胞转化AD,使用高效液相色谱(HPLC)和薄层层析(TLC)分析产物ADD。结果表明:将重组菌C.crenatum SYPA 5-5/pXMJ19-ksdd的产物进行HPLC分析后,发现重组菌的产物在ADD标样处具有特征吸收峰,重组菌C.crenatum SYPA 5-5/p XMJ19-ksdd最大转化率为83.87%。通过对不同时间段转化样品的TLC分析,证明了随着重组菌钝齿棒杆菌SYPA 5-5转化时间的增加,在一定的时间范围内转化体系中的底物AD逐渐减少而产物ADD逐渐增多。综上所述重组的C.crenatum SYPA 5-5/pXMJ19-ksdd具有转化AD为ADD的能力。更多还原

【Abstract】 Steroidal medicine refers to their molecule structures containing steroid nucleus structure. It was regarded as “the key of life” because of its strong allergy, anti-infection, anti-shock and antiviral. Now Steroidal medicine has been widely used in the pharmaceutical industry. The main synthesis method of steroidal medicine is semisynthesis, which use the natural steroidal compounds as a skeleton and conbinate the chemical and biological transformation method to modify its structure. As an alternative and modest synthesis method, biocatalysis production of ADD has become a right alternative, mainly because it provides a superb combination of cost-effectiveness, sustainability and scalability. Such as Mycobacterium neoaurum can degradate phytosterol to yield much valuable steroidal derivatives, such as 4-androstene-3,17-dione(AD), androst-1,4-diene-3,17-dione(ADD), and 9α-OH-AD. ADD has been acknowledged to be a worthwhile precursor in the synthesis of steroid pharmaceuticals such as progesterone, norethindrone and estrone. As a cosolvent, hydroxypropyl-β-cyclodextrins(HP-β-CD) can improve the solubility of phytosterol and increase the conversion rate of phytosterol to ADD. This thesis will concentrate on the effect of HP-β-CD on the ADD synthesis by M. neoaurum JC-12. The main results are as follows:(1) 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). 22 proteins were identified by 2-DE and MS. Therein, the expression level of enoyl-Co A 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-Co A hydratase(TCEH) acetyl-Co A acetyltransferase, short-chain dehydrogenase and monooxygenase were improved, which participated in the synthesis of ADD; The expression level of glyoxalase, 3-oxoacyl-ACP synthase, chorismate synthase, 3-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydratase, glycine oxidase and phosphoribosylformylglycinamidine synthase which involved in the metabolism of carbohydrate, lipid, nucleic acid and amino acid were improved, however the expression level of phosphoglyceromutase was decrease; The expression level of DNA-binding protein, pyridoxamine 5’-phosphate oxidase, F0F1 ATP synthase subunit alpha, riboflavin synthase subunit alpha, transcriptional regulator and elongation factor Tu were improved too.(2) The genes of different expression proteins and key proteins in the metabolic pathway of ADD synthesis were analyzed by RT-q PCR at transcriptional level. The results showed that the transcriptional levels of these genes were improved with HP-β-CD. The RT-q PCR experiment was further applied to verify the results of 2-DE, and there was a notable positive correlation between the protein expression levels and the transcriptional levels.(3) Corynebacterium crenatum SYPA 5-5 was chosen as a new host for heterologous expression of 3-ketosteroid-Δ1-dehydrogenase(KSDD) from M. neoaurum JC-12. SDS-PAGE indicated that the KSDD was successfully expressed in recombinant C. crenatum SYPA 5-5/p XMJ19-ksdd. After purification of the recombinant KSDD, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and p H for KSDD activity were 30℃ and p H 7.0, respectively. The purified KSDD activity was strongly stimulated by 1 m M K+, Na+, Ca2+ and EDTA, while 1 m M Ag+, Mn2+, Cu2+ and Fe3+ can inhibite its activity. The Km and Vmax values of purified KSDD were 8.91 μmol·L-1 and 6.43 μmol·L-1·min-1.(4) When using the whole-cell of the recombinant C. crenatum to transform AD, the product ADD was assayed by HPLC and TLC. The results proved that the recombinant cells could efficiently catalyze the transformation from AD to ADD and the maximum conversion of AD was 83.87% in 10 h. As a control, the strain C. crenatum SYPA 5-5 harboring p XMJ19 could not convert AD to ADD. From above results, the recombinant C. crenatum has been surely identified to have the capability to produce ADD when using AD as substrate.更多还原