Comparative Proteomic Analysis Of Mycobacterium Neoaurum JC-12 By Two-dimensional Electrophoresis And Heterologous Expression The Key Enzyme Of Synthesis ADD

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.