改进底物添加体系制备氢化可的松

在蓝色梨头霉和新月弯孢霉协同转化制备氢化可的松(HC)过程中,常规的底物热溶液体系由17α羟基孕甾4-烯-3,20-二酮-21-醋酸酯(RSA)和乙醇组成,其中m(RSA)∶V(乙醇)=1∶25;改进后的底物溶液体系由Tween-80、丙三醇、RSA和磷酸盐缓冲溶液(PBS)组成,其中V(Tween-80)∶V(丙三醇)∶m(RSA)∶V(PBS)=1∶3∶1∶25。RSA质量浓度从2g/L起,累加到5g/L,RSA全部被转化,且产物氢化可的松(HC)产率与常规低浓度投料相当;在RSA质量浓度3g/L时添加底物,协同菌丝体能重复利用达3次,HC产率稳定在70%左右;经3批次实验室摇瓶放大制备实验,产物HC平均收率为52%,重现性较好,工艺操作稳定。Tween-80/丙三醇/RSA/PBS底物体系较常规RSA/乙醇构成的底物添加体系,可显著提高HC生产收率,有工业应用价值。     

有机溶剂对黑根霉甾体11α-羟基化的影响

考察了有机溶剂对黑根霉甾体11α-羟基化反应中转化底物16α,17α-环氧孕甾酮生成11 α, 16α,17α-环氧孕甾酮的转化率和细胞色素P450酶浓度的影响.向培养28 h的培养液中添加终浓度0.035 mol/L的丙酮和1.75 g/L的底物,继续转化48 h.与未添加丙酮相比,添加丙酮后的底物转化率和细胞色素P450酶表达量分别提高了6.8%和30%.说明丙酮添加可明显提高黑根霉甾体11α-羟基化能力和细胞色素P450酶的表达.     

静息细胞连续两批次生物催化生产氢化可的松

以新月弯孢霉（Curvularia lunata）的Ⅱ级培养18h的活菌丝作为C11β位羟基化生物催化剂,在选定的含有微粒结晶甾体底物（0．2％,质量分数）17α,21-二羟基孕甾-4-烯-3,20-二酮的磷酸缓冲液（0．05mol／L,pH6．0）介质体系中,在经过连续两批次约55h的转化反应,底物转化率可维持在较高水平,产物氢化可的松净收率可达60％;从菌丝回收的底物RS可再利用。此工艺提高了生物催化剂的利用率,具有工业应用价值。     

拟结晶投料环氧黄体酮犁头霉羟化过程研究

环氧黄体酮羟化产物是多种甾体激素药物的中间体,其11-羟化过程利用犁头霉(Absidiacoerulea)所得转化率远高于其他菌株。采用拟结晶投料方式,将环氧黄体酮颗粒细化后加一定量水,β-环糊精,吐温-80,超声波乳化后投入发酵液中。这种投料方式可避免传统投料中使用有机溶剂毒害细胞的缺点,更利于底物转化。采用多层前传神经网络建立培养基和投料成分配比与转化率关系模型,并将具有全局寻优性能的粒子群优化算法(PSO)应用于培养基和投料成分配比的优化,收敛速度快,效果好。在优化的操作条件下,摇瓶中投料浓度为10g/L时底物转化率达到87.5%,在3.7L发酵罐中投料浓度提高到20g/L时底物转化率仍高达86.6%。     

转化5a-娠烷-3β，17a-二羟基一20一酮-21-醋酸酯为氢化可的松诺卡氏菌62-1菌株和蓝色梨头霉AS3．65的协同转化

本文研究了诺卡氏菌(Noeardia) 62-1 菌株和蓝色梨头霉(Absidia coerulea) AS3.65在一起,转化5a-娠烷-3β,17a-二羟基-20-酮-21-醋酸酯一步生成氢化可的松的协同转化作用。由于诺卡氏菌62一l菌株的水解酶活性弱,A1一脱氢酶的活性强,所以在单独转化中形成RSA,P．S和ALRS等多种产物,但蓝色梨头霉有很强的水解酶活性并具11β一羟化酶,所以在和诺卡氏菌的协同转化中,只测到少量RSA和微量不欲得的△1-RS,生成的主要中间产物是RS,它紧接着进一步转化成氢化可的松,这是一种理想的转化方法。     

转化RSA为氢化可的松的新月弯胞霉筛选

从土样中筛选到 1株能转化甾体化合物 RSA(17α-羟基孕甾 - 4 -烯 - 3,2 0 -二酮 - 2 1-醋酸酯 )生成氢可的松 (11β,17α,2 1-三羟基孕甾 - 4 -烯 - 3,2 0 -二酮 )的 C4菌株 ,经菌落形态与个体形态观察 ,初步鉴定为新月弯胞霉 (curvularia lunata)。正交实验确定该菌株的最适培养基为 :葡萄糖 10 g,黄豆粉 10 g,自来水 10 0 0 m L,p H6 .5.经硅胶层析和高交液相色谱测定 ,氢化可的松     

犁头霉11α-羟基化制备16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯-3,20-二酮

选育到一株对16β-甲基,17α,21-二羟基孕甾-1,4-二烯-3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21-乙酰化(Ⅱb)可明显提高11α-羟基化的能力.在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,11α,21-三羟基孕甾-1,4-二烯-3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认.     

犁头霉11α-羟基化制备16β-甲基-11α，17α，21 三羟基孕甾-1，4 二烯 3，20 二酮

选育到一株对16β-甲基-17α,21-二羟基孕甾-1,4-二烯3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21乙酰化(Ⅱb)可明显提高11α-羟基化的能力。在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认。     

微生物学方法制备16α-甲基-11α，17α，21-三羟基孕甾-1，4-二烯3，20-二酮

本文报道了简单节杆菌A69-2和球孢白僵菌AS69同时存在下对16α-甲基-17α-羟基孕甾-4．烯-3,20-二酮-21-醋酸酯(16MRSA)的协周转化作用。 这种协同转化怍用既能解除16α-甲基-11α,17a,21-三羟基孕甾-4-烯-3,20-二酮(16MllaHC)对球孢白僵菌AS69的11α羟化酶的抑制作用,又可降低高浓度的16M11aHC对节杆菌A69—2脱氢酶活性的影响,同时还能抑制节杆菌脱氢过程的副反应。在底物浓度为0.15％(W／V)时,l6α-甲基-11α,17a,21-三羟基孕甾-1,4-二烯-3,20-二酮(16MDHC)的收率约50%,故是制备1 6MDHC的一种理想的微生物学方法。     

用新月弯孢霉11β-羟基化16a-甲基化合物RS-21醋酸酯

对新月弯孢霉AS3．4381的菌丝体转化16a-甲基Reicbstein's 化合物S21-醋酸酯(I)生成16a-甲基氢化可的松(II)进行了研究。培养24h的菌丝体的11β-羟基化活性最高;乙醇对此羟基化活性的抑制作用明显。当(1)浓度为0.15％,转化72h,产物(II)的重量收率为55.4％。     

甾体化合物RSA的11β-羟基化反应

原生质体在甾体中的应用起始于Ｄｌｕｇｏｎｓｋｉ在 1984年采用Ｃｕｎｎｉｎｇｈａｍｅｌｌａｅｌｅｇａｎｓ转化可的松龙 ( 17α ,2 1 二羟基孕甾 4 烯 3 ,2 0 二酮 )和Ｈｙｐｈｏｄｅｒｍａｒｏｓｅｕｍ转化 6α 氟 可的松龙 16,17 醋酸酯 ( 6α Ｆｌｕ 17α ,2 1 二羟基孕甾 4 烯 3 ,2 0 二酮 16,17 醋酸酯 ) ,发现原生质体具有甾体转化能力[1 ,2 ] 。Ｓｅｄｌａｃｚｅｋ进一步采用等重的原生质体和菌丝体进行比较 ,原生质体的羟基化能力较后者提高了 3倍 ,表现出很高的转化能力[3] ,从而引起人们的关注。随后展开了有关原生质…     

Linkage mapping of powdery mildew and greenbug resistance genes on recombinant 1RS from 'Amigo' and 'Kavkaz' wheat-rye translocations of chromosome 1RS.1AL.

Cultivated rye (Secale cereale L., 2n = 2x = 14, RR) is an important source of genes for insect and disease resistance in wheat (Triticum aestivum L., 2n = 6x = 42). Rye chromosome arm 1RS of S. cereale 'Kavkaz' originally found as a 1BL.1RS translocation, carries genes for disease resistance (e.g., Lr26, Sr31, Yr9, and Pm8), while 1RS of the S. cereale 'Amigo' translocation (1RSA) carries a single resistance gene for greenbug (Schizaphis graminum Rondani) biotypes B and C and also carries additional disease-resistance genes. The purpose of this research was to identify individual plants that were recombinant in the homologous region of.1AL.1RSV and 1AL.1RSA using both molecular and phenotypic markers. Secale cereale 'Nekota' (1AL.1RSA) and S. cereale 'Pavon 76' (1AL.1RSV) were mated and the F1 was backcrossed to 'Nekota' (1AL.1AS) to generate eighty BC1F2:3 families (i.e., ('Nekota' 1AL.1RSA x 'Pavon 76' 1AL.1RSV) x 'Nekota' 1AL.1AS). These families were genotyped using the secalin-gliadin grain storage protein banding pattern generated with polyacrylamide gel electrophoresis to discriminate 1AL.1AS/1AL.1RS heterozygotes from the 1AL.1RSA+V and 1AL.1AS homozygotes. Segregation of the secalin locus and PCR markers based on the R173 family of rye specific repeated DNA sequences demonstrated the presence of recombinant 1AL.1RSA+V families. Powdery mildew (Blumeria graminis) and greenbug resistance genes on the recombinant 1RSA+V arm were mapped in relation to the Sec-1 locus, 2 additional protein bands, 3 SSRs, and 13 RFLP markers. The resultant linkage map of 1RS spanned 82.4 cM with marker order and spacing showing reasonable agreement with previous maps of 1RS. Fifteen markers lie within a region of 29.7 cM next to the centromere, yet corresponded to just 36% of the overall map length. The map position of the RFLP marker probe mwg68 was 10.9 cM distal to the Sec-1 locus and 7.8 cM proximal to the powdery mildew resistance locus. The greenbug resistance gene was located 2.7 cM proximal to the Sec-1 locus.     

用新月弯孢霉11β——羟基化16α——甲基化合物Rs—21醋酸酯

The conversion of 16 alpha-methyl-Reichstein's compound S 21-acetate (I) to 16 alpha-methyl-hydrocortisone (II) by the mycelium Curvularia lunata AS3.4381 was studied. Maximal 11 beta-hydroxylating activity of the mycelium was found during cultivation of the microorganism by 24h. Inhibition of the ethanol to the 11 beta-hydroxylating activity was obvious. The yield of 55.4% (II) (W/W) could be achieved during conversion of 0.15% substrate at 72 h.     

Microbial hydroxylation of 16α,17α-dimethyl-17β-(l-oxopropyl)androsta-l,4-dien-3-one to rimexolone by Curvularia lunata AS 3.4381

A novel synthetic route to rimexolone, a corticosteroid for treatment of ocular inflammation without significant elevation of intraocular pressure, was described. An investigation has been undertaken of the microbial transformation of 16α,17α-dimethyl-17β-(l-oxopropyl)androsta-l,4-dien-3-one by microorganisms known to hydroxylate conventional steroids, using Curvularia lunata AS 3.4381 gave rimexolone, the product of 11β-hydroxylation, respectively. The target compound was characterized with reference substance rimexolone by TLC, HPLC, elemental analysis, MS, IR, and NMR.     

11α-Hydroxylation of 16α,17-epoxyprogesterone by Rhizopus nigricans in a biphasic ionic liquid aqueous system

11α-Hydroxylation of 16α,17-epoxyprogesterone (EP) by Rhizopus nigricans is an essential step in the synthesis of many steroidal drugs, while low conversion of the biohydroxylation is a tough problem to be solved urgently in industry. Two ionic liquids (ILs) of [BMIm][PF(6)] and [BMIm][NTf(2)] were used in the biotransformation of EP by R. nigricans. The results indicated that the conversion carried out in [BMIm][PF(6)]-aqueous biphasic system was greatly increased to above 90% at 18 g/L feeding concentration. A simplified mechanism was proposed to explain the improvement of the bioconversion in a biphasic ionic liquid aqueous system. Besides, successive three batches of bioconversion were carried out in the biphasic system with a total conversion of 87% at phase ratio 10 and 75% at phase ratio 5, respectively. Since recycling of the [BMIm][PF(6)] is quite easy, there is a great potential for the application of ILs in fungi biotransformation to implement green production.     

一株源于醇化烟叶表面高效降解TSNA 菌株AS97 的分离筛选、鉴定及应用

摘要:【目的】烟草特有亚硝胺(Tobacco-specific nitrosamines,简称TSNA)是烟叶中的主要致癌物质。本研究从筛选建立的特有菌库中发现了1 株可有效降解TSNA 的菌株AS97,并对其进行了鉴定及初步应用研究。【方法】采用富集驯化及选择培养基筛选得到硝酸盐与亚硝酸盐转化能力最强的菌株AS97;根据菌株的形态特征、生理生化特性及16S rRNA 基因序列分析对其进行鉴定;并将AS97 自制发酵液喷施于烟丝表面,确定适宜接种量和发酵条件,采用LC-MS /MS(液相色谱串联质谱)方法检测TSNA 中四种主要成分的含量。【结果】菌株AS97 源于云南玉溪烤烟样品表面,经分析确定其为荧光假单胞菌(Pseudomonas fluorescens,GenBank 登录号:JF449445)。将AS97 自制发酵液以5% 的接种量喷施于烟丝,30℃(相对湿度是60%)条件下发酵10 d 检测烟叶中硝酸盐、亚硝酸盐、TSNA、4-(N-甲基-亚硝基) -1-(3-吡啶基)-1-丁酮(NNK)、N-亚硝基去甲基烟碱(NNN)、N-亚硝基新烟草碱(NAT) 及N-亚硝基假木贼碱(NAB) 的转化率分别达到68. 77%、45. 57%、45. 47%、59. 08%、38. 79%、21. 41% 及11. 76%。相关性分析结果表明烤烟中硝酸盐、亚硝酸盐与TSNA 的含量均呈极显著相关(P＞0.01),进一步证实硝酸盐与亚硝酸盐是TSNA 的主要前体物质。【结论】醇化烟叶表面的荧光假单胞菌AS97 能够显著降低TSNA 的含量。本文首次报道了源于醇化烤烟表面对TSNA 有良好转化能力的Pseudomonas fluorescens。可将其开发成新型微生物制剂,应用于低害卷烟制品的生 产实践中。     

Targeting FoxO1 with AS1842856 Suppresses Adipogenesis

Hyperplasia (i.e., increased adipogenesis) contributes to excess adiposity, the hallmark of obesity that can trigger metabolic complications. As FoxO1 has been implicated in adipogenic regulation, we investigated the kinetics of FoxO1 activation during adipocyte differentiation, and tested the effects of FoxO1 antagonist (AS1842856) on adipogenesis. We found for the first time that the kinetics of FoxO1 activation follows a series of sigmoid curves, and reveals the phases relevant to clonal expansion, cell cycle arrest, and the regulation of PPARγ, adiponectin, and mitochondrial proteins (complexes I and III). In addition, multiple activation-inactivation transitions exist in the stage of terminal differentiation. Importantly, persistent inhibition of FoxO1 with AS1842856 almost completely suppressed adipocyte differentiation, while selective inhibition in specific stages had differential effects on adipogenesis. Our data present a new view of FoxO1 in adipogenic regulation, and suggest AS1842856 can be an anti-obesity agent that warrants further investigation.