细胞色素P450酶对拟无枝酸菌转化洛伐他汀的影响

无锡他汀是胆固醇合成途径中限速酶羟甲基戊二酰辅酶A(HMG—CoA)还原酶抑制剂,由洛伐他汀经拟无枝酸菌(Amycolatopsis sp.ST2710)羟基化转化产生,为研究无锡他汀转化过程,本文利用CO差光谱法在摇瓶水平考察了底物浓度、温度、pH值、溶氧等因素对具有羟基化功能的细胞色素P450酶活的变化情况,以及细胞色素P450酶变化对洛伐他汀羟基化过程中的影响。研究表明,细胞色素P450酶可能是Amycolatopsis sp.ST2710转化洛伐他汀为无锡他汀代谢途径的一个关键酶,对洛伐他汀羟基化有重要作用,这为进一步对微生物转化洛伐他汀的研究打下了基础。     

Bioconversion of lovastatin to a novel statin by Amycolatopsis sp.

3-Hydroxy-3-methylglutaryl–coenzyme A (HMG–CoA) reductase catalyzes the conversion of HMG–CoA to mevalonic acid, which plays a significant role in cholesterol synthesis. Several statins, inhibitors of HMG–CoA reductase, can be synthesized and converted by microorganisms. Among 700 strains obtained from culture collections, one strain could convert lovastatin to a novel statin, wuxistatin. The strain was identified as a member of the genus Amycolatopsis based on 16S rRNA gene sequence, morphology analysis, and chemotaxonomic properties. Wuxistatin, a novel HMG–CoA reductase inhibitor, was purified by chromatography, and the structure was determined by electrospray ionization mass and nuclear magnetic resonance spectroscopy. The results show that wuxistatin was butanoic acid, 2-methyl-,1,2,3,5,8,8a-hexahydro-5-hydroxy-7-methyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) ethy]-1-naphthalenyl ester. An additional hydroxyl group was added to lovastatin at the 5-position to yield wuxistatin. This modification enhanced the intrinsic inhibitory activity (IC50) of wuxistatin (41 ± 5 nM) for fourfold compared with lovastatin (160 ± 10 nM). A stoichiometric conversion of lovastatin to wuxistatin occurred.     

Evaluation of the antibiotic biosynthetic potential of the genus Amycolatopsis and description of Amycolatopsis circi sp. nov., Amycolatopsis equina sp. nov. and Amycolatopsis hippodromi sp. nov

Aims: To describe three new Amycolatopsis strains and assess the antibiotic biosynthetic potential of the genus. Methods and Results: Three strains, designated S1·3^T, S3·6^T and SE(8)3^T, belonging to the genus Amycolatopsis were isolated and found to cluster together by 16S rRNA and gyrB gene‐based phylogenetic analysis. Genetic distance values, based on the gyrB gene, were calculated between the strains and their closest relatives and were all above the threshold value of 0·02 that has been proposed to distinguish Amycolatopsis type strains. DNA–DNA hybridization experiments against related type strains confirmed that strain S3·6^T represents a unique genomic species. Strain S3·6^T was also found to be distinct from strains S1·3^T and SE(8)3^T, the latter two of which were also shown to be distinct from each other. Antibiotic biosynthetic genes were identified from multiple Amycolatopsis strains, and their presence was found to be phylogenetically associated. Conclusions: The data presented in this study indicate that strains S1·3^T, SE(8)3^T and S3·6^T belong to three novel species, for which the names Amycolatopsis circi sp. nov. (= DSM 45561^T = NRRL B‐24841^T), Amycolatopsis equina sp. nov. (= DSM 45563^T = NRRL B‐24842^T) and Amycolatopsis hippodromi sp. nov. (= DSM 45562^T = NRRL B‐24843^T) are proposed. Significance and Impact of the Study: Three new species of Amycolatopsis are described, and the knowledge of the antibiotic biosynthetic potential of the genus has been extended.     

Effect of bioconversion conditions on vanillin production by Amycolatopsis sp. ATCC 39116 through an analysis of competing by-product formation

This study investigated the effects of transformation conditions such as initial pH, the initial concentration of glucose and yeast extract in the medium, and the separate addition of ferulic acid and vanillic acid, on the production of vanillin through an analysis of competing by-product formation by Amycolatopsis sp. ATCC 39116. The extent and nature of by-product formation and vanillin yield were affected by initial pH and different initial concentrations of glucose and yeast extract in the medium, with a high yield of vanillin and high cell density obtained at pH 8.0, 10 g/l glucose, and 8 g/l yeast extract. High concentrations of ferulic acid were found to negatively affect cell density. Additional supplementation of 100 mg/l vanillic acid, a metabolically linked by-product, was found to result in a high concentration of vanillin and guaiacol, an intermediate of vanillin. Via an analysis of the effect of these transformation conditions on competing by-product formation, high concentrations of ferulic acid were transformed with a molar yield to vanillin of 96.1 and 95.2 %, by Amycolatopsis sp. ATCC 39116 and Streptomyces V1, respectively, together with a minor accumulation of by-products. These are among the highest performance values reported in the literature to date for Streptomyces in batch cultures.     

黑曲霉CGMCC0774和朱红密孔菌CGMCC1115两步转化阿魏酸制备生物香兰素

在25 L发酵罐中黑曲霉Aspergillus niger CGMCC0774转化阿魏酸可生成香草酸2.24 g/L,摩尔转化率64.6%;朱红密孔菌Pycnoporus cinnabarinus CGMCC1115转化提取的香草酸可生成香草醛1.45 g/L,摩尔转化率为79.9%。将两步微生物转化有机串联,即用黑曲霉转化液加预先培养的朱红密孔菌Pycnoporus cinnabarinus CGMCC1115菌丝体继续转化,可产香草醛1.06 g/L,对原料阿魏酸的摩尔转化率34.0%。用米糠提取的天然阿魏酸做原料,两步串联微生物转化制备的生物香兰素经13C同位素的分析,符合生物香草素的等同要求。     

rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

[背景]苯乙醇(2-Phenylethanol,2-PE)是一种具有玫瑰香气味的高级香料添加剂,被广泛应用于香水、化妆品、食品和医药等领域.目前,利用工程菌合成苯乙醇有很好的应用前景.我们分离到一株肠杆菌(Enterobactersp.)CGMCC 5087,其可以通过苯丙酮酸途径合成2-PE.然而该菌的生长受到不同环...     

Nitrile-metabolizing potential of Amycolatopsis sp. IITR215

Strain Amycolatopsis sp. IITR215 was isolated from a sewage sample using polyacrylonitrile powder as the sole nitrogen source. Identification was performed by 16S rDNA analysis. The isolated strain harbored multiple nitrile-metabolizing enzymes having a wide range of substrate specificities. It metabolized nitrile and amide compounds with constitutive enzymes. Studies using an amidase inhibitor showed that hydrolysis of acrylonitrile and acrylamide occurred due to nitrile hydratase and amidase, respectively, while hydrolysis of hexanenitrile was due to the action of either nitrilase or a second nitrile hydratase/amidase system. The inhibitory effects of N-bromosuccinimide and N-ethylmaleimide on enzymes of this culture were also studied and this further indicated the involvement of either a nitrilase or a second nitrile hydratase/amidase system for hydrolysis of hexanenitrile. Interestingly, hexanenitrile hydrolysis exhibited an optimum temperature of 55 °C, whereas acrylonitrile and acrylamide hydrolysis showed an optimum temperature of 45 °C. The optimum pH was 5.8 for hexanenitrile hydrolysis and 7.0 for acrylonitrile and acrylamide hydrolysis. Hexanenitrile hydrolysis by enzymes of this strain showed better organic solvent tolerance in the presence of alcohols. The maximum enzyme activity of nitrile-metabolizing enzymes was found using media containing isobutyramide as the nitrogen source. This is the first report on constitutive multiple enzymes from the Amycolatopsis genus.     

利用Pseudonocardia sp.微生物转化制备骨化醇类化合物

【背景】近年来骨化醇类化合物(活性维生素D及其类似物)在肿瘤治疗、免疫调节中的作用逐渐被发现,药用价值显著,但目前化学合成法制备困难,因此高效制备骨化醇类化合物成为研究热点。【目的】利用假诺卡氏菌(Pseudonocardiasp.)转化骨化醇类底物分子并提高转化率。【方法】从化学合成方法常用的原料及中间体中筛选能被有效转化的底物分子,利用单因素及正交试验对转化条件进行优化。【结果】筛选到阿法骨化醇(Alfacalcidol,1α-(OH)VD3)、艾地骨化醇中间体(Eldecalcitolintermediate, AD-M07)、帕立骨化醇中间体(Paricalcitol intermediate,PC-M07) 3种底物分子,分别被转化为骨化三醇(Calcitriol,1α,25(OH)2VD3)、艾地骨化醇中间体(Eldecalcitol intermediate,AD-M08)、帕立骨化醇(Paricalcitol,PC)。确定最优转化条件:蛋白胨20.0 g/L,葡萄糖15.0 g/L,部分甲基化的β-环糊精(PMCD) 0.5%(质量体积比),转化温度25-30°C,接种量5%-10%(体积比),转化时间72、72、96 h,底物浓度1.2、0.6、0.6mg/mL,初始pH6.0-8.0。在此条件下3种底物的最高转化率分别达到85%、96%、75%。【结论】通过转化条件的优化3种产物的转化率大幅提高,为更加快速高效地利用微生物转化法制备骨化醇类化合物提供参考。     

一株分枝杆菌降解胆固醇制备AD(D)的转化条件

通过分枝杆菌(Mycobacteriumsp.)M3限制性降解胆固醇侧链获得了产物雄甾-4-烯-3,17-二酮(AD)和雄甾-1,4-二烯-3,17-二酮(ADD)。优化了胆固醇的投料时间、投料方式、培养基初始pH和葡萄糖浓度等工艺参数。将羟丙基-β-环糊精(HP-β-CD)应用于转化反应中,确定了HP-β-CD的最佳添加时间和添加量,使AD(D)生成率由初始对照的30%提高到60%,转化至72 h时AD(D)生成率达48%,是同期对照的4.0倍,生成率与生成速率均得到显著提高。在添加HP-β-CD的最佳转化条件下,AD(D)生成率达到70%,是初始对照的2.3倍。     

Advances in bioconversion of anthracycline antibiotics

During the last decade new anthracycline-type structures with potential usefulness in cancer treatment have been supplied both by new microbial strains and by bioconversions of precursor molecules employing cells or enzymes. We highlight recent advances in bioconversion of anthracycline structures with the main focus on late transformations such as are carried out by oxidoreductases.     

Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.     

Degradation of Polycarbonate by a Polyester-Degrading Strain, Amycolatopsis sp. Strain HT-6

Amycolatopsis sp. strain HT-6, a poly(tetramethylene succinate) (PTMS)-degrading actinomycete, was observed to degrade poly(tetramethylene carbonate) (PTMC). In a liquid culture with 150 mg of PTMC film, 59% degradation was achieved, but with a low yield of cell growth. On the other hand, PTMS copolymerized with a small amount of PTMC, forming a copolyester carbonate (PEC) that was completely and rapidly degraded with a high yield of cell growth.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Optimization of bioconversion conditions for vitamin D3 to 25-hydroxyvitamin D using Pseudonocardia autotrophica CGMCC5098

The optimal culture conditions for bioconversion of vitamin D₃ to calcifediol (25(OH)D₃) were investigated by varying carbon and nitrogen sources, metal salt concentrations, initial pH, temperature, solvents, surfactants, and agitation speed. In the process of this microbial hydroxylation, the timing of the addition of vitamin D₃, which is dissolved in ethanol, is of critical importance. Besides, the concentration of ethanol in zymotic fluid is the key factor to get high conversion ratio of vitamin D₃. In particular, the optimal culture conditions were 1.5% glucose, 1.5% soybean cake meal, 0.5% yeast extract, 0.5% corn steep liquor, 0.3% CaCO₃, 0.1% NaCl, 0.2% KH₂PO₄, pH 7.2 at 27?°C and the timing of the addition of vitamin D₃ dissolved in 5% (v/v) ethanol was 48?h followed by the inoculation of seed culture broth. Under the optimized conditions, the conversion of vitamin D₃ (1?g/L) by Pseudonocardia autotrophica CGMCC5098 in 50?L fermenter resulted in about 61.31% bioconversion ratio (639?mg/L) of 25(OH)D₃ on the 5th day.     

Glucose and lactose effect on AD and ADD bioconversion by Mycobacterium sp.

Summary The glucose and lactose effect on the steroid biotransformation reactions by Mycobacterium sp. NRRL B-3683 was studied. The reduction reactions increased at pH 7 and when sugars are added. The oxidation reactions are highers at pH 8 without any sugar added. The dehydrogenation reaction is inhibited when an endogenous carbon source is present in the media.