雄甾-4-烯-3,17-二酮(4-AD)转化菌种的筛选及鉴定

从土壤中筛选能将植物甾醇转化为雄甾-4-烯-3,17-二酮(4-AD)的菌种。采用富集培养基富集能降解植物甾醇的菌种、采用摇瓶进行发酵、采用薄层层析的方法对发酵产物进行检测、采用高效液相方法测定发酵液中4-AD的含量、采用PCR方法扩增菌种的16S rDNA序列。筛选出了一株转化能力最强的菌株,命名为:3-12。将这株菌的16S rDNA序列与GeneBank中收载的序列进行比对,3-12号菌株为分支杆菌。     

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。     

胆固醇氧化酶PsCO_4异源表达、纯化及酶学性质分析

胆固醇氧化酶专一性催化胆固醇为胆甾-4-烯-3-酮,广泛的应用于临床以及食品加工行业。本论文将来源于Pimelobacter simplex的胆固醇氧化酶PsCO_4,分别转化到大肠杆菌宿主BL21(DE3)、Rosetta(DE3)和C41(DE3)中,在不同温度(15℃、25℃、37℃)及IPTG诱导浓度(0.01mmol/L、0.1mmol/L、0.5mmol/L)下异源表达Ps CO_4。结果表明,转入Rosetta(DE3)菌株的PsCO_4蛋白,在IPTG浓度为0.1mmol/L、15℃下经18h诱导表达,PsCO_4可溶性表达量最高(0.63mg/ml)。异源表达的胆固醇氧化酶PsCO_4最适温度为30℃,最适pH为7.5。通过TLC,GC-MS检测出Ps CO_4催化胆固醇生成胆甾-4-烯-3-酮。以胆固醇和β-谷甾醇、豆甾醇和孕烯醇酮为底物,测定PsCO_4对四种底物的催化反应动力学参数,胆固醇k_(cat)/K_m为0.08s~(-1)·μM~(-1)分别高于β-谷甾醇(0.04s~(-1)·μM~(-1))、豆甾醇(0.005s~(-1)·μM~(-1))和孕烯醇酮(0.02s~(-1)·μM~(-1))。     

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。     

黑曲霉催化雄甾-4-烯-3,17-二酮16β-羟基化

为进一步确定黑曲霉菌株TCCC41650的生物转化能力,以雄甾-4-烯-3,17-二酮(Androstenedione)为底物,利用黑曲霉菌株TCCC41650进行催化,产物经纯化、重结晶后,通过单晶衍射鉴定为16β-羟基雄甾-4-烯-3,17-二酮。转化条件为:培养液pH 6.0,乙醇添加量为2%,投料浓度为1‰时,72 h转化率为85.8%。目前甾体研究领域对于C16β-羟基化的微生物转化未见报道,研究结果为C16β-羟基甾体药物的研发奠定了基础。     

可产酸快生小菌的分离鉴定

目的：鉴定在实验过程中分离到的一株生长快速,并且可以将L-山梨糖转化为2-酮基-L-古龙酸的菌株。方法：将快生小菌传代,并进行产酸、抗菌谱、山梨糖脱氢酶活性等分析,通过PCR方法扩增并分析16S rDNA。结果：在传代过程中还分离得到了不产酸菌株;从快生小菌中扩增得到了普通酮古龙酸菌16S rDNA;从产酸菌中能够扩增得到包含酮古龙酸菌和乙酸钙不动杆菌的16S rDNA序列;在不产酸菌中只检测到乙酸钙不动杆菌的16S rDNA序列。结论：产酸的快生小菌可能是普通酮古龙酸菌和乙酸钙不动杆菌形成的融合细胞,这种融合细胞基因组表现为很不稳定,普通酮古龙酸菌基因组容易丢失,且丢失后也失去了产酸能力。     

A环饱和甾体的微生物转化作用II.降解5a一△16一3β一羟基-孕甾烯-20-酮-3β-醋酸酯为△1,4-雄甾二烯-3，17一二酮

节杆菌9—2能彻底降解5a一△16一3β一羟基一孕甾烯一20一酮一3β一醋酸酯(I)形成二氧化碳和水。在它的培养基质中添加钴离子可抑制甾核的进一步降解,从而积累中间产物△1,4-雄甾二烯-3,17一二酮(VI),     

来源于红球菌胆固醇氧化酶ChOG的异源表达、纯化及催化反应结构分析

胆固醇氧化酶是胆固醇代谢过程中的关键酶,临床上用胆固醇氧化酶作为检测血清胆固醇含量的应用潜力巨大。将来源于红球菌Rhodococcus ruber的胆固醇氧化酶Ch OG,分别转化到大肠杆菌宿主BL21(DE3)和Rosetta(DE3)中,在不同条件下进行诱导表达,结果表明:BL21(DE3)菌株在诱导温度为16℃、IPTG浓度为0.1 mmol/L时,Ch OG可溶性表达量最高(0.49 mg/ml)。Ch OG的最适反应温度为30℃,最适反应p H为7.5。最适反应条件下,酶活性达到8.0 U/mg。利用TLC、HPLC对Ch OG催化产物胆甾-4-烯-3-酮进行了鉴定分析。三维结构及定点突变分析表明Glu406及Arg408、Glu261在进行胆固醇C3羟基的脱氢、质子传递,以及底物异构化方面发挥着重要作用。     

胆固醇转化菌株的筛选及发酵条件优化

【目的】从土壤中筛选及鉴定具有转化胆固醇能力的菌株SE-1,对转化产物进行结构鉴定,并通过一定的工艺条件优化提高转化产率。【方法】利用胆固醇为唯一碳源筛选能转化胆固醇的菌株SE-1,对菌株进行形态、生理生化特征试验及16S rRNA基因序列同源性分析确定该菌株的系统发育学地位。发酵转化产物经氯仿萃取,对转化产物进行硅胶板薄层层析法分析,用硅胶柱层析法、Sephadex LH20分离产物,通过1H-NMR、13C-NMR分析确定转化产物的化学结构。对菌株转化胆固醇的发酵培养基的碳源、氮源、底物添加方式及发酵条件进行优化。【结果】菌株SE-1为革兰氏阴性菌,生理生化特征与洋葱伯克霍尔德氏菌(Burkholderia cepacia)相似,16S rRNA序列与洋葱伯克霍尔德氏菌(GenBank No.U96927)相似性为99%。硅胶薄层层析显示转化产物为两种产物。发酵转化时,在胆固醇-吐温乳化液的添加量为1 g/L,碳源糖蜜5%,氮源(NH4)2SO40.3%,接种量4%,发酵液pH7.5,36℃发酵的条件下,7β-羟基胆固醇的产率最高,达到34.4%。【结论】分离得到的菌株SE-1鉴定为Burkholderia cepacia。菌株SE-1转化胆固醇的主产物为7β-羟基胆固醇,次产物为7-酮基胆固醇,胆固醇7β-羟基化转化率在最适的转化条件下比优化前提高了20.8%。     

节杆菌9—2对皮质甾类C20-酮基的还原作用II．转化3β，17a，21-三羟基-5a-孕甾-20酮

根据多项物理性质,包括熔点、比旋值、分子旋光度、紫外光谱,红外光谱、核磁共振、质谱等的测定,确定节杆菌(Arthrobacter)9-2生物转化3β,17a,21-三羟基-5a-孕甾-3-酮(I)所生成的产物(Ⅱ)的分子结构为17a,20,21-三羟基-孕甾-1,4-二烯-3-酮,其中C20-羟基的构型是β-型。并讨论了该菌转化(I)的可能途径。     

The substrate specificity and stereochemistry, reversibility and inhibition of the 3-oxo steroid delta 4-delta 5-isomerase component of cholesterol oxidase.

1. 5-Cholesten-3-one was shown to be an intermediate in the conversion of cholesterol into 4-cholesten-3-one by Nocardia cholesterol oxidase. 2. The absence of a C-17 side chain from 5-androstene-3,17-dione slightly increased the Vmax. of the isomerase activity relative to 5-cholesten-3-one (1.7-fold), but greatly increased the Km. 3. Incubations of [4alpha-2H]-and [4beta-2H]-cholesterol with cholesterol oxidase showed that the 4beta-hydrogen atom can be transferred to the 6beta-position. However, incubations of cholesterol, 5-cholesten-3-one and 4-cholesten-3-one with the enzyme in 2H2O led to some incorporation of 2H into the 4-cholesten-3-one products, mostly at position 6beta. 4. Both the isomerase and the oxidase activities of cholesterol oxidase were inhibited by 5,10-seco-19-nor-5-cholestyne-3,10-dione.     

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

Characterization of production of cholesterol oxidases in three Rhodococcus strains

The production of cholesterol oxidase in two strains of Rhodococcus equi No. 23 from butter, and Rhodococcus sp. No. 33 from bacon, which had properties on biochemical and physiological tests almost similar to the strains of R. equi, was compared with that of the type strain (ATCC 6939) of R. equi. The intensity of cholesterol oxidase activity, both extracellular and membrane-bound, from the three strains was in the order No. 23, ATCC 6939 and No. 33. More extracellular enzyme was produced by strain No. 23 than by the other two strains. Halo formation on the agar medium containing cholesterol depended on the conversion of cholesterol to 4-cholesten-3-one by the extracellular cholesterol oxidase.     

Characterization of production of cholesterol oxidases in three Rhodococcus strains

The production of cholesterol oxidase in two strains of Rhodococcus equi No. 23 from butter, and Rhodococcus sp. No. 33 from bacon, which had properties on biochemical and physiological tests almost similar to the strains of R. equi , was compared with that of the type strain (ATCC 6939) of R. equi. The intensity of cholesterol oxidase activity, both extracellular and membrane-bound, from the three strains was in the order No. 23, ATCC 6939 and No. 33. More extracellular enzyme was produced by strain No. 23 than by the other two strains. Halo formation on the agar medium containing cholesterol depended on the conversion of cholesterol to 4-cholesten-3-one by the extracellular cholesterol oxidase.     

Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus

The recombinant plasmid pNCO937 (8.1 kbp) containing a Streptomyces sp. cholesterol oxidase gene was introduced into Streptococcus thermophilus by electrotransformation. Transformation frequency was 7.2 x 10(5) colony forming units/micrograms of DNA. The presence of the cholesterol oxidase gene in S. thermophilus was confirmed with Southern blot analysis using a biotinylated probe. Thin-layer chromatographic analysis showed the expression of the Streptomyces cholesterol oxidase gene resulting in the oxidation of cholesterol to 4-cholesten-3-one. S. thermophilus may be a suitable host for the expression of other genes regulating prokaryotic cholesterol metabolism.     

Cytotoxicity of cholesterol oxidase to cells of hypercholesterolemic guinea pigs

1. A high cholesterol diet caused guinea pig erythrocytes to become sensitive to lysis by cholesterol oxidase (CO), a protein not hemolytic to normal cells. 2. Lysis was associated with conversion of membrane cholesterol to its oxidation product (delta-4-cholesten-3-one). 3. Intravenous injection of CO to hypercholesterolemic guinea pigs produced a reduction in serum cholesterol, but was not lethal as it was in rabbits. 4. Homogenized spleen, liver and kidney from the hyperlipidemic animals were sensitive to in vitro cholesterol oxidation while tissues from non-lipemic animals were resistant to modification.     

Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione by cholesterol-degrading bacteria

Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione, which are intermediates of microbial conversion of cholesterol, by cholesterol-degrading bacteria (12 strains of the genus Rhodococcus isolated from food of animal origin and 12 culture collection strains) were examined. All strains had the ability to degrade 4-cholesten-3-one without necessarily being able to degrade cholesterol. On the other hand, the bacteria were divided into three groups with little or no (0-10%), intermediate (10-70%) and high (70-100%) degradation abilities for 1,4-androstadiene-3,17-dione.     

Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione by cholesterol-degrading bacteria

Degradations of 4-cholesten-3-one and 1,4-androstadiene-3,17-dione, which are intermediates of microbial conversion of cholesterol, by cholesterol-degrading bacteria (12 strains of the genus Rhodococcus isolated from food of animal origin and 12 culture collection strains) were examined. All strains had the ability to degrade 4-cholesten-3-one without necessarily being able to degrade cholesterol. On the other hand, the bacteria were divided into three groups with little or no (0–10%), intermediate (10–70%) and high (70–100%) degradation abilities for 1,4-androstadiene-3,17-dione.     

Effect of 4-cholesten-3-one on lecithin-cholesterol acyltransferase activity and the lipid concentration in the serum of normocholesterolaemic and hypercholesterolaemic rats

Male Wistar strain rats and PHHC (Prague hereditary hypercholesterolaemic) rats received an intraperitoneal injection of 4-cholesten-3-one for five days. Lecithin-cholesterol acyltransferase activity and total cholesterol, triglyceride and phospholipid levels were determined in their serum. A significant drop in the total cholesterol level was found in normocholesterolaemic Wistar rats after the administration of cholestenone. The serum triglyceride content remained unaltered and the phospholipid concentration showed a downward trend. Lecithin-cholesterol acyltransferase activity was also significantly reduced. In PHHC rats, no significant changes occurred in total cholesterol, triglyceride and phospholipid levels, or in lecithin-cholesterol acyltransferase activity, after the administration of 4-cholesten-3-one. A comparison of serum 4-cholesten-3-one concentrations in the two groups of experimental animals shows that the turnover time for this substance in hypercholesterolaemic rats is only half as long as in normocholesterolaemic rats.     

Polyethylene glycol derivative-modified cholesterol oxidase soluble and active in benzene

Cholesterol oxidase from Nocardia sp. was modified with a synthetic copolymer of polyoxyethylene allylmethyldiether (PEG) and maleic acid anhydride (MA anhydride), poly(PEG-MA anhydride). The modified cholesterol oxidase, in which 64% of the amino groups in the protein molecule were coupled to poly(PEG-MA), was soluble in organic solvents and catalyzed the oxidation reaction of cholesterol in benzene to form 4-cholesten-3-one with the enzymic activity of 0.6 mumol/min/mg protein. Using the modified cholesterol oxidase together with polyethylene glycol-modified peroxidase, coupled reactions shown below took place in Cholesterol + O2----4-Cholesten-3-one + H2O2 H2O2 + o-Phenylenediamine----H2O + Oxidized o-Phenylenediamine transparent benzene solution, not in an emulsified system. The oxidation of cholesterol was directly determined in benzene by measuring the absorbance of oxidized o-phenylenediamine at 490 nm.