Coenzyme Q(10) production by immobilized Sphingomonas sp. ZUTE03 via a conversion-extraction coupled process in a three-phase fluidized bed reactor

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ(10) production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion-extract coupled process. In the reactor, the CoQ(10) yield reached 46.99 mg/L after 8 h of conversion; a high-level yield of about 45 mg/L was maintained even after 15 repetitions (8 h/batch). To fully utilize the residual precursor (para-hydroxybenzoic acid, PHB) in the aqueous phase, the organic phase was replaced with new solution containing 70 mg/L solanesol for each 8 h batch. The CoQ(10) yield of each batch was maintained at a level of about 43 mg/L until the PHB ran out. When solid solanesol was fed to the organic phase for every 8 h batch, CoQ(10) could accumulate and reach a yield of 171.52 mg/L. When solid solanesol and PHB were fed to the conversion system after every 8 h batch, the CoQ(10) yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion-extract coupled process could enhance CoQ(10) production in the TPFBR.     

茄尼醇转化高产辅酶Q10菌株的分离鉴定与发酵条件的研究

采用以异戊二烯为唯一碳源的选择性平板筛选模型,从钱塘江沿岸杭州市九堡段土壤中新筛选到一株产辅酶Q10的细菌菌株E03,经形态、生理生化、Biolog碳源利用试验和16S rDNA序列分析,确定E03属于鞘氨醇属(Sphingomonas sp.),命名为Sphingomonas sp.ZUTE03.摇瓶试验确定了该菌发酵生产辅酶Q10的最佳碳源为葡萄糖15 g/L,氮源为硫酸铵10 g/L,初始pH8.0,发酵温度25℃,并考察了该菌转化茄尼醇产辅酶Q.0的发酵工艺,以合适溶剂为溶解体系,于发酵培养基中摇床培养12 h后,加入终浓度为0.75 g/L的茄尼醇粗品,转化12 h,辅酶Q10产值可达96.88 mg/L.     

Association of colony morphology with coenzyme Q(10) production and its enhancement from Rhizobium radiobacter T6102W by addition of isopentenyl alcohol as a precursor

Rhizobium radiobacter T6102 was morphologically purified by the aniline blue agar plates to give two distinct colonies; white smooth mucoid colony (T6102W) and blue rough colony (T6102B). The coenzyme Q(10) (CoQ(10)) was produced just by T6102W, showing 2.0 mg/g of CoQ(10) content, whereas the T6102B did not produce the CoQ(10). All of the used CoQ(10) biosynthetic precursors enhanced the CoQ(10) production by T6102W. Specifically, the supplementation of 0.75 mM isopentenyl alcohol improved the CoQ(10) concentration (19.9 mg/l) and content (2.4 mg/g) by 42% and 40%, respectively.     

Recovery of solanesol from tobacco as a value-added byproduct for alternative applications

Solanesol in the waste streams of a bioprocess designed for alternative applications of low-alkaloid tobacco was recovered using three different extraction methods. Compared to the conventional heat-reflux extraction (HRE) and ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) using 1:3 hexane:ethanol (v/v) as the solvent after saponification treatment of tobacco biomass was found the most effective in terms of solanesol yield, processing time, and volume of solvent consumed. Quantification of solanesol was achieved by optimizing the mobile phase at 60/40 acetonitrile–isopropanol and lowering the oven temperature to 22 °C using a standard reverse-phase high performance liquid chromatography (RP-HPLC). The total solanesol recovered from tobacco biomass and chloroplast accounted for 30% (w/w) of the total solanesol in the fresh leaves. Since solanesol is the precursor of metabolically active quinones such as coenzyme Q10 and vitamin K analogues, extraction of solanesol from tobacco bioprocess waste is a feasible operation and could leverage the overall profitability of biorefining tobacco for alternative, value-added uses.     

对沼泽红假单胞菌生产CoQ10添加前体物的优化

CoQ10因其具有多种生理生化功能而不仅用于药物也用作食品添加剂.本研究采用全因析中心复合设计及响应面法对茄尼醇、对羟基苯甲酸及甲硫氨酸三种CoQ10前体物的添加量进行了优化,以达到沼泽红假单胞菌J001最大量地生产CoQ10的目的.结果表明:经过对所得模型进行响应曲面法分析,当茄尼醇、对羟基苯甲酸及甲硫氨酸最佳添加量分别为124.8mg1-1,267.7mg1-1,130.2mg1-1时,得最大CoQ10产量预测值40.6[(mgCoQ10)(g干细胞)-1].对上述最佳组合进行试验验证得40.5±0.2[(mg CoQ10)(g干细胞)-1],很接近预测值,比对照(未加三种CoQ10前体物)CoQ10产量提高了109.8%.     

类球红细菌添加前体物产辅酶Q_(10)的工艺优化

目的:考察摇瓶与发酵罐水平下类球红细菌CU20-9产辅酶Q10添加前体物质的最优配比。方法:通过正交试验优化发酵前体配比,考察摇瓶发酵多种前体物质的添加对辅酶Q10产量的影响,并利用发酵罐进行中试规模实验。结果:优化后发酵前体配比为维生素B1 10 mg/L、乙酸钠100 mg/L、对羟基苯甲酸20 mg/L、花生油0.01%。结论:前体添加及优化配比后,摇瓶及中试发酵水平下辅酶Q10的产量均有显著提高。     

酶解破壁促进废次烟叶中茄尼醇溶浸

协同应用纤维素酶和木质素酶催化降解废次烟叶,探讨清洁高效的酶解破壁效应及浸提茄尼醇工艺条件。结果发现复配酶催化裂解溶浸茄尼醇效果明显优于单一酶,酶解时间、温度、pH值以及酶投加量等条件均影响酶破壁浸提茄尼醇能效。结果表明,采用纤维素酶：木质素酶酶活比15∶1 (U/U) 的复配酶,在体积为5倍烟草质量的水介质环境中,当复配酶投加量为175 U/g,水浴温度40 ℃,pH=6时,催化酶解烟叶8 h后,茄尼醇溶浸浓度可达0.33 g/L。在此条件下,茄尼醇平均提取率可达96.53％,是化学回流浸提方法的1.68倍。该方法为有效提取废次烟草中茄尼醇提供了一种新途径。     

代谢工程改造大肠杆菌生产辅酶Q10

辅酶Q10(CoQ10)是一种脂溶性抗氧化剂,具有提高人体免疫力、延缓衰老和增强人体活力等功能,广泛应用于制药行业和化妆品行业。微生物发酵法能可持续性生产辅酶Q10,具有越来越多的商业价值。本研究首先将来自类球红细菌的十聚异戊二烯焦磷酸合成酶基因(dps)整合到大肠杆菌ATCC 8739染色体上,敲除内源的八聚异戊二烯焦磷酸合成酶基因(ispB),使内源的辅酶Q8合成途径被辅酶Q10合成途径取代,得到稳定生产辅酶Q10的菌株GD-14,其辅酶Q10产量达0.68 mg/L,单位细胞含量达0.54 mg/g DCW。随后用多个固定强度调控元件在染色体上对MEP途径的关键基因dxs和idi基因以及ubiCA基因进行组合调控,将辅酶Q10单位细胞含量提高2.46倍(从0.54到1.87 mg/g)。进一步引入运动发酵单胞菌Zymomonas mobilis的Glf转运蛋白代替自身的磷酸烯醇式丙酮酸:碳水化合物磷酸转移酶系统(PTS),使辅酶Q10产量进一步提高16%。最后,对高产菌株GD-51进行分批补料发酵,辅酶Q10产量达433 mg/L,单位细胞含量达11.7 mg/g DCW。这是目前为止文献报道的大肠杆菌产辅酶Q10最高菌株。     

Effects of coenzyme Q10 and alpha-tocopherol administration on their tissue levels in the mouse: elevation of mitochondrial alpha-tocopherol by coenzyme Q10.

Coenzyme Q (CoQ) was previously demonstrated in vitro to indirectly act as an antioxidant in respiring mitochondria by regenerating alpha-tocopherol from its phenoxyl radical. The objective of this study was to determine whether CoQ has a similar sparing effect on alpha-tocopherol in vivo. Mice were administered CoQ10 (123 mg/kg/day) alone, or alpha-tocopherol (200 mg/kg/day) alone, or both, for 13 weeks, after which the amounts of CoQ10, CoQ9 and alpha-tocopherol were determined by HPLC in the serum as well as homogenates and mitochondria of liver, kidney, heart, upper hindlimb skeletal muscle and brain. Administration of CoQ10 and alpha-tocopherol, alone or together, increased the corresponding levels of CoQ10 and alpha-tocopherol in the serum. Supplementation with CoQ10 also elevated the amounts of the predominant homologue CoQ9 in the serum and the mitochondria. A notable effect of CoQ10 intake was the enhancement of alpha-tocopherol in mitochondria. alpha-Tocopherol administration resulted in an elevation of alpha-tocopherol content in the homogenates of nearly all tissues and their mitochondria. Results of this study thus indicate that relatively long-term administration of CoQ10 or alpha-tocopherol can result in an elevation of their concentrations in the tissues of the mouse. More importantly, CoQ10 intake has a sparing effect on alpha-tocopherol in mitochondria in vivo.     

Liposomal membranes. XIV. Fusion of liposomal membranes induced by polyisoprenoids as monitored by fluorescence quenching method

Fusion of the single-walled liposomes of egg phosphatidylcholine as induced by the polyisoprenoids such as solanesol, trans-ethyl decaprenoate (EDP), coenzyme Q10, and dolichol has been investigated adopting the fluorescence quenching method. Relative efficiency of the polyisoprenoids employed on the induced fusion of liposomes was a sequence of solanesol less than or equal to EDP much less than CoQ10, dolichol, which was consistent with the result previously obtained by the dye-release method.     

Rapid determination of solanesol in tobacco by high-performance liquid chromatography with evaporative light scattering detection following microwave-assisted extraction

Solanesol is the starting material for many high-value biochemicals, including coenzyme Q(10) and Vitamin-K analogues. The aim of the current study was to develop a reliable and fast analytical procedure for the determination of solanesol in tobacco using high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique. The HPLC conditions were Agilent C18 column using acetonitrile-isopropanol (60:40, v/v) as mobile phase at a flow rate of 1 ml/min. ELSD conditions were optimized at nebulizer-gas flow rate of 1.5 l/min and drift tube temperature of 65 degrees C. The method was validated to achieve the satisfactory precision and recovery, and the calibration range was 0.1-1.5 mg/ml. The developed analytical procedure was successfully applied to determine solanesol content in tobacco samples from different growing regions in China.     

Coenzyme Q<Subscript>10</Subscript> production directly from precursors by free and gel-entrapped <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. ZUTE03 in a water-organic solvent,two-phase conversion system

In a water-organic solvent, two-phase conversion system, CoQ₁₀ could be produced directly from solanesol and para-hydroxybenzoic acid (PHB) by free cells of Sphingomonas sp. ZUTE03 and CoQ₁₀ concentration in the organic solvent phase was significantly higher than that in the cell. CoQ₁₀ yield reached a maximal value of 60.8 mg l⁻¹ in the organic phase and 40.6 mg g⁻¹-DCW after 8 h. CoQ₁₀ also could be produced by gel-entrapped cells in the two-phase conversion system. Soybean oil and hexane were found to be key substances for CoQ₁₀ production by gel-entrapped cells of Sphingomonas sp. ZUTE03. Soybean oil might improve the release of CoQ10 from the gel-entrapped cells while hexane was the suitable solvent to extract CoQ₁₀ from the mixed phase of aqueous and organic. The gel-entrapped cells could be re-used to produce CoQ₁₀ by a repeated-batch culture. After 15 repeats, the yield of CoQ₁₀ kept at a high level of more than 40 mg l⁻¹. After 8 h conversion under optimized precursor’s concentration, CoQ₁₀ yield of gel-trapped cells reached 52.2 mg l⁻¹ with a molar conversion rate of 91% and 89.6% (on PHB and solanesol, respectively). This is the first report on enhanced production of CoQ₁₀ in a two-phase conversion system by gel-entrapped cells of Sphingomonas sp. ZUTE03.     

半导体激光和紫外线对辅酶Q10产生菌的复合诱变效应

采用紫外线、半导体激光及紫外线与半导体激光复合作用的方法,诱变产辅酶Q10红酵母菌SY-3,以提高辅酶Q10的产量。结果表明,紫外线和半导体激光单独作用,诱变效果不佳,而二者的复合作用却能产生很好的效果。用紫外照射120 s再经半导体激光辐射8 min,得到一株叠氮钠和维生素K3双抗性突变株,产辅酶Q10的量达到157.7 mg/L,比原始菌株提高了88.1%,并具有良好的遗传稳定性。     

正交试验法优选烟草中辅酶Q10的匀浆提取工艺

以烟草叶片为原料,用正交试验法优化了烟草中辅酶Q10的匀浆提取工艺,考察了匀浆时间、乙醇体积分数、液料比和提取次数4个因素对辅酶Q10提取率的影响,确立了烟草中辅酶Q10的优化匀浆提取工艺条件为:提取溶剂为85%乙醇,匀浆时间6 min,液料比9∶1(mL/g),提取次数2次。将该法与索氏提取进行了对比。结果表明,匀浆提取烟草中辅酶Q10的提取率为12.82μg·g-1,与索氏提取的提取率相当,但匀浆提取所用提取时间短,提取溶剂用量少,因此匀浆法具有明显的优势。     

Coenzyme Q improves LDL resistance to ex vivo oxidation but does not enhance endothelial function in hypercholesterolemic young adults

Oxidative modification of low-density lipoprotein (LDL) may cause arterial endothelial dysfunction in hyperlipidemic subjects. Antioxidants can protect LDL from oxidation and therefore improve endothelial function. Dietary supplementation with coenzyme Q (CoQ(10)) raises its level within LDL, which may subsequently become more resistant to oxidation. Therefore, the aim of this study was to assess whether oral supplementation of CoQ(10) (50 mg three times daily) is effective in reducing ex vivo LDL oxidizability and in improving vascular endothelial function. Twelve nonsmoking healthy adults with hypercholesterolemia (age 34+/-10 years, nine women and three men, total cholesterol 7.4+/-1.1 mmol/l) and endothelial dysfunction (below population mean) at baseline were randomized to receive CoQ(10) or matching placebo in a double-blind crossover study (active/placebo phase 4 weeks, washout 4 weeks). Flow-mediated (FMD, endothelium-dependent) and nitrate-mediated (NMD, smooth muscle-dependent) arterial dilatation were measured by high-resolution ultrasound. CoQ(10) treatment increased plasma CoQ(10) levels from 1.1 +/-0.5 to 5.0+/-2.8 micromol/l (p =.009) but had no significant effect on FMD (4.3+/-2.4 to 5.1+/-3.6 %, p =.99), NMD (21.6+/-6.1 to 20.7+/-7.8 %, p = .38) or serum LDL-cholesterol levels (p = .51). Four subjects were selected randomly for detailed analysis of LDL oxidizability using aqueous peroxyl radicals as the oxidant. In this subgroup, CoQ(10) supplementation significantly increased the time for CoQ(10)H(2) depletion upon oxidant exposure of LDL by 41+/-19 min (p = .04) and decreased the extent of lipid hydroperoxide accumulation after 2 hours by 50+/-37 micromol/l (p =.04). We conclude that dietary supplementation with CoQ(10) decreases ex-vivo LDL oxidizability but has no significant effect on arterial endothelial function in patients with moderate hypercholesterolemia.     

Reinvestigation of coenzyme Q10 isolation from Sporidiobolus johnsonii

There is considerable current interest in coenzyme Q10 (CoQ10) from a medical perspective. CoQ10 has been shown to alleviate the side effects of statin drugs, for instance, and so there is a push to find naturally high producers of the compound. Sporidiobolus johnsonii (S. johnsonii) has been reported to produce CoQ10 in studies that used only standards on thin‐layer chromatography (TLC) and also suggested the production of coenzyme Q9 (CoQ9). This work set out to verify CoQ9/CoQ10 production in S. johnsonii and quantify as appropriate. We show that S. johnsonii produces CoQ10 but found no evidence for CoQ9 biosynthesis. The specific production of CoQ10 was noted at 10 mg/g dry cell weight (DCW) in media supplemented with 4‐hydroxybenzoic acid (HBA). This makes S. johnsonii a naturally high CoQ10 producer. New methods for extraction and purification of CoQ10 are also discussed, and identification of a closely eluting side product under normal phase isolation is reported.     

Ubiquinone (coenzyme Q10) prevents renal mitochondrial dysfunction in an experimental model of type 2 diabetes

Cardiovascular benefits of ubiquinone have been previously demonstrated, and we administered it as a novel therapy in an experimental model of type 2 diabetic nephropathy. db/db and dbH mice were followed for 10 weeks, after randomization to receive either vehicle or ubiquinone (CoQ10; 10mg/kg/day) orally. db/db mice had elevated urinary albumin excretion rates and albumin:creatinine ratio, not seen in db/db CoQ10-treated mice. Renal cortices from db/db mice had lower total and oxidized CoQ10 content, compared with dbH mice. Mitochondria from db/db mice also contained less oxidized CoQ10(ubiquinone) compared with dbH mice. Diabetes-induced increases in total renal collagen but not glomerulosclerosis were significantly decreased with CoQ10 therapy. Mitochondrial superoxide and ATP production via complex II in the renal cortex were increased in db/db mice, with ATP normalized by CoQ10. However, excess renal mitochondrial hydrogen peroxide production and increased mitochondrial membrane potential seen in db/db mice were attenuated with CoQ10. Renal superoxide dismutase activity was also lower in db/db mice compared with dbH mice. Our results suggest that a deficiency in mitochondrial oxidized CoQ10 (ubiquinone) may be a likely precipitating factor for diabetic nephropathy. Therefore CoQ10 supplementation may be renoprotective in type 2 diabetes, via preservation of mitochondrial function.     

Coenzyme Q10: is there a clinical role and a case for measurement?

Coenzyme Q(10) (CoQ(10)) is an essential cofactor in the mitochondrial electron transport pathway, and is also a lipid-soluble antioxidant. It is endogenously synthesised via the mevalonate pathway, and some is obtained from the diet. CoQ(10) supplements are available over the counter from health food shops and pharmacies. CoQ(10) deficiency has been implicated in several clinical disorders, including but not confined to heart failure, hypertension, Parkinson's disease and malignancy. Statin, 3-hydroxy-3- methyl-glutaryl (HMG)-CoA reductase inhibitor therapy inhibits conversion of HMG-CoA to mevalonate and lowers plasma CoQ(10) concentrations. The case for measurement of plasma CoQ(10) is based on the relationship between levels and outcomes, as in chronic heart failure, where it may identify individuals most likely to benefit from supplementation therapy. During CoQ(10) supplementation plasma CoQ(10) levels should be monitored to ensure efficacy, given that there is variable bioavailability between commercial formulations, and known inter-individual variation in CoQ(10) absorption. Knowledge of biological variation and reference change values is important to determine whether a significant change in plasma CoQ(10) has occurred, whether a reduction for example following statin therapy or an increase following supplementation. Emerging evidence will determine whether CoQ(10) does indeed have an important clinical role and in particular, whether there is a case for measurement.     

Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

Micro-analysis for coenzyme Q10 in endomyocardial biopsies of cardiac patients and data on bovine and canine hearts

A micro-analysis was designed for determining the levels of coenzyme Q10 (CoQ10) in specimens of ca. 500 nanograms of myocardial tissues. The prime steps included acetone extraction, purification by thin-layer chromatography, and HPLC. CoQ11 was the internal standard. This methodology was successful for human endomyocardial biopsies from cardiac patients before and after therapy with CoQ10 and has now been used for canine and bovine tissues. The mean canine CoQ10 level from six specimens from the left ventricle (l.v.) of a single animal is 0.99 +/- 0.06 micrograms/mg/d.w. The mean levels of the tissues from nine animals is 1.04 +/- 0.17 (l.v.) and 1.11 +/- 0.16 (r.v.). The mean bovine level from seven animals was 0.48 +/- 0.12 (l.v.) and 0.68 +/- 0.06 (r.v.).