Purification of solanesyl-diphosphate synthase from Micrococcus luteus. A new class of prenyltransferase.

The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography. By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa. In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate. HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner. Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis.     

Possible involvement of an enzymatic system for superoxide generation in lepidopteran larval haemolymph

The superoxide generative system in Pseudaletia separata larval haemolymph plasma was shown to consist of at least two components, low (LMF) and high (HMF) molecular weight factors using ultrafiltration, dialysis, and gel filtration. The LMF was concluded to be a molecule(s) smaller than 5 kDa while the HMF to be a protein(s) larger than 100 kDa. The total amount of superoxide produced depended on the amount of LMF and was independent of that of HMF added to the reaction mixture. Both the LMF and HMF were required for O₂^? generation. From results in the present article, it was hypothesized that the LMF was a substrate(s) discharging electrons and HMF was an enzyme(s) to mediate the electron transfer to O₂ forming O₂^?. Superoxide production was detected in the haemolymph plasma of 5 lepidopteran species in addition to P. separata. It was concluded that superoxide production is a common phenomenon at least in these lepidopterans. © 1995 Wiley-Liss, Inc.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Reaction pathways of glucose during esterification: effects of reaction parameters on the formation of humin type polymers

The formation of humin-type polymers and other products during exposure of glucose to methanol/water mixtures with methanol/water mass ratios from 10 to 0.22 in the presence of the acid catalyst Amberlyst 70 was investigated. In water-rich medium (methanol/water mass ratio: 0.22), dehydration of glucose produced 5-(hydroxymethyl)furfural (HMF), furfural, and substantial amounts of polymer. In methanol-rich medium (methanol/water mass ratio: 10), the hydroxyl and carbonyl groups of glucose, HMF or furfural were protected via etherification and acetalisation. These protections stabilized these reactive compounds and significantly lowered the polymer formation (1.43% of the glucose loaded). The polymerization of glucose and HMF was also favored at high temperatures and long residence times. Conversely, high catalyst dosage mainly accelerated the conversion of glucose to methyl levulinate. Thus, the polymerization of glucose and HMF can be suppressed in methanol/water mixtures with high methanol ratios, at low temperatures and short residence times.     

On-line control of fed-batch fermentation of dilute-acid hydrolyzates

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).     

Conversion of biomass into 5-hydroxymethylfurfural using solid acid catalyst

5-Hydroxymethylfurfural (HMF) was produced from monosaccharide (fructose and glucose), polysaccharide (inulin) and the Jerusalem artichoke juice by a simple one-pot reaction including hydrolysis and dehydration using solid acid under mild condition. Hydrated niobium pentoxide (Nb(2)O(5)·nH(2)O(2)) after pretreatment showed high catalytic activities for dehydration of mono- and polysaccharide to HMF at 433 K in water-2-butanol (2:3 v/v) biphasic system, giving high HMF yield of 89% and 54% from fructose and inulin, respectively. The HMF yield was up to 74% and 65% when inulin and Jerusalem artichoke juice were hydrolyzed by exoinulinase. The solid acid made the process environment-friendly and energy-efficient to convert carbohydrates into bio-fuels and platform chemicals.     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

松杨栅锈菌遗传多样性初步分析

采用ITS-nrDNA-RELP、测序技术、RAPD(随机扩增多态性DNA)分子标记技术,对我国松杨栅锈菌不同地域的5个生理小种11个菌系进行了遗传多样性分化研究.结果表明,该菌在我国的遗传分化与地理来源相关,可分为西部地理群和北方地理群.西部地理群又可分为高山森林生态型(HMF)和平原生态型(WPL).小种遗传分化不一定与致病性分化一致.t检验表明,各生理小种RAPD遗传多样性指数无明显差异,高山森林小种遗传多样性指数(0.5172)略高于平原小种遗传多样性指数(0.5089).核糖体基因转录间隔区高度保守,不适合该菌种内群体遗传多样性分化研究.     

亚磁场及其生物响应机制

根据亚磁生物学的研究历史和空间亚磁环境的实际情况,本文定义磁感应强度总量在“0<|B|≤5 μT”区间内的静态弱磁场为亚磁场．亚磁场能对生命活动的多个方面,特别是中枢神经系统产生负面影响．随着月球与火星航天计划的开展,航天员将长期暴露于亚磁空间中．这可能对宇航员的身心健康带来潜在的危害．亚磁场生物学效应及其机制的研究,将为相关载人航天的空间防护提供理论基础,已成为空间生物科学以及航天医学等相关领域的新热点．     

Production of 5-hydroxymethylfurfural in ionic liquids under high fructose concentration conditions

Acid-promoted, selective production of 5-hydroxymethylfurfural (HMF) under high fructose concentration conditions was achieved in ionic liquids (ILs) at 80 °C. A HMF yield up to 97% was obtained in 8 min using 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) catalyzed with 9 mol % hydrochloric acid. More significantly, an HMF yield of 51% was observed when fructose was loaded at a high concentration of 67 wt % in [C₄mim]Cl. Water content below 15.4% in the system had little effect on HMF yield, whereas a higher water content was detrimental to both reaction rate and HMF yield. In situ NMR analysis suggested that the transformation of fructose to HMF was a highly selective reaction that proceeded through the cyclic fructofuranosyl intermediate pathway. This work increased our capacity to produce HMF, and should be valuable to facilitate cost-efficient conversion of biomass into biofuels and bio-based products.     

Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol.     

Characterization and Functional Properties of Sub-Fractions of Soluble Soybean Polysaccharides

Soluble soybean polysaccharide (SSPS) was fractionated into two sub-fractions, a high-molecular-weight fraction (HMF) and a low-molecular-weight fraction (LMF) by the ethanol-extraction method. Characterization of the sub-fractions, that is, analysis of chemical composition, gel filtration, and SDS–PAGE, revealed that the main component of HMF was a large polysaccharide molecule with covalently-attached peptides, possibly corresponding to the intact SSPS molecule. LMF consisted of free peptides and saccharides of small size, which might have occurred as by-products during the production process of SSPS. HMF exhibited high ability to emulsify oil droplets and stabilize α-casein dispersions in an acidic pH region, but this ability of LMF was inferior to HMF. On the other hand, LMF had higher activity to prevent the oxidation of emulsified lipids than HMF. These results suggest that HMF and LMF had different characteristics and functional properties, and that the combination of the two sub-fractions generates the multi-functions of commercial SSPS.     

Securing a furan-based biorefinery: disclosing the genetic basis of the degradation of hydroxymethylfurfural and its derivatives in the model fungus Aspergillus nidulans

Hydroxymethylfurfural (HMF) is a promising lignocellulosic-derived source for the generation of diverse chemical building blocks constituting an alternative to fossil fuels. However, it remains unanswered if ubiquitous fungi can ensure their efficient decay, similar to that observed in highly specialised fungi. To disclose the genetic basis of HMF degradation in aspergilli, we performed a comprehensive analysis of Aspergillus nidulans ability to tolerate and to degrade HMF and its derivatives (including an HMF-dimer). We identified the degradation pathway using a suite of metabolomics methods and showed that HMF was modified throughout sequential reactions, ultimately yielding derivatives subsequently channelled to the TCA cycle. Based on the previously revealed hmfFGH gene cluster of Cupriavidus basilensis, we combined gene expression of homologous genes in Aspergillus nidulans and functional analyses in single-deletion mutants. Results were complemented with orthology analyses across the genomes of twenty-five fungal species. Our results support high functional redundancy for the initial steps of the HMF degradation pathway in the majority of the analysed fungal genomes and the assignment of a single-copy furan-2,5-dicarboxylic acid decarboxylase gene in A. nidulans. Collectively our data made apparent the superior capacity of aspergilli to mineralise HMF, furthering the environmental sustainability of a furan-based chemistry.     

长期亚磁场处理降低成年雄性小鼠肌肉组织的线粒体功能和运动能力*

亚磁场是深空载人航天任务中的一个关键风险因素。研究表明,亚磁场影响动物多种运动相关行为,但长期亚磁场处理对成年个体运动能力的影响还需要进一步的研究,以评估深空飞行任务中亚磁场的潜在风险。本研究利用三轴亥姆霍兹线圈系统模拟的亚磁环境,长期（一个月）曝露处理成年雄性C57BL/6小鼠,并从行为,组织,细胞,分子水平研究其对小鼠运动能力的影响。相比于地磁组对照,亚磁组小鼠的耐力显著下降。并且,其骨骼肌中柠檬酸水平和肌膜下线粒体数量的下降,以及骨骼肌线粒体形态的变化,表明亚磁场诱导了肌肉线粒体功能抑制,并可能导致其与耐力密切相关的能量代谢的下降。我们的研究结果为线粒体直接响应亚磁场提供了体内证据,并且提示线粒体相关指标可能用于亚磁场效应的风险评估和干预药物的开发。     

Mechanism of formation of 5-(hydroxymethyl)-2-furaldehyde from D-fructose an sucrose

The literature contains two alternative hypotheses for the mechanism of dehydration of fructose to 5-(hydroxymethyl)-2-furaldehyde (HMF), namely (1) a sequence of reactions commencing with and retaining the fructofuranose ring intact, and (2) a succession of reactions proceeding mainly via open-chain intermediates. The existing evidence for hypotheses (1) and (2) is reviewed and found to favor (1). The major products from fructose in water at 250 degrees, (with and without acid catalysis) have been investigated on a time-resolved basis and analysis of the results was found to confirm the first hypothesis. A necessary fructofuranosyl-cation intermediate in this hypothesis is produced directly by the hydrolysis of sucrose, and reacts to produce HMF in high yields.     

Removal and recovery of furfural, 5-hydroxymethylfurfural, and acetic acid from aqueous solutions using a soluble polyelectrolyte

In the cellulosic ethanol process, furfural, 5-hydroxymethylfurfural (HMF), and acetic acid are formed during the high temperature acidic pretreatment step needed to convert biomass into fermentable sugars. These compounds can inhibit cellulase enzymes and fermentation organisms at relatively low concentrations (≥ 1 g/L). Effective removal of these inhibitory compounds would allow the use of more severe pretreatment conditions to improve sugar yields and lead to more efficient fermentations; if recovered and purified, they could also be sold as valuable by-products. This study investigated the separation of aldhehydes (furfural and HMF) and organic acid (acetic acid) inhibitory compounds from simple aqueous solutions by using polyethyleneimene (PEI), a soluble cationic polyelectrolyte. PEI added to simple solutions of each inhibitor at a ratio of 1 mol of functional group to 1 mol inhibitor removed up to 89.1, 58.6, and 81.5 wt% of acetic acid, HMF, and furfural, respectively. Furfural and HMF were recovered after removal by washing the polyelectrolyte/inhibitor complex with dilute sulfuric acid solution. Recoveries up to 81.0 and 97.0 wt% were achieved for furfural and HMF, respectively. The interaction between PEI and acetic acid was easily disrupted by the addition of chloride ions, sulfate ions, or hydroxide ions. The use of soluble polymers for the removal and recovery of inhibitory compounds from biomass slurries is a promising approach to enhance the efficiency and economics of an envisioned biorefinery.     

The influence of the initial and catalyst concentrations on the dehydration of d-fructose

The dehydration at 95° of d-fructose (0.25m-1.0m) to 5-hydroxymethyl-2-furaldehyde (HMF) and the rehydration of HMF (0.25-1.0m) to levulinic and formic acids in 0.5-2m HCl has been studied. The conversion rate of d-fructose was proportional to the Hammett activity. The acidity had a smaller influence on the conversion rate of HMF, although it was not proportional to the catalyst concentration. The rehydration of HMF was faster in the presence of d-fructose. The yield of levulinic acid was independent of the catalyst concentration, but was lower at higher initial concentrations of d-fructose and HMF, and a kinetic model has been derived. The formation of humin was of an overall order 1.3 in an intermediate between d-fructose and HMF, and of an order 1.7 in an intermediate between HMF and levulinic acid.     

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH?HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).     

Oxidation of 5-hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601

Bio-based 5-hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5-furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil-based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5-Hydroxymethylfurfural was converted to 5-formyl-2-furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30–40°C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3-fold higher k_cat/K_m and ~1.7-fold lower K_m with HMF.