米根霉利用木糖与葡萄糖的代谢差异

[目的]木质纤维素是世界上储量最丰富、最廉价的可再生生物质资源,以米根霉为研究对象,探讨对木质纤维素中主要单糖成分--木糖和葡萄糖的代谢差异,为木质纤维素的高效利用提供科学依据.[方法]分别以木糖和葡萄糖为碳源,考察米根霉的生物量、细胞大分子组分、胞内还原力(NADH/NAD+)、ATP含量以及有机酸积累的差异.[结果...     

离子注入选育高产L-乳酸的米根霉突变株及其发酵特性研究

通过氮离子注入获得米根霉突变株RQ4012,其利用木糖的能力比出发菌株提高了1.6倍;通过多次传代,证明其具有良好的遗传稳定性。试验测定菌株RQ4012发酵木糖生产L-乳酸的最佳发酵条件:木糖10%,生理盐水浸泡孢子9 h,(NH4)2SO43 g/L,接种量4%,CaCO3添加量6%,装液量20%,温度37℃,转速200 r/min,在此条件下,乳酸产量达到79.51 g/L。对混合糖的发酵进行了探索,结果表明该菌能高效利用混合糖生产L-乳酸,在利用植物纤维素水解液生产L-乳酸上有良好的应用前景。     

米根霉富马酸酶三维结构的同源建模与分析

利用Modeller7v7软件对米根霉（Rhizopus oryzoe）富马酸酶（fumarase）进行了三级结构的同源建模并对结果的空间和能量上的合理性进行了验证,进一步对酶的结构域和催化活性位点进行了研究。结果表明：富马酸酶由三个结构域组成,中心区域为一个由五个几乎平行的α螺旋组成的独特的束型结构,其催化活性位点是由三个亚基上的氨基酸相互靠近共同组成的。为以后有针对性的进行富马酸酶的定点突变提高富马酸产量提供分子水平上的理论指导。     

米根霉ME—F12发酵产富马酸的菌体形态控制

丝状真菌发酵体系中菌体形态对产量有着重要影响。考察富马酸产生菌Rhizopus oryzae ME—F12种子培养过程中不同pH条件、孢子悬浮液密度以及CaCl2添加量对其形态的影响。结果表明,当控制种子培养液pH2．3～2．7、接种孢子的终密度为1．5×10^8～3．0×10^8／L和添加0．5g／LCaCl2时,培养可获得直径约为0．65mm光滑规整茵球,后继的产酸发酵中富马酸量高达58．9g／L。正交实验表明,pH是影响菌球形成的最主要因素,孢子液密度主要影响菌体生物量,而CaCl2则是菌球表面光滑度的主要影响因素。     

米根霉利用纯糖和不同预处理玉米秸秆酶解糖生产L-乳酸

通过单因素实验设计,优化米根霉摇瓶发酵产L-乳酸。在此基础上,以蒸气爆破和碱处理玉米秸秆酶解液为混合C源,与纯糖对比,研究不同预处理玉米秸秆混合C源对米根霉发酵产L-乳酸的影响。结果显示:在初始葡萄糖质量浓度100g/L、(NH4)2SO4质量浓度2g/L、接种量6%(体积分数)、转速170r/min、发酵12h后添加30g/LCaCO3的条件下,米根霉发酵产L-乳酸质量浓度为69.15g/L。米根霉发酵不同预处理玉米秸秆酶解混合C源,木糖的存在影响了米根霉的C代谢网络,降低L乳酸的产量。     

膜反应器固定化米根霉发酵产富马酸的工艺研究

以膜反应器固定化米根霉发酵产富马酸为研究对象,以Na2CO3为中和剂,考察固定化米根霉在5L搅拌式发酵罐中的发酵特征,采用智能可视化软件(IVOS)优化发酵工艺条件。结果表明,在80g/L初始糖浓及最优工艺下,富马酸产量、生产速率及转化率分别为21.1g/L、0.25g/(L·h)和28%;采用40g/L初始糖浓及连续批次发酵工艺时,富马酸产量、生产速率及转化率最高分别为10.8 g/L、0.36g/(L·h)和27%。搅拌式反应器中,固定化米根霉的膜反应器比表面积有限,以及菌膜的空间阻隔效应对传质传氧的限制作用,显著影响了富马酸的生产强度和转化率。因此,亟需发掘新的固定化方法及反应器形式,达到既解决米根霉形态控制问题,又有助于生产性状提升的目标。     

碳源与氮源限制下细菌代谢调节研究进展

营养限制是微生物最常面临的环境胁迫之一。除了在营养物质匮乏的海洋、冰川、沙漠、深层地表等自然环境中,越来越多的人工环境也出现了营养限制的特征,例如各类微污染水体、提标改造的废水生物处理系统等。基质浓度极大地影响着包括细菌在内的许多微生物的生长、代谢及群落结构,最终导致其功能的改变。为了在营养限制条件下维持生存,微生物首先需感知营养供给的减少,其后通过基因、蛋白质、信号分子、代谢产物等对各代谢过程进行全局调控,最后改变基质亲和力、生长速率、运动能力、形态等以适应营养不足。胞内各种信号物质及其触发的响应是微生物应对营养胁迫的关键。本文分别梳理了以细菌为代表的微生物应对碳源、氮源限制时的关键信号物质、受体蛋白/调控过程及响应结果,并分析了碳氮限制响应过程中的相互作用,以期为极端环境微生物的认识、营养限制条件下微生物的应用,尤其是低浓度污染物生物处理、生物监测等领域提供理论基础。     

利用固定化米根霉在三相流化床中发酵生成L-乳酸

用聚氨酯泡沫吸附固定米根霉菌丝,在三相流化床中对葡萄糖、木糖以及木糖渣的纤维素酶解液等不同碳源进行L乳酸发酵研究,并对游离菌丝和固定化菌丝发酵L乳酸进行了比较。结果表明,聚氨酯泡沫是米根霉的良好载体,具有经济、高效等特点。实验条件下,不同碳源的乳酸转化率分别为：葡萄糖,82.5%;木糖,53.8%;木糖渣酶水解液,71.9%。三相流化床中固定化米根霉产酸速率(对葡萄糖)为191g.h-１.L(bead)-1。     

积累L-苹果酸的米根霉突变株酶活性初探

对富马酸产生菌株—米根霉ME-F10进行诱变育种的过程中, 得到一株性能稳定的高效积累L-苹果酸的突变株ME-M15。该菌株发酵96 h平均L-苹果酸产量达16.3 g/L, 较出发菌株L-苹果酸积累量平均提高3倍, 而富马酸和乙醇的积累量大幅下降。对突变株代谢途径关键酶活研究表明, 突变株富马酸酶胞质途径同功酶和乙醇脱氢酶活力较之出发菌株酶活力明显减弱, 而丙酮酸羧化酶活力无明显差别。     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

Dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in Ralstonia eutropha

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation. The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.     

Prenyltransferases as key enzymes in primary and secondary metabolism

Attachment of isoprene units to various acceptors by prenylation plays an important role in primary and secondary metabolism of living organisms. Protein prenylation belongs to posttranslational modification and is involved in cellular regulation process. Prenylated secondary metabolites usually demonstrate promising biological and pharmacological activities. Prenyl transfer reactions catalyzed by prenyltransferases represent the key steps in the biosynthesis and contribute significantly to the structural and biological diversity of these compounds. In the last decade, remarkable progress has been achieved in the biochemical, molecular, and structural biological investigations of prenyltransferases, especially on those of the members of the dimethylallyltryptophan synthase (DMATS) superfamily. Until now, more than 40 of such soluble enzymes are identified and characterized biochemically. They catalyze usually regioselective and stereoselective prenylations of a series of aromatic substances including tryptophan, tryptophan-containing peptides, and other indole derivatives as well as tyrosine or even nitrogen-free substrates. Crystal structures of a number of prenyltransferases have been solved in the past 10 years and provide a solid basis for understanding the mechanism of prenyl transfer reactions.     

Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

高温对早籼稻花后剑叶氮代谢关键酶活性及籽粒蛋白质含量的影响

为探讨高温对不同类型早籼稻开花后剑叶中氮代谢关键酶活性及籽粒蛋白质含量的影响,利用人工气候室在籽粒灌浆成熟期进行高温(日均温31.5 ℃)和适温(日均温23.5 ℃)处理,对2个早籼稻品种(湘早籼24和株两优611)灌浆成熟期籽粒中蛋白质含量及剑叶中GS和GOGAT活性的动态变化进行了分析.结果表明:供试2个品种在高温...     

Effect of cobalt chloride on the activity of key enzymes of heme metabolism in the rat liver

The effects of actinomycin D and cycloheximide on the original and CoCl2-induced delta-aminolevulinate-synthase and heme-oxygenase activities in rat liver were investigated. It was shown that 1.5 hours after CoCl2 administration the delta-aminolevulinate-synthase activity diminishes, then increases up to the original level within the subsequent 4.5 hours, showing a further increase thereafter. The heme-oxygenase activity does not change within 1,5 hours and is then increased. Actinomycin D and cycloheximide suppress the increase in the CoCl2-induced heme-oxygenase activity, whereas that of the delta-aminolevulinate-synthase activity is blocked only by cycloheximide. Hence, the increase in the CoCl2-induced delta-aminolevulinate-synthase activity in the liver is a result of activation of translation. The degree of tryptophan pyrrolase saturation with heme decreases already by the 6th hour, whereas the level of heme in liver mitochondria and microsomes decreases only by the 15th hour following CoCl2 injection. The heme content in the liver shows a further decrease irrespective of the increase in the delta-aminolevulinate synthase activity induced by CoCl2. It may be concluded that under the given experimental conditions this enzyme is not a rate-limiting step in the terminal reaction of heme biosynthesis.     

Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation.

To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame. Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths. These genes were expressed in Saccharomyces cerevisiae. The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2. The specific activity of the XDH part of the complexes increased when longer peptide linkers were used. Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers. No degradation products were detected by Western blot analysis. S. cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation. When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S. cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed. The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually. Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol. However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher. The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction.     

Insulin-dependent production of low-molecular-weight compounds that modify key enzymes in metabolism

The mechanism by which the metabolic effects of insulin are transmitted is yet to be resolved. Second messengers mediating the action of insulin have been proposed and recently an insulin-dependent, peptide-like, heat- and acid-stable substance (Mr approx. 1000-3000) released from plasma membranes has been described which regulates the activity of key enzymes such as pyruvate dehydrogenase by altering its state of phosphorylation. It has been suggested that this material is the elusive second messenger of insulin and its discovery, generation, properties, isolation and mode of action are reviewed.     

On the stability of key enzymes of energy metabolism in muscle biopsies

Enzymes of energy metabolism were tested for stability depending on different storage conditions (-20, -80 degrees C). To avoid problems due to the different fiber type composition of human muscle, we selected two muscles from rabbit. The m. psoas consists almost exclusively of type 2B fibers, and the m. soleus consists almost exclusively of type 1 fibers. Enzyme activities were measured from small aliquots of these muscles at various time points up to 1 year after sacrificing the animal. Enzymes from anaerobic metabolism were stable for more than 1 year, independent of whether the muscle was stored at -20 or -80 degrees C. Oxidative enzymes, such as succinate dehydrogenase, citrate synthetase, or cytochrome c oxidase (COX) decrease in activity at -20 degrees C and, to a lesser degree, at -80 degrees C. In addition, mitochondria were isolated from freshly taken muscle and stored at -80 degrees C. Oxidative enzymes were surprisingly stable for more than 1 year, with the exception of COX which decreased by 60% of its original activity in mitochondria from m. soleus.