海藻糖合酶的分子生物学研究进展

海藻糖合酶能够将麦芽糖转化为海藻糖,在海藻糖的工业生产中具有十分重要的意义。本文从海藻糖合酶的基因克隆、基因工程应用、结构和催化机制的研究以及其在微生物体内的功能等方面讨论了海藻糖合酶的研究进展。     

鲁氏酵母胞内海藻糖积累过程的代谢特征分析

海藻糖合成是微生物对抗环境逆境的一种重要途径。研究10L发酵罐中的分批、分批补料及分批补料控温三种不同的海藻糖发酵调控策略下酱油风味形成微生物鲁氏酵母CCTCC M2013310的代谢特征。色谱结果表明,乳酸、丙酮酸和α-酮戊二酸受到不同发酵调控模式的显著影响,但谷氨酸和谷氨酰胺总含量在三种发酵调控模式间却无显著差异。这些结果表明,细胞还原力平衡途径和碳氮调控代谢均对胞内海藻糖的积累产生影响。研究结果为鲁氏酵母CCTCC M2013310的高浓度内源性海藻糖细胞代谢工程改造提供了新思路。     

海藻糖对微生物谷氨酰氨转胺酶热稳定性研究

本实验目的是研究海藻糖对微生物谷氨酰胺转胺酶（TGase）热稳定性的作用。糖类对TGase的保护作用根据糖种类不同有所差异,海藻糖和蔗糖的保护作用优于葡萄糖对TGase的保护作用。在45℃、50℃、55℃、60℃、65℃下研究了海藻糖对TG酶的保护作用。结果表明,在50～65℃下海藻糖使谷氨酰胺转胺酶受热时的稳定性提高了约20％。海藻糖与酶复合的最合适浓度约为14％,浓度低时保护作用不明显,加入过高浓度的糖对酶的活性维持不利。50℃下处理一段时间内,海藻糖对酶的保护作用随时问变化很小。     

相容性溶质对强化耐盐苯酚降解菌群的影响

文章探究了两种不同相容性溶质（甜菜碱、海藻糖）对强化耐盐苯酚降解菌群的影响。结果表明，在驯化过程中，海藻糖的促进效果优于甜菜碱，添加5 mM海藻糖驯化得到的微生物菌群在1%的盐度条件下，18 d后对200 mg/L苯酚的降解率为100%，累计产气8 m L，与对照组相比，显著提升了微生物菌群的降解效果。这表明，在驯化过程中添加海藻糖可以提高微生物降解效率。     

海藻糖代谢途径相关基因及生物工程

海藻糖(Trehalose)是一种由两个葡萄糖分子通过α,α-1,l糖苷键连接的非还原性双糖。最早的记录是在19世纪初期作为黑麦的麦角菌的一种成分而被描述,后来发现海藻糖广泛存在于微生物、动物和植物体内,特别是在那些能抗脱水作用的生物中起着重要作用。这些特殊生物具有在脱水条件下存活多年的性质,包括所谓的“复苏植物”(Selaginella lepidophylla)、某些咸水虾、线虫及面包酵母等。当它们体内99％的水分被去掉之后,仍保持着能在获水后迅速复活的能力^[1]。研究表明,海藻糖对于生物抗逆具有重要的保护作用。海藻糖的应用研究因此得到了人们的广泛关注和重视,目前海藻糖已被用作酶、其它蛋白、生物制品甚至移植器官的保护剂。海藻糖作为生物体对抗环境胁迫的重要应激保护物质,在不同生物中存在多种合成和分解代谢途径,相关基因已相继被克隆和分析。海藻糖合成、分解及其调控是生物抗逆的重要机制,其相关基因的研究也是海藻糖生物工程的重要基础。     

月产100公斤海藻糖的设备

日商岩井系砂糖制造厂——富士制糖公司9月底完成月产100公斤海藻糖的制造设备,首先向食品企业等使用者供应。该公司与静风县沼津工业技术中心和静冈大学理学部等合作开发了用一种新发现的酶作用于麦芽糖生产海藻糖的技术。曾于20年前使用磷酸化酶进行海藻糖的酶合成等研究的熊本工业大学应用微生物工学科教授村尾泽夫指导该项技术开发。     

微生物生产海藻糖及其应用前景

微生物生产海藻糖及其应用前景罗明典（中国北京科学院微生物研究所,北京１０００８０）在自然界有些生物能够在脱水情况下连续数年保持生命状态,一旦遇水又重新恢复生机,其重要原因在于这些生物细胞含有大量海藻糖之故,这类糖由两个葡萄糖分子聚合组成的双糖,分子式...     

海藻糖强化高盐胁迫下异养硝化-好氧反硝化菌群的代谢机制

异养硝化-好氧反硝化(heterotrophic nitrification-aerobic denitrification, HN-AD)菌是一类可在高盐环境脱氮的好氧微生物,但其工程应用效果不理想。海藻糖作为相容性溶质,通过参与调节细胞渗透压帮助微生物抵抗高盐胁迫,对提升高盐环境菌群的脱氮效率起重要作用。本研究通过启动膜曝气生物膜反应器(membrane aerobic biofilm reactor, MABR)富集HN-AD菌,设计添加150μmol/L海藻糖的C150实验组和未添加海藻糖的C0对照组,开展了外源性海藻糖对高盐胁迫下HN-AD菌群代谢的强化机制研究。反应器运行性能及群落结构分析结果显示,C150组相较C0组,NH4+-N、总氮(total nitrogen, TN)和化学需氧量(chemical oxygen demand, COD)去除率分别提高29.7%、28.0%和29.1%;以不动杆菌属(Acinetobacter)和假黄褐藻属(Pseudofulvimonas)为优势菌属的耐盐型HN-AD菌群总相对丰度在C150组达到66.8%、较C0组提高了18.2...     

组学技术在产油微生物中的应用

微生物油脂是未来燃料和食品用油的重要潜在资源。近年来,随着系统生物学技术的快速发展,从全局角度理解产油微生物生理代谢及脂质积累的特征成为研究热点。组学技术作为系统生物学研究的重要工具被广泛用于揭示产油微生物脂质高效生产的机制研究中,这为产油微生物理性遗传改造和发酵过程控制提供了基础。文中对组学技术在产油微生物中的应用概况进行了综述,介绍了产油微生物组学分析常用的样品前处理及数据分析方法,综述了包括基因组、转录组、蛋白(修饰)组及代谢(脂质)组等在内的多种组学技术,以及组学数据基础上的数学模型在揭示产油微生物脂质高效生产机制中的研究,并对未来发展和应用进行了展望。     

海藻糖及其在微生物领域中的应用

海藻糖是生物体低湿休眠及微生物、生物大分子耐热抗干燥的稳定物质,８０年代以来受到国内外广泛关注并做了大量细致研究,本文详细介绍了这种独特双糖的理化及分子生物学特性、功能和作用机理以及它的制备和应用前景。     

The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state

This essay is an introduction to a series of papers arising from a symposium on stabilization of cells in the dry state. Nearly all of these investigations have utilized the sugar trehalose as a stabilizing molecule. Over the past two decades a myth has grown up about special properties of trehalose for stabilization of biomaterials. We review many of such uses here and show that under ideal conditions for drying and storage trehalose has few, if any, special properties. However, under suboptimal conditions trehalose has some distinct advantages and thus may remain the preferred excipient. We review the available mechanisms for introducing trehalose into the cytoplasm of living cells as an introduction to the papers that follow.     

Internal trehalose protects endocytosis from inhibition by ethanol in Saccharomyces cerevisiae

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.     

Internal Trehalose Protects Endocytosis from Inhibition by Ethanol in Saccharomyces cerevisiae

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.     

Trends in bacterial trehalose metabolism and significant nodes of metabolic pathway in the direction of trehalose accumulation

The current knowledge of trehalose biosynthesis under stress conditions is incomplete and needs further research. Since trehalose finds industrial and pharmaceutical applications, enhanced accumulation of trehalose in bacteria seems advantageous for commercial production. Moreover, physiological role of trehalose is a key to generate stress resistant bacteria by metabolic engineering. Although trehalose biosynthesis requires few metabolites and enzyme reactions, it appears to have a more complex metabolic regulation. Trehalose biosynthesis in bacteria is known through three pathways – OtsAB, TreYZ and TreS. The interconnections of in vivo synthesis of trehalose, glycogen or maltose were most interesting to investigate in recent years. Further, enzymes at different nodes (glucose‐6‐P, glucose‐1‐P and NDP‐glucose) of metabolic pathways influence enhancement of trehalose accumulation. Most of the study of trehalose biosynthesis was explored in medically significant Mycobacterium, research model Escherichia coli, industrially applicable Corynebacterium and food and probiotic interest Propionibacterium freudenreichii. Therefore, the present review dealt with the trehalose metabolism in these bacteria. In addition, an effort was made to recognize how enzymes at different nodes of metabolic pathway can influence trehalose accumulation.     

Inhibition of yeast glutathione reductase by trehalose: possible implications in yeast survival and recovery from stress

Accumulation of trehalose has been implicated in the tolerance of yeast cells to several forms of stress, including heat-shock and high ethanol levels. However, yeast lacking trehalase, the enzyme that degrades trehalose, exhibit poor survival after exposure to stress conditions. This suggests that optimal cell viability also depends on the capacity to rapidly degrade the high levels of trehalose that build up under stress. Here, we initially examined the effects of trehalose on the activity of an important antioxidant enzyme, glutathione reductase (GR), from Saccharomyces cerevisiae. At 25 degrees C, GR was inhibited by trehalose in a dose-dependent manner, with 70% inhibition at 1.5M trehalose. The inhibition was practically abolished at 40 degrees C, a temperature that induces a physiological response of trehalose accumulation in yeast. The inhibition of GR by trehalose was additive to the inhibition caused by ethanol, indicating that enzyme function is drastically affected upon ethanol-induced stress. Moreover, two other yeast enzymes, cytosolic pyrophosphatase and glucose 6-phosphate dehydrogenase, showed temperature dependences on inhibition by trehalose that were similar to the temperature dependence of GR inhibition. These results are discussed in terms of the apparent paradox represented by the induction of enzymes involved in both synthesis and degradation of trehalose under stress, and suggest that the persistence of high levels of trehalose after recovery from stress could lead to the inactivation of important yeast enzymes.     

Trehalose: A Key Organic Osmolyte Effectively Involved in Plant Abiotic Stress Tolerance

Trehalose is a natural non-reducing sugar that is found in the vast majority of organisms such as bacteria, yeasts, invertebrates and even in plants. Regarding its features, it is considered as a unique compound. It plays a key role as a carbon source in lower organisms and as an osmoprotectant or a stabilizing molecule in higher animals and plants. Although in plants it is present in a minor quantity, its levels rise upon exposure to abiotic stresses. Trehalose is believed to play a protective role against different abiotic stressful cues such as temperature extremes, salinity, desiccation. Moreover, it regulates water use efficiency and stomatal movement in most plants. Detectable endogenous trehalose levels are vital for sustaining growth under stressful cues. Exogenously applied trehalose in low amounts mitigates physiological and biochemical disorders induced by various abiotic stresses, delays leaf abscission and stimulates flowering in crops. External application of trehalose also up-regulates the stress responsive genes in plants exposed to environmental cues. The genetically modified plants with trehalose biosynthesis genes exhibit improved tolerance against stressful conditions. An increased level of trehalose has been observed in transgenic plants over-expressing genes of microbial trehalose biosynthesis. However, these transgenic plants display enhanced tolerance to heat, cold, salinity, and drought tolerance. Due to multiple bio-functions of this sugar, it has gained considerable ground in various fields. However, exogenous use of this bio-safe sugar would only be possible under field conditions upon adopting strategies of low-cost production of trehalose. In short, trehalose is a unique chemical that preserves vitality of plant life under harsh ecological conditions. Certainly, the new findings of this disaccharide will revolutionize a wide array of new avenues.

     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.     

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.