谷氨酸棒杆菌H+-ATPase基因失活提高谷氨酸产生量

为了证实在谷氨酸棒杆菌中,利用H⁺-ATPase基因失活构建高产谷氨酸基因工程菌的应用可行性,通过重组PCR技术部分缺失H⁺-ATPaseγ亚基基因序列,采用插入失活方法构建H⁺-ATPase失活的谷氨酸棒杆菌。考察了其谷氨酸产生能力及对生长速率的影响。实验结果表明,H⁺-ATPase失活的谷氨酸棒杆菌在含有100g/L的葡萄糖培养基中摇瓶发酵,其谷氨酸最大累积量为51.6g/L, 比野生菌株提高了42.9%。生长速率研究结果表明,H⁺-ATPase失活的谷氨酸棒杆菌生长速率略低于野生谷氨酸棒杆菌。证实了H⁺-ATPase基因失活对提高谷氨酸产量的作用,为利用H⁺-ATPase基因构建高产谷氨酸基因工程菌株提供了科学依据。     

奥利司他低温溶析结晶条件的优化

为了寻找一种高效的奥利司他结晶工艺,对溶剂种类、晶种、反溶剂加入量、待结晶溶液浓度、搅拌转速和降温速度等因素进行了研究。通过静态结晶实验,确立了甲醇/水为最佳的结晶体系及其比例,加入晶种能明显提高奥利司他的析晶温度,提高产品含量和纯度。在此基础上,经正交试验设计L9(34),通过50 L结晶罐动态考察了各因素对奥利司他结晶产品含量和收率的影响,并进一步优化了加水方式。结果表明:按照2℃/h速率降温,初始奥利司他质量浓度为30 g/L,反溶剂水的加量为25%(体积分数),分2次加入,析晶前加入产品质量0.1%的晶种,搅拌转速250 r/min条件下,所得奥利司他晶体含量和收率最高。经高效液相色谱(HPLC)检测,奥利司他含量可达98.6%,收率可达85.3%。     

TCA循环关键节点对L-谷氨酸合成的影响

谷氨酸是一种重要的氨基酸,其衍生出来的高值化产品具有广泛的应用,市场需求量巨大。文中通过对出发菌株谷氨酸棒杆菌Corynebacterium glutamicum E01和谷氨酸高产菌C. glutamicum G01进行转录组测序与重测序分析,挑选中心代谢途径中转录水平和基因水平上存在差异的基因进行研究,以挖掘出对谷氨酸合成影响较大的基因进一步提高谷氨酸的产量。草酰乙酸节点和α-酮戊二酸节点在谷氨酸合成中扮演着重要角色,探索研究了草酰乙酸节点和α-酮戊二酸节点对谷氨酸生产的扰动影响。综合以上实验结果构建的整合菌株,5 L发酵罐发酵过程中其菌体生长速率较原始菌略有降低,但48h的谷氨酸产量高达(136.09±5.53)g/L,较原始菌的(93.53±4.52)g/L提高了45.5%;糖酸转化率提高至58.9%,较原始菌的45.2%提高了13.7%。可见,上述实验策略的应用在一定程度上提高了谷氨酸产量和糖酸转化率,为谷氨酸棒杆菌的代谢工程改造提供了理论基础。     

外源精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下生长和发酵特性的影响

【目的】探究外源添加不同氨基酸和相容性溶质对谷氨酸棒杆菌(Corynebacterium glutamicum)在高糖胁迫环境下生长的影响及可能的作用机理。【方法】通过在培养基中外源添加各种氨基酸和相容性溶质,研究其对谷氨酸棒杆菌在高葡萄糖和高蔗糖胁迫下生长的影响,并分析添加精氨酸对高葡萄糖胁迫下菌株糖转运和代谢途径中关键酶转录水平的影响,以及对菌株发酵产氨基酸的影响。进一步探究了碱性氨基酸在其它棒状杆菌属中抵御高葡萄糖胁迫的潜在作用。【结果】在高葡萄糖胁迫条件下,外源添加赖氨酸、精氨酸和组氨酸后谷氨酸棒杆菌的生物量分别提高54.7%、50.0%和37.6%;而在高蔗糖胁迫条件下,添加脯氨酸和四氢嘧啶后菌株生物量增加20%以上。进一步研究表明,在高葡萄糖胁迫下,外源添加精氨酸后谷氨酸棒杆菌的葡萄糖利用速率提高约2.5倍,谷氨酸的发酵产量也增加了127.5%。此外,碱性氨基酸对其它4种棒状杆菌也具有一定的渗透保护效应。【结论】精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下具有良好的渗透保护作用,可能归因于其能促进葡萄糖的转运和代谢能力,同时发现碱性氨基酸的渗透保护效应对棒状杆菌属具有一定的普遍性。     

谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

溶解氧对γ-聚谷氨酸合成的影响

在5L发酵罐上研究了溶解氧(DO)对地衣芽孢杆菌分批发酵生产γ-聚谷氨酸(γ-PGA)的影响并考察在8h、32h、56h时,葡萄糖激酶、6-磷酸葡萄糖脱氢酶、丙酮酸脱氢酶、异柠檬酸脱氢酶和谷氨酸脱氢酶的活性及对应时间点上γ-PGA的生产速率。通过溶解氧电极和搅拌转速的串联控制发酵过程中溶解氧水平,发现高溶解氧(60%)水平和低溶解氧(10%)水平均不能高效积累γ-PGA。6-磷酸葡萄糖脱氢酶活性的提高对产物的积累有抑制作用,葡萄糖激酶和谷氨酸脱氢酶的酶活提高对产物积累有促进作用,过高的丙酮酸脱氢酶和异柠檬酸脱氢酶的活性在一定程度上可以促进菌体生长但不利于产物的积累。此外,通过对三种不同DO水平下γ-PGA生物合成途径中相关代谢流量的计算表明,在p H 6.5的条件下,对于谷氨酸依赖型生产菌株,提高外源谷氨酸利用率可以促进γ-PGA的生物合成。     

界面立构复合诱导聚乳酸/弹性体复合材料的超韧化及快速结晶

为了提高聚乳酸(PLA)的韧性和结晶速率,笔者对环氧化乙烯-醋酸乙烯酯弹性体(EVMG)接枝右旋聚乳酸(PDLA)并与左旋聚乳酸(PLLA)基体熔融共混,制备复合材料。结果表明:接枝的PDLA与PLLA基体在相界面原位形成了立构复合晶体,显著提高了弹性体与基体之间的界面作用力,从而赋予共混物优异的韧性,使PLLA的缺口冲击强度由1.1 kJ/m~2提高至68.7 kJ/m~2。同时,立构复合晶体还显著提高了PLLA基体的结晶速率,使PLLA在120℃条件下的半结晶时间缩短了81%。     

超声波对木质纤维素糖化过程影响的研究

将超声波应用在木质纤维素预处理及其酶解糖化过程中,通过SEM、FTIR研究了处理前后纤维素的形态结构和结晶性能,并考察了不同预处理方式对原料 成分的影响和超声波对酶解糖化率的影响。结果表明,超声波作用能有效的破坏纤维素分子中的氢键,降低其结晶程度,而且能有效地提高木质素的脱除率和酶解糖化率。对超声波作用于酶解过程中的机理进行了初步探讨     

利用超声波促进苯胺生物降解

报道了用超声波降解苯胺的试验 ,探讨了超声波辐射时间、溶液中苯胺的初始浓度、溶液温度、pH等因素对苯胺降解效果的影响。试验表明 ,超声波辐射的较适时间为 8min ,苯胺的较适浓度为 15 33mg/L ,适宜降解的培养条件为 ,温度 30℃ ,pH 7.5 ,培养时间 12d。此条件下的苯胺最高降解率可达 94 .2 %。     

超声强化生化分离技术应用进展

将超声波的空化作用、热效应、机械作用应用于生化分离技术中,可提高分离效率、缩短时间、简化操作过程。综述了超声波在生化分离如天然成分提取、沉淀、萃取、结晶等各种分离方法中的应用现状,并探讨了超声波与分离方法结合的发展前景。     

Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Metabolites of -[¹⁴C]proline were found in the trichloroacetic acid-soluble fraction of 16-day-old chick embryo frontal bones. In several ion-exchange procedures these metabolites interfered with the analysis of hydroxyproline derived from the metabolic breakdown of collagen. The major metabolite was identified as glutamic acid by its chromatographic and crystallization properties. It was eluted from AG50 cation-exchange resin with 1.0 HCL in the hydroxyproline region, but was separated from hydroxyproline on a DC-6A column in the amino acid analyzer. Another metabolite was identified as aspartic acid. It was not separated from hydroxyproline on either AG50 using 1 HCL for elution or on DC-6A using 0.1 sodium citrate, pH 3.25, for elution, but adequate separation was obtained by elution with 0.2 sodium citrate buffer at pH 2.91. Formation of these metabolites was not related either to protein synthesis or proline hydroxylation. Therefore, it is possible to analyze for hydroxyproline accurately by using a separate unhydroxylated sample to correct for the presence of the metabolites. The formation of glutamic acid suggested that proline oxidase activity might be present in bone tissue, but none was detected using a sensitive radioisotopic assay. Although the amount of radioactivity found in the metabolites was 36% of the amount of [¹⁴C]proline incorporated into protein, no radioactive glutamic or aspartic acid was present in protein hydrolyzates. This observation suggests that the metabolites did not enter the major amino acid pool used for protein synthesis.     

Biliary secretion of cefoperazone and its inter-relationship with bile acid transport in the rat

Cefoperazone is a third generation cephalosporin which is secreted predominantly in bile. This study set out to examine the effect of stimulating bile choleresis on the biliary secretion of cefoperazone. Stimulation of both bile acid-dependent and independent bile flow (phenobarbitone pretreatment) hastened the peak appearance of a pulse of cefoperazone into bile. Although the biliary secretion rate of cefoperazone was enhanced by bile acid infusion, the % recovery and maximal biliary concentration were reduced. The reciprocal effect of continuous cefoperazone infusion on the rate of biliary transport of a pulse of bile acid was examined. Cefoperazone infusion hastened the biliary transport of glycocholate. Net recovery of glycocholate was unaffected.     

Characterization and in vitro mineralization function of a soluble protein complex P60 from the nacre of Pinctada fucata

A soluble protein complex P60 from the powdered nacre of Pinctada fucata was extracted and partially characterized. The biological activity of the P60 on pre-osteoblast cell line MC3T3-E1 and bone marrow stroma cells (MSCs) was investigated. The P60 protein from the decalcified powered nacre was solubilized with acetic acid and then purified by liquid chromatography. The P60 protein was a protein complex composed of several subunits with disulfide bridges. The known protein nacrein, and its two derivatives, N28 and N35, were included in the P60 protein complex. The most abundant amino acids in the P60 that account for 68.3% of the total residues are glycine (32.1%), aspartic acid (17.4%), alanine (13.6%), and glutamic acid (5.2%). The in vitro study of the crystallization showed that this protein complex could control the formation and size of calcium carbonate. The assay of biological activity of the P60 protein complex on the pre-osteoblast cell line MC3T3-E1 and MSCs suggested that the P60 could stimulate the formation of mineralized nodules.     

Hormone mediated changes of brain glutamic acid system in amino acid imbalance

The effects of adrenal cortical hormone and thyroxine on brain glutamic acid, gamma-amino butyric acid (GABA) and glutamine were studied in rats fed on the amino acid imbalanced diet (8% casein diet supplemented with 0.3% L-threonine). The studies revealed that the decrease in brain glutamic acid and GABA levels in threonine imbalance was recovered by hydrocortisone supplementation. The increased level of brain glutamine in threonine imbalance could not, however, be reversed by hydrocortisone supplementation. Thyroxine supplementation was found to have no impact on any of the members of glutamic acid family in the brain of rats receiving the threonine-imbalanced diet. It was suggested that the decreased levels of brain glutamic acid and GABA in threonine imbalance were caused by diminished adrenal cortical function and the influence of adrenal cortical hormone could be suggested to reside at the level of formation of both glutamic acid and GABA.     

Effects of Chemical Components on the Browning of Tomato Juice

The effects of eleven components in tomato juice on browning by heating were examined at the concentrations found in juice, About 70% of the browning of tomato juice was attributable to three constituents; fructose, galacturonic acid and glutamic acid. The interaction between glutamic acid and galacturonic acid or fructose was also found to accelerate the browning of tomato juice. The browning by heating of galacturonic acid with or without amino acids was larger than that of sugars or amino acids. The browning mechanism of galacturonic acid on heating at high temperature was discussed.     

氮源对聚γ-谷氨酸合成与分泌的影响

研究了无机与有机氮源对聚γ-谷氨酸合成与分泌的影响。分别在基础培养基及进入生物合成期的发酵液中添加氯化铵、硝酸钠和各种氨基酸,基础培养基中添加氯化铵质量浓度为1 g.L-1时,合成量提高了22.7%,同时,氯化铵对聚γ-谷氨酸组分也有一定影响。在培养至24 h添加8 g.L-1硝酸钠,合成量提高54.6%,而在基础培养基中分泌率提高了32.7%。在生物合成期添加8 g.L-1谷氨酸,合成量提高了64.2%。同时在静息细胞培养基中进行了验证实验。     

Effect of hyperoxia on glutathione levels and glutamic acid uptake in endothelial cells

Intracellular glutathione was increased by 80% after exposure of bovine pulmonary arterial endothelial cells to 80% O2 (hyperoxia) for 24 h. No change in glutathione occurred in cells exposed to hypoxia (3% O2) for a corresponding period of time. The rate of uptake of [3H]glutamic acid also increased by 35-55% after 24 h of exposure of cells to hyperoxia, whereas exposure to hypoxia had no effect on the [3H]glutamic acid uptake. The increase in glutamic acid uptake reflected a specific effect on amino acid transport systems rather than a change in cell membrane permeability. The major portion of the increased uptake was inhibited by the elimination of sodium and the addition of the competitive inhibitor, cystine, to the incubation medium. Thus increases in glutamic acid uptake parallel increases in cellular glutathione, and glutamic acid may be a regulating factor in the increase in glutathione after exposure to hyperoxia.     

Effects of static and low-frequency alternating magnetic fields on the ionic electrolytic currents of glutamic acid aqueous solutions

A current peak has been observed and measured in ionic electrolytic current of a glutamic acid aqueous solution, placed in a static magnetic field of flux density of 40 microT, with a superimposed low-frequency alternating magnetic field of flux density of 40 nT. The peak occurs at the frequency of the cyclotronic resonance of the molecular mass of a single charged glutamic acid ion, placed in a magnetic flux density equal to that of the static field. The amplitude of the current peak is about 30% of the background electrolytic current. Qualitative considerations and a listing of unsolved problems related to the phenomenology are given. The result is the first contribution to the study we have undertaken on the effects of low-frequency alternating electromagnetic fields on the ionic current of amino acid aqueous solutions which are the basic structural units of the proteins.     

Effect of nicotine on the level of extracellular amino acids in the hippocampus of rat

Using microdialysis, we compared intracerebral and subcutaneous administration of nicotine for the effect on the levels of extracellular amino acids in the hippocampus of anesthetized rats. Administration by microdialysis of 10 mM nicotine, resulting in a nicotine concentration of 0.134 μmol/g in the hippocampus, increased the extracellular levels of aspartic acid, glutamic acid, and serine by 26–60%. At 50 mM nicotine the increases in the levels of aspartic acid, glutamic acid, serine, glycine, and glutamine were between 76% and 141%. Subcutaneous administration of nicotine at a dose of 6 μmol/kg caused a 57% increase in the extracellular level of glutamic acid. After a dose of 12 μmol/kg that resulted in a nicotine level of 0.015 μmol/kg in the hippocampus, the extracellular level of glutamic acid was increased by 100%, and that of aspartic acid by 24%. Thus, higher cerebral nicotine levels were needed with intracerebral than with subcutaneous administration to obtain similar amino acid changes. Prior administration of mecamylamine or L-kynurenine prevented the subcutaneous nicotine-induced elevation of the extracellular levels of aspartic acid and glutamic acid. Our results indicate that receptor interactions modulate nicotine effects and that both nicotinic cholinergic and NMDA/glycine glutamatergic receptors participate in the action of nicotine in increasing extracellular amino acid levels.     

Operating parameters for glutamic acid crystallization in displacement ion exchange chromatography

Glutamic acid can be crystallized inside cation exchange column when displacer NaOH concentration is high enough to concentrate displaced glutamic acid beyond its solubility limit. Resulting crystal layer of glutamic acid was moved with liquid phase through the column, and thus could be eluted from the column and recovered in fraction collector. For the purpose of enhancing crystal recovery, effects of operating parameters on the crystal formation were investigated. The increase in the degree of crosslinking of resin favored crystal recovery because of its low degree of swelling. Higher concentration of displacer NaOH was advantageous. If NaOH concentration is too high, however, crystal recovery was lowered due to the solubility-enhancing effects of high pH and ionic strength. The decrease of mobile phase flow rate enhanced crystal recovery because enough time to attain local equilibrium could be provided, but film diffusion would control the overall crystal formation with extremely low flow rate. Lower temperature reduced solubility of glutamic acid and thus favored crystal formation unless the rate of ion exchange was severely reduced. The ion exchange operated by displacement mode coupled with crystallization was advantageous in reducing the burden of further purification steps and in preventing purity-loss resulted from overlapping between adjacent bands.