温度调控大肠杆菌发酵合成L-丙氨酸

正L-丙氨酸是最小的手性分子之一,被用于医药和兽药行业,与其他L-型氨基酸共同用作手术前和手术后的营养剂[1]。由于L-丙氨酸具有甜味,也被用于食品添加剂[2]。目前,L-丙氨酸全球需求年增长率为20%,主要增长地区是亚洲、北美等。然而,L-丙氨酸产量和价格基本被日本垄断和控制。国内企业生产规模小,生产菌种陈旧低效,L-丙氨酸的供应量远低于市场需求量。     

温度对大肠杆菌L-色氨酸发酵过程的影响

目的：研究变温控制对大肠杆菌TRTH L-色氨酸补料分批发酵过程中生物量、色氨酸产量、比生长速率及质粒稳定性的影响。方法：利用5L自控发酵罐对L-色氨酸补料分批发酵过程进行温度控制,对不同温度下相关参数进行分析比较,确定优化的温度控制方案。结果：以30-36％顺序升温的工艺进行发酵得到理想结果,与单一温度控制策略相比,L-色氨酸产量提高了15．4％;色氨酸的比合成速率提高了21．6％;质粒稳定性增加,未出现质粒丢失现象,质粒拷贝数保持在恒定水平。结论：温度对大肠杆菌L-色氨酸发酵有重要影响。     

模块化工程改造大肠杆菌生产L-色氨酸

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein, trpR)、替换l-色氨酸弱化子(trpL)、引入抗反馈调节的aroG^fbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid, SA)途径至分支酸(chorismic acid, CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为l-色氨酸的规模化生产奠定了良好的基础。     

大肠杆菌发酵生产L-色氨酸工艺简析

本文对L-色氨酸进行了简要概述,指出利用大肠杆菌工程菌直接发酵生产L-色氨酸为国内主流方法,并对其成熟的发酵工艺控制、提取工艺进行了简析,并指出部分可进一步优化的工艺点。其中发酵工艺简析包括菌种培养基增加一定溶度抗生素和控制发酵温度来控制质粒稳定性;分析物料作用并提出优化后的种子、发酵培养基组成;菌种无需控制溶氧,而发酵则用溶氧反馈补料;控制乙酸和氨氮浓度、顺序升温缩短周期降低抑制性副产物作用。分离提取工艺简析包括硫酸酸化p H2-3,陶瓷膜过滤并控制滤液平均单位为14000-18000u/ml,阳离子树脂纯化,醋酸调p H5.89,0.5%活性炭60℃脱色20-30min,蒸发浓缩结晶,纯化水洗涤整条工艺路线。     

家蚕丙氨酸转氨酶的纯化与鉴定

应用细胞匀浆,硫酸铵分段盐析,DEAE-纤维素柱层析和羟基磷灰石柱层析等方法,从家蚕后部丝腺中成功地分离制备了高纯度的丙氨酸转氨酶,经聚丙烯酰胺凝胶电泳分析鉴定,本法制备的丙氨酸转氨酶已达到均一的纯度。     

重组大肠杆菌生产L-色氨酸发酵条件优化

为了提高重组大肠杆菌FB讲／pSV－04发酵生产L－色氨酸的产量,减少代谢副产物乙酸的生成,考察了比生长速率和无机盐对重组大肠杆菌发酵生产L．色氨酸的影响。在确定了合适的比生长速率和无机盐浓度之后,乙酸积累很少,L-色氨酸的产量为53．4g／L,比优化前提高了141．6％。经30L发酵罐初步放大,L－色氨酸的产量达53．6g／L,发酵结果稳定,具有工业化应用前景。     

大肠杆菌色氨酸转运系统多基因敲除对色氨酸生产的影响

大肠杆菌中色氨酸向胞内的转运主要是由mtr、tnaB和aroP 3个基因编码的通透酶进行调控.利用Red重组技术,在mtr单基因敲除菌的基础上,成功构建了mtr.tnaB和mtr.aroP双基因敲除菌以及mtr.tnaB.aroP三基因敲除菌,并通过发酵实验首次考察了色氨酸转运系统多基因缺失对大肠杆菌合成色氨酸的影响.发酵结果表明,mtr.tnaB和mtr.aroP双基因缺失后,色氨酸产量分别达到1.38 g/L和1.27 g/L,与出发菌株相比分别提高了17％和9％,而mtr.tnaB.aroP三基因缺失后,菌体生长受到了明显抑制,发酵后色氨酸产量仅为0.63 g/L.在补料分批发酵实验中,mtr.tnaB双基因敲除菌的色氨酸产量进一步提高至12.2 g/L,与出发菌株相比色氨酸产量提高了27％.     

代谢副产物乙酸对L-色氨酸发酵的影响

分析了重组大肠杆菌(E.coli TRTH/pSV-709)发酵生产L-色氨酸的发酵过程,检测结果表明发酵液中有大量代谢副产物乙酸的积累。利用外源添加试验研究了乙酸对L-色氨酸发酵的影响,结果表明乙酸浓度高于2g/L时对L-色氨酸生产菌的生长和产酸均有抑制作用。分析了乙酸的产生机制,并采取了调节溶氧水平、确定合适初始葡萄糖浓度、限制葡萄糖流加及控制菌体比生长速率等措施来减少乙酸的生成。在优化条件下,乙酸含量与原工艺相比降低了51.35%,菌体生物量和L-色氨酸产量分别提高了51.07%和46.54%,实现了高密度发酵培养的目的。     

大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统改造对产L-色氨酸的影响

L-色氨酸是芳香族氨基酸的一种,被广泛应用于医药、食品和饲料等领域。大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统(PTS系统)在葡萄糖转运和磷酸化过程中起重要作用,是糖代谢基因表达调控的核心。利用Red同源重组系统,构建包含两类典型PTS系统突变(ptsHIcrr~-glf-glk~+和ptsG~-)的L-色氨酸生产菌,并对相关菌株进行补料分批发酵研究。结果表明,不同类型PTS系统突变对菌体生长、L-色氨酸产量、糖酸转化率及副产物生成均有较大影响。与出发菌相比,ptsHIcrr~-glf-glk~+突变株最高OD_(600)达到125,提高47.0%,产酸38.5 g/L,提高25.9%,糖酸转化率16.7%,提高26.5%,乙酸生成略有增加;ptsG~-突变株最高OD_(600)达到100,提高17.6%,产酸33.4 g/L,提高9.4%,糖酸转化率15.5%,提高17.4%,乙酸生成略有减少。对葡萄糖转运系统的进一步研究将为大肠杆菌合成L-色氨酸效率的提升提供帮助。     

L-丙氨酸厌氧发酵关键技术及产业化

传统氨基酸制造主要是通过化学合成或好氧发酵实现。相对于化学合成,微生物发酵可以实现以可再生资源为原料直接生产氨基酸,减少了对石油基原料的依赖,解决了化学合成高污染、高能耗等问题。好氧发酵具有生长快、产量高等特点,但好氧发酵中大量碳源用于细胞生长容易造成糖酸转化率低、能耗高等问题。厌氧发酵是近年来新出现的氨基酸生产模式,具有操作简单、无需通氧、糖酸转化率高容易接近理论最大值等优势。L-丙氨酸是国际上首个实现厌氧发酵产业化生产的氨基酸。本文以L-丙氨酸为例,综述了氨基酸厌氧发酵过程中的关键问题及其在产业化实施中的应用。未来,随着厌氧发酵关键技术在更多化合物生物制造技术中的突破,这种低成本、高效、低碳环保型发酵方式将会带来更大的经济价值和社会效益。     

Influence of Convulsants on Rat Brain Activities of Alanine Aminotransferase and Aspartate Aminotransferase

There exist differences between 12-day-old and adult rats in the onset of seizures induced by some inhibitors of glutamate decarboxylase (GAD). The aim of study was to investigate if there are differences between both groups in activities of rat brain alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the enzymes involved in glutamate metabolism, after the administration of 3-mercaptopropionic acid as specific GAD inhibitor or isoniazid as less specific general inhibitor of pyridoxal enzymes. Activities of both aminotransferases in a supernatant 20,000 g of the whole brain (containing predominantly cytosolic isoforms of enzymes) were increased at the beginning of 3-mercaptopropionic acid-induced generalized tonic-clonic seizures. At isoniazid-induced generalized tonic-clonic seizures, a significant increase in both enzyme activities was observed in adult rat brain. In the 12-day-old rat brain, ALT and AST activities reached about 40% and about 50–60% of adult control levels, respectively. In in vitro experiments, no influence of 3-mercaptopropionic acid on transaminase activities was found and an inhibitory effect of isoniazid on the enzymes was confirmed. Increased aminotransferase activities might participate in the enhanced synthesis of excitatory amino acid neurotransmitters in the nervous system, which may take a part in the initiation of epileptic seizures. Alternatively, the increased AST activity may be connected with an increased transport of NADH from the cytosol to mitochondria, while the increased ALT activity would represent the transformation of pyruvate to alanine as a consequence of increased glycolysis.     

Influence of Proline on Rat Brain Activities of Alanine Aminotransferase,Aspartate Aminotransferase and Acid Phosphatase

Hyperprolinemia type II (HPII) is an autosomal recessive disorder caused by the severe deficiency of enzyme ¹-pyrroline-5-carboxylic acid dehydrogenase leading to tissue accumulation of proline. Chronic administration of Pro led to significant reduction of cytosolic ALT activity of olfactory lobes (50.57%), cerebrum (40%) and medulla oblongata (13.71%) only. Whereas mitochondrial ALT activity was reduced significantly in, all brain regions such as olfactory lobes (73.23%), cerebrum (70.26%), cerebellum (65.39%) and medulla oblongata (65.18%). The effect of chronic Pro administration on cytosolic AST activity was also determined. The cytosolic AST activity from olfactory lobes, cerebrum and medulla oblongata reduced by 75.71, 67.53 and 76.13%, respectively while cytosolic AST activity from cerebellum increased by 28.05%. The mitochondrial AST activity lowered in olfactory lobes (by 72.45%), cerebrum (by 78%), cerebellum (by 49.56%) and medulla oblongata (by 69.30%). In vitro studies also showed increase in brain tissue proline and decrease in glutamate levels. In vitro studies indicated that proline has direct inhibitory effect on these enzymes and glutamate levels in brain tissue showed positive correlation with AST and ALT activities. Acid phosphatase (ACP) activity reduced significantly in olfactory lobes (40.33%) and cerebrum (20.82%) whereas it elevated in cerebellum (97.32%) and medulla oblongata (76.33%). The histological studies showed degenerative changes in brain. Following proline treatment, the animals became sluggish and showed low responses to tail pricks and lifting by tails and showed impaired balancing. These observations indicate influence of proline on AST, ALT and ACP activities of different brain regions leading to lesser synthesis of glutamate thereby causing neurological dysfunctions.     

Alanine Aminotransferase in Bovine Brain: Purification and Properties

Abstract: Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140- and 180-fold, respectively) from bovine brain cortex by means of (NH₄)₂SO₄ precipitation, gel filtration on Sephadex G-150, and ion-exchange chromatography on DEAE A-50 and characterized. The enzymes exhibited identical molecular weights (110,000 ± 10,000) and pH optima (7.8), but were eluted from CM Sephadex C-50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pi value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The K _m values of the mitochondrial enzyme were 5.1 m M , 6.6 m M , 0.7 m M , and 0.4 m M and of the cytosolic isozyme were 30.3 m M , 4.3 m M , 0.7 m M , and 0.5 m M for alanine, glutamate, 2-oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.     

Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Abstract: Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high-and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na⁺. Rapid accumulation of the Na⁺-alanine complex by synaptosomes stimulated activity of the Na⁺/K⁺ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na⁺ also caused a small depolarization of the plasma membrane, a rise in [Ca²⁺]₁, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [¹⁵N]alanine formation from [¹⁵N]glutamine was seven to eight times higher than the rate of [¹⁵N]glutamate generation from [¹⁵N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.     

荧光假单胞菌天冬氨酸转氨酶的基因克隆及其在大肠杆菌中的表达

采用克隆基因测序技术,从荧光假单胞菌GcM5-1A基因组文库中筛选到了天冬氨酸转氨酶的编码基因aspC。通过聚合酶链式反应（PCR）扩增目的基因,插入pET-15b构建重组表达质粒pET-15bAAT,转化E.coli BL21(DE3),IPTG诱导天冬氨酸转氨酶在大肠杆菌中高效表达,利用亲和层析法初步分离纯化了重组蛋白。生物活性分析表明,纯化的重组天门冬氨酸转氨酶具有氨基转移活性。     

Stabilization of Rat Liver Mitochondrial Alanine Aminotransferase with Ethanol and Trehalose

Rat liver mitochondrial alanine aminotransferase (mALT) is known to be a very unstable enzyme, a property that has hindered efforts to purify it. In this report we examine the possibility of stabilizing mALT with ethanol, trehalose, and protease inhibitors. The presence of ethanol was shown to slow down the inactivation of mALT, increasing its half-life from 1 to 4 h. Trehalose was found to greatly enhance the stability of mALT in a concentration-dependent manner. In the presence of 36.5% trehalose, the half-life of mALT was 85 h. Of the protease inhibitors tested only antipain and chymostatin slowed down the inactivation of mALT but only within the first 24 h following preparation of the crude enzyme. It is concluded that the inclusion of ethanol and trehalose in purification protocols could aid the purification of the enzyme. It is also concluded that the inclusion of protease inhibitors in purification protocols of mALT may not be necessary as its inactivation does not seem to be due to protease activity.