芳香族氨基酸添加提高井冈霉素产量的研究

目的 通过添加芳香族氨基酸促进井冈霉素生物合成起始单位的供应,提高其发酵产量.方法 优化苯丙氨酸添加时间和浓度确定最优添加条件,分析前体代谢相关的酶活考察其影响机制.结果 确定苯丙氨酸最佳添加条件为在发酵第24h添加0.1g/L.发现37℃时苯丙氨酸的添加提高了井冈霉素A产量,但在42℃条件下提高幅度大大降低.通过分析6-磷酸葡萄糖脱氢酶酶活,发现芳香族氨基酸添加并未影响其酶活.结论 苯丙氨酸的添加能有效提高井冈霉素发酵产量近60％.苯丙氨酸的添加可能通过反馈抑制降低了戊糖磷酸途径中中间体的消耗,从而为井冈霉素合成提供了更加充足的7-磷酸景天庚酮糖.     

利福霉素B产生菌的推理选育

利福霉素B由地中海拟分枝酸菌产生，本文对工业生产菌株A.mediterraneiX1－02进行进一步筛选。采用推理选育的方法使产生菌减轻由芳香族氨基酸（色氨酸（tup）、苯丙氨酸、酪氨酸)和对羟基苯甲酸（PHBA，pbh）引起的对利福霉素B生成合成的反馈抑 制作用，并提高对前体丙酸（prp）的耐受量。通过UV诱变提高诱变株的耐受性，获得了高产菌株A.mediterraneiXC9－25（trp′，phb′，prp′），其生产能力达到10000u/ml，较原始出发菌株A.mediterraneiX1－02提高了1．385倍。动力学试验结果利用福霉素B的生物合成与菌体生长不同步，属于非生长偶联型。     

氨基酸制剂在肝病临床上的应用

氨基酸是人体不可缺少的营养成分之一,是合成激素、酶和蛋门质的原料,又是维持生命的物质基础。当患者不能经口从食物中摄取蛋白质或摄取量不足时,可采用胃肠道外途径供给氨基酸。目前用于肝病的氨基酸制剂可归纳为;1.用于肝性脑病的特殊氨基酸制剂l 2.营养性复方氨基酸溶液。现就常用的氨基酸制剂在肝病的临床应用作一介绍。一、用于盱性脑病的特殊氮基酸制剂(一)肝性脑病时氯基酸代谢异常根据临床和实验研究表明,肝性脑病时血浆氨基酸代谢有明显改变,主要表现为芳香族氨基酸(AAA)如苯丙氨酸、酪氨酸增多和支链氨基酸(BCAA)如亮氨酸、异亮氨酸以及缬氨酸减少。由于BCAA减少和AAA增高,导致BCAA/AAA克分子浓发比值降『氐。在肝硬化并发脑病时,AAA、游离色氨酸和蛋氨酸较     

L-苯丙氨酸

L-苯丙氨酸属于8种必需氨基酸之一，也是生物体内合成酪氨酸的重要原料，人和动物自身不能合成而必须从外界摄取。L-苯丙氨酸的最重要用途是用作合成阿斯巴甜以及用作营养强化剂、氨基酸输液和复合氨基酸制剂的成分。近年来，随着阿斯巴甜的广泛生产与使用，以及氨基酸类药物、营养保健品的开发应用，全球对L-苯丙氨酸的需求量相应迅速放大。     

利福霉素B产生菌─—地中海诺卡氏菌的推理选育

利福霉素Ｂ的生物合成受芳香族氨基酸（Ｔｒｐ、Ｔｙｒ、Ｐｈｅ）的反馈抑制。用紫外线处理Ｎ．ｍｅｄｉｔｅｒｒａｎｅｉＸＣ－１０２菌丝悬浮液后，并在含０．１％芳香族氨基酸的琼脂平板上分离芳香族氨基酸抗性变种。结果表明，芳香族氨基酸抗性变种的发酵效价高于自然分离株和紫外线诱变株；色氨酸抗性变种（Ｔｒｐｒ）的发酵效价又高于酪氨酸抗性变种（Ｔｙｒｒ）和苯丙氨酸抗性变种（Ｐｈｅｒ）；０．５％Ｔｒｐｒ变种比０．１％Ｔｒｐｒ变种更高产。其中０．５％Ｔｒｐｒ变种ＸＣ－５４０的生产能力较出发菌株ＸＣ－１０２增加４２．１６％。传代试验表明ＸＣ－５４０的遗传特性稳定，在７ｍ３罐做发酵放大，与出发菌株相比，发酵效价和发酵指数分别提高５１．１１％和５１．０２％。     

治疗肝昏迷新药——14氨基酸注射液-800

<正> 肝昏迷是由于严重肝功能障碍所致的一系列神经精神症状,其发病机制尚有待于进一步研究,除氨中毒学说、假神经递质学说外,近年来对肝脏病时血浆氨基酸异常图谱的研究表明,氨基酸代谢异常与肝功能障碍和出现昏迷的程度有密切关系。Sherwin指出,肝硬化时血浆苯丙氨酸、酪氨酸等芳香族氨基酸(AAA)以及蛋氨酸等含硫氨基酸与色氨酸增加,而亮氨酸、异亮氨酸、缬氨酸等支链氨基酸(BCAA)含量明显减少。Fischer     

生物技术文摘

B38-13在大肠杆菌中表达来源于酵母毛孢子菌属的酪氨酸诱导型酪氨酸氨解酶Vannelli T等[Enzyme Microb Technol,2007,41(4):413]芳香族氨基酸苯丙氨酸/酪氨酸的脱氨作用需要苯丙氨酸氨解酶(PAL)/酪氨酸氨解酶(TAL)的参与。由于酪氨酸脱氨后的产物对羟基桂皮酸具有广阔的应用前景,因此TAL成为研究     

中国氨基酸工业生产状况、差距及今后方向

人类的必需氨基酸有下列8种:赖氨酸、苯基丙氨酸、缬氨酸、蛋氨酸、色氨酸、亮氨酸、异亮氨酸、苏氨酸.如果人体缺乏这些氨基酸,就会影响人体的正常的生长和健康.另外,由于人体合成胱氨酸、组氨酸、精氨酸、酪氨酸及甘氨酸等的能力较低,当需要它们的量增多时,必须从食物中摄入或者摄取人工合成氨基酸,以提高自身免疫能力,这类氨基酸被称为人体"半必需氨基酸".     

蕨藻素对醋酸所致小鼠扭体反应的影响

蕨藻素(caulerpin),小鼠口服的最大耐受量为134.74mg/kg,灌胃给药或静脉注射caulerpin对腹腔注射醋酸所致小鼠的扭体反应无影响;皮下注射caulerpin对扭体反应有一定抑制作用,但镇痛百分率<50％;腹腔注射caulerpin对醋酸所致小鼠的扭体反应有显著的抑制作用,其ID_(50)=1.088±0.125mg/kg,当给药剂量≥10mg/kg时,镇痛百分率为100％。     

苯丙氨酸对恩霉素产生菌S.refuineus生物合成酪氨酸的调节作用

HurLey等发现,抗肿瘤抗生素恩霉素(Anthram-ycin)是由色氨酸,酪氨酸和蛋氨酸衍生而来。本文作者试图研究产生恩霉素时有关芳香氨基酸的部份调节机制。用N-甲基-N′-硝基-N-亚硝基奎尼定(NTG)或青霉素G处理S.refuineus得到酪氨酸缺陷型变株,然后用变株与野生型菌株同时进行比较试验,发现该变株能生长在添加酪氨酸的最低营养培基(MM)上,而不生长在仅含MM培养基上,说明它是单一营养缺陷株。变株在含对-羟苯丙酮酸的MM培基上不生长,因而估计它在对-羟苯丙酮酸转氨酶部位受损,致使它不能使用该前体物。但野生型和缺陷型变株两者都能利用谷酰胺,而且两者的对-羟苯丙氨酸转氨酶的活性都     

Disorders of tetrahydrobiopterin metabolism and their treatment

Tetrahydrobiopterin (BH4) deficiencies are disorders affecting phenylalanine metabolism in liver and neurotransmitters biosynthesis in brain. BH4 is the essential cofactor in the enzymatic hydroxylation of 3 aromatic amino acids (phenylalanine, tyrosine, and tryptophan). BH4 is synthesized from guanosine triphosphate (GTP) catalyzed by GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase (SPR), and in aromatic amino acids hydoxylating system is regenerated by pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR). To date, 4 enzyme deficiencies (GTPCH, PTPS, DHPR, PCD) have been reported and they all follow an autosomal recessive mode of inheritance. The incidence of BH4 deficiency is at 1 in 1,000,000, except that in Taiwanese (much higher than in Japanese and Caucasians). BH4 deficiency has been diagnosed in patients with hyperphenylalaninemia (HPA) by neonatal mass-screening based on BH4 oral loading tests, analysis of urinary or serum pteridines, and measurement of dihydropterindine reductase (DHPR) activity in blood from a Guthrie card. BH4 deficiency without treatment causes combined symptoms of HPA and neurotransmitter (dopamine, norepinephrine, epinephrine, and serotonin) deficiency, such as red hair, psychomotor retardation, and progressive neurological deterioration. Treatment of BH4 deficiencies consists of BH4 supplementation (2-20 mg/kg per day) or diet to control blood phenylalanine concentration and replacement therapy with neurotransmitters precausers (L-dopa/CarbiDOPA and 5-hydroxytryptophan), and supplements of folinic acid in DHPR deficiency.     

偏振荧光光度法同时测定氨基酸注射液及动、植物浸出液中酪氨酸、色氨酸的研究

酪氨酸、色氨酸和苯丙氨酸具有荧光,本文使用互相垂直的偏振片有效地消除了苯丙氨酸的干扰和溶剂散射光的影响;附加滤光片,进一步提高了酪氨酸与色氨酸荧光测定的选择性。线性范围分别为0.02～12.0mg·L^-1(酪氨酸)与0.01～2.5mg·L^-1(色氨酸)。此法可直接用于氨基酸注射液和动、植物浸出液中游离酪氨酸、色氨酸的分析,结果满意。     

Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases

Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.     

An amino acid mixture deficient in phenylalanine and tyrosine reduces cerebrospinal fluid catecholamine metabolites and alcohol consumption in vervet monkeys

 An amino acid mixture devoid of tryptophan, given orally, was previously shown to reduce cerebrospinal fluid levels of tryptophan and 5-hydroxyindoleacetic acid in vervet monkeys, as compared to a control mixture containing all essential amino acids. In the present study, we tested the possibility that a similar amino acid mixture containing tryptophan, but devoid of phenylalanine and tyrosine (the amino acid precursors of catecholamine neurotransmitters), would influence dopamine and noradrenaline metabolism. Five hours after the administration of this mixture to vervet monkeys, cerebrospinal fluid levels of homovanillic acid and 3-methoxy-4-hydroxyphenylethylene glycol were reduced by 27.4% and 26.9%, respectively. Both effects were statistically significant. Plasma tyrosine (-30%) and the ratio of tyrosine to the sum of other large neutral amino acids (ΣLNAA) were also significantly reduced. The behavioral efficacy of phenylalanine/tyrosine depletion was compared with that of tryptophan depletion in a primate model of voluntary alcohol consumption. All three drinks lowered alcohol consumption, but the effects of the tryptophan-deficient amino acid mixture were not different from those of the balanced amino acid control. The phenylalanine/tyrosine-deficient drink differentially lowered alcohol consumption, consistent with other data in this species and elsewhere implicating dopamine in the rewarding effects of alcohol. Received: 14 January 1997 / Final version: 10 September 1997     

Beta-adrenergic regulation of amine precursor amino acid transport across the blood-brain barrier.

Beta-Adrenoceptor agonists were administered i.p. into rats and amino acid levels in brain and plasma were then determined to assess the effects on transport across the blood-brain barrier. Isoproterenol (10 mumol/kg) caused significant increases in aromatic amino acid (tyrosine, phenylalanine and tryptophan) levels in cerebral cortex and decreases in almost all amino acid concentrations in plasma. This effect of isoproterenol on brain tyrosine level was dose-dependent with an ED50 of 0.25 mumol/kg. Salbutamol (beta 2-adrenoceptor agonist, 10 mumol/kg) showed similar effects, but dobutamine (beta 1-adrenoceptor agonist, 50 mumol/kg) failed to increase brain amino acid levels. When 1-threo-3,4-dihydroxyphenylalanine (L-DOPA, 100 mumol/kg) was i.p. loaded, beta-adrenoceptor agonists promoted the transport of L-DOPA into brain without increasing the clearance rate of plasma L-DOPA. Moreover, significant increases in dopamine and its metabolites were observed in rat brain. These findings suggest that the transport of aromatic amino acids across the blood-brain barrier may be regulated through beta 2-adrenoceptors and that co-administration of beta 2-adrenoceptor agonists with L-DOPA may enhance the therapeutic efficacy of L-DOPA.     

Determination of the ratios of the aromatic amino acid residues by first- or second-derivative UV spectrometry for a simple characterization of peptides

A method for the simultaneous determination of the ratios of the three aromatic amino-acid residues in peptides was set up in acidic conditions. Binary and ternary mixtures of these amino acids were prepared, and first- and second-derivative spectra then calculated from their 0.1 nm resolution spectra between 240 and 320 nm. Certain spectral bands were chosen to differentiate tyrosine from tryptophan on the first-derivative spectra, and phenylalanine from tyrosine and tryptophan on the second-derivative spectra. Variation of the amplitude of the chosen bands was shown to be a linear function of the ratio of the aromatic amino acids in the mixture. This technique was validated with peptides whose sequence was known. The difference between theoretical and experimentally determined ratios was lower than 10%. Since the results are obtained as ratios, neither the concentration nor the nature of the peptide has to be known. The feasibility of application using a photodiode array detector with high resolution in reversed-phase high-performance liquid chromatography is discussed. © Munksgaard 1995.     

CHAINS OF ALTERNATING SULFUR AND π-BONDED ATOMS IN EIGHT SMALL PROTEINS

This paper demonstrates the existence of regions in eight small globular proteins in which the side chains of sulfur-containing amino acids (cysteine and methionine) alternate in space with side chains of aromatic amino acids (histidine, phenylalanine, tryptophan and tyrosine). The proteins are: rubredoxin, high potential iron protein, cytochrome c, flavodoxin, deoxyhemoglobin, trypsin inhibitor, ribonuclease-S, and lysozyme. The sulfur-φ-bonded ‘chains’ involve a minimum of five and a maximum of 10 amino acids, and contain the most polarizable atoms within proteins. S-φ-chains give extra stability to the folding of proteins; they may also afford paths for the step-wise movement of electrons.     

Effects of the beta 2-adrenoceptor agonist clenbuterol on tyrosine and tryptophan in plasma and brain of the rat

The beta 2-adrenoceptor agonist, clenbuterol (initially 5 mg/kg), was found to significantly reduce plasma tyrosine and raise brain tryptophan levels (P less than 0.01). By comparison, decreases in plasma tryptophan and increases in brain tyrosine were small and often nonsignificant. Amino acid levels measured in different brain regions revealed that the elevations were similar among the cerebellum, striatum, and cortex. These effects were partially blocked by propanolol but not by atenolol. The ED50 was estimated from dose-response curves to be about 0.05 mg/kg for both the decrease in plasma tyrosine and the increase in brain tryptophan. The effects of low doses of clenbuterol were prevented completely by propranolol. Peripheral organs displayed strikingly different patterns of change in amino acid concentrations. Only the spleen had any accumulation of tryptophan, but that was much less than in brain. In contrast, tyrosine and tryptophan were decreased in heart and unaltered in liver; tyrosine was decreased in lung. The elevation in brain tryptophan levels was attenuated by the beta 2-antagonist, ICI 118,551, but not by the beta 1-antagonist, betaxolol; but the reduction in plasma tyrosine was unaffected by either drug. The serotonin antagonist, methysergide, failed to block the effects of clenbuterol. We conclude that changes in amino acid concentrations produced by clenbuterol are mediated by beta 2-adrenoceptor stimulation. Although the increases in brain tyrosine and tryptophan were similar to increases in the plasma ratios of these amino acids to the sum of the other large neutral amino acids competing for transport into the brain, the disparity between the effects of ICI 118,551 in brain and plasma suggests that clenbuterol may also have a direct action in brain to regulate levels of aromatic amino acids. Since clenbuterol has been purported to have an antidepressant effect and since other antidepressants also increase brain tryptophan, this may be a common feature of antidepressant drug action.     

Effect of aminophylline on tryptophan and other aromatic amino acids in plasma, brain and other tissues and on brain 5-hydroxytryptamine metabolism.

1 Aminophylline and other methylxanthines increase brain tryptophan and hence 5-hydroxytryptamine turnover. The mechanism of this effect of aminophylline was investigated. 2 At lower doses (greater than 100 mg/kg i.p.) the brain tryptophan increase could be explained by the lipolytic action of the drug, i.e. increased plasma unesterified fatty acid freeing plasma tryptophan from protein binding so that it became available to the brain. 3 Plasma unesterified fatty acid did not increase when aminophylline (109 mg/kg i.p.) was given to nicotinamide-treated rats but as both plasma total and free tryptophan rose, a tryptophan increase in the brain still occurred. 4 The rise in brain tryptophan concentration following the injection of a higher dose of the drug (150 mg/kg i.p.) could no longer be explained by a rise of plasma free tryptophan as the ratio of brain tryptophan to plasma free tryptophan rose considerably. Plasma total tryptophan fell and the plasma insulin concentration rose. 5 The increase of brain tryptophan concentration after injection of 150 mg/kg aminophylline appeared specific for this amino acid as brain tyrosine and phenyllanine did not increase. However as their plasma concentrations fell the brain/plasma ratio for all three amino acids rose. 6 The higher dose of aminophylline increased the muscle concentration of tryptophan but that of tyrosine fell and that of phenylalanine remained unaltered. The liver concentrations were not affected. 7 The aminophylline-induced increase of the ratio of brain tryptophan of plasma free tryptophan no longer occurred when the drug was given to animals injected with the beta-adrenoreceptor blocking agent propranolol or the diabetogenic agent streptozotocin. 8 The changes in brain tryptophan upon aminophylline injection may be explained by (a) increased availability of plasma tryptophan to the brain due to increased lipolysis and (b) increased effectiveness of uptake of tryptophan by the brain due to increased insulin secretion.