苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达及酶法合成L—苯丙 …

利用基因重组技术,在大肠杆菌中克隆并表达苯丙氨酸脱氨酸（ＰＡＬ）（ＥＣ４．３．１．５）,并应用此酶转化肉桂酸生成Ｌ－苯丙氨酸。方法是将欧芹苯丙氨酸脱氨酶ｃＤＮＡ亚克隆到组成型表达载体ｐＭＧ３６ｅ启动子Ｐ３２下游,以菌落ＰＣＲ法鉴定插一段的大小和方向都正确的克隆,进而以ＨＰＬＣ检测肉桂酸浓度的方法鉴别重组质粒有催化肉桂酸生成Ｌ－苯丙氨酸的酶活力。结果获得能表达ＰＡＬ酶活性的阳性克隆,在ＰＨ１０,含１     

粘红酵母产L-苯丙氨酸解氨酶的条件和酶法合成苯丙氨酸的研究

研究了粘红酵母(Rhodotorula glutinis)中L-苯丙氨酸解氨酶(PAL)(EC4.3.1.5)的产酶条件及用此酶把反式肉桂酸转化成苯丙氨酸的条件.结果表明,在下列培养基(g/L)及培养条件下PAL的活力较高:酵母膏10.0,蛋白胨10.0,NaCl5.0,KH_2PO_4 0.5,苯内氨酸0.5,(NH_4)_2SO_41.0,葡萄糖5.0,pH6.0—6.5,培养温度为30℃.转化过程中,[NH_4~+]对初速度的影响符合米氏方程,其K_m和V_(max)分别为16.85mol/L和5.96 g·L~(-1)·h~(-1),最适pH为10.0.底物肉桂酸对反应初速度的影响,在低浓度时有激活作用,在高浓度下则有抑制作用.肉桂酸转化为苯丙氨酸的转化率在60.0%以上.     

几种效应物对苯丙氨酸解氨酶稳定性的影响

为了对利用苯丙氨酸解氨酶（PAL）转化肉桂酸生成L-苯丙氨酸的生物转化反应条件进行优化,采用添加效应物的方法来提高苯丙氨酸解氨酶的稳定性,通过单因素实验研究了谷氨酸钠,海藻酸钠,聚乙二醇,甘油,锌粉,氮气等对PAL的稳定性影响,通过正交实验和方差分析,确定在转化液中添加1.0g/L锌粉和20g/L谷氨酸钠作为效应物,L-苯丙氨酸积累浓度提高55%,该两种效应物对PAL的稳定性增加显著。     

红冬孢酵母苯丙氨酸解氨酶基因pal在大肠杆菌中的克隆与表达

以红冬孢酵母总RNA为模板反转录获得其苯丙氨酸解氨酶基因pal,测序与已公布蛋白序列进行比对,相似度为99％.并以含有T7强启动子的pET - 28a(+)为载体构建重组质粒pET - 28a(+)- pal,通过异丙基硫代-β-D-半乳糖苷(IPTG)诱导实现苯丙氨酸解氨酶(PAL)在大肠杆菌中的表达.对诱导条件进行初步优化后,重组茵PAL比酶活可达到42.99 U/g.转化实验中肉桂酸的转化率为48.52％,L-苯丙氨酸生成量为1.73 g/L.结果表明,红冬孢酵母pal基因通过表达载体pET - 28a(+)在E.coli中获得了高效表达.     

粘红酵母产L-苯丙氨酸解氨酶发酵培养基的优化

通过单因子和正交试验 ,对粘红酵母产 L -苯丙氨酸解氨酶 ( PAL )培养基进行优化 ,L-苯丙氨酸的积累浓度可以从 2 .0 g/1 0 0 ml提高到 3 .3 g/1 0 0 ml,最终得到了 L-苯丙氨酸解氨酶发酵的最适条件     

利用酵母菌细胞转化肉桂酸生成L—苯丙氨酸

对利用酵母菌转化肉桂酸生成L-苯丙氨酸的方法进行了菌株筛选、菌体细胞培养、转化反应条件以及产物提取等方面的探索。从13个属的71株酵母菌中选到转化生成L-苯丙氨酸较高的粘红酵母(Rhodosorula glusinis)As 2.102菌株。经实验得出该菌株的最佳培养条件为:在含有1.5%酵母膏、1%葡萄糖、1.5%蛋白胨、0.05%L-苯丙氨酸、0.05% KH_2PO_4、0.5%NaCl、pH5.0的培养基中,30℃振荡培养20小时;最佳转化条件为1.5%肉桂酸、8mol/L氨、0.1?sO_4 pH10.0 30℃振荡转化反应24小时。每升转化液可生成10.78克L-苯丙氨酸,肉桂酸重量转化率为71.8%。产物用阳离子交换树脂法进行提取,所得结晶经纸层析单斑试验、生物测定、熔点、旋光、元素分析、红外光谱等项鉴定结果证实是L-苯丙氨酸,纯度超过99%。     

使用偏爱密码子大幅度提高苯丙氨酸脱氨酶在食品级乳酸乳球菌中的表达

为获得苯丙氨酸脱氨酶(PAL)在食品级乳酸乳球菌中的高效表达,将欧芹palcDNA(palnat)及根据乳酸乳球菌偏爱密码子设计人工合成的pal基因(palart)重组并转化到两种乳酸乳球菌NICE诱导表达系统中,测定基因工程菌表达PAL酶的量及活性,对比分析密码子偏爱性对乳酸乳球菌表达外源蛋白的影响。结果表明在两种乳酸乳球菌NICE表达系统中,使用偏爱密码子均可显著提高PAL酶的表达效率,使NZ9000/pNZ8048表达系统表达量提高22.23倍,NZ3900/pNZ8149系统提高35.90倍。此研究获得了安全高效表达PAL,可用于治疗苯丙酮尿症的基因工程菌。     

酵母细胞生物转化反式—肉桂酸生产L—苯丙氨酸的研究

据文献调查,搜集了国内可能相关的30株酵母,进行生物转化反式-肉桂酸(t-Ca) 生产L-苯丙氨酸 (L-Phe) 的微生物筛选研究,并对部分菌株生物转化能力,即苯丙氨酸解氨酶 (PAL,EC _(4、3、1、5) 活性水平进行了初步评估。筛选结果是:22株酵母具有转化 t-Ca 生成 L-Phe 的能力,转化率在2—67％范围。选出7株酵母研究在液体培养条件下细胞生长和PAL活性的时间过程关系,PAL 活性范围在 2.3—14.4x10~(-s)u/m g细胞干重。深红酵母 (Rhodotorularubra) AS2.166作为生物转化制备实经菌株,在静止细胞和固定化细胞批式反应条件下,结果获得L-Phe分离产率分别为42.0％,28.7％。     

麻疯树苯丙氨酸解氨酶启动子的克隆和表达载体的构建

苯丙氨酸解氨酶（phenylalanine ammonia lyase, PAL）是苯丙烷类代谢途径的关键酶,催化苯丙氨酸转化为肉桂酸,促进黄酮、香豆素等次生代谢物的生成。本文根据已克隆的麻疯树苯丙氨酸解氨酶基因JcPAL的序列设计引物,通过DNA步移技术,克隆出长度为1 334 bp的JcPAL基因起始密码子上游序列。序列分析显示其不仅具备CAAT、TATA盒这些保守元件,而且包含多种胁迫诱导元件,特别是在序列中发现一些苯丙氨酸解氨酶特有的元件。为了鉴定JcPAL基因的启动子元件,分别将长度不同的5′端侧翼区缺失体定向插入载体pBI121中, 取代原有的CaMV35S启动子,构建了4个驱动报告基因GUS的植物表达载体。     

苯丙氨酸稀土配合物与磷脂脂质体相互作用的研究

用ＤＳＣ、ＦＴ－ＩＲ和Ｒａｍａｎ光谱法研究了苯丙氨酸（Ｐｈｅ）及其稀土配合物Ｌｎ（Ｐｈｅ）Ｃｌ３．６Ｈ２Ｏ（Ｌｎ＝Ｓｍ．ｌａ．Ｇａ．Ｙｂ）对ＤＰＰＣ热致性的影响,并从分子水平上探讨了它们与ＤＰＰＣ相互作用的机理。结果表明：Ｐｈｅ对ＤＰＰＣ从凝胶态到液晶态的相变温度Ｔｍ影响不大,但磷脂酰链的构象有序度降低;Ｌｎ（Ｐｈｅ）Ｃｌ３．６Ｈ２Ｏ像Ｌｎ３＋一样,使ＤＰＰＣ的Ｔｍ显著升高,但前者的升高程度小于后者,同时磷脂链的构象有序度升高。上述ＤＰＰＣ脂质体二元体系的铸膜ＦＴＩＲ结果与ＤＰＰＣ脂质体基本上相同,配合物Ｌｎ（Ｐｈｅ）Ｃｌ３．６Ｈ２Ｏ的配合键是离子型的．     

半胱氨酸、苯丙氨酸对慢性缺氧大鼠肺循环和脑血流的影响

本实验研究了半胱氨酸（ＣＹＳ）与苯丙氨酸（ＦＨＥ）对慢性缺氧大鼠肺循环和脑血流的影响及其可能机制。结果ＣＹＳ（３００ｍｇ·ｋｇ￣（－１）·ｄ￣（－１））与ＰＨＥ（４００ｍｇ·ｋｇ￣（－１）·ｄ￣（－１））能缓解慢性缺氧大鼠肺动脉高压、肺血管阻力增加和右心肥厚,并抑制慢性缺氧大鼠急性缺氧性肺血管收缩反应（ＨＰＶ）,但对脑血流无显著影响;ＣＹＳ还可减少慢性缺氧大鼠肺脑组织中的丙二醛。ＣＹＳ的作用可能与清除自由基有关;ＰＨＥ的作用不是通过减少中枢神经的正常递质去甲肾上腺素。     

大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ,ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶,为了通过基因工程手段来增加本丙氨酸生物的产量,在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上,采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因,进行克隆表达,并与ｐｈｅＡ基因串联,以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式,实现了２种酶基因在大肠杆菌中的表达, ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带,经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。     

DL-苯丙氨酸酶法拆分

ＤＬ－苯丙氨酸的拆分是以Ｎ－乙酰－ＤＬ－苯丙氨酸铵为起始原料,经猪肾酰化酶不对称水解作用,再经离子交换色谱分离,减压浓缩得到Ｌ－苯丙氨酸和Ｎ－乙酰－Ｄ－苯丙氨酸结晶。     

Studies on the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase

The uncoupled portion of the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase can be described by the same model as we have recently derived for the fully uncoupled reaction (Davis, M.D. and Kaufman, S. (1989) J. Biol. Chem.264, 8585–8596). Although essentially no hydrogen peroxide is formed during the fully coupled oxidation of tetrahydrobiopterin or 6-methyltetrahydropterin by phenylalanine hydroxylase when phenylalanine is the amino acid substrate, significant amounts of hydrogen peroxide are formed during the partially uncoupled oxidation of 6-methyltetrahydropterin whenpara-fluorophenylalanine orpara-chlorophenylalanine are used in place of phenylalanine. Similarly, during the partially uncoupled oxidation of the unsubstituted pterin, tetrahydropterin, even in the presence of phenylalanine, hydrogen peroxide formation is detected. The 4a-carbinolamine tetrahydropterin intermediate has been observed during the fully uncoupled tyrosine-dependent oxidations of tetrahydropterin and 6-methyltetrahydropterin by lysolecithin-activated phenylalanine hydroxylase, suggesting that this species is also a common intermediate for uncoupled oxidations by this enzyme.Abbreviations BH₄ 6-[dihydroxypropyl-(L-erythro)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) - 6MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PH₄ 5,6,7,8-tetrahydropterin - BH₃OH 4a-hydroxytetrahydropterin (4a-carbinolamine) - qBH₂ quinonoid dihydrobiopterin - q6MPH₂ quinonoid dihydro-6-methylpterin - qPH₂ quinoid dihydropterin - PAH phenylalanine hydroxylase - DHPR dihydropteridine reductase - PHS phenylalanine hydroxylase stimulating enzyme which is 4a-carbinolamine dehydratase - SOD superoxide dismutase - HPLC high performance liquid chromatography - R.T. retention time Special issue dedicated to Dr. Santiago Grisolia.     

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.     

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites (phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.     

Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae

Suspension cultured cells of a blast-resistant rice genotype (Oryza sativa L. cv. Gigante Vercelli) were treated with cell wall hydrolysates prepared from the fungal pathogen Magnaporthe oryzae. As a consequence, a complex pattern of phenylalanine ammonia lyase time course specific activity levels was evident. Ion-exchange chromatographic fractionation of crude extracts suggested that the early (6 h) and the late (48-72 h after elicitation) increase of activity relied upon the sequential induction of two different isoenzymes. The relative expression levels of 11 genes putatively coding for a phenylalanine ammonia lyase were measured by semi-quantitative capillary gel electrophoresis of RT-PCR products. Two genes were indeed found to be induced by treatments with the hydrolysate, and data were validated by real-time PCR. Conversely, only the early-responsive enzyme form was observed following elicitation in a blast-sensitive rice genotype (cv. Vialone nano). Therefore, the late-responsive isoform may represent a candidate gene to select for decreased sensitivity to blast.