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。     

一菌双酶法制备L-4-氟苯丙氨酸

利用重组E．coli产天冬氨酸酶和天冬氨酸转氨酶催化生产L-4-氧苯丙氨酸的工艺。实验结果表明最佳转化条件为-37℃,pH值4．5—8．5,菌体与酮酸的质量浓度比为1.5,CTAB的质量分数为0．04％,酮酸的质量浓度11．28g／L,富马酸铵与酮酸的摩尔比为3．0：1．0,添加1mmol／L的Fe^2＋,L-天冬氨酸与酮酸的摩尔比为0．4：1。在最适条件下,经过14h酶转化反应达到平衡,酮酸转化率可达到95％以上,L-4-氟苯丙氨酸得率也可达到80％以上。此法原料简单易得,为L-4-氟苯丙氨酸的制备提供了一种新方法：     

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%。     

苯丙氨酸解氨酶(PAL)的生产:适用于L-苯丙氨酸商品化生产的酵母菌株的分离与评价

含有苯丙氨酸解氨酶的微生物已从各种自然环境中分离得到,以L—苯丙氨酸和t—苯丙烯酸盐为底物对丙氨酸解氨酸双向酶活性初步筛选出最好的21株,其中的12株对细胞总产量和来丙氨酸解氨酶活性进行了比较,并选出7株来作了在各种培养基中的PAL诱导程度的试验.根据上述的筛选,对分离株SPA10(已鉴定为Rh-odotorula rudra)的最适条件进行了筛选,在28℃和pH5.0是最适的生长条件,但细胞的苯丙氨酸解氨酶活性已表明在亚适生长温度(36℃)和pH(8.0)时较高。当机体在有各种糖和铵离子的条件下生长时,苯丙氨酸解氨酶的合成受到抑制,控制发酵条件能使苯丙氨酸解氨酶的合成发生在最大生物量水平,在糖耗尽时出现.到达最大合成之后,短时间内细胞内苯丙氨酸解氨酶迅速失活,若补充D.L—异亮氨酸和低浓度D.L—本丙氨酸,改变发酵温度,能使酶催化发酵稳定在100小时以上.上述结果证明了SPA10分离株从反式来丙烯酸生产L—苯丙氨酸进行商业化生产是可行的.     

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-苯丙氨酸

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酶 (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pMG3 6e启动子P3 2下游 ,以菌落PCR法鉴定插入片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pH1 0 ,含 1 .0 %肉桂酸、8.0mol/L氨的转化液中 ,3 0℃反应 2 0h ,肉桂酸重量转化率可达 60 %。该基因工程菌有希望用于工业化生产L 苯丙氨酸。     

农杆菌介导Txpam基因转化烟曲霉TMS-26及其产紫杉醇效果评价

为研究红豆杉紫杉醇合成途径限速酶基因功能及其对内生真菌烟曲霉TMS-26发酵产紫杉醇的影响,以曼地亚红豆杉愈伤组织制备cDNA作为模板扩增苯丙氨酸氨基变位酶基因（Txpam）,构建重组质粒pGEX-4T-1-Txpam,转入大肠杆菌中进行异源诱导表达,经亲和层析纯化,获取重组酶TxPAM并验证其酶活性。构建pCAMBIA1302-Txpam质粒,转化农杆菌感受态细胞,利用农杆菌介导的转化体系获得转化子并优化转化条件,结合插入片段携带的分子标记和目的基因进行转化子验证,同时培养转化菌株并检测紫杉醇产量。结果表明：纯化获取的重组酶TxPAM,经HPLC检测具有将α-苯丙氨酸催化为β-苯丙氨酸的功能;在最优转化条件下,转化子数目达到471个/10⁶个孢子;根据基因hyg和Txpam的克隆以及测序结果,说明成功构建了基因工程菌株,通过对其发酵条件进行优化,紫杉醇产量达到721.87μg/L。     

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.     

基因重组菌质粒稳定性的提高与苯丙氨酸发酵的研究

本研究用具有苯丙氨酸生产抗反馈抑制基因pheA^FR、aroF^FR及温度敏感型阻遏基因CI857的质粒pSYl30—14和具有分配机能的低拷贝质粒pSYl6,重组构建了具有苯丙氨酸生产基因系统的质粒：psY200一14,然后使其转化到大肠杆菌AT2471中,育成了基因重组菌株AT247l／psY200—14。试验表明,该菌株质粒稳定性比原菌株AT2471／psYl30—14有较大的提高,当存在选择压时,在30～42℃范围内维持100％的高稳定性。应用此重组菌株,在2．5L通气搅拌罐进行发酵试验,在搅拌转速850rpm,通气速率1．Ovvm,38．5℃和pH7．O的条件下,发酵48h苯丙氨酸生成量达14．2g／L,比原株增产l1．8％。     

双波长紫外吸收法测定L-苯丙氨酸含量

采用双波长紫外吸收法测定转化液中L 苯丙氨酸含量 ,测定波长为 2 5 8nm ,参比波长为 2 78nm。由Acr2 78=138.18C+0 .0 0 85γ =0 .9995得到肉桂酸浓度Cr ,再由A2 58=ACr2 58+Acp2 5 8=76 .5 31Cr +.94 4 6Cp +0 .0 36 6 ,计算出L 苯丙氨酸含量Cp。此方法快速、简便、准确度高、除杂效果好 ,适用于生产过程控制。     

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% KH₂PO₄、0.5%NaCl、pH5.0的培养基中,30℃振荡培养20小时;最佳转化条件     

The conversion of L-phenylalanine into benzoic acid on the thylakoid membrane of higher plants.

The conversion of L-phenylalanine into benzoic acid and other aromatic carboxylic acids was investigated in Nasturtium officinale (watercress), Astilbe chinensis, and Hydrangea macrophylla in vivo and in vitro. Comparative feeding experiments with radioactively labelled L-phenylalanine and cinnamic acid administered to intact leaf discs of A. chinensis indicated a rapid formation of benzoic acid from L-phenylalanine, whereas cinnamic acid was a poor precursor. Using a pulse-chase labelling technique followed by a fractionation of the tissue into subcellular components, chloroplasts could be identified as the predominant, if not exclusive, site of benzoic acid formation in A. chinensis. Experiments in vitro with chloroplasts and thylalkoids of N. officinale, H. macrophylla, and A. chinensis demonstrate the capacity of thylakoid membranes to catalyze the degradation of L-phenylalanine to benzoic acid. The results obtained upon stimultaneous incubation with [4'-3H]L-phenylalanine and [3-14C]cinnamic acid lead to the hypothesis that the reaction of L-phenylalanine to benzoic acid proceeds via a cinnamic acid pool which is different from that of soluble cinnamic acid.     

Enzymatic and nonenzymatic dehydration reactions of L-arogenate

L-Arogenate, an immediate precursor of either L-tyrosine, L-phenylalanine, or both in many microorganisms and plants, may undergo two types of dehydration reactions that yield products of increased stability. Under acidic conditions, a facile aromatization attended by loss of the C-4 hydroxyl and the C-1 carboxyl moieties results in quantitative conversion to L-phenylalanine. When aromatization was largely prevented by maintaining pH in the range of 7.5-12, a second dehydration reaction occurred in which the alanyl side chain and the carboxyl group at C-1 formed a lactam ring to yield spiro-arogenate. The latter reaction occurs at 100 degrees C, roughly 50% conversion being obtained in 2 h. The product formed from L-arogenate was authentic spiro-arogenate, as demonstrated by high-performance liquid chromatography and thin-layer chromatography identification procedures. Further confirmation was obtained by 1H nuclear magnetic resonance, ultraviolet spectroscopy, and mass spectrometry. Thus far, the conversion of L-arogenate to spiro-arogenate is not known to be enzyme catalyzed. The other dehydratase reaction, however, is catalyzed in nature by an enzyme denoted arogenate dehydratase. An improved assay is described for this in which [3H]dansyl derivatives of L-arogenate (substrate) and L-phenylalanine (product) are separated by using bidimensional thin-layer chromatography. The radioactive reaction product is then quantitated. This assay was used to study partially purified arogenate dehydratase from Pseudomonas diminuta, an organism that depends upon the arogenate pathway for L-phenylalanine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural-based screening of L-phenylglycine aminotransferase using L-phenylalanine as the optimal amino donor: Recycling of L-phenylalanine to produce L-phenylglycine

The aproteinogenic amino acid, L-phenylglycine, is an important side chain building block for some drugs. It would be of great commercial and environmental value to biocatalyse L-phenylalanine to L-phenylglycine, and thus replace the organic synthesis method. To produce L-phenylglycine from L-phenylalanine, an L-phenylglycine aminotransferase was screened and characterized. HpgT_AO showed high homology to α-aminoadipate aminotransferase. The L-phenylalanine binding site was near the residues S26, R401, N201, and G46 in HpgT_AO, and L-phenylalanine formed a hydrogen bond with Asn20, which was similar to the substrate binding mechanism of α-aminoadipate aminotransferase. HpgT_AO showed increased activity in alkalescent environment below 40°C. The kinetic analysis showed that L-phenylalanine had the highest affinity to HpgT_AO, which ensured the recycle biosynthesis of Lphenylglycine from L-phenylalanine. To date, it was the only aminotransferase using L-phenylalanine as an optimal amino donor. The L-phenylglycine biocatalysis operon was also constructed by co-expressing the hmaS, hmo and hpgT by a single plasmid. The first in vitro conversion of L-phenylalanine to L-phenylglycine was achieved by directly using the L-phenylalanine fermentation broth as the raw material.     

A model of closely assembled consecutive enzymes on membranes: formation of hydroxycinnamic acids from L-phenylalanine on thylakoids of Dunaliella marina.

p-Hydroxycinnamic acid was found to be located within the plastids of the green alga Dunaliella marina. Thylakoid fractions desintegrated by ultrasonic treatment were capable of converting L-phenylalanine into o- and p-hydroxycinnamic acids; the hydroxylation reaction was increased by addition of NADPH. Hydroxycinnamic acids produced when [3-14C]cinnamate was incubated with varying amounts of [4'-3H]L-phenylalanine exhibited a 3H/14C ratio 10-150 times higher than that of the cinnamic acid reisolated from the incubation mixture. The lack of equilibration between cinnamate formed from L-phenylalanine and cinnamate added to the solution supports the hypothesis that cinnamate as an intermediate in hydroxycinnamate formation remains bound to the membrane enzyme complex. A model of membrane-bound multienzyme complexes is proposed for the conversion of aromatic amino acids into phenols.     

Metabolic engineering of the E. coli L-phenylalanine pathway for the production of D-phenylglycine (D-Phg)

D-phenylglycine (D-Phg) is an important side chain building block for semi-synthetic penicillins and cephalosporins such as ampicillin and cephalexin. To produce d-Phg ultimately from glucose, metabolic engineering was applied. Starting from phenylpyruvate, which is the direct precursor of L-phenylalanine, an artificial D-Phg biosynthesis pathway was created. This three-step route is composed of the enzymes hydroxymandelate synthase (HmaS), hydroxymandelate oxidase (Hmo), and the stereoinverting hydroxyphenylglycine aminotransferase (HpgAT). Together they catalyse the conversion of phenylpyruvate via mandelate and phenylglyoxylate to D-Phg. The corresponding genes were obtained from Amycolatopsis orientalis, Streptomyces coelicolor, and Pseudomonas putida. Combined expression of these activities in E. coli strains optimized for the production of L-phenylalanine resulted in the first completely fermentative production of D-Phg.     

Inhibition of intestinal absorption of phenylalanine by phenylalaninol.

Plasma phenylalanine and tyrosine levels in rats which had been orally administered L-phenylalaninol and L-phenylalanine were determined. Since these amino acid levels in rats administered L-phenylalanine solution containing L-phenylalaninol were significantly lower than those in rats administered L-phenylalanine alone. L-phenylalaninol appears to inhibit the intestinal absorption of L-phenylalanine. This effect was more potent than that of cycloleucine. L-phenylalaninol inhibited the phenylalanine transport of everted sacs. The Km value of L-phenylalanine was 3.44 X 10(-3) M and the Ki value of L-phenylalaninol was 7.69 M 10(-3) M from Lineweaver-Burk plots. From these two curves, it appeared that L-phenylalaninol may competitively inhibit the intestinal transport of L-phenylalanine. The effects of L-phenylalanine, L-phenylalaninol and cycloleucine on the urinary excretions of Na+ and K+ in rats were also examined. Potassium excretion which increased on oral administration of L-phenylalanine, was suppressed by the administration of L-phenylalaninol but not administration of cycloleucine. L-phenylalaninol alone enhanced Na+ excretion in urine. These results confirmed that L-phenylalaninol shows inhibitory effects as potent as those of cycloleucine on the intestinal absorption of L-phenylalanine.     

The contribution of phenylalanine to tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat.

1. Rates of appearance and oxidation of plasma L-leucine, L-phenylalanine and L-tyrosine, as well as conversion of plasma phenylalanine into plasma tyrosine, were determined in 90-120 g rats after overnight starvation and while receiving 115-120 mumol of L-phenylalanine/h. 2. In the post-absorptive state, plasma tyrosine and phenylalanine appearances were similar, despite the fact that 22% of plasma tyrosine appearance could be attributed to the hydroxylation of phenylalanine. 3. A constant infusion of 115-120 mumol of L-phenylalanine/h did not significantly alter plasma leucine kinetics, but increased appearance of plasma phenylalanine and tyrosine. The percentage of phenylalanine and tyrosine appearance that was oxidized increased from 12.1% and 24.4% to 37.3% and 48.0% respectively. In phenylalanine-loaded rats, 72% of plasma tyrosine appearance could be attributed to the conversion of phenylalanine. 4. Whole-body tyrosine oxidation measured from a continuous infusion of either L-[14C]tyrosine or L-[14C]phenylalanine differed by 165%. 5. It can be concluded that, in the post-absorptive state, phenylalanine hydroxylation makes a substantial contribution to the plasma appearance of tyrosine and is significantly increased when phenylalanine is administered. The disposal of excess infused phenylalanine is a result of a greater percentage of plasma phenylalanine being converted into tyrosine and a greater proportion of tyrosine being further oxidized. However, apparent tyrosine oxidation rates estimated from plasma tyrosine specific radioactivities and appearance of expired 14CO2 during administration of [14C]tyrosine are underestimates of true rates, in part because tyrosine generated from phenylalanine hydroxylation is catabolized without freely equilibrating with the plasma compartment.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Inhibition of Leaf Processes by p-Fluorophenylalanine During Induction of Flowering in the Cocklebur

The amino acid antimetabolite, DL-p-fluorophenylalanine (FPA), inhibited induction of flowering in the short-day cocklebur plant, Xanthium pensylvanicum Wall., primarily by interfering with processes occurring during the inductive dark period. At the concentrations used the inhibitor had little effect on subsequent vegetative development of the plant.The inhibition was largely reversed (internally) by L-phenylalanine, but not by D-phenylalanine nor by DL-tyrosine. The FPA strongly inhibited the absorption of labeled phenylalanine, leucine, and glycine, and inhibited the conversion of phenylalanine into protein in experiments where incorporation was separated in time from effects upon absorption. The FPA, too, was incorporated into protein, at nearly half the rate of phenylalanine. Neither D- nor L-phenylalanine significantly interfered with absorption of FPA, showing the FPA did not affect amino acid absorption by simple competition for a common carrier site. It was concluded that FPA may affect flower induction because of its interference with normal enzyme synthesis, although effects on other processes might also be involved.     

大肠杆菌EP8—10转化苯丙酮酸生成L—苯丙氨酸的研究

E. coli EP8-10 was selected from the soil. It was able to produce the transaminase with high activity when it was cultivated on the medium containing peptone and beef extract. Optimum conditions of enzyme reaction was: phenylpyruvic acid's concentration of 0.3-0.5 mol/L, L-Asptaric acid used as amino donor, pH 8.5 37 degrees C. When phenylpyruvic acid was 0.3 mol/L, 48 g/L L-phenylalanine was produced after 6 h with 97% conversion rate.