反应条件下苯丙氨酸解氨酶的活力稳定性

在苯丙氨酸解氨酶(PAL)的作用下由肉桂酸和氨合成L-苯丙氨酸(L-Phe)是酶法合成该氨基酸的重要途径,国外已利用该途径进行L-苯丙氨酸的工业生产,但是该过程仍存在着转化率低和酶活力稳定性差的问题。为解决这些问题,有必要在现有基础上开展提高酶活力稳定性的研究。     

植物苯丙氨酸解氨酶基因的研究进展

苯丙氨酸解氨酶(phenylalanineammonia-lyase,PAL)是连接植物初级代谢和苯丙烷类代谢、催化苯丙烷类代谢第一步反应的酶。综述植物PAL基因的研究进展,主要包括PAL基因的结构特点、表达特点和PAL基因表达的调控机制,并指出今后对PAL基因的研究方向。     

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％。     

苯丙氨酸代谢途径关键酶:PAL、C4H、4CL研究新进展

主要阐述了苯丙氨酸代谢途径中三种关键酶:苯丙氨酸解氨酶(PAL)、肉桂酸4-羟基化酶(C4H)、4-香豆酸辅酶A连接酶(4CL)的研究进展,希望能为研究植物次生代谢途径的研究工作者提供一些帮助。     

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.     

水稻黄化苗PAL内源性抑制物的初步纯化及其基本性质

从去胚乳水稻黄化苗中提取、并部分纯化了苯丙氨酸解氨酶的抑制因子(PAL-I)。它是非透析性的蛋白质;大部分活力可被蛋白酶(胰蛋白酶、链霉蛋白酶)所破坏。动力学实验表明PAL-I对PAL的抑制作用是竞争性的。水稻PAL-I不仅能抑制水稻PAL,而且能抑制从玉米、小麦、马铃薯块茎切片中提取的PAL;但不能抑制从水稻中提取的多酚氧化酶、α-淀粉酶(过氧化物酶除外)。     

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.     

大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系,利用 RT-PCR 和 RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长 cDNA。此 cDNA 长 1 300 bp,包含一个长为 1 191 bp,编码 397 个氨基酸的完整开放阅读框(ORF),推导的氨基酸序列与水稻 PAL 基因氨基酸序列同源性达 89%,将此基因命名为 M-PAL。Southern杂交结果表明大蕉中存在一个包含 4-5 个 PAL基因的基因家族,将此基因克隆到大肠杆菌表达载体 pET32(a )中,表达的蛋白质分子量大小与推导的相一致,并且表达的蛋白质表现出 PAL 酶活性。对接种香蕉枯萎病菌 4 号生理小种(Fusarium oxysporumf. sp. cubense (FOC) race 4 )后大蕉叶片中 M-PAL基因的转录谱进行研究表明,在接种枯萎病菌后,M-PAL基因在叶片中的转录水平提高,因此推测 M-PAL基因的表达可能与香蕉枯萎病抗性相关。     

水稻抗稻瘟病与苯丙氨酸解氨酶及过氧化物酶活性的相关性

已知苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)与水稻抗稻瘟病有密切相关,水稻受到稻瘟病菌(Pyricularia oryzae)侵染时,体内PAL与POD活性会发生显著变化。本文采用30个水稻(Oryza sativa)品种为材料,选择经常规浸种、催芽、生长较一致的种子,每一品种     

竹子木质素合成酶基因克隆与分析

苯丙氨酸解氨酶(Phenylalanine Ammonia-Lyase,PAL;EC 4.3.1.5)是木质素生物合成过程的关键酶和限速酶,竹材中的木质素含量的降低有利于提高竹浆的品质.采用RACE技术分别从黄古竹(Phyllostachys angusta)等4种竹子中克隆了PAL基因的全长序列,并进行了生物信息学分析.结果显示,PAL基因的开放读码框长度为2 136 bp,共编码712个氨基酸,具有2个外显子和1个内含子;对PAL蛋白单体的三维结构分析显示,PAL蛋白均含有大量的α螺旋和β折叠结构;基于邻接法的进化树对31种植物的PAL基因的氨基酸序列分析表明,竹类植物PAL的氨基酸序列之间同源性较高,与单子叶植物禾本科(除竹亚科植物外)的玉米和甘蔗等的亲缘关系较近,而与双子叶植物的辣椒、烟草、红参、莴笋等亲缘关系较远.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Caenorhabditis elegans and Ascaris suum: fragmentation of isocitrate lyase in crude extracts

It is well known that proteolysis often occurs after rupture of metazoan cells. Thus proteins isolated from extracts may not be representative of their native cellular counterparts. In the present research, extensive proteolysis was observed in crude extracts of the freeliving soil nematode Caenorhabditis elegans and the parasitic nematode Ascaris suum. Phenylmethylsulfonyl fluoride (PMSF) reduced the loss in activity of isocitrate lyase (EC 4.1.3.1), fumarase (EC 4.2.1.2), and citrate synthase (EC 4.1.3.7) in extracts of C. elegans but had little or no effect upon loss of malate synthase (EC 4.1.3.2). Catalase (EC 1.11.1.6) was stable. The loss of isocitrate lyase and citrate synthase was less pronounced in extracts of 22-day-old embryos of A. suum. Catalase decayed in these extracts. The addition of PMSF reduced the loss in all three of these activities. Fumarase was stable. The number of active fragments of isocitrate lyase recovered after filtration on Sephadex G-200 increased with the length of storage of crude extracts in the absence of PMSF at 4 C. Even in the presence of PMSF five activity peaks were observed after storage of extracts of C. elegans at 4 C for 72 hr. The molecular weights of active species ranged between 549,000 and 128,000 for isocitrate lyase in extracts of either C. elegans or A. suum. The 549,000- and 214,000-dalton species of isocitrate lyase from A. suum were much more labile at 50 C than the 543,000- and 195,000-dalton species from C. elegans.     

Lipid synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid-specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl-galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.     

In Vitro and Cellular Effects of 4-Pyridone-3-Carboxamide Riboside on Enzymes of Nucleotide Metabolism

4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that has recently been identified as a substrate for intracellular phosphorylation to form nucleotide derivatives. Low level of 4PYR is normally present in human plasma, but 4PYR massively accumulates in patients with renal failure. This study aimed to evaluate effects of 4PYR and its monophosphate derivative (4PYMP) on several enzymes of nucleotide metabolism in homogenates and intact cells. Activities of adenosine monophosphate deaminase (AMPD), adenosine deaminase, ecto-5′-nucleotidase (e5NT), adenine phosphoribosyltransferase (APRT), hypoxanthine/guanine phosphoribosyltransferase, purine nucleoside phosphorylase, and S-adenosylhomocysteine hydrolase (SAHH) were evaluated in erythrocyte lysates, rat heart homogenates, and in the intact rat cardiomyocytes by high performance liquid chromatography–based assays. 4PYMP caused significant inhibition of AMPD in both erythrocyte lysate and heart homogenate with 50% inhibitory concentration (IC50) of 74 and 55 μM, respectively. Inhibition of e5NT in heart homogenates was also noted with IC50 of 63 μM. 4PYMP slightly inhibited APRT and 4PYR caused moderate activation of SAHH. No effects on other enzymes studied were noted. Inhibition of AMPD by 4PYMP in homogenates was confirmed in the intact cell experiments with isolated cardiomyocytes that were allowed to accumulate 4PYMP by incubation with 4PYR. We conclude that among pathways studied, most important is the effect of 4PYMP on AMPD and that such effect could be one of the consequences of elevated plasma 4PYR concentration.     

Effect of chitin hexamer and thuricin 17 on lignification-related and antioxidative enzymes in Soybean Plants

Inducers of disease resistance in crop plants have a role in sustainable agriculture. We describe a set of bacteriocins that can potentially improve plant growth by controlling specific pathogens and inducing generalized resistance. Solutions of the bacteriocin thuricin 17 and/or a chitin hexamer (a known inducer and positive control) were applied to leaves of two-week-old soybean plants, and levels of lignification-related and antioxidative enzymes were monitored. Phenyl ammonia lyase (PAL) activity in thuricin 17-treated leaves was highest at 60 h after treatment, being 61.8% greater than the control. PAL activity also was increased 18.1% at 72 h after treatment with the chitin hexamer. Tyrosine ammonia lyase (TAL) activity in leaves was 57.0% higher than the control at 48 h after treatment with thuricin 17, while such activity in chitin hexamer-treated leaves was increased by 23.8% at 72 h. At 36 h after treatment with the chitin hexamer or chitin hexamer + thuricin 17, the total concentration of phenolic compounds was 15.3 or 19.3%, respectively, greater than the control. At 72 h, total phenolic concentrations increased by 23.2 and 19%, respectively, in response to thuricin 17 and chitin hexamer + thuricin 17. POD activity in thuricin 17-treated leaves increased by 74.6 and 81.2% at 48 and 72 h, respectively, whereas SOD activity increased by 24.9 and 79.9%, respectively, in chitin hexamer- and thuricin 17-treated leaves at 48 h. A peroxidase isozyme (31 kDa isomer) was induced in thuricin 17-treated leaves at 60 h, while catalase (59 kDa isomer) was induced in chitin hexamer-treated leaves. PAGE showed that two major SOD bands (Fe-SODs) were produced by both types of treatment. Collectively, these results indicate that the bacteriocin thuricin 17 can act as an inducer of plant disease defenses (i.e., activated lignification-related enzymes, antioxidative enzymes, and related isozymes) and that this induction is similar, but not identical, to that of the chitin hexamer elicitor. Although treatment with thuricin 17 + chitin hexamer also induced those responses, it did not present a clear pattern of additivity or synergy.     

Glucose transporter 4 can be inserted in the membrane without exposing its catalytic site for photolabeling from the medium

Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4 from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100nmol/L insulin and 10μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabelling and possibly glucose uptake after treatment with PI(3,4,5)P3.     

Molecular structure of malate synthase and structural changes upon ligand binding to the enzyme

Malate synthase has a molecular weight of about 170 000 as shown by ultracentrifugation, sucrose gradient centrifugation, and thin layer gel-chromatography. High dilution, extremes of pH, succinylation, and treatment with sodium dodecylsulfate suggest the enzyme to be a tetramer. The CD spectrum is typical for a globular protein with moderate helical content (～30 %), and shows anomalous Cotton effects at 250–290 nm. Binding of substrates (acetyl-CoA, glyoxylate) or the substrate analog pyruvate causes slight conformational changes which are reflected in alterations of the CD bands in the range of aromatic absorption; binding of Mg²⁺ causes no structural effects, suggesting the metal ion to be involved in enzymatic catalysis rather than structural alterations.     

Glucose transporter 4 can be inserted in the membrane without exposing its catalytic site for photolabeling from the medium

Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100 nmol/L insulin and 10 μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabeliing and possibly glucose uptake after treatment with PI(3,4,5)P3.