酶法生产L-苯丙氨酸

<正> 一、前言直到几年前,L—苯丙氨酸(L-phe)主要用作输液,一年不过需要100～200吨。但近来作为人工甜味剂天冬甜精(L-苯丙氨酸甲酯和L-天冬氨酸的肽)的合成原料,对它的需求量显著增加,预测到1990年年需要量近8000吨。L-phe过去主要采用两种方法提供:直     

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。     

草鱼肠道对L-亮氨酸和L-苯丙氨酸的吸收

利用离体灌注法研究草鱼肠道对Ｌ－亮氨酸和Ｌ－苯丙氨酸的吸收规律。结果表明,草鱼肠道对两种氨基酸的吸收是一种逆浓度、需要转运载体的主动吸收方式。通过动力学特征分析表明,草鱼肠道对Ｌ－亮氨酸的吸收率强于Ｌ－苯丙氨酸,而肠道对Ｌ－苯丙氨酸的跨壁运输能力强于Ｌ－这氨酸。由氨酸酸浓度对吸收率的影响建立的动力学参数为Ｌｅｕ：Ｊｍａｘ＝０．７３２μｍｏｌ／ｇ．ｍｉｎ,ｋｔ＝５１．６０ｍｍｏｌ／Ｌ;Ｐｈｅ：Ｊｍａ     

L-苯丙氨酸与血管平滑肌细胞增殖

本文用氚标胸腺嘧啶核苷掺入ＤＮＡ合成法测定自发性高血压大鼠（ＳＨＲ）与正常对照鼠的培养主动脉血管平滑肌细胞（ＶＳＭＣ）增殖,观察Ｌ－苯丙氨酸对细胞增殖、细胞生长及原癌基因ｃ－ｆｏｓ、ｃ－ｍｙｃ表达的影响。结果显示：（１）Ｌ－苯丙氨酸剂量依赖性地抑制血清、碱性成纤维细胞生长因子及凝血酶诱导的ＤＮＡ合成;（２）Ｌ－苯丙氨酸剂量依赖性地抑制细胞对血清的增殖反应;（３）Ｌ－苯丙氨酸抑制血清诱导的ｃ－ｆｏｓ     

深红酵母转化反式肉桂酸生成L-苯丙氨酸的研究

研究了深红酵母Ａｓ２．２７９产生Ｌ－苯丙氨酸解氨酸（ＰＡＬ）的条件、转化反式 桂酸（ｔＣａ）生成Ｌ－苯丙氨酸（Ｌ－Ｐｈｅ）的条件以及几种因素对ＰＡＬ稳定性的影响,结果表明,最佳转化条件为：１．０％ｔ－Ｃａ,８ｍｏｌ／Ｌ氨,ｐＨ１０．０,３０℃。在转化液中加入还原剂和充入Ｎ２有利于提高酶的稳定性,在此条件下可一次转化６４％的ｔ－Ｃａ,保留６０％的酶活。生成Ｌ－Ｐｈｅ浓度为５．８ｇ／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.     

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。     

浊度法测定发酵液中L－苯丙氨酸含量

本文介绍浊度测定发酵液中Ｌ－苯丙氨酸含量的方法。根据指示菌生长的细胞密度与生长培养基内所含的苯丙氨酸量在一定浓度范围内呈线性关系的原理,摸索了浊度测定的最适条件。结果表明浊度法测定发酵液中的苯丙氨酸含量,简便、准确性高。     

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.     

定点突变提高苯丙氨酸羟化酶的热稳定性

嗜热菌中,蛋白质存在Ala替换Gly以及Arg替换Lys的趋势。为了提高紫色色杆菌来源的苯丙氨酸羟化酶的热稳定性,将该酶中所有Gly突变成Ala,Lys突变成Arg,筛选获得热稳定性提高的突变体,并进行组合突变,对突变酶的酶学性质进行研究。结果表明,突变酶K94R和G221A在50℃的半衰期分别为26.2 min、16.8 min,比原始酶(9.0 min)分别提高了1.9倍、0.9倍,同时组合突变酶K94R/G221A在50℃处理1 h后仍保留65.6%的酶活,比原始酶(8.6%)高出6.6倍。圆二色谱结果显示原始酶和突变酶K94R、G221A及K94R/G221A的T_m值分别为51.5℃、53.8℃、53.1℃和54.8℃。蛋白三维结构模拟推测突变体热稳定性提高机理为:突变体K94R中Arg94与Ile95之间形成额外氢键,稳定其所在的柔性区域;突变体G221A中Ala221与Leu281产生疏水作用,稳定酶分子C-端柔性区。该研究结果为蛋白质热稳定性改造提供了参考,也为苯丙氨酸羟化酶在功能性食品领域的应用奠定了基础。     

Development of an interference-free biosensor for l-glutamate using a bienzyme salicylate hydroxylase/l-glutamate dehydrogenase system

An amperometric biosensor was developed for the interference-free determination of l-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l-glutamate consuming NAD⁺. The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of l-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate with a detection limit of 3.0 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and electroactive substances.     

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase.

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.     

The spectrum of mutations and methods for their detection in the phenylalanine hydroxylase gene in phenylketonuria patients from the novosibirsk region

Phenylketonuria (PKU) is a widespread autosome recessive hereditary disease caused by a deficiency of the liver enzyme phenylalanine hydroxylase, which results in distortion of metabolism of phenylalanine and accumulation of toxic metabolites. The knowledge of molecular bases of PKU is of a high social importance as it enables phenotypic correction of the disease in the case of its early diagnostics. This disease is known to be associated with mutations in the phenylalanine hydroxylase gene, the distribution and mutation spectrum having pronounced ethnic and regional features. We studied the spectrum of mutations in the phenylalanine hydroxylase gene in a group of patients with PKU from the Novosibirsk region to reveal 10 missense point mutations, 1 mutation in the splice donor site, and 1 microdeletion. For these mutations, most widely distributed in the region, we used straightforward detection methods basing on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR, and denaturing gradient gel electrophoresis (DGGE).     

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.     

Molecular epidemiology and BH4-responsiveness in patients with phenylalanine hydroxylase deficiency from Galicia region of Spain

Knowledge of hyperphenylalaninemia (HPA) mutational spectrum in a population allows in many cases an accurate prediction of the phenotype and tetrahydrobiopterin (BH4) responsiveness, thus selecting an adequate treatment. In this work, we have performed the molecular characterization of 105 HPA patients from Galicia, in the northwest region of Spain, evaluating their phenotype and BH4 response.     

Development of a mutation hotspot detection kit for the phenylalanine hydroxylase gene by ARMS-PCR combined with fluorescent probe technology

To develop a screening kit for detecting mutation hotspots of the phenylalanine hydroxylase (PAH) gene. Thirteen exons of the PAH gene were sequenced in 84 cases with phenylketonuria (PKU) diagnosed during neonatal genetic and metabolic disease screening in Shaanxi province, and their mutations were analyzed. We designed and developed a screening kit to detect nine mutation sites covering more than 50% of the PAH mutations found in Shaanxi province (c.728G>A, c.1197A>T, c.331C>T, c.1068C>A, c.611A>G, c.1238G>C, c.721C>T, c.442-1G>A, and c.158G>A) by using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) combined with fluorescent probe technology. Peripheral blood and dried blood samples from PKU families were used for clinical verification of the newly developed kit. PAH gene mutations were detected in 84 children diagnosed with PKU. A total of 159 mutant alleles were identified, consisting of 100 missense mutations, 28 shear mutations, 24 nonsense mutations, and 7 deletion mutations. Exon 7 had the highest mutation frequency (32.08%). Among them, the mutation frequency of p.R243Q was the highest, accounting for 20.13% of all mutations, followed by p.R111X, IVS4-1G>A, EX6-96A>G, and p.R413P; these five loci accounted for 47.17% (75/159) of all mutations. In addition, we identified three previously unreported PAH gene mutations (p.C334X, p.G46D, and p.G256D). Fifteen mutation sites were identified in the 47 PAH carriers identified by next-generation sequencing (NGS), which were verified by the newly developed kit, with an agreement rate of 100%. This newly developed kit based on ARMS-PCR combined with fluorescent probe technology can be used to detect common PAH gene mutations.