林生山黧豆幼苗2,4—二氨基丁酸的合成及其与胁迫条件的关系

林生山黧豆幼苗用［3H］－天门冬氨酸标记后,高丝氨酸在6h内迅速增加。高丝氨酸合成速率降低后,2,4－二氨基丁酸的合成量上升,于18h达到高峰。赖氨酸和苏氨酸与二氨基丁酸的合成表现有协同反馈机制。结果支持了天门冬半醛转氨生成二氨基丁酸的假说。盐胁迫、渗透胁迫和热激增加了二氨基丁酸的合成,可能是因为不同胁迫条件都造成了细胞脱水,从而促进了二氨基丁酸的合成。     

大田生长的林生山黧豆体内游离氨基酸含量

林生山黧豆（Lathyrussylvestris）是一种耐干旱抗贫瘠的多年生草本植物，含有丰富的游离氨基酸，特别是含有一些非蛋白质游离氨基酸，如2，4-二氨基丁酸，γ-氨基丁酸和高丝氨酸。国外文献报道的均是各种逆境条件下生长的林生山黧豆，本文则是大田生长的林生山黧豆，种子从美国引进。数年的栽培试验，表明它能适应我国北方气候。但其体内游离氨基酸的组成特点如何，尚不清楚。为此我们采用本实验室建立的聚酰胺薄膜层析荧光定量法，结合氨基酸自动分析仪，测定了其体内的游离特殊非蛋白质氨基酸和常规氨基酸。样品采自中国农业大学科学园…     

林生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究

以林生山黧豆为材料,利用硫酸按分段盐析,丙酮沉淀,DEAE-SepharoseFF离子交换柱层析,SephacrylS300凝胶过滤柱层析及FPLC-MonoQ柱层析技术,以聚酰胺薄膜层析荧光定量法为酶活力检测手段,分离纯化了谷氨酸脱羧酶,达到电泳银染纯．纯化后的林生山黧豆谷氨酸脱羧酶活力达375．09U·mp-1,纯化倍数38．2倍,经SDS-PAGE测定,其亚基分子量为70kD,经梯度PAGE确定,天然分子量为140kD,表明该酶是由两个亚基组成的二聚体．酶学研究表明,纯化的林生山黧豆谷氨酸脱羧酶的最适pH值为5．4,对谷氨酸的Km值为1．62×10-3mol·L-1,酶的最适温度为40℃,酶特异性地使谷氨酸脱羧,不能使天门冬氨酸等其它氨基酸脱羧．     

一株食用菌生物转化富集γ-氨基丁酸条件研究

从本实验室保存的食用菌中筛选到菌株CSP501,其菌丝体可以谷氨酸钠为底物转化富集γ-氨基丁酸。通过单因素试验和正交试验,菌株CSP501转化富集γ-氨基丁酸的最适条件：转化温度45℃,转化时间5h,体系pH4．0,底物浓度1％。γ-氨基丁酸的含量可以达到干重的1．05％,达到较高的含量。     

苏云金芽胞杆菌γ-氨基丁酸代谢途径相关功能基因的克隆、表达及同源性分析

γ-氨基丁酸代谢旁路作为三羧酸循环的一个分支,在真核、原核生物中广泛存在。在这条代谢途径中,涉及γ-氨基丁酸分解代谢的主要有两种酶:一种是γ-氨基丁酸转氨酶,能将γ-氨基丁酸转变成琥珀酸半醛;另一种是琥珀酸半醛脱氢酶,该酶能将琥珀酸半醛氧化形成琥珀酸,后者进入三羧酸循环。从国内分离得到的苏云金芽胞杆菌G03菌株中克隆了gabT和gabD基因。其中gabT基因含有1440bp,编码一个大小为52.6kD的蛋白质,而gabD基因大小为1449bp,编码一个52.2kD的蛋白质。这两个基因都分别在大肠杆菌中进行了表达和纯化。通过酶活测定结果表明,GabT和GabD蛋白分别呈现出γ-氨基丁酸转氨酶和琥珀酸半醛脱氢酶的活性。氨基酸序列同源性比对分析发现,这两个蛋白质在蜡样芽胞杆菌群(B.cereus group)中具有较高的相似性,而与枯草芽胞杆菌的相似性较低则分别为58%、51%。为进一步深入研究γ-氨基丁酸代谢旁路在苏云金芽胞杆菌中的生物学功能及其转录调控机制奠定了基础。     

亮氨酸脱氢酶偶联NADH再生体系合成L-2-氨基丁酸

文中以大肠杆菌BL21(DE3)为宿主,构建两株分别共表达亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/甲酸脱氢酶(FDH,来源水生弯杆菌)和亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/醇脱氢酶(ADH,来源红球菌)的重组大肠杆菌。通过偶联两种不同NADH再生体系,以L-苏氨酸为起始原料,利用苏氨酸脱氨酶(L-TD)与LDH-FDH或LDH-ADH一锅法合成L-2-氨基丁酸,并对LDH-FDH工艺和LDH-ADH工艺进行对比优化。LDH-FDH工艺的最适反应pH为7.5,最适反应温度为35℃,通过加入50 g/L甲酸铵、0.3 g/L NAD+、10%LDH-FDH粗酶液(V/V)和7 500 U/L的L-TD酶液,对L-苏氨酸进行分批补加,以便控制2-丁酮酸浓度小于15 g/L,反应28 h,实现了L-2-氨基丁酸的产量为161.8 g/L,产率97%。LDH-ADH工艺的最适pH为8.0,最适反应温度为35℃,通过加入0.3 g/L NAD+、10%LDH-ADH粗酶液(V/V)及7 500 U/L的L-TD酶液,分批补加L-苏氨酸及1.2倍摩尔量异丙醇,以便控制2-丁酮酸浓度小于15g/L,且每生成约40g/L的L-2-氨基丁酸,抽真空去除丙酮,反应24h,实现了L-2-氨基丁酸的产量为119.6 g/L,产率98%。文中所采用的工艺及结果可为L-2-氨基丁酸的工业化提供一定的参考依据。     

南瓜叶中γ-氨基丁酸的提取及薄层扫描测定

本文在单因素试验基础上,通过正交试验优化了南瓜叶中γ-氨基丁酸的提取条件,并对γ-氨基丁酸的薄层扫描测定方法进行了探索.结果表明,南瓜叶中γ-氨基丁酸的最佳提取条件为:以20％乙醇为溶剂、料液比1∶17(w/v)、提取时间1h,此条件下得到的γ-氨基丁酸的含量为209 mg/100 g.所建立的薄层扫描测定方法在0.125 ～2.0 mg/mL范围内呈良好线性关系(R2=0.9991),平均加样回收率(n=4)为99.61％.     

植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成,反应体系成分简单、环境友好。然而,绝大多数GAD酶催化pH偏酸性且反应范围狭小,需要加入无机盐维持最适催化环境,增加了生产附加成分。此外,随着产物γ-氨基丁酸的生成,溶液pH会逐渐上升,不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD,基于酶蛋白表面电荷修饰,选择9个位点进行定点突变及组合突变,酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高,尤其拓宽了在偏中性pH 6.0下的酶活,为野生酶的1.68倍。接下来,通过分子动力学模拟解析了酶活提高的机理。此外,将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达,通过优化确定了摇瓶最适转化条件为反应温度40 ℃,菌体量OD₆₀₀=20,底物L-谷氨酸100.0 g/L,5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中,不调节pH,通过分批投料底物L-谷氨酸,γ-氨基丁酸产量高达402.8 g/L,较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活,提高了γ氨基丁酸的转化效率,为实现其规模化工业生产奠定了基础。     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

比色法快速测定酶转化反应中γ-氨基丁酸质量分数

建立了谷氨酸脱羧酶催化L-谷氨酸生成γ-氨基丁酸过程中产物质量分数快速测定的比色检测方法,其原理为Berthelot显色测定ω-氨基酸的反应。结果表明,该方法灵敏度较高,准确度好,快捷有效,可替代氨基酸分析仪分析法。取不同时段反应液300μL在冰浴中加200μL 0.2 mol.L-1硼酸缓冲液(pH10.0)终止反应,加入体积分数6%重蒸苯酚100μL和质量分数10%次氯酸钠400μL,沸水浴加热10 m in后冰浴冷却5 m in,630 nm测定吸光度值,以标准曲线法确定γ-氨基丁酸的质量分数。该方法适合大批量样品的快速分析,但需排除游离氨的影响。     

In Vitro Enzymological Studies on Anabolism of 2,4-diaminobutyric Acid in Lathyrus sylvestris

An in vitro synthetic reaction system was established with 2,3-3H-aspartic acid (Asp) as a substrate and the homogenate of fiatpea ( Lathyrus sylvestris L. ) leaves as the crude enzyme extract. The results showed that 3H-Asp was incorporated into 2,4-diaminobutyric acid (DABA). The incorporation was inhibited by the addition of glutamic acid (Glu). 3H-Asp was also incorporated into DABA after the cmde enzyme was dialyzed, indicating that Asp as a substrate for DABA synthesis was catalyzed by a group of enzymes which converted Asp to DABA in flatpea. From the in vitro reactions it was proved that DABA and γ-aminobutyric acid (GABA) could not be mutually substituted as substrates.     

Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue

The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var `Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells.     

The presence of l-2,4-diaminobutyric acid decarboxylase activity in Vibrio species: A new biosynthetic pathway for 1,3-diaminopropane

Abstract A new enzyme activity, which catalyzes decarboxylation of l -2,4-diaminobutyric acid (DABA) to yield 1,3-diaminopropane (DAP), has been found in dialyzed crude extracts prepared from Vibrio alginolyticus . The pH optimum for the activity was 8.0–8.5, and the enzyme showed a pyridoxal 5'-phosphate (PLP) requirement. Mg²⁺ caused about 30% stimulation in activity. The enzyme was active to only l -DABA among the diamino acids examined, and the K _m value for l -DABA was 0.13 mM. Ammonium sulfate fractionation of a dialyzed crude extract followed by HPLC separation allowed us to conclude that this enzyme differed from the decarboxylase which occurs in Vibrio spp. to produce norspermidine (Nspd) for carboxynorspermidine (C-Nspd) having a moiety similar in structure to DABA. The same enzyme activity was detected in several other Vibrio species.     

Amino Acid Neurotransmitters in the CNS: Properties of Diaminobutyric Acid Transport

Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of gamma-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 +/- 12 microM and a Vmax of 1.3 +/- 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]c) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of +2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.     

EVIDENCE FOR THE HETEROGENEITY OF L-2,4-DIAMINOBUTYRIC ACID UPTAKE IN RAT CORTEX

The uptake of L-[³H]DABA by rat cerebral cortex slices was studied. Analysis of the kinetic data obtained provides evidence that DABA entry is mediated by both high and low affinity carriers. When cortical slices were incubated in the presence of equimolar [³H]DABA and [¹⁴C]GABA the ratio of entry of the two radionuclides was found to depend upon the loading concentration. The specificity of the uptake of 1 μM and 1 mM-L-DABA was examined: GABA and DABA were relatively potent inhibitors of 1 μM-DABA uptake whereas an equal concentration of histidine did not produce significant inhibition. In contrast, DABA and histidine were markedly more potent as inhibitors of 1 mM-DABA uptake than was GABA. It is concluded from these experiments that L-DABA is transported into cortical slices by a carrier which has high affinities for both DABA and GABA and by a second lower affinity carrier which prefers DABA as a substrate to GABA. On the basis of a comparison of the effects of inhibitors on [³H]DABA and [³H]GABA uptake it is estimated that approx 26% of DABA uptake at 1 μM does not occur by the high affinity carrier whereas at 1 mM-DABA this proportion rises to 62–67%.     

Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

Fatty acid biosynthesis in Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin. Purification and characterization of a novel fatty acid synthase, mycocerosic acid synthase, which elongates n-fatty acyl-CoA with methylmalonyl-CoA

A crude extract from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin was previously shown to incorporate methylmalonyl-CoA into mycocerosic acids, exemplified by 2,4,6,8-tetramethyloctacosanoic acid, and malonyl-CoA into n-fatty acids (Rainwater D. L., and Kolattukudy, P. E. (1983) J. Biol. Chem. 258, 2979-2985). The presence of several fatty acid synthases with differences in substrate preference and product chain length was detected in the crude extract of M. tuberculosis var. bovis. Among them was a mycocerosic acid synthase which was purified to homogeneity using anion-exchange chromatography, gel filtration, affinity chromatography, and hydroxylapatite chromatography. This fatty acid synthase elongated long-chain fatty acyl-CoA primers using methylmalonyl-CoA and NADPH to produce multimethyl-branched mycocerosic acids. The enzyme was specific for methylmalonyl-CoA and would not incorporate malonyl-CoA into fatty acids. It elongated n-C6 to n-C20 CoA esters to generate primarily the corresponding tetramethyl-branched mycocerosic acids. Exogenous [1-14C]acyl-CoA and trideuteromethylmalonyl-CoA were incorporated into the multimethyl-branched fatty acids. Dodecyl sulfate electrophoresis showed that the enzyme had a molecular weight of 238,000, whereas gel filtration showed a native molecular weight of 490,000, indicating that the enzyme is composed of two monomers of identical molecular weight. The enzyme contained an acyl carrier protein-like segment as indicated by incorporation of [1-14C] pantothenate into the 238-kDa protein and production of 1 mol of taurine/mol of the monomer upon hydrolysis of performic acid-oxidized enzyme. It is concluded that the mycocerosic acid synthase is a multifunctional enzyme similar to the well-characterized multifunctional fatty acid synthases except for the substrate specificity.     

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.     

Influence of drought on the concentration and distribution of 2,4-diaminobutyric acid and other free amino acids in tissues of flatpea (Lathyrus sylvestris L.)

2,4-Diaminobutyric acid (A₂bu) may be responsible for the apparent toxicity of flatpea (Lathyrus sylvestris L.) forage to some livestock. To obtain information relative to environmental regulation of A₂bu, 3-month old flatpea plants, cv. “Lathco”, were subjected to water-deficit stress for 1, 2, and 4 weeks. A₂bu, the most abundant free amino acid in roots, stems, and leaves, increased nearly 100% in roots of stressed plants. Increases in the concentrations of asparagine (Asn), proline (Pro), and arginine (Arg) occurred in roots; Asn, Pro, and 4-aminobutyric acid (Abu) in stems; and Pro and homoserine (Hse) in leaves also occurred in response to drought stress. Proline was a minor constituent of the free amino acid pool, even under water-deficit stress. The distribution of A₂bu and Pro in the stressed plants (roots > stems > leaves) was the reverse of that in plants supplied with adequate water (roots < stems < leaves). As concentrations of Asn and Abu decreased from roots to leaves in control tissues, concentrations of Hse and A₂bu increased in roughly the same proportions. This observation suggests that Abu and Asn may be precursors of A₂bu and Hse, respectively. The increase in A₂bu levels in aerial parts of drought-stressed flatpea plants is probably not sufficient to lower the feed value of the forage.