L-天冬氨酸脱羧酶研究进展

根据作用于L-天冬氨酸的位置不同,L-天冬氨酸脱羧酶可分为L-天冬氨酸α-脱羧酶和L-天冬氨酸β-脱羧酶,本文综述了两种酶在结构特点、酶学性质、基因表达、酶的应用等方面的研究进展。     

谷氨酸棒杆菌L-天冬氨酸α-脱羧酶在大肠杆菌中的表达及酶转化生产β-丙氨酸

【目的】克隆谷氨酸棒杆菌来源L-天冬氨酸α-脱羧酶基因, 实现其在大肠杆菌中的异源表达, 并进行酶转化L-天冬氨酸合成β-丙氨酸的研究。【方法】PCR扩增谷氨酸棒杆菌L-天冬氨酸α-脱羧酶基因pand, 构建表达载体pET24a(+)-Pand, 转化宿主菌大肠杆菌BL21(DE3), 对重组菌进行诱导表达, 表达产物经DEAE离子交换层析和G-75 分子筛层析纯化后进行酶学性质研究, 然后进行酶转化实验, 说明底物和产物对酶转化的影响。【结果】重组菌SDS-PAGE分析表明Pand表达量可达菌体总蛋白的50%以上, AccQ·Tag法检测酶活达到94.16 U/mL。该重组酶最适反应温度为55 °C, 在低于37 °C时保持较好的稳定性, 最适pH为6.0, 在pH 4.0?7.0范围内有较好的稳定性。酶转化实验说明: 底物L-天冬氨酸和产物β-丙氨酸对转化反应均有抑制作用; 实验建立了较优的酶转化反应方式, 在加酶量为每克天冬氨酸3 000 U时, 以分批加入固体底物L-天冬氨酸的形式, 使100 g/L底物转化率达到97.8%。【结论】重组L-天冬氨酸α-脱羧酶在大肠杆菌中获得高效表达, 研究了酶转化生产β-丙氨酸的影响因素, 为其工业应用奠定了基础。     

产L-天冬氨酸α-脱羧酶细菌的分离、鉴定及发酵条件优化

【目的】从葡萄园土壤中分离L-天冬氨酸α-脱羧酶的产生菌株,对其进行分类鉴定,优化其产生L-天冬氨酸α-脱羧酶的发酵条件,为β-丙氨酸的生物合成提供基础。【方法】采用变色圈法和液体复筛培养基分离筛选具有L-天冬氨酸α-脱羧酶活力的菌株,对菌株进行形态、生理生化特征试验及16S r RNA序列同源性分析鉴定菌株的系统发育学地位,采用单因素及正交设计试验优化培养基及发酵条件。【结果】筛选到一株L-天冬氨酸α-脱羧酶高产菌株Pan D37,其亲缘关系和特基拉芽孢杆菌(Bacillus tequilensis)较近,且形态与培养特征、生理生化特性与特基拉芽孢杆菌基本相符。研究表明其最佳发酵配方和培养条件为:蔗糖22.5 g/L、富马酸7.5 g/L、蛋白胨20 g/L、L-天冬氨酸6 g/L、Triton X-100 2g/L,起始p H为7.0,装液量50 m L/500 m L,摇床转速220 r/min,种子液接种量为5%(V/V),35°C培养28h。在最优条件下L-天冬氨酸α-脱羧酶活力可达44.57 U/m L,比初筛时提高2.57倍。【结论】分离并获得一株特基拉芽孢杆菌(Bacillus tequilensis)Pan D37,经条件优化后具有较高的L-天冬氨酸α-脱羧酶产生能力,有望应用于β-丙氨酸的工业生产。     

L-天冬氨酸-α-脱羧酶高产菌株初筛方法的建立

通过试管法、平板法、Berthelot法和纸层析法对L-天冬氨酸-α-脱羧酶(PanD)高产菌株的初筛方法进行研究。分别检测转化体系中底物L-天冬氨酸减少引起的pH变化及产物CO2和β-丙氨酸的增加量,并用高效液相色谱法比较这四种方法的准确性。结果表明:CO2能溶于转化液,难以用倒置管收集完全;PanD系胞内酶,难以用平板点种法改变平板的pH;β-丙氨酸和L-天冬氨酸在Berthelot检测中吸收值均偏低,不适用于PanD高产菌的筛选;纸层析法可以直接检测β-丙氨酸和L-天冬氨酸,成本低,操作简单,具一定的精确度,可以较大规模地筛选PanD高产菌株。     

L-天冬氨酸-α-脱羧酶的重组表达、定点突变及高通量检测方法的建立

将枯草芽孢杆菌L-天冬氨酸-α-脱羧酶基因进行了克隆和异源表达,并通过定点突变构建了2个突变体。针对该酶活力检测时存在的检测通量低、周期长和成本高等缺点,旨在建立一种简单高效的酶活力高通量检测方法。采用氯酚红(CPR)指示剂和4-吗啉乙磺酸(MES)缓冲液体系,并对检测条件进行优化,提高检测的准确性和灵敏性,建立了基于比色法的微孔板高通量检测方法,然后以L-天冬氨酸-α-脱羧酶及其突变体作为模型酶,对高通量检测方法进行了验证。优化后的酶活检测条件为MES缓冲液2 mmol/L,CPR指示剂75 μmol/L,L-天冬氨酸75 mmol/L,pH 6.5,温度37℃,反应时间10 min,检测波长为567 nm。采用3种模型酶对微孔板高通量检测方法进行了验证,结果显示该方法与HPLC法测得的结果一致。高通量检测方法具有操作简便易行、灵敏度高等优点,能够用于L-天冬氨酸-α-脱羧酶的快速检测。该方法的建立将为L-天冬氨酸-α-脱羧酶进行定向进化及突变体的高通量筛选奠定基础。     

双酶偶联转化富马酸制备β-丙氨酸的工艺的建立与优化

酶转化法是生产β-丙氨酸的重要途径,但单一酶法转化存在底物价格较高的问题。通过构建双酶催化体系制备β-丙氨酸,即将来源于大肠杆菌的天冬氨酸酶(AspA)和来源于谷氨酸棒杆菌的L-天冬氨酸α-脱羧酶(PanD)偶联,以富马酸和氨为底物进行酶促反应合成β-丙氨酸。催化反应中AspA与PanD的最适加酶比例为1∶80,其中AspA的浓度为10μg/mL,转化温度为37℃,pH为7.0;浓度为100 mmol/L的富马酸可在8 h内被完全转化,转化率为100%,摩尔产率为90.9%,β-丙氨酸的产量为90 mmol/L,约为7 g/L;浓度为200 mmol/L的富马酸在反应8 h后,体系中β-丙氨酸的产量为126 mmol/L,约合9.8 g/L,继续延长反应时间,转化率并没有明显提高。根据该研究提出的双酶偶联转化工艺可将价格低廉的富马酸一步转化为具有高附加值的β-丙氨酸。     

杰氏棒杆菌L-天冬氨酸α脱羧酶半理性改造及全细胞催化合成β-丙氨酸

β-丙氨酸是多个药物合成的重要砌块，可以通过天冬氨酸α脱羧酶（Pan D）催化L-天冬氨酸脱羧来合成，但普遍在用的Pan D酶活性不高是制约全细胞催化合成β-丙氨酸的瓶颈。因此，本研究通过酶的挖掘，选择将杰氏棒杆菌来源（Corynebacterium jeikeium）Pan D在Escherichia coli中异源表达。对杰氏棒杆菌来源Pan D进行Alaph Fold2建模和分子对接，采用Rosetta虚拟突变确定突变热点，结合薄层层析初筛和纯化后复筛，最终筛选到突变体L39A，其比酶活为13.45 U/mg，相比野生型酶的比酶活（9.6 U/mg）提升了1.4倍。酶学性质表征数据表明，野生型酶和L39A突变体最适p H均为6.5，且在p H 6.0-7.0之间酶活性稳定；两者最适温度为55℃，但L39A热稳定性较野生型提高；突变体酶的催化效率比野生型提升了1.4倍。对突变体进行结构解析发现，39位取代为侧链基团更小的丙氨酸，亲水性增强，增加了关键催化氨基酸58位酪氨酸与其他氨基酸的相互作用，使活性中心周围的区域稳定性提高，从而提高了催化活性。全细胞催化数据表明，在OD600=4...     

蚕氨基酸代谢之研究 Ⅲ.丝腺体L-天门冬氨酸及α-酮戊二酸形成丙氢酸之机制

研究了家蚕(Bombyx mori L.),天蚕蛾科之蓖麻蚕(Philosama cynthia ricini B.)及柞蚕(Antheraea pernyi G.)丝腺体后部自L-天门冬氨酸与α-酮戊二酸形成丙氨酸的机制。以上各种蚕的丝腺体组织都可利用L-天门冬氨酸与α-酮戊二酸形成丙氨酸,谷氨酸及CO_2。当存在DL-环丝氨酸(10~(-4)M)时,形成较多的谷氨酸与丙酮酸,而丙氨酸之量显著地减少。以L-天门冬氨酸与α-酮戊二酸或以L-谷氨酸与丙酮酸为底物,对丙氨酸之形成具有相同的抑制程度。DL-环丝氨酸(10~(-4))并不抑制谷-天转氨酶与草酰乙酸脱羧酶,但在同样条件下,可显著抑制谷-丙转氨酶的活力(～90%)。此外,若以L-天门冬氨酸或其与小量α-酮戊二酸为底物,尤其是用透析后之酶液,并无显著的丙氨酸与CO_2形成。我们认为,自L-天门冬氨酸与α-酮戊二酸形成之丙氨酸,并非通过Bheemeswar提出的L-天门冬氨酸β-脱羧酶之作用,而是经过三个相继的反应,即在谷-天转氨酶催化下,形成谷氨酸与草酰乙酸,后者除非酶促分解外,在草酰乙酸脱羧酶作用下,形成丙酮酸与CO_2;由以上两反应所形成之谷氨酸与丙酮酸,在蚕丝腺普遍存在的谷-丙转氨酶催化下形成丙氨酸(见图8)。     

L-天冬氨酸-β-脱羧酶发酵条件的响应面优化

利用Plackett-Burman设计和响应面分析方法对L-天冬氨酸-β-脱羧酶产生菌德阿昆哈假单孢菌XG-2-81的发酵条件进行优化.得出影响产L-天冬氨酸-β-脱羧酶的3个重要的因素为:玉米浆,多肽朊和装液量.通过对实验结果的方差分析,得到其最适条件分别为:玉米浆浓度0.64%,多肽朊浓度1.21%,装液量14.7...     

重组大肠杆菌转化富马酸生产L-丙氨酸

通过克隆来源于睾丸酮丛毛单胞菌的L-天冬氨酸-β-脱羧酶基因,在一株具有L-天冬氨酸酶生产能力的大肠埃希氏菌CICC 11022S中异源表达,构建转化富马酸生产L-丙氨酸的重组工程菌。结果发现:重组工程菌9 h转化富马酸产生112.7 g/L的L-丙氨酸,生产速率12.5 g/(L·h),转化率93.8%。富马酸价格较低,有效降低L-丙氨酸生产的原料成本。通过构建重组工程菌,以富马酸为底物,高效生产L-丙氨酸,结果表明该方法具有较好的工业应用潜力。     

Crystal structure of the schiff base intermediate prior to decarboxylation in the catalytic cycle of aspartate alpha-decarboxylase

l-Aspartate alpha-decarboxylase (ADC), encoded by the panD gene, catalyzes the conversion of l-aspartate into beta-alanine. In the microorganisms, beta-alanine is required for the synthesis of pantothenate (vitamin B(5)), which is the precursor of 4'-phosphopantetheine and coenzyme A. We have determined the crystal structure of Helicobacter pylori ADC, a tetrameric enzyme, in two forms: the apo structure at 2.0 A resolution and the isoasparagine complex structure at 1.55 A resolution. All subunits of the tetramer are self-processed at the Gly24-Ser25 linkage, producing the smaller beta chain (residues 1-24) and the larger alpha chain (residues 25-117). Each subunit contains nine beta-strands and three alpha-helices; it is folded into the double-psi beta-barrel structure. In the apo structure, the new amino terminus of the alpha chain, Ser25, is converted into a pyruvoyl group. In the isoasparagine complex structure, the substrate analog is covalently attached to the pyruvoyl group. This structure represents the enzyme-substrate Schiff base intermediate that was proposed to form prior to the decarboxylation step in the catalytic cycle of ADC. Thus our study provides direct structural evidence for the reaction mechanism of ADC.     

Analysis of pyrimidine catabolism in Drosophila melanogaster using epistatic interactions with mutations of pyrimidine biosynthesis and beta-alanine metabolism

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.     

Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine

Pantothenic acid and beta-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for beta-alanine and pantothenic acid biosynthesis. Overexpression of FMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required beta-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 and YIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in beta-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for beta-alanine biosynthesis; spe1Delta, spe2Delta, spe3Delta, and spe4Delta are all beta-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the beta-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms.     

Beta-alanine transport in the isolated hepatocytes of the elasmobranch Raja erinacea

Cells were isolated from the liver of the skate and the uptake of beta-alanine followed using [14C]-beta-alanine. The isolated hepatocytes showed good viability, were found to accumulate beta-alanine from the incubation medium, and did so in a manner indicating a transport system involving a saturable carrier. The data for the rate of beta-alanine uptake suggest that this may be a rate-limiting step in the oxidation of the amino acid by the liver. Experiments indicated that the transport system could distinguish beta-alanine from certain structurally similar molecules (L-alanine and taurine, but not gamma-amino butyrate). Cells isolated from fish adapted to a diluted environment (50% seawater) showed no significant change in the uptake rate. However, evidence indicates that, over the range of beta-alanine concentrations occurring in the fish, the uptake rate would be acutely sensitive to small changes in the concentration in the blood, thus forming a self-regulating system for the metabolism of beta-alanine.     

Which mechanisms are involved in taurine-dependent granulocytic immune response or amino- and α-keto acid homeostasis?

We examined the effects of beta-alanine (taurine analogue and taurine transport antagonist), taurine (regarding its role in neutrophil (PMN) immunonutrition) and taurine combined either with L-NAME (inhibitor of *NO-synthase), SNAP (*NO donor), DON (glutamine-analogue and inhibitor of glutamine-requiring enzymes), DFMO (inhibitor of ornithine-decarboxylase) and beta-alanine on neutrophil amino- and alpha-keto acid profiles or important PMN immune functions in order to establish whether taurine transport-, nitric oxide-, glutamine- or ornithine-dependent mechanisms are involved in any of the taurine-induced effects. According to the present findings, the taurine-mediated effect appears to be based primarily on a modulation of important transmembraneous transport mechanisms and only secondarily on directly or indirectly induced modifications in intragranulocytic amino- and alpha-keto acid homoeostasis or metabolism. Although a direct relation to the parallel observed immunological modifications can only be presumed, these results show very clearly that compositional modifications in the free intragranulocytic amino- and alpha keto-acid pools coinciding with changes in intragranulocytic taurine levels are relevant metabolic determinants that can significantly influence the magnitude and quality of the granulocytic immune response.     

D-Alanine dehydrogenase. Its role in the utilisation of alanine isomers as growth substrates by Pseudomonas aeruginosa PA01.

Pseudomonas aeruginosa PA01 was found to utilise both the D- and L-isomers of alpha-alanine and also beta-alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation of D-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation of L-alanine required its conversion to the directly oxidisable D-form by a soluble racemase; (iii) utilisation of beta-alanine, like L-alanine, involves both the racemase and D-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity of which is still speculative; (iv) P. aeruginosa, like Escherichia coli, appears to take up D-alanine and L-alanine by means of two specific permeases.     

Regulation of coenzyme A biosynthesis.

Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine. Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium. Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E. coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content. Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis. Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis. Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E. coli.     

Specialization of function among aldehyde dehydrogenases: the ALD2 and ALD3 genes are required for beta-alanine biosynthesis in Saccharomyces cerevisiae

The amino acid beta-alanine is an intermediate in pantothenic acid (vitamin B(5)) and coenzyme A (CoA) biosynthesis. In contrast to bacteria, yeast derive the beta-alanine required for pantothenic acid production via polyamine metabolism, mediated by the four SPE genes and by the FAD-dependent amine oxidase encoded by FMS1. Because amine oxidases generally produce aldehyde derivatives of amine compounds, we propose that an additional aldehyde-dehydrogenase-mediated step is required to make beta-alanine from the precursor aldehyde, 3-aminopropanal. This study presents evidence that the closely related aldehyde dehydrogenase genes ALD2 and ALD3 are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to beta-alanine in vivo. While deletion of the nuclear gene encoding the unrelated mitochondrial Ald5p resulted in an enhanced requirement for pantothenic acid pathway metabolites, we found no evidence to indicate that the Ald5p functions directly in the conversion of 3-aminopropanal to beta-alanine. Thus, in Saccharomyces cerevisiae, ALD2 and ALD3 are specialized for beta-alanine biosynthesis and are consequently involved in the cellular biosynthesis of coenzyme A.     

Beta-alanine suppresses heat inactivation of lactate dehydrogenase

Beta-Alanine exhibits neurotransmitter activity and is a component of the anti-glycation agent carnosine. We propose that beta-alanine may have additional properties which may be of physiological significance. Interestingly, stress modulates the level of beta-alanine, which regulates excitotoxicity responses and prevents neuronal cell death. We hypothesize that beta-alanine's protective role may involve preservation of enzyme structure and function, suggesting that beta-alanine may act as a chemical chaperone. We used light scattering, enzyme activity and intrinsic fluorescence to monitor heat-induced changes in lactate dehydrogenase (LDH) in the presence and absence of beta-alanine. We observed that beta-alanine suppressed heat-induced LDH inactivation, prevented LDH aggregation, ameliorated the decrease in intrinsic fluorescence and reactivated thermally denatured LDH. These observations support the hypothesis that beta-alanine has chaperone-like activity and may play a cellular role in the preservation of enzyme function.     

Two omega-amino acid transaminases from Bacillus cereus.

Bacillus cereus strain K-22 produced two distinct omega-amino acid transaminases, one catalyzing the transamination between beta-alanine and pyruvic acid and the other that between gamma-aminobutyric acid and alpha-ketoglutaric aic. The two enzymes were partially purified and separated from each other by various chromatographies. beta-Alanine:pyruvic acid transaminase and gamma-aminobutyric acid:alpha-ketoglutaric acid transaminase were induced by the addition of beta-alanine and gamma-aminobutyric acid, respectively, to the growth medium. beta-Alanine transaminase showed an optimum pH of 10.0 and optimum temperature of 35 degrees C, and its Km values for beta-alanine and pyruvic acid were both 1.1 mM. gamma-Aminobutyric acid, epsilon-aminocaproic acid, 2-aminoethylphosphonic acid, and propylamine showed about 30-40% of the activity of beta-alanine as amino donors, and oxalacetic acid was as good an amino acceptor as pyruvic acid. The optimum pH and temperature of gamma-aminobutyric acid transaminase were 9.0 and 50 degrees C, respectively, and its Km value for gamma-aminobutyric acid was 2.8 mM, while that for alpha-ketoglutaric acid was 2.3 mM. gamma-Aminobutyric acid and delta-aminovaleric acid were good amino donors but other omega-amino acids were virtually inactive with gamma-aminobutyric acid transaminase; alpha-ketoglutaric acid, and to a lesser extent glyoxylic acid, were active amino acceptors. Sulfhydryl reagents specifically activated gamma-aminobutyric acid transaminase.