D型氨基酸氧化酶活性对于D-硝基精氨酸手性转化的影响

D-硝基精氨酸(D-NNA)可在大鼠体内发生手性转化生成其L型异构体,即L-NNA,后者可抑制一氧化氮合酶活性,减少一氧化氮生成,升高动脉血压.研究了D型氨基酸氧化酶(DAAO)在D-NNA手性转化中的作用及DAAO对不同(包括已报道在体内可发生手型转化的)D型氨基酸的选择活性.体内实验显示,DAAO的选择性抑制剂苯甲酸钠(400mg/kg)或肌酐(400mg/kg)均可在不同程度上抑制D-NNA升压作用,进一步研究发现,肾脏或肝脏DAAO酶液在外加DAAO后可提高D-NNA的手性转化约2倍,表明DAAO对于D-NNA在体内的手性转化是必需的.DAAO酶液对可在体内发生手性转化且转化率相似(30%～50%)的D型氨基酸(D-Phe,D-Leu和D-NNA)的选择性表现出显著差异(Kcat/Km相差可达约15倍左右),这从另一方面表明体内D-硝基精氨酸氧化是其发生手性转化的前提条件但非决定因素.     

D-氨基酸检测方法研究进展

自然界中存在20种基本氨基酸,除甘氨酸外都有两种互成镜像的对映体D-型和L-型。长期以来,人们认为D-氨基酸仅存在于微生物中,参与构成细胞壁肽聚糖层。但随着分析方法的发展,人们相继在多种生物体中(包括人体)发现了多种D-氨基酸,D-氨基酸才引起人们的高度重视,并意识到它们在医药、农药和食品等的组成中起着重要作用。D-氨基酸检测方法还需要不断发展与完善,以便更好地分析和认识D-氨基酸的重要作用。主要介绍酶法、气相层析法、高效液相法及生物传感器等检测方法。     

三角酵母D-氨基酸氧化酶基因的克隆、测序及表达*

利用跨越内含子的PCR技术,三角酵母(Trigonopsos variabilis)变种FA1-10中扩增得到D-氨基酸氧化酶基因(daao),并通过TA克隆的方法将其克隆至pGEM—T载体。序列测定结果表明,所得daao基因的5’端内含子已被删除,基因总长度为1071bp,它与Trigonopsis variabilis D-氨基酸氧化酶同源性达98.3％,与Fusarium solanii和Rhodotoru-la gracilis的同源性分别是38.9％和30.8％。为提高表达水平,又将此基因转移至高表达载体pET-28b上．在大肠杆菌BL-2l(DE3)中进行诱导表达。经IPTG诱导,目的蛋白的产生量可占菌体总蛋白量的46％,分子量约为38kD。D-氨基酸氧化酶的活力可达802u／L。     

三角酵母菌D-氨基酸氧化酶的纯化和一些基本性质的研究

1．比较了17株霉菌和36株酵母菌的D-氨基酸氧化酶活力,其中以三角酵母的酶活力最高,比较适宜作为研究D-氨基酸氧化酶的材料。 2．用硫酸铵、聚乙二醇(PEG 6000)分部沉淀,Sephadcx G-200、羟基磷灰石柱层析分离纯化了三角酵母D-氨基酸氧化酶,聚丙烯酰胺凝胶电泳为单一组份。 以聚丙烯酰胺凝股梯度浓度电泳测得D-氢基酸氧化酶的分子量为170,000,sDs凝胶电泳测得其亚基分子量为42,000,表明三角酵母D-氨基酸氧化酶由四个相同亚基组或。 3．三角酵母D-氨基酸氧化酶在以D-丙氨酸为底物时,最适pH为8．3,米氏常数(Km)为3．3mM。三角酵母D-氨基酸氧化酶具有较宽的底物专一性,多种D或DL型氨基酸都可以做它的底物。     

中枢神经系统D-氨基酸氧化酶的研究进展

新近研究证实,哺乳动物神经系统中存在内源性D-氨基酸氧化酶,参与脑内D-氨基酸的代谢.遗传学研究发现,D-氨基酸氧化酶基因与精神分裂症的发生密切相关.分子生物学研究表明,在D-氨基酸氧化酶基因启动子区域存在一些转录因子的结合位点.这些研究结果提示,中枢神经系统的D-氨基酸氧化酶除了参与D-氨基酸的代谢以外,可能还具有其它的生理功能.本文就中枢神经系统D-氨基酸氧化酶的研究进展作一综述.     

D-乳酸脱氢酶基因克隆及其表达

构建了一株产D ,L 乳酸的乳杆菌 (Lactobacillussp .)MD 1的基因文库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的EscherichiacoliFMJ1 4 4作为宿主 ,在厌氧条件下通过互补筛选获得乳酸脱氢酶基因 (ldh) ,非变性聚丙烯酰胺凝胶电泳 (Native PAGE)检测证明其阳性克隆表现出D 乳酸脱氢酶 (D LDH)活性。核酸序列分析表明 ,ldhD的ORF编码 331个氨基酸残基组成的蛋白质有两个保守区域 ,其中V1 47～D1 76 区是NADH的结合位点 ,R77～E1 0 7区据报道是酶的活性部位。该菌株D LDH和D羟基异己酸脱氢酶 (D HicDH)属于NADH依赖性脱氢酶家族 ,ldhD和其他乳杆菌属的ldhD及D HicDH基因和编码的氨基酸序列相似性较低 ,核酸序列相似性最高达 4 9 33% ,氨基酸序列相同性最高为 4 2 % ,是一个新的D 乳酸脱氢酶基因     

D- 型氨基酸氧化酶抑制剂的发展

D-型氨基酸氧化酶(D-Amino acid oxidase,DAAO)抑制剂可以阻止D-型氨基酸(主要是D-型丝氨酸)的降解和过氧化氢的生成,在治疗精神分裂症阴性症状和认知障碍与镇痛等方面均表现出较好的疗效。从第一个DAAO抑制剂芳香羧酸类的苯甲酸到经过烯醇互变的α-羟基酮喹类抑制剂喹诺林-2,3-二酮,DAAO抑制剂结构上总共经历了3代变化,抑制剂与酶之间的相互作用模式逐渐加强,其抑制活性升高了数万倍,脂溶性增加,酸性减弱,理化性质逐渐优化。本文就近10年DAAO抑制剂的结构发展与生物活性之间的关系进行综述。     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

类芽孢杆菌来源D-甘露醇氧化酶的性质及制备D-甘露糖的应用

D-甘露糖具有多种功能活性，在食品、医药、农业等行业应用广泛。D-甘露醇氧化酶可以高效地将D-甘露醇转化为D-甘露糖，在D-甘露糖的酶法制备中具有应用潜力。从类芽孢杆菌(Paenibacillus sp.) HGF5中发掘出一个D-甘露醇氧化酶(PsOX)，与天蓝链霉菌(Streptomyces coelicolor)来源的D-甘露醇氧化酶(AldO)氨基酸序列相似性为50.94%，分子量约为47.4 kDa，构建了重组表达质粒pET-28a-PsOX并在大肠杆菌BL21(DE3)中表达，PsOX对D-甘露醇的K_m、k_cat/K_m值分别为5.6 mmol/L、0.68 L/(s∙mmol)，最适pH和温度分别为7.0和35 ℃，在60 ℃以下保持稳定。PsOX对400 mmol/L D-甘露醇的摩尔转化率为95.2%。利用PsOX与AldO全细胞分别催化73 g/L D-甘露醇，PsOX反应9 h后反应完全，生成70 g/L D-甘露糖，相较于AldO具有更高的催化效率。PsOX作为新型D-甘露糖氧化酶为D-甘露糖的酶法制备提供了依据。     

内消旋-二氨基庚二酸脱氢酶不对称合成非天然手性D-氨基酸的研究进展

内消旋-二氨基庚二酸脱氢酶不对称合成非天然的手性D-氨基酸是目前生物催化领域的研究热点。内消旋-二氨基庚二酸脱氢酶具有优良的立体选择性,利用其进行酶催化不对称合成光学纯的手性D-氨基酸,被广泛用于医药、食品、化妆品、精细化学品等领域。为了促进生物催化法在合成手性D-氨基酸方向的进一步发展,本文对内消旋-二氨基庚二酸脱氢酶催化合成D-氨基酸的现状进行了综述。重点介绍了Corynebacterium glutamicum、Ureibacillus thermosphaericus、Symbiobacterium thermophilum来源的内消旋-二氨基庚二酸脱氢酶在新酶的挖掘、催化性能、晶体结构解析、分子改造、功能与催化机制、合成D-氨基酸新途径等方面的研究进展,并对内消旋-二氨基庚二酸脱氢酶的未来研究方向及策略进行了展望。本综述将进一步加深人们对内消旋-二氨基庚二酸脱氢酶的认识,也为具有挑战性的生物合成任务提供信息借鉴。     

Evidence for the presence of two D-amino acid oxidases inPseudomonas aeruginosa PAO

By the isolation of mutants that were unable to grow on L-hydroxyproline or DL-valine, it has been possible to demonstrate the presence of two different types of D-amino acid oxidase activities inPseudomonas aeruginosa PAO. One was the D-amino acid dehydrogenase, probably involved in the oxidation of a number of D-amino acids such as D-alanine, D-phenylalanine and D-valine. The other was the inducible oxidase, specific to the oxidation of allohydroxy-D-proline formed from L-hydroxyproline during its oxidation. Thus, it has been possible to delink the involvement of the general D-amino acid dehydrogenase in the oxidative breakdown of allohydroxy-Dsproline.     

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.     

Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase

When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.     

Oxidation of 3,4-dehydro-D-proline and other D-amino acid analogues by D-alanine dehydrogenase from Escherichia coli

3,4-Dehydro-DL-proline is a toxic analogue of L-proline which has been useful in studying the uptake and metabolism of this key amino acid. When membrane fractions from Escherichia coli strain UMM5 (putA1::Tn5 proC24) lacking both L-proline dehydrogenase and L-Delta(1)-pyrroline-5-carboxylate reductase were incubated with 3,4-dehydro-DL-proline, pyrrole-2-carboxylate was formed. There was no enzyme activity with 3,4-dehydro-L-proline, but activity was restored after racemization of the substrate. Oxidation of 3,4-dehydro-DL-proline by membrane fractions from strain UMM5 was induced by growth in minimal medium containing D- or L-alanine, had a pH optimum of 9, and was competitively inhibited by D-alanine. An E. coli strain with no D-alanine dehydrogenase activity due to the dadA237 mutation was unable to oxidize either 3,4-dehydro-D-proline or D-alanine, as were spontaneous Dad(-) mutants of E. coli strain UMM5. Membrane fractions containing D-alanine dehydrogenase also catalyzed the oxidation of D-2-aminobutyrate, D-norvaline, D-norleucine, cis-4-hydroxy-D-proline, and DL-ethionine. These results indicate that d-alanine dehydrogenase is responsible for the residual 3,4-dehydro-DL-proline oxidation activity in putA proC mutants of E. coli and provide further evidence that this enzyme plays a general role in the metabolism of D-amino acids and their analogues.     

Occurrence of D-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum

The contents of D-enantiomers of serine, alanine, proline, glutamate (glutamine) and aspartate (asparagine) were examined in the membrane fractions, soluble proteins and free amino acids from some species of archaea, Pyrobaculum islandicum, Methanosarcina barkeri and Halobacterium salinarium. Around 2% (D/D+L) of D-aspartate was found in the membrane fractions. In the soluble proteins, the D-amino acid content was higher in P. islandicum than that in the other archaeal cells: the concentrations in P. islandicum were 3 and 4% for D-serine and D-aspartate, respectively. High concentrations of free D-amino acids were found in P. islandicum and H. salinarium; the concentrations of D-serine (12-13%), D-aspartate (4-7%) and D-proline (3-4%) were higher than those of D-alanine and D-glutamate. This result showed a resemblance between these archaea and not bacterial, but eukaryotic cells. The presence of D-amino acids was confirmed by their digestion with D-amino acid oxidase and D-aspartate oxidase. The occurrence of D-amino acids was also confirmed by the presence of activities catalyzing catabolism of D-amino acids in the P. islandicum homogenate, as measured by 2-oxo acid formation. The catalytic activities oxidizing D-alanine, D-aspartate and D-serine at 90 degrees C were considerably high. Under anaerobic conditions, dehydrogenase activities of the homogenate were 69, 84 and 30% of the above oxidase activities toward D-alanine, D-aspartate and D-serine, respectively. Comparable or higher dehydrogenase activities were also detected with these D-amino acids as substrate by the reduction of 2, 6-dichlorophenolindophenol. No D-amino acid oxidase activity was detected in the homogenates of M. barkeri and H. salinarium.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.