苏氨酸醛缩酶的催化机理、分子改造及合成应用

苏氨酸醛缩酶催化醛和甘氨酸羟醛缩合,一步反应即可构建产物β-羟基-α-氨基酸的两个手性中心,从原子经济性和环境影响角度,是非常具有潜力的绿色合成光学纯β-羟基-α-氨基酸的方式之一.多个不同生物来源的苏氨酸醛缩酶得到分离和表征,较低的β-碳立体选择性以及反应过程中动力学和热力学控制难题,使其在合成应用中面临很大挑战.文...     

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。     

荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。     

L—乳酸脱氢酶基因克隆及功能分析

构建了一株产D,L-乳酸的乳杆菌(Lactobaeillus sp．)MD—1的基因库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJ144作为宿主,通过互补筛选分离克隆到乳酸脱氢酶基因(ldhL)。核酸序列分析表明,该基因以ATG为起始密码子编码316个氨基酸残基组成的蛋白质,预测的分子量为33．84kD;5′端存在典型的启动子结构,3′端的终止子是不依赖于ρ因子的转录终止子。ldhL编码的蛋白质有3个保守区域,其中Gly13～Asp50保守区域是NADH的结合位点,Asp73～Ile100和Asn123～Arg154保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低,核苷酸序列相似性最高仅为64．1％,氨基酸序列相似性最高仅为68．9％,是新的L—乳酸脱氢酶基因。     

氨基酸不对称合成

<正> 氨基酸不对称合成不仅对不对称合成本身有理论上及实践上的重要性,对生物活性多肽的研究也有很大意义。人们发现,用某些非蛋白氨基酸代替蛋白氨基酸,会大大提高多肽的活性。例如,在TRH中用六员环的L-焦-α-氨基己二酸代替五员环的L-焦谷氨酸大大提高它的中枢神经系统活     

内消旋-二氨基庚二酸脱氢酶关键氨基酸位点的改造提高对烷基取代2-酮酸的催化活力

【背景】高效实现D-氨基酸的生物合成一直是人们关注的热点。内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,DAPDH)能够直接催化2-酮酸和氨合成D-氨基酸。【目的】提高DAPDH对烷基取代2-酮酸的催化活力,并解释其催化机制。【方法】以来源于嗜热共生杆菌(Symbiobacteriumthermophilum)的内消旋-二氨基庚二酸脱氢酶(StDAPDH)为模板,在前期结构分析结合被选择位点突变结果的基础上,确定对H227位进行定点饱和突变,并以D-丙氨酸、D-2-氨基丁酸、D-正缬氨酸、D-谷氨酸为底物进行筛选。【结果】获得突变体H227Q和H227N。突变体H227Q对丙酮酸、2-氧代丁酸、2-氧代戊酸、2-酮戊二酸的比活力比野生型分别提高了10.9、11.5、8.6和7.6倍。动力学参数表明,突变体H227Q同时提高了酶对底物的亲和力及催化常数,使其对丙酮酸的催化效率(k_(cat)/K_m)相较于野生型提高了9.4倍。利用分子模拟技术分析突变体H227Q与产物氨基酸之间的相互作用表明,227位的谷氨酰胺通过与氨基酸的羧酸形成氢键,使得氨基酸产物Cα上的氢和辅酶烟酰胺环C4原子之间的距离缩短。【结论】利用定向进化技术提高DAPDH对烷基取代2-酮酸的催化活力,有助于开发新型的高效生物催化剂,这些工作也为下一步继续进行更具挑战性的D-氨基酸研究提供了基础。     

氨基酸L-和D-异构体对离体小麦胚植株生长的影响

5种氨基酸L-和D-异构体对离体小麦胚植株生长的影响结果表明,在1~7 mmol.L-1浓度范围内,脯氨酸异构体均不抑制植株生长;缬氨酸和蛋氨酸的L-型严重抑制生长,D-型不抑制或轻微抑制;丙氨酸则相反,L-型无抑制作用,D-型是严重抑制类型;丝氨酸的L-型轻微抑制生长,D-型严重抑制生长。生长结果也表明,DL-异构体的抑制作用介于D-型和L-型之间。     

氨基酸酰胺类化合物的合成

阐述了氨基酸氨基保护的常用方法和试剂,氨基酸酰胺类化合物合成的基本原理和方法以及在合成中需要注意的问题。重点阐述了氨基酸酰胺类化合物的合成机理和合成方法。展望了氨基酸酰胺类衍生物的合成方向。     

酶的分子设计、改造与工程应用

酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。     

漆酶的性质、功能、催化机理和应用

漆酶是一种结合多个铜离子的蛋白,是铜蓝氧化酶蛋白家族的一员。本文叙述漆酶的分子结构、底物特异性及其物理化学特性,并讨论漆酶的酶促反应机理和生物学功能,包括植物漆酶参与细胞壁的形成以及漆酶与病原菌毒力的关系。本文还着重介绍了漆酶在环境生物修复方面的应用。     

两种UDP-葡萄糖脱氢酶对透明质酸生物转化的影响

分别从大肠杆菌和化脓链球菌中扩增出编码UDP-葡萄糖脱氢酶基因ecohas B和spyhas B,并将其插入T7表达载体p RX2构建重组质粒p RXEB和p RXSB。在大肠杆菌BL21(DE3)中重组表达,并对经镍柱纯化后的UDP-葡萄糖脱氢酶的酶学性质进行分析。酶学性质研究表明:spy Has B的最适反应温度是30℃,最适p H 10,最适条件下的比活力是12.2 U/mg;eco Has B的最适反应温度是30℃,最适p H 9,最适条件下的比活力是5.55 U/mg。从多杀巴氏杆菌扩增出的透明质酸合成酶基因pmuhas A分别与ecohas B和spyhas B构建共表达载体p BPAEB和p BPASB。将其转化到大肠杆菌BW25113中,经生物转化生产透明质酸(HA),并对转化条件进行了优化。结果表明:重组菌株进行透明质酸转化时,UDP-葡萄糖脱氢酶酶活力越高,稳定性越好,HA产量越高;转化条件优化后,p BPAEB/BW25113和p BPASB/BW25113在摇瓶中的产量分别是1.52和1.70 g/L,比之前报道的提高了2-3倍。     

Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Five different immobilized NAD+ derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S6-, N6-, N1-, 8'-azo-, and pyrophosphate-linked immobilized NAD+. The amino acid dehydrogenases were NAD+-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P)+-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart L-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N6-linked NAD+ but was biospecifically adsorbed to S6-linked NAD+ derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS.     

大肠杆菌合成启动子的构建及在顺,顺-粘康酸生物合成中的应用

启动子是基因表达调控的重要元件.在代谢工程和合成生物学研究中,经常需要利用不同强度的启动子对代谢途径进行精细调控,来实现代谢平衡,降低中间产物积累,提高目标产物合成.然而目前可获得的启动子难以满足以上要求,而且不同来源的启动子通用性差,缺乏标准化.针对这些问题,设计了1条88个碱基对的启动子,包含典型的-35区、-10区以及核糖体结合区.同时,在转录起始位点上游6个碱基、-35与-10区间隔区14个碱基对中引入简并序列,构建了合成启动子文库.利用合成启动子控制红色荧光蛋白mCherry的表达强度,经过两轮筛选,从5 000多个克隆中获得了720个不同强度的启动子.随机挑选35条不同强度的启动子进行测序分析,结果表明不同强度的启动子具有碱基偏好性.对于强启动子,-13位点嘌呤碱基出现频率高,转录起始区除-4位点外,嘧啶碱基出现的频率高于嘌呤碱基,而-10区与-35区间14个位点的嘌呤碱基与嘧啶碱基出现频率大致相当.最后选取5条不同强度启动子应用于顺,顺-粘康酸合成途径调控优化,结果显示不同强度的启动子可以调节目标产物顺,顺-粘康酸的合成和中间产物儿茶酚的积累.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Semi-rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures

Enzymes are industrially applied under increasingly diverse environmental conditions that are dictated by the efforts to optimize overall process efficiency. Engineering the operational stability of biocatalysts to enhance their half-lives under the desired process conditions is a widely applied strategy to reduce costs. Here, we present a simple method to enhance enzyme stability in the presence of monophasic aqueous/organic solvent mixtures based on the concept of strengthening the enzyme's surface hydrogen-bond network by exchanging surface-located amino acid residues for arginine. Suitable residues are identified from sequence comparisons with homologous enzymes from thermophilic organisms and combined using a shuffling approach to obtain an enzyme variant with increased stability in monophasic aqueous/organic solvent mixtures. With this approach, we increase the stability of the broad-spectrum amino acid racemase of Pseudomonas putida DSM 3263 eightfold in mixtures with 40% methanol and sixfold in mixtures with 30% acetonitrile.     

代谢工程改造野生耐酸酵母生产L-乳酸

以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5 (乳酸调节) 的培养基中生长且不利用乳酸的酵母 (初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因 (ldhA) 插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C. magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C. magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最     

基于二级结构氨基酸组成识别酸性、中性及碱性酶

本研究系统分析了酸性、碱性和中性酶在二级结构氨基酸组成上的差异。结果发现在形成特定二级结构过程中,酸性酶和碱性酶有着不同的氨基酸使用偏向;同时,在酸性和碱性酶中,中性氨基酸和侧链微小的氨基酸含量明显较高,这可能是它们适应极端pH的普遍机制。基于此,提出了一种提取蛋白质序列特征值的新方法,其10倍交叉验证的精度可达80.3%。与其他常见特征值提取方法相比,其精度提高了9.4%到18.7%不等;而随机森林算法比其他机器学习算法识别精度也高出2.7%到21.8%不等。     

系统代谢工程改造树干毕赤酵母生产琥珀酸

为提高树干毕赤酵母发酵生产琥珀酸的产量,借助基因组规模代谢网络模型iTL885获得琥珀酸合成的最佳代谢途径为扩增icl1基因和敲除sdh1基因。在此基础上,借助代谢工程策略构建过量表达异柠檬酸裂解酶基因icl1的重组菌株FPLicl、缺失琥珀酸脱氢酶基因sdh1的重组菌株FPLΔsdh和缺失sdh1基因同时过量表达icl1基因的重组菌株FPLΔsdh-icl。结果表明:3株重组菌的异柠檬酸裂解酶活性由0.33 U/mg分别增加为1.6、5.6和6.6U/mg;而琥珀酸脱氢酶活性则从13.8 U/mg分别降为10.7、0.3和0.3 U/mg。在以木糖为C源的培养基中,3株重组菌生产琥珀酸的能力分别是0.30、1.20和1.60 g/L。     

Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases

The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2-oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1·0 mM concentration of the drug. This effect was explained by inhibition of 2-oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0·4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX. © 1997 John Wiley & Sons, Ltd.     

Metabolic engineering of Pichia pastoris for malic acid production from methanol

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP‐CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45‐fold increase compared to the parent strain. To improve the efficiency of site‐directed gene knockout, NHEJ‐related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by‐product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose‐6‐phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value‐added chemicals from methanol.