酶法转化DL-ATC合成L-半胱氨酸的酶促反应条件研究

目的:考察酶源保存方式、酶促反应时间、底物pH值、底物浓度、酶浓度、金属离子等因素对酶活力的影响。方法:以假单胞菌(Pseudomonassp.)TS1138为供试菌株,采用酸式茚三酮法测定L-半胱氨酸含量,研究了酶法转化DL-ATC合成L-半胱氨酸的酶促反应条件。结果:TS1138菌株中L-半胱氨酸脱巯基酶具有较高的活性,而且Mg2 、Mn2 、Fe2 、Zn2 、Cu2 等5种金属离子对DL-ATC水解酶酶系有不同程度的抑制,其中Cu2 对该酶系的抑制作用很大。结论:确定了TS1138菌株酶法转化DL-ATC合成L-半胱氨酸的最适酶促反应条件,为酶促反应动力学的研究奠定了基础。     

假单胞菌生物合成L-半胱氨酸途径中脱巯基酶的研究

通过PCR方法扩增得到假单胞菌TS1138L-半胱氨酸脱巯基酶基因（cd）,将其克隆至pBlueseript SKII载体,测定了含有L-半胱氨酸脱巯基酶基因的1．2kbDNA片段序列,并与其它菌株的脱巯基酶基因进行了同源性比较;同时,将其克隆至表达载体pET-21a（＋）,IPTG诱导表达,表达产物经Ni-NTA柱亲合层析后,得到纯化的重组蛋白。利用脱巯基酶的活性染色方法对重组表达的L-半胱氨酸脱巯基酶进行了鉴定,并探讨了L-半胱氨酸脱巯基酶的酶学性质,以及在生物转化合成L-半胱氨酸途径中的关键作用。     

微生物酶法合成L-半胱氨酸和L-胱氨酸

从土壤中分离到一株假单胞菌Pseudomonas sp.TS1138菌株,其胞内含有DL-2-氨基-Δ²-噻唑啉-4-羧酸(DL-2-Amino-Δ²-Thiazoling-4-Carboxylic Acid,缩写为DL-ATC)水解酶,以培养16h的细胞为酶源,可转化DL-ATC合成L-半胱氨酸。该菌株生长及产酶的最佳碳、氮源为葡萄糖和尿素,DL-ATC对酶的产生具有诱导作用。酶促反应后的产物经薄层层析、旋光度法和高效液相色谱鉴定为L-半胱氨酸。     

恶臭假单胞菌TS1138转化生产L-胱氨酸的工艺研究

对以DL-2-氨基-?2-噻唑啉-4-羧酸(DL-2-amino-?2-thiazoline-4-carboxylic acid, DL-ATC)为底物原料, 经微生物酶法催化合成L-半胱氨酸, 并进一步氧化和分离纯化产物L-胱氨酸的生产工艺和条件进行了研究。建立了以恶臭假单胞菌TS1138 (Pseudomonas putida TS1138)全细胞为酶源, 反复多次催化底物合成L-半胱氨酸, 并以2.0%二甲基亚砜(DMSO)为氧化剂氧化生成L-胱氨酸, 进而通过001×7型阳离子交换树脂纯化胱氨酸的新工艺。采用高效液相色谱法考察该方法L-胱氨酸的总收率可以达到78.55%, 纯度为99.12%。该方法简单高效, 解决了酶稳定性差不能重复使用, 而固定化酶方法繁琐成本高的问题, 为我国L-半胱氨酸和L-胱氨酸的生产开辟一条新途径。     

微生物酶法转化生产L-半胱氨酸的工艺研究

L-半胱氨酸(Cys)侧链巯基是构成蛋白质活性基团的重要氨基酸,在生物化学、医药、食品、饲料、化妆品等行业具有广泛的用途,国内外的需求量逐年增长.然而,Cys难以通过单纯的微生物发酵来进行生产;由于化学合成的步骤繁多,也很难进行化学合成.传统生产方法沿用毛发酸解制取L-半胱氨酸,收率低,能耗高,水解过程产生难闻气体及大量废酸,环境污染严重.     

恶臭假单胞菌TS1138转化生产L-胱氨酸的工艺研究

对以DL-2-氨基-△2-噻唑啉-4-羧酸(DL-2-amino-△2-thiazoline-4-carboxylic acid,DL-ATC)为底物原料,经微生物酶法催化合成L-半胱氨酸,并进一步氧化和分离纯化产物L-胱氨酸的生产工艺和条件进行了研究.建立了以恶臭假单胞菌TS1138(Pseudomonas putida TS1138)全细胞为酶源,反复多次催化底物合成L-半胱氨酸,并以2.0%二甲基亚砜(DMSO)为氧化剂氧化生成L-胱氨酸,进而通过001×7型阳离子交换树脂纯化胱氨酸的新工艺.采用高效液相色谱法考察该方法L-胱氨酸的总收率可以达到78.55%,纯度为99.12%.该方法简单高效,解决了酶稳定性差不能重复使用,而固定化酶方法繁琐成本高的问题,为我国L-半胱氨酸和L-胱氨酸的生产开辟一条新途径.     

北戴河海洋沉积物中L-半胱氨酸脱硫细菌的多样性及其特性

【背景】脱硫细菌对有机硫的脱硫作用在硫的生物地球化学循环以及脱硫工业中都起着重要的作用。【目的】了解海洋沉积物中可分解有机物产生硫化氢的细菌多样性。【方法】对我国北戴河海洋沉积物中可培养的L-半胱氨酸脱硫细菌进行分离与筛选,通过对其16SrRNA基因序列测定与分析,构建系统发育树,并对其脱硫、脱氮能力进行检验。【结果】从海洋沉积物中分离得到97株细菌,从以L-半胱氨酸为硫源的培养基中筛选出62株有机脱硫专一型细菌。根据脱硫细菌的形态及其特征,从中选取12株作为典型代表做进一步分析,它们分别属于芽孢杆菌属(Bacillus)、赖氨酸芽孢杆菌属(Lysinibacillus)、动性球菌属(Planococcus)和红球菌属(Rhodococcus)。结果表明,这12株细菌均可产生半胱氨酸脱巯基酶,能够将半胱氨酸分解为丙酮酸、硫化氢和氨,即同时具备脱硫与脱氮的能力。其中有5株菌脱硫能力较强,分别属于赖氨酸芽孢杆菌属、动性球菌属和芽孢杆菌属。【结论】海洋沉积物中存在着丰富的L-半胱氨酸脱硫细菌,为进一步研究海洋中硫的生物地球化学循环提供了素材。     

代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域.前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素.文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究L-半胱氨酸供给模块强化对杆菌肽合成的影响.首先,构建了L-半胱氨酸合...     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

Synthesis of DL-2,3-diacyloxypropylphosphonyl-cholines from DL-2,3-diacyloxyiodopropanes.

The chemical synthesis of racemic diacyloxypropylphosphonylcholines having octanoyl, myristoyl, oleoyl and stearoyl groups is described. The route involved reaction of dioactanoyloxy-dimyristoyloxy-dioleoyloxy-, and distearoyloxpropyliodide with tris (trimethylsilyl) phosphite to yield the corresponding bis (trimethylsilyl) phosphonate. Removal of the trimethylsilyl groups by neutral aqueous hydrolysis gave the free diacylpropylphosphonic acids, which, when treated with choline toluenesulfonate, yielded the desired dioctanoyloxy-, dimyristoyloxy-, dioleoyloxy-, and distearoyloxypropylphosphonylcholines. The paper also describes the synthesis of 2-octadecyleicosylphosphorylcholine.     

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

Crystallization and preliminary X-ray crystallographic studies of L-2-haloacid dehalogenase from Pseudomonas sp. YL

The dimeric L -2-haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 Å, b = 62.78 Angst; c = 50.84 Å, and β = 122.4°, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 Å resolution.     

Enzymatic dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 in organic solvents

Summary 4-Chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 showed dehalogenating activity in various organic solvents. In alcohols like methanol (150%) or ethanol (120%) higher activities than in water (100%) were obtained. In apolar solvents like petroleum ether (5%) and nhexane (5%) only trace activities were observed. The solvents did not increase the stability of the enzyme. 4-Chlorobenzoic acid methylester, a substance not soluble in water, was not dehalogenated in organic solvents.     

Microbial Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino Acids by Pseudomonas sp. Strain NS671

A bacterial strain, NS671, which converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine, was isolated from soil and was classified into the genus Pseudomonas. With growing cells of Pseudomonas sp. strain NS671, DL-5-(2-methylthioethyl)hydantoin was effectively converted to L-methionine. Under adequate conditions, 34g of L-methionine per liter was produced with a molar yield of 93% from DL-5-(2-methylthioethyl)hydantoin added successively. In addition to L-methionine, other amino acids such as L-valine, L-leucine, L-isoleucine, and L-phenylalanine were also produced from the corresponding 5- substituted hydantoins, but these L-amino acids produced were partially consumed by strain NS671. The hydantoinase, by which 5-substituted hydantoin rings are opened, was ATP-dependent. The N-carbamylamino acid amidohydrolase was found to be strictly L-specific, and its activity was inhibited by high concentration of ATP.     

一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh 的克隆、表达及酶学性质

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。     

Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys¹⁰¹) that is required for catalysis and interacts with Asp¹⁷, Thr¹⁰², and an ordered water molecule in the active site. Asp¹⁷ has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys¹⁰¹ and Asp¹⁷, Thr¹⁰² is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 Å C-α root mean square deviation), structural differences in the vicinity of Cys¹⁰¹ disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue.     

Biotin-vitamer Formation from Salicylic Acid by Pseudomonas sp.

Biotin-vitamer formation from salicylic acid was investigated. Strains of Pseudomonas sp., No. 102 and No. 362, isolated from soil samples utilized well salicylic acid as a sole source of carbon, and formed biotin-vitamers in culture broth. The metabolites were partially purified by the methods of active carbon adsorption and anion-exchange column chromatography, and clarified as desthiobiotin, bisnordesthiobiotin and 7-keto-8-aminopelargonic acid.