Arthrobacter K1108乙内酰脲酶反应条件和立体选择性研究

研究了ArthrobacterK110 8乙内酰脲酶的反应条件 ,结果表明 ,K1108乙内酰脲酶的最适反应温度为 55℃ ,最适pH为 70 ,Co²⁺ 和Fe²⁺ 对该酶有激活作用 ,而Ca²⁺ 有严重抑制作用。K1108乙内酰脲酶的底物专一性较强 ,其最适底物为 5 苄基乙内酰脲 ,5 苯基乙内酰脲和 5 吲哚甲基乙内酰脲均不能作为其有效底物。对K1108乙内酰脲酶立体反应机制研究结果表明 ,其乙内酰脲水解酶不具立体选择性 ,决定产物立体构型的酶是N 氨甲酰氨基酸水解酶。     

Arthrobacter K1108乙内酰脲酶反应条件和立体选择性研究

研究了Arthrobacter K1108乙内酰脲酶的反应条件,结果表明,K1108乙内酰脲酶的最适反应温度为55℃,最适pH为7.0,Co^2 和Fe^2 对该酶有激活作用,而Ca^2 有严重抑制作用。K1108乙内酰脲酶的底物专一性较强,其最适底物为5－苄基乙内酰脲,5－苯基乙内酰脲和5－吲哚甲基乙内酰脲均不能作为其有效底物。对K1108乙内酰脲酶立体反应机制研究结果表明,其乙内酰脲水解酶不具立体选择性,决定产物立体构型的酶是N－氨甲酰氨基酸水解酶。     

一株乙内酰脲酶产生菌Arthrobacter K1108的筛选及鉴定

从沈阳市浑河地区污泥中分离得到了一株乙内酰脲酶产生菌 ,薄层色谱和氨基酸自动分析仪的分析结果表明 ,该菌的完整细胞可催化 5 -苄基乙内酰脲水解产生苯丙氨酸。对该菌进行了细菌分类学鉴定 ,确定该菌为节杆菌属的一个种 ,故命名为 Arthrobacter sp.K1 1 0 8     

节杆菌K1108乙内酰脲酶产酶条件研究

研究了乙内酰脲酶产生菌节杆菌K1108的产酶条件。该菌乙内酰脲酶为诱导酶,存在于细胞内,乙内酰脲水解酶和N氨甲酰氨基酸水解酶是同时被诱导产生。最适诱导物为5苄基乙内酰脲,而5吲哚甲基乙内酰脲和5苯基乙内酰脲等不能诱导其酶的产生。筛选到一种安慰诱导物,诱导活性提高了2倍多。对产酶培养基进行了筛选和优化,在最适条件下,K1108产酶能力可达108U/mL。     

节杆菌K1108乙内酰脲酶三维结构的模建和分析

利用同源模建技术,以节杆菌DSM3745乙内酰脲酶的晶体结构为模板,模建了节杆菌K1108乙内酰脲酶的三维结构。模建的节杆菌K1108乙内酰脲酶结构由一个中心的(α/β)g捅状结构域和富含β-折叠的结构域两个区域组成,富含β-折叠的结构域在中心(α/β)g捅状结构域的侧面,由分子的N端和C端组成。根据K1108乙内酰脲酶和其它酶在结构和活性部位的保守性,确定了K1108乙内酰脲酶的底物结合部位,并对酶的活性中心的特征进行了分析,对L-Hyd的底物选择性进行了解释。     

节杆菌BT801基因文库构建及其乙内酰脲酶基因分离与表达

L-乙内酰脲酶产生菌节杆菌 BT801的染色体DNA经Sau3A I 部分酶切后分离30kb左右的片段,与经HpaI和PstI酶切的黏粒载体Pkc505进行连接,将连接产物用包装蛋白包装,转染大肠杆菌DH5α得到10 000多个转化子,构建成节杆菌BT801的基因组文库。通过薄层层析等方法筛选得到了1个阳性克隆,通过亚克隆得到了乙内酰脲酶的完整基因,该基因能分别利用自身的启动子和T5启动子在大肠杆菌中进行表达产生有活性的蛋白。     

微生物乙内酰脲酶及其研究进展

乙内酰脲酶是广泛分布在微生物中的一类可降解乙内酰脲酶类化合物的酶系 ,包括乙内酰脲水解酶、N-氨甲酰氨基酸水解酶及乙内酰脲消旋酶。微生物的乙内酰脲酶在结构与组成、立体选择性、底物专一性、反应条件和作用机制等方面有所不同 ,在各种 L-及 D-型氨基酸的酶法生产中具有良好的应用前景。本文对乙内酰脲酶研究及应用的一般情况作了概述 ,并讨论了有关乙内酰脲酶研究的主要研究进展     

腈水合酶转化反应的影响因子

棒状杆菌(corynebactcrium sp.)ZBB-21腈水合酶能高效地将丙烯腈转化为丙烯酰胺,其转化反应的最遣PH为8．0,最适转化反应温度为25℃。反应体系中加入微量的K+、Na+、Mg2+和Fe3+对酶的转化反应有明显的促进作用。过量丙烯腈(浓度为0．3mol／L以上)对酶活性有抑制作用,转化产物丙烯酰胺及其结构类似物丙烯酸是腈水合酶的竞争性抑制剂,其抑制常数K．分别为0．06mol／L和0．70mol,L,游离氰离子(CN-)的存在严重抑制丙烯酰胺的形成(K;=1．25 x 10-3mol／L)。     

乙内酰脲水解酶基因在大肠杆菌中的克隆表达

节杆菌BT801的乙内酰脲酶系能够水解5-苄基乙内酰脲生成L-苯丙氨酸,其中乙内酰脲水解酶负责乙内酰脲的水解开环。乙内酰脲水解酶的表达对于乙内酰脲酶的催化机制研究及氨基酸的生物不对称合成都具有重要意义。通过PCR技术扩增得到乙内酰脲水解酶基因(hyuH),置于表达载体pT221的,17启动子下游,将构建的重组质粒引入大肠杆菌BL21(DE3)。SDS-PAGE分析在相对分子量50kD处有一较强的表达带,经薄层扫描分析目的蛋白占全菌蛋白的40％,主要以可溶性形式存在,活性分析表明表达产物具有天然的酶活性。     

N-氨甲酰基氨基酸水解酶在毕赤酵母中的表达

N-氨甲酰氨基酸水解酶是乙内酰脲酶系的组成部分,催化N-氨甲酰氨基酸水解为相应氨基酸。节杆菌BT801的N-氨甲酰氨基酸水解酶是该菌乙内酰脲酶系中惟一具立体专一性的酶,也是整个反应体系的限速酶。通过PCR从携带乙内酰脲酶系完整操纵子的亚克隆质粒pUC18-169上扩增得到N-氨甲酰氨基酸水解酶基因(hyuC)片段,连接到载体pPIC3.5K上,经BglⅡ酶切线性化,通过PEG法转化导入毕赤酵母GS115感受态细胞,利用G418抗性筛选得到插入多拷贝目的基因的转化子。酶活性分析表明所得转化子具     

L-海因酶与D-海因酶的序列及结构比较研究

运用生物信息学的研究方法,从序列及结构上对L型及D型海因酶进行了初步的比较。研究了两种类型的海因酶在序列、骨架结构及活性中心的区别,并探讨了产生这些差异的理论基础,为海因酶进一步的理论及应用研究提供一定的指导。     

Burkholderia piCkettii中D-乙内酰脲酶基因(dha)的克隆、测序及表达

对一株能转化D,L－对羟基苯乙内酰脲为D－对羟基苯甘氨酸的菌株MMR003进行了细菌分类学鉴定,该菌为皮氏伯克霍尔德氏菌（Burkholderia pickettii)。实验通过Southern杂交,部分文库构建和筛选,并经一系列亚克隆分析,获得一长度为1374bp的完整开放阅读框,编码458个氨基酸的D－乙内酰脲酶基因。用该基因序列构建的高表达质粒xXZPH2转化E.coliBL21(DE3),经IPTG诱导后,检测到D－乙内酰脲酶活性。该基因编码的氨基酸序列经Blast同源比较分析与放射形土壤杆菌NRRL B11291所产相应酶有85％的同源性。以D,L－对羟基苯乙内酰脲为底物测得的表达酶的活力为0.66u/mL,比相同条件下所测出发菌株MMR003的酶活提高了2倍。     

Production of hydantoinase fromPseudomonas fluorescens strain DSM 84

Summary Pseudomonas fluorescens strain DSM 84 was selected as a good hydantoinase (dihydropyrimidinase E.C. 3.5.2.2.) producer from a screening involving 60 collection strains. Optimization of the culture and growth conditions were performed in order to increase the enzyme production. A mineral medium supplemented with 10 g/l of yeast extract having an initial pH of 7.1±0.1 but containing no additional carbon source or inducer was devised. The strain DSM 84 was found to produce the maximal level of hydantoinase in the defined mineral medium within 15 h of incubation at 27°C. When using 5-isopropylhydantoin as substrate, N-carbamyl-valine was detected as the end product of the crude hydantoinase. Conditions leading to the isolation and conservation of a crude hydantoinase as well as its temperature and pH stability are described.     

Enzymatic production of <Emphasis Type="SmallCaps">l</Emphasis>-amino acids from the corresponding <Emphasis Type="SmallCaps">dl</Emphasis>-5-substituted hydantoins by <Emphasis Type="Italic">Bacillus fordii</Emphasis> MH602

Bacillus fordii MH602 was newly screened from soil at 45 °C and exhibited high activities of hydantoinase and carbamoylase, efficiently yielding l-amino acids including phenylalanine, phenylglycine and tryptophan with the bioconversion yield of 60–100% from the corresponding dl-5-substituted hydantoins. Hydantoinase activity was found to be cell-associated and inducible. The optimal inducer was dl-5-methylhydantoin with concentration of 0.014 mol L⁻¹ and added to the fermentation medium in the exponential phase of growth. In the production of optically pure amino acids from dl-5-benylhydantoin, the optimal temperature and pH of this reaction were 45–50 °C and 7.5 respectively. The hydantoinase was non-stereoselective, while carmbamoylase was l-selective. The hydantoinase activity was not subject to substrate inhibition, or product inhibition by ammonia. In addition, The activities of both enzymes from crude extract of the strain were thermostable; the hydantoinase and carbamoylase retained about 90% and 60% activity after 6 h at 50 °C, respectively. Since reaction at higher temperature is advantageous for enhancement of solubility and for racemization of dl-5-substituted hydantoins, the relative paucity of l-selective hydantoinase systems, together with the high level of hydantoinase and carbamoylase activity and unusual substrate selectivity of the strain MH602, suggest that it has significant potential applications.     

Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and l-N-carbamoylase from Arthrobacter aurescens for the production of optically pure l-amino acids

2The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements.In comparison to the free enzymes, temperature and pH-optima were increased by 10 degrees C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degrees C (60 degrees C).In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

Thermostable hydantoinase from a hyperthermophilic archaeon, Methanococcus jannaschii

A hyperthermophilic hydantoinase from Methanococcus jannaschii with an optimum growth at 85°C was cloned and expressed in E. coli. The recombinant hydantoinase was purified by affinity and anion-exchange chromatography and determined to be homotetrameric protein by gel filtration chromatography. The best substrate for the hydantoinase was D,L-5-hydroxyhydantoin, which has the specific activity of 183.4 U/mg. The optimum pH and temperature for the hydantoinase activity was 8.0 and 80°C, respectively. The half-life of the hydantoinase was measured to be 100 min at 90°C in the buffer containing 500 mM KCl. Manganese ions were the most effective for the hydantoinase activity. Stereospecificity was determined to be L-specific for the 5-hydroxymethylhydantoin and 5-methylhydantoin by chiral TLC. The activity yields as well as the operational stabilities of the thermostable M. jannaschii hydantoinase could be significantly improved by immobilization method.     

Purification of industrial hydantoinase in one chromatographic step without affinity tag

Hydantoinase is used in industry as a biocatalyst for the production of optically pure D- or L-amino acids. Previously, homogeneous hydantoinase was obtained by multi-chromatographic purification procedures. Here, we reported a process that contained only a single chromatographic step to purify a recombinant hydantoinase to homogeneity. Hydantoinase from Agrobacterium radiobacter NRRL B11291 was expressed in Escherichia coli. The recombinant enzyme was purified following heat treatments, high concentration alcohol precipitation, and chelating Sephacel chromatography. The recombinant hydantoinase did not contain any affinity tags from the plasmid. This simplified procedure provided a convenient way to obtain hydantoinase in high yield (71%) and high purity. It should be very useful for further industrial application and for the study of the structure-function of hydantoinase.     

Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase

Bacterial hydantoinase possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. How the carboxylated lysine and metal binding affect the activity of hydantoinase was investigated. A significant amount of iron was always found in Agrobacterium radiobacter hydantoinase purified from unsupplemented cobalt-, manganese-, or zinc-amended Escherichia coli cell cultures. A titration curve for the reactivation of apohydantoinase with cobalt indicates that the first metal was preferentially bound but did not give any enzyme activity until the second metal was also attached to the hydantoinase. The pH profiles of the metal-reconstituted hydantoinase were dependent on the specific metal ion bound to the active site, indicating a direct involvement of metal in catalysis. Mutation of the metal binding site residues, H57A, H59A, K148A, H181A, H237A, and D313A, completely abolished hydantoinase activity but preserved about half of the metal content, except for K148A, which lost both metals in its active site. However, the activity of K148A could be chemically rescued by short-chain carboxylic acids in the presence of cobalt, indicating that the carboxylated lysine was needed to coordinate the binuclear ion within the active site of hydantoinase. The mutant D313E enzyme was also active but resulted in a pH profile different from that of wild-type hydantoinase. A mechanism for hydantoinase involving metal, carboxylated K148, and D313 was proposed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.