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。     

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。     

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     

产环氧化物水解酶的黑曲霉菌种分离和发酵条件的研究

从土壤中筛选出一株能将苯基环氧乙烷立体选择性水解为R－苯基乙二醇的环氧化物水解酶的黑曲霉SQ－6。对其产酶发酵条件进行了研究,最佳碳,氮源分别为2．0％蔗糖和2．0％玉米浆,最适初始pH为4．0,该酶不需诱导,同时还研究了其他发酵条件对产酶的影响,使用含酶细胞进行底物苯基环氧乙烷转化。产物（R）－苯基乙二醇转化率为41％,ee值为99％。     

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     

微生物酶拆分方法生产D-泛酸的手性中间体D-泛解酸内酯

筛选到一株产D－泛解酸内酯水解酶的串珠镰孢霉菌（Fusarium moniliforme SW-902)。产酶条件研究表明,用甘油作碳源,蛋白胨作氮源,初始pH8.0,温度26℃,摇瓶培养3d,产酶量最高。在60L和1000L发酵罐中通风发酵45－47h,产酶量为6－8g干菌体／L,D－泛解酸内酯水解酶酶活力达到0．87－0．92IU／g干菌体。该酶的最适反应温度为55℃,最适反应pH为7．0－7．5。在酶不对称水解泛解酸内酯过程中,对溶液加酶量5％－10％,底物浓度10％－20％,控制水解率20％－30％,水解效果最好。     

突变型环酰亚胺水解酶的底物专一性

摘要 目的：研究环酰亚胺水解酶（CIH293）C-末端区残基对其底物专一性的影响。方法：通过缺失或替代获得了环酰亚胺水解酶C-末端剔除2个或3个氨基酸残基及C-末端两个Lys替代为两个Glu的突变型酶CIH291、CIH290以及KK292,293EE,用比色法与高效液相色谱法分析了重组野生型酶与突变型酶的底物专一性和动力学参数。结果：突变型酶与野生型酶相比,底物专一性未发生显著改变,最适底物仍为琥珀酰亚胺,然突变型酶对最适底物的亲和力略有降低,导致反应速度减小。结论：环酰亚胺水解酶（CIH293）C-末端区残基的改变对其底物专一性的影响不大,但影响了酶对底物的亲和力。     

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。     

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

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.     

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.     

Substrate- and stereospecificity,induction and metallo-dependence of a microbial hydantoinase

Summary D, L-5-monosubstituted hydantoins can be used as substrates for a two-step-enzymatic production of optically active aminoacids. The substrate- and stereospecificity of the first enzyme — a hydantoinase -, investigations on its induction and on its dependence upon metallo-ions are described. It is shown, that the activity of this hydantoinase, which is not identical with the well-known enzyme D-hydantoinase, depends on manganese-ions. Of synthetic and natural compounds tested as inductors, D, L-5-indolylmethylhydantoin showed the best effect. The hydantoinase has a wide substrate-specificity. Its stereoselectivity seems to depend on the structure of the side chain in 5-position of the hydantoin.     

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4°C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40°C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50°C.     

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.     

Optimization of a fermentation medium using neural networks and genetic algorithms

Artificial neural networks and genetic algorithms are used to model and optimize a fermentation medium for the production of the enzyme hydantoinase by Agrobacterium radiobacter. Experimental data reported in the literature were used to build two neural network models. The concentrations of four medium components served as inputs to the neural network models, and hydantoinase or cell concentration served as a single output of each model. Genetic algorithms were used to optimize the input space of the neural network models to find the optimum settings for maximum enzyme and cell production. Using this procedure, two artificial intelligence techniques have been effectively integrated to create a powerful tool for process modeling and optimization.     

Purification and characterization of an acetyl-CoA hydrolase from Saccharomyces cerevisiae

Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 1080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 +/- 5 kDa by gel-filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 +/- 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase. Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent.     

Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine

Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate. The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.