紫外光谱法实时测定腈水合酶的催化动力学

采用紫外光谱法研究了腈水合酶催化丙烯腈水合的过程,在不同丙烯腈初始浓度下,测定了催化过程中275nm紫外吸光度的变化,计算出丙烯酰胺的生成速率.用Michaelis-Menten方程对不同丙烯腈浓度下的Nocardiasp.腈水合酶催化速率进行了拟合,得到该酶以丙烯腈为底物的米氏常数(Km)为8.46mmol/L,单位质量腈水合酶的催化速率常数(kcat)为2398μmol/(min.mg).     

游离细胞法制备丙烯酰胺

采用新工艺游离细胞法半连续操作制备丙烯酰胺，探讨了反应的最佳条件。结果表明，在温度高于20℃时酶易失活，底物丙烯腈体积分数高于4％和产物丙烯酰胺质量浓度高于300g／L时，将对酶活性产生强烈的抑制作用；在较低温度下，采用底物流加及酶流加方法能保持较高的酶活性。当菌悬液体积分数为10％、反应温度为20℃、流动加入时，最终生成丙烯酰胺的质量浓度为366g/L，与菌悬液一次性加入相比，丙烯酰胺质量浓度提高23％。     

恶臭假单胞菌整细胞的腈水合酶催化特性

丙烯酰胺是一个重要的精细化工产品,其生产工艺早期采用硫酸法,因存在产品精制困难、环境污染等问题而被淘汰.近年来,美、日等国开发了使用高效铜系催化剂直接水合法来生产丙烯酰胺,目前该方法已成为丙烯酰胺的主要生产工艺,七十年代以后,国外相继开展了分离筛选水合丙烯腈生成丙烯酰胺的菌种并对具有腈水合酶的微生物细胞进行固定化的研究.近年来,我们开展了腈水合酶菌种的分离筛选和发酵条件的试验研究,分离筛选出优良菌株恶臭假单胞菌JP-1.本文报道恶臭假单胞菌游离细胞丙烯腈水合酶催化的特性.     

单一粒度法研究固定化酶内扩散限制的酶应动力学

本文提出了用一种粒度的固定化酶,利用在高底物浓度与低底物浓度时分别表现为零级反应与一级反应的特点,研究在连续式反应器中内扩散限制下酶反应动力学的方法。通过在棒条状异构化酶上葡萄糖转化为果糖的反应,测求了酶反应的本征参数以及一级反应速度常数、表观米氏常数、底物扩散系数、效率因子等动力学参数。     

嗜热古细菌Sulfolobus tokodaii脱卤酶在D-乳酸生产中的应用

将来源于嗜热古菌Sulfolobus tokodaii的脱卤酶(L-HADST)基因克隆到载体p ET28b,转化大肠杆菌BL21(DE3)进行表达,在蛋白的N末端带有6个组氨酸融合标签,纯化后经聚丙烯酰胺凝胶电泳显示融合蛋白的分子量约为25000.融合蛋白催化2-氯丙酸(2-CPA)的最适反应温度为70℃,最适p H值为9.5.以外消旋2-CPA为底物生产D-乳酸,利用HPLC检测反应液中2-CPA及乳酸的变化,发现L-HADST只催化L-2-CPA脱氯反应.对酶催化反应条件进行了优化,结果表明,在p H值为9.5,温度为60℃的条件下,当反应体系中缓冲液浓度为3 mol/L,底物浓度为0.5 mol/L,酶浓度为3×104U/L时有较高的底物转化率及乳酸生成量.依据条件优化结果可知,影响反应速度的因素有底物浓度、缓冲液浓度以及酶浓度,其中底物浓度变化对转换率的影响最明显.     

大孔载体对米曲霉氨基酰化酶的固定化研究

用合成的大孔丙烯酸甲酯-—二乙烯苯交联共聚物(聚丙烯酸甲酯)、丙烯酰胺-N,N′-亚甲基双丙烯酰胺交联共聚物(聚丙烯酰胺)及它们的功能基化产物作为固定化氨基酰化酶的载体。考察了载体性质对固定化氨基酰化酶效果的影响。比较了底物浓度、pH、磷酸缓冲液浓度、温度对氨基酰化酶溶液酶及固定化酶的影响。利用其中一种载体制成固定化酶柱,对DL-蛋氨酸进行了连续拆分,考察固定化酶的操作稳定性。     

贝壳状革耳菌漆酶酶活测定方法分析

酶活测定方法中所用的反应底物、反应条件以及酶活单位定义对漆酶酶活的测定结果有很大的影响。在对贝壳状革耳菌 (Panusconchatus)漆酶酶活测定方法的研究中发现 ,该酶与 2 ,2’ -连氮 -双 ( 3-乙基并噻- 6 -磺酸 ) (ABTS)反应的最适pH值为 3.0 ,而与紫丁香醛连氮、2 ,6-二甲氧基苯酚和邻甲联苯胺反应的最适pH值均为 4.0。上述底物与Panusconchatus漆酶反应的Km (米氏常数 )分别为 0 .0 1 1 6(mmol·L) - 1 ,1 4.1 0 95 (mmol·L) - 1 ,0 .1 0 1 1 (mmol·L) - 1 ,0 .1 41 5 (mmol·L) - 1 。该酶与ABTS、2 ,6-二甲氧基苯酚和邻甲联苯胺反应均表现为零级反应 ,而与紫丁香醛连氮反应时只在一定范围的酶浓度以及反应时间内才表现为零级反应。     

胶体铜催化丙烯腈水合的高选择性及其活性中心结构的研究

研究了胶体铜催化丙烯腈水合制丙烯酰胺的高选择性与活性中心结构的关系. 在聚乙烯吡咯烷酮(PVP)保护下, 用肼和氢氧化钠混合液还原CuCl2制得胶体铜, 用其催化丙烯腈水合反应, 选择性达到100％， 产生高选择性的原因如下: (1) 胶体铜的活性中心不是胶粒表面的点缺陷, 而是胶体铜颗粒表面的位错端点. (2) 由于胶体铜具有高硬度和高强度的力学特性, 保证了活性中心结构的稳定性; 胶体铜颗粒的平均粒径(45 nm)超过晶粒的特征长度, 进一步保证了活性中心的稳定性.     

酶促反应分子马达的研究进展

传统观点认为酶促反应并不会影响酶本身的扩散运动.最近的研究表明,在酶促反应过程中,酶分子的扩散系数会增大,而且其增大强度具有底物依赖性,即随着底物浓度的增加而增大.酶促反应分子马达,是利用酶促反应过程中产生的能量驱动纳米或微米级物体的运动.尽管在几种不同的酶体系中的研究已经证实了酶在催化过程中的底物依赖性,但是造成酶扩散增强的原因至今仍不清楚.本文从酶促反应过程中酶自身扩散系数的变化、酶自身扩散系数变化的可能机理及其应用等3个方面,对酶在催化过程中的底物依赖性以及酶促分子马达的研究进展进行了综述.     

固相PGA酶催化一步法合成氨苄西林的工艺研究

采用吸附法将青霉素酰化酶(PGA)固定于AB-8树脂上,通过固相酶催化D-苯甘氨酸甲酯与6-氨基青霉烷酸(6-APA)反应制备氨苄西林,对酰化反应中的最适pH条件、溶剂体系、反应温度、反应时间、底物浓度进行实验研究。以氨苄西林收率和水解比为评价条件,固相酶最适催化pH值为6.5,通过正交实验分析表明,在异丙醇-PBS缓冲体系中,350mg PGA/AB-8(固定化酶活性113U·g~(-1))催化下,反应温度10℃,底物浓度(D-PGME∶6-APA)为3.5∶1,反应时间8h,氨苄西林收率达到71.3%。固相PGA酶重复回用6次,氨苄西林收率由71.3%降至63.6%。     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.     

Synthesis of pyrrolo[1,2-a]pyrazines and pyrazino[1,2-a]indoles by Curtius reaction in Morita-Baylis-Hillman derivatives

Synthesis of substituted pyrrolo[1,2-a]pyrazines and pyrazino[1,2-a]indoles from the Morita-Baylis-Hillman derivatives of acrylates via saponification followed by Curtius reaction is described.     

Cu0-catalysed 1,3-dipolar cycloadditions of α-amino acid derived N,N-cyclic azomethine imines to ynones

A series of 20 CuAIAC reactions between eight 4-acylamino substituted pyrazolidine-3-one-1-azomethine imines and four terminal ynones were performed using Cu⁰ as catalyst. The corresponding fluorescent cycloadducts were obtained in very high yields upon simple workup. Thus, Cu-metal turned out to be a better catalyst than Cu^I in terms of yield and ease of isolation. Availability of azomethine imines, mild reaction conditions, and simple workup enable a “click” access to libraries of densely substituted 2,3-dihydro-1H,5H-pyrazolo[1,2-a]pyrazol-1-ones. Reactivity of differently substituted dipoles was evaluated experimentally and by quantum chemical methods (DFT).     

Synthesis and antioxidative/anti-inflammatory activity of novel fullerene–polyamine conjugates

(E)-4-(Fullerenopyrrolidin-1-yl)-3-methylbut-2-enoic acid and its corresponding succinimidyl ester, readily obtained through Prato-type modification of C₆₀, were used for the selective N-acylation of polyamines. The thus obtained conjugates were evaluated for their antioxidative and anti-inflammatory activity and their cytotoxicity was determined. Members of this family of compounds showed interesting anti-lipid peroxidation, anti-lipoxygenase and anti-inflammatory activity and comparable cytocompatibility to spermidine.     

Copper-catalyzed multicomponent reactions of 2-iodoanilines,benzylamines, and elemental sulfur toward 2-arylbenzothiazoles

A relatively cheap copper salt-catalyzed, three-component approach providing 2-arylbenzothiazoles in good to excellent yields from readily available 2-iodoanilines, benzylamines, and sulfur powder is reported. This methodology allows preparation of various classes of 2-arylbenzothiazoles and provides a general, reliable approach.