水化层影响酸酐酶内CO2扩散行为的分子动力学模拟

气相中酶分子表面的水化层对其催化行为具有显著的影响。本文采用全原子分子动力学模拟方法考察了气相体系碳酸酐酶表面的水化层对酶结构以及CO₂在酶分子中扩散行为的影响。首先展现了水分子在酶分子及其活性中心周围的分布,研究了水化层厚度对于酶结构以及CO₂扩散速率的影响;发现最有利于CO₂扩散进入酶分子的水化层厚度为0.7 nm。确认了碳酸酐酶内CO₂的吸附位点,通过对其开合状态统计,显示出碳酸酐酶中CO₂扩散通道中的瓶颈位置。上述结果对设计和优化碳酸酐酶催化气相体系中CO₂的吸附和转化提供了依据和启示。     

Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas,Vanua-Lava and Viangchan

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.     

二氧化钛基催化剂催化氧化甲醛的研究进展

甲醛（HCHO）是一种主要的室内空气污染物,具有高致癌性和致突变性,催化氧化是一种理想而有效的室内消除甲醛的方法。二氧化钛作为载体负载贵金属催化剂在甲醛催化氧化反应中表现出优良的催化性能。本文综述了近年来二氧化钛基催化剂催化氧化甲醛的研究进展,主要总结了二氧化钛的吸附机理,二氧化钛晶体结构、表面形貌及微观结构、表面活性氧物种和还原性等对二氧化钛基负载贵金属催化剂催化氧化甲醛性能的影响,重点关注了催化剂的化学结构性质与甲醛氧化活性的关系,并归纳了一部分甲醛催化氧化反应机理。最后指出二氧化钛基催化剂在甲醛催化氧化反应中未来研究方向为：合理设计TiO₂各种缺陷和晶面暴露,同时提高贵金属的分散性和原子利用效率,继而进一步提高催化剂的性能;在保持催化剂高活性、高稳定性的同时,推进设计实际应用整体式催化剂的研究进程。     

Three in One: Temperature,Solvent and Catalytic Stability by Engineering the Cofactor‐Binding Element of Amine Transaminase

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate‐induced inactivation mechanism involving dissociation of the enzyme–cofactor intermediate. Here, we report on engineering the cofactor‐ring‐binding element, which also shapes the active‐site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme–cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′‐phosphate supply, thus making large‐scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural “hotspot” is a common feature of other transaminases it could serve as a general engineering target.     

A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO₂. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability.     

Kinetics of the reaction of propylene oxide with carbon dioxide in the presence of tetrasubstituted ammonium and phosphonium halogenides

Reactions of alkylene oxides with carbon dioxide underlie the industrial technology of ethylene- and propylene carbonates. Looking for new catalytic systems for these processes remains of interest due to the possibility of creating a new energy-saving process for the production of ethylene and propylene glycols. This work is aimed at comparing the catalytic activity of halogenides in the reaction of propylene oxide (PO) and carbon dioxide in the presence of tetrasubstituted ammonium and phosphonium halogenides, and evaluating the feasibility of using them in designing industrial technologies for the production of alkylene carbonates and glycols as active catalytic systems. Triphenylphosphine halogenides have been shown to possess high catalytic activity as compared to triethanolamine-based analogs. In terms of efficiency, triphenylphosphonium bromides are as good as the familiar catalysts based on potassium iodide. The high activity of these catalysts in the reaction for propylene carbonate (PC) production and their good solubility in the reaction medium allow us to propose them for the development of industrial technology for the subsequent production of alkyl carbonates and alkylene glycols.     

Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme

Directed evolution was used to enhance the activity of the glycerophosphodiesterase enzyme from Enterobacter aerogenes, GpdQ, toward bis(para-nitrophenol) phosphate (BpNPP), a substrate that is frequently used to assay phosphodiesterases. Native GpdQ has a low level of activity toward BpNPP while the evolved enzymes exhibited k(cat) values that were well over 100 times better while improvements in k(cat)/K(m) of around 500 times were observed along with improved activity we observed a change in the oligomeric structure in the evolved enzymes. The native enzyme is a hexamer with tightly associated dimers related by a 3-fold axis. The stability of the dimer was attributed in part to the cap domain that forms a disulfide bond with its 2-fold-related subunit and in part due to the fact that dimerization results in burying 23.6% of the monomer's accessible surface area. The cap domain also forms the top of the active site and contributes an essential part of the interface between 3-fold-related molecules. The evolved proteins quickly lost one of the cysteine residues that formed the disulfide bond and other mutations that might stabilize the cap domain. The likely effect of these mutations was to open up the active site for the new substrate and to favor the formation of dimeric molecules. The breakdown of the oligomeric structure was accompanied by a reduction in the thermal stability of the protein-as monitored by the residual activity of the native and mutant proteins following pre-incubation at elevated temperatures. A discussion on the evolutionary implications of these studies is presented.     

Catalytic reduction of carbon dioxide. 1. Reduction of carbon dioxide with carbon carrying potassium carbonate

The catalytic behaviour of potassium carbonate doped onto active carbon was investigated with respect to reduction of carbon dioxide. The distribution of potassium on the carbon surface was found by an X-ray microanalyzer to be uniform. The amount of oxygen trapped on the carbon surface during reaction was nearly proportional to the surface concentration of potassium. The catalytic activity with carbon doped with less than 2% potassium carbonate was proportional to the amount of trapped oxygen, while excessive doping by potassium was found to form trapped oxygen independently of the gasification of carbon. Disproportionation of carbon monoxide into carbon dioxide and carbon was found to take place readily at 700 °C, with carbon doped with 4.0 wt % potassium carbonate.     

Site-directed mutagenesis study of a conserved residue in family 10 glycanases: histidine 86 of xylanase A from Streptomyces lividans

Xylanases from family 10 glycanases contain three conserved histidine residues in their active site. The role of H86 in the structure- function of xylanase A from Streptomyces lividans (XlnA) was studied by site-directed mutagenesis. Six mutant proteins (H86A/E/F/K/Q/W) were produced, purified and characterized. The six mutations reduced the affinity of XlnA towards xylan without having any major effect on the catalytic constant. All these mutations also lowered the pKa of the acid-base catalyst by 0.46-1.94 pH units. The mutations decreased the enzyme stability at 60 degrees C by up to 95% and the transition temperature by 2.2-5.8 degrees C. Unfolding of the protein with guanidine hydrochloride (GdnxHCl) showed that five out of six mutations decreased the concentration required to denature 50% of the XlnA, confirming the importance of H86 for the stability of the enzyme. The increase in m value m=d(deltaG)/d[GdnxHCl] also suggested the involvement of residue H86 in the structure of the denatured state of XlnA. It can be concluded from this study that this active site residue was conserved in family 10 glycanases for its function in maintaining the elevated pKa of the acid-base catalyst and in the stability of the protein, while being of little importance for the activity.      

多元催化体系中Cd的助催化机制对低汞触媒性能的影响

以活性炭为载体,采用多重合成工艺复配出特定的多元催化剂,并用自制模拟工业氯乙烯生产装置进行试验,结合SP-6890型气相色谱仪对该多元催化反应体系进行催化活性评价。研究发现:在该多元催化反应体系中,Cd2+和Hg2+与Cl-形成较大分子的双核桥式络合物催化活性中心,这种大分子结构不仅增大了催化体系活性组分的比表面积和催化活性中心结构的稳定性,有效地提高了整个体系的催化活性,同时也增大了催化体系中活性组分与载体活性炭的结合力,有利于抑制汞的流失。     

环氧乙烷非均相催化水合动力学及均温反应器热稳定性分析

采用碳纳米管增强复合材料催化剂,在等温积分反应器中获得环氧乙烷非均相催化水合宏观反应动力学实验数据,建立了幂函数型宏观反应动力学方程,采用Levenberg-Marquardt法对动力学模型参数进行估算,并以该动力学模型为基础,分析了均温反应器的热稳定性。结果表明,生成乙二醇主反应的表观活化能为71.7 k J/mol,与两个典型的串联副反应的活化能接近。模型参数统计检验结果表明,该宏观动力学方程参数是适定的,可用于工业反应器的设计。给出的反应器关键参数的计算方法,可为乙二醇合成反应器的模拟计算和设计开发提供必要的依据。     

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli

When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.      

Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes

It is widely agreed that new enzymes evolve from existing ones through the duplication of genes encoding existing enzymes followed by sequence divergence. While evolution is an inherently random process, studies of divergently related enzymes have shown that the evolution of new enzymes follows one of three general routes in which the substrate specificity, reaction mechanism, or active site architecture of the progenitor enzyme is reused in the new enzyme. Recent developments in structural biology relating to divergently related (beta/alpha)8 enzymes have brought new insight into these processes and have revealed that conserved structural elements play an important role in divergent evolution. These studies have shown that, although evolution occurs as a series of random mutations, stable folds such as the (beta/alpha)8 barrel and structural features of the active sites of enzymes are frequently reused in evolution and adapted for new catalytic purposes.     

Mechanisms of the alkali metal catalysed gasification of carbon

The catalytic effects of alkali metal salts in the gasification of carbonaceous materials by oxygen, steam and carbon dioxide are described. The most effective catalysts are generally the carbonates, oxides and hydroxides; other active salts tend to convert to these species under gasification conditions. Current theories of the mechanism of this type of catalysis are reviewed. Thermodynamic considerations, the results of thermal analysis and the magnitude of kinetic isotope effects suggest that cyclic sequences of elementary reactions are responsible for the catalytic phenomena.     

Variation of the morphological and the catalytic properties of active carbon-nickel catalysts

The relation between the morphological and the catalytic properties of active carbon-nickel catalysts was investigated in connection with using these catalysts as cathodes in air-zinc cells. The samples, containing different amounts of deposited nickel nitrate were activated in carbon dioxide. During the process of activation changes in texture porosity, surface area and helium density appeared. These properties are discussed in relation to the catalytic activity of the active carbon-nickel catalysts. It was found that the properties mentioned above may be modified according to the nickel content. The dispersion and the texture of nickel on the surface of active carbon were examined by scanning electron microscopy.     

超临界二氧化碳改性活性炭及其对载体性能的影响

以氯化钌为活性前驱体,活性炭为载体,采用超临界CO2处理制备钌/炭催化剂。以超临界二氧化碳流体对活性炭进行预处理,并使用联碱中和法、XPS等手段表征活性炭表面官能团数量,发现处理后活性炭表面羧基和酚羟基的数量明显减少,内酯基的数量增多,这是由于在超临界二氧化碳条件下活性炭表面的羧基和酚羟基发生酯化反应生成内酯基造成的。活性炭表面官能团的改变,改善了它的负载性能。结果表明,使用超临界CO2预处理活性炭作催化剂载体,能提高葡萄糖加氢制备山梨醇的反应催化活性。     

Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4‐Oxalocrotonate Tautomerase More Effectively than Distant Mutations

The enzyme 4‐oxalocrotonate tautomerase (4‐OT), which catalyzes enol–keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon–carbon bond‐forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4‐OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000‐fold improvement in catalytic efficiency (k_cat/K_m) and a >10⁷‐fold change in reaction specificity, by exploring small libraries in which only “hotspots” are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4‐OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.     

Influence of support composition on the structure and reactivity of strontium base catalysts

Strontium was supported on a variety of carriers, including silica, alumina, titania and carbon, by impregnation and decomposition of an acetate precursor at 773 K. These supported samples were characterized by surface area measurements, stepwise temperature-programmed desorption of carbon dioxide and activity for the catalytic decomposition of 2-propanol. In some cases, infrared and X-ray absorption spectroscopy were used to identify surface species. Results from these techniques suggested that strontium supported on silica forms a weakly basic surface silicate phase that had low activity for 2-propanol dehydrogenation. On alumina and titania, strontium acetate decomposed to form supported basic carbonates that were moderately active for 2-propanol dehydrogenation. When rates are normalized by the base site density determined from CO₂ desorption, strontium supported on carbon was the most active sample for dehydrogenation of 2-propanol. These results suggest that the nature of supported alkaline earth catalysts is strongly dependent on the composition of the carrier. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alumina/silica aerogel with zinc chloride alkylation catalyst: Influence of supercritical drying conditions and aerogel structure on alkylation catalytic activity

Alumina/silica aerogel with zinc chloride alkylation catalyst, was obtained using one step sol–gel synthesis and subsequent drying with the supercritical carbon dioxide. The high density supercritical carbon dioxide drying conditions resulted in zinc chloride removal from the catalyst surface, surface area and pore volume increase and catalytic activity decrease. The low density supercritical carbon dioxide drying conditions, pore size distribution centred around 6 nm pore radius and high degree of mixed Al–O–Si bonds in the alumina/silica aerogel network, were found to increase the catalytic activity of the obtained aerogel catalysts.     

Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol

2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.