Third Generation ATP Sensor with Enzymatic Analyte Recycling

For the first time the direct electron transfer of an enzyme ‐ cellobiose dehydrogenase, CDH ‐ has been coupled with the hexokinase catalyzed competition for glucose in a sensor for ATP. To enhance the signal output for ATP, pyruvate kinase was coimmobilized to recycle ADP by the phosphoenolpyruvate driven reaction. The new sensor overcomes the limit of 1 : 1 stoichiometry of the sequential or competitive conversion of ATP by effective enzymatic recycling of the analyte. The anodic oxidation of the glucose converting CDH proceeds at electrode potentials below 0 mV vs. Ag|AgCl thus potentially interfering substances like ascorbic acid or catecholamines do not influence the measuring signal. The combination of direct electron transfer of CDH with the enzymatic recycling results in an interference‐free and oxygen‐independent measurement of ATP in the lower µmolar concentration range with a lower limit of detection of 63.3 nM (S/N=3).     

纤维二糖与葡萄糖催化转化制备乙二醇(英文)

选用纤维二糖作为探针分子,探索纤维素催化转化制备乙二醇过程的反应路径.分别考察了纤维二糖和葡萄糖在双组分催化剂H2WO4和Ru/C下的催化反应活性.结果表明,乙二醇不仅来自于纤维二糖水解产物葡萄糖的逆羟醛缩合作用,同时也可以来自于纤维二糖的直接逆羟醛缩合过程.而且,纤维二糖的直接逆羟醛缩合作用对糖苷键的水解也有一定的促进作用.比较发现,钨基催化剂作用下纤维二糖的逆羟醛缩合反应活性比葡萄糖要低,因此乙醇醛可以缓慢产生并在Ru/C催化剂上迅速加氢生成乙二醇.使得以纤维二糖作为原料比以葡萄糖作为原料时获得更高的乙二醇收率.     

Optimization of Oligosaccharide Synthesis from Cellobiose by Dextransucrase

There is a growing market for oligosaccharides as sweeteners, prebiotics, anticariogenic compounds, and immunostimulating agents in both food and pharmaceutical industries. Interest in novel carbohydrate-based products has grown because of their reduced toxicity and low immune response. Cellobiose is potentially valuable as a nondigestible sugar. The reaction of cellobiose, as an acceptor with a sucrose as a donor, catalyzed by a dextransucrase from Leuconostoc mesenteroides B-512FMCM, produced a series of cellobio-oligosaccharides. This production system was optimized using a Box–Behnken experimental design for 289 mM of sucrose and 250 mM of cellobiose and 54 U of the enzyme at pH 5.2 and 30 °C, to produce maximum yields of oligosaccharide.     

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.     

Understanding the Mechanocatalytic Conversion of Biomass: A Low‐Energy One‐Step Reaction Mechanism by Applying Mechanical Force

On the way to establishing biomass as a renewable and environmentally friendly source to cover the ever‐increasing global demand on energy and chemicals, one great challenge is the efficient depolymerization of cellulose. Enhanced conversion rates have been discovered in ball‐milling experiments, thus opening a mechanocatalytic approach. However, an understanding of the impact of mechanical forces on the acid‐catalyzed cleavage of glycosidic bonds at the molecular level is still missing. Herein, we contribute such fundamental insight based on atomistic modeling. Mechanically stressing the macromolecular backbone radically changes the depolymerization pathway from a complex high‐barrier reaction upon thermal activation to a low‐energy single‐step mechanocatalytic process. In addition to revealing a regioselective increase in basicity under a mechanical force, our results provide molecular‐level explanations of the experimental findings and might therefore guide rational ways to improve such mechanocatalytic processes.     

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 μM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e⁻/H⁺ acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.     

7Li NMR as probe for solvent–cellulose interactions in cellulose dissolution

Molten salt hydrates proved to be alternative solvents to cellulose. Toinvestigate the reasons for this dissolving ability, information about thesolvent–cellulose interactions is essential. As well as ¹³CHR NMR, ⁷Li NMR was used to obtain further insight into thisproblem.After comparing several molten salt hydrates, the ⁷Li NMR spectrarevealed a smaller shielding of the lithium cation for not dissolving than fordissolving and swelling cellulose systems. In most solvent systems theshieldingat the ⁷Li nuclei increases with the cellulose concentration. 2D⁷Li-¹H HOESY NMR was successfully applied to verify thepresence of cellobiose, used as a model compound for cellulose, in the firstco-ordination sphere of the lithium cation.     

Synthesis and acid catalyzed hydrolysis of B2,5 type conformationally constrained glucopyranosides: incorporation into a cellobiose analogue

Isopropyl and p-nitrophenyl α- and β-d-glucopyranosides, restrained in a conformation close to B_2,5 via an oxymethylene bridge have been synthesized. These four glucopyranosides were found to be hydrolyzed at similar rates, close to those observed for the parent unconstrained glucosides. In such derivatives, either α or β, the exocyclic cleaved bond is synperiplanar to an endocyclic oxygen lone pair. This conformationally locked glucopyranosyl moiety was also incorporated into a disaccharide, affording a conformationally restrained cellobiose analogue which was assayed against various glycosidases.     

The effect of agitation speed,enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 °C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2³ full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.     

Cu催化羟基自由基氧化断裂纤维二糖分子糖苷键反应及其在纤维素低温解聚中的应用

纤维素是葡萄糖通过β-1,4-糖苷键链接而成的高聚物,在木质纤维素中含量最高,结构稳定,较难水解.糖苷键的解聚主要有三种方式:酶水解、酸水解以及碱降解.酶解的优点是反应条件温和、副产物少,但存在成本高、活性低等缺点,限制了其大规模的工业化生产.碱水解纤维素的同时伴随着葡萄糖的peeling-off反应得到异变糖酸,需要消耗大量的碱,并且强碱也存在腐蚀性强和回收难等问题.酸水解是目前工业上常用的纤维素水解方法,在保持较高葡萄糖选择性的同时,通过对反应条件的控制(提高反应温度和酸浓度)来提高纤维素的水解效率,但是硫酸对设备的腐蚀性强,也难以回收,不符合绿色化学的发展要求.固体酸是近年来研究较多的纤维素水解催化剂.固体酸虽然腐蚀性弱、易回收,但是其活性低,水热稳定性较差,目前还不具备大规模生产的条件.本文发展了一种羟基自由基活化断裂糖苷键的方法,利用羟基自由基的高活性在低温下实现糖苷键的选择性断裂,同时羟基自由基与糖苷键作用后转化为无毒无害的水和氧气,将不会对环境造成污染.我们首先以纤维二糖作为纤维素的模型分子,通过羟基自由基能够优先与糖苷键反应得到葡萄糖和葡萄糖酸的实验证实所提出的方法的可行性.实验表明,来自H2O2的·OH自由基能够在铜基催化剂作用下选择性氧化断裂其糖苷键,生成葡萄糖和葡萄糖酸.比如:采用均相CuSO4体系,纤维二糖转化率约为20%时,葡萄糖和葡萄糖酸的选择性分别为28.5%和32.3%.采用多相CuO/SiO2(4 wt%CuO)体系,纤维二糖转化率约为20%时,葡萄糖和葡萄糖酸的选择性约分别为23.3%和25.7%,并且该催化剂具有良好的循环使用性能.与·OH类似,CuSO4催化过硫酸钾生成的·SO4-自由基也能够有效转化纤维二糖,在纤维二糖转化率为20%时,葡萄糖和葡萄糖酸的选择性分别为36.6%和39.9%.利用这种·OH和·SO4-自由基氧化的方法,也能够在较低温度下(333 K)解聚纤维素中的糖苷键.我们发展了H2O2浸渍预处理纤维浸渍预处理纤维素的方法,通过部分破坏纤维素糖苷键,提高了纤维素的水解活性.比如:处理后的纤维素在413 K条件下反应12 h,纤维素转化率和葡萄糖选择性分别达到约36.1%和42.5%.XRD结果表明,处理后的纤维素的晶体结构未发生明显的变化.FT-IR表征结果显示处理后的纤维素表面生成了大量的羧酸基团.