Modulation of the catalytic behavior of alpha-chymotrypsin at monolayer-protected nanoparticle surfaces

Amino-acid-functionalized gold clusters modulate the catalytic behavior of alpha-chymotrypsin (ChT) toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (k(cat)/K(M)) of ChT-nanoparticle complexes increases by approximately 3-fold for the cationic substrate but decreases by 95% for the anionic substrate as compared with that of free ChT, providing enhanced substrate selectivity. Concurrently, the catalytic constants (k(cat)) of ChT show slight augmentation for the cationic substrate and significant attenuation for the anionic substrate in the presence of amino-acid-functionalized nanoparticles. The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the active site and release of product through electrostatic interactions, leading to the observed substrate specificities and catalytic constants.     

Transportation and mixing of droplets by surface acoustic wave

Unit operations for complicated biochemical analysis cannot usually be integrated into one substrate. A possible solution to solve this problem is to integrate multi-unit operations into two or more substrates. In this case, transporting droplets from one substrate to another is essential. In this work, a new method to transport droplets from a hydrophobic glass substrate to a piezoelectric substrate is proposed. An interdigitated transducer (IDT) and reflectors were fabricated on an optic grade 128° YX-cut lithium niobate (LiNbO₃) substrate, and its working surface between the IDT and a reflector was modified to be hydrophobic. Droplets to be transported were first pipetted onto a glass substrate. Adjust the glass substrate so that the droplets could contact the working surface of the piezoelectric substrate, and then was moved down. These droplets could be successfully transported from the glass surface to the piezoelectric substrate because of their “adhesion work” difference. By using this mechanism, water and red dye droplets were successfully transported from glass substrate to piezoelectric substrate. As an application, droplets mixing process was demonstrated in the piezoelectric substrate by using surface acoustic wave after they have been transported from the glass substrate.     

DMF竞争性抑制大豆脂氧酶好氧催化及其松弛底物抑制的综合作用

在大豆脂氧酶催化亚油酸的氧化反应中加入溶剂二甲基甲酰胺DMF(lgP为-1.01)可将底物亚油酸浓度提升到232 mmol/L而不产生底物抑制作用, 并使平衡产率从38.93%提高到66.09%. 在有底物存在时, 质量分数为5%的DMF基本不影响酶活; 此时体系具有最大的Kss与Ki值, 表明5%DMF对底物抑制作用的松弛效应最强, 而对酶的抑制作用最小.     

Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw

In this study, the applicability of a “fed-batch” strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.     

Toward the Achievement of Substrate Melting and Controlled Solidification in Thermal Spraying

The substrate is usually kept at a distant location in traditional thermal spraying, and substrate melting, which can improve splat adhesion usually does not happen. By moving the substrate close to the plasma flame and attaching a temperature control device to the backside of the substrate, as well as by additional heating from the molten droplets, substrate melting may occur and directional splat solidification becomes possible. In this proposed design, the substrate temperature is controlled by spray distance, flame temperature and initial substrate temperature. The variations of particle in-flight characteristics and contact interface temperature on spray distance are investigated. Optimal operating conditions are determined.     

A designed synthetic analogue of 4-OT is specific for a non-natural substrate

The substrate specificity of 4-oxalocrotonate tautomerase (4-OT) is characterized by electrostatic interactions between positively charged arginine (Arg) side chains on the enzyme and the dianionic substrate, 4-oxalocrotonate. To generate specific hydrogen-bonding interactions with a monoanionic substrate analogue, we have introduced a urea functional group into the active site by replacing arginine side chains with isosteric citrulline (Cit) residues. This design was based on the complementarity between the urea functionality of citrulline and the uncharged amide function of the substrate, as opposed to the guanidinium-carboxylate electrostatic interaction between the wild-type enzyme and the natural substrate. Indeed, the synthetic (Arg39Cit)4-OT analogue catalyzed the tautomerization of the non-natural monoamide-monoacid substrate while it was a poor catalyst for the natural diacid substrate. The specificity of (Arg39Cit)4-OT for the monoamide-monoacid substrate relative to that of the diacid substrate was found to be 740-fold greater than that of the wild-type enzyme for tautomerization of the non-natural substrate as compared with the natural one. The role of electrostatic interactions in the tautomerization of the monoamide-monoacid substrate was probed in detail with several other Arg to Cit analogues of this enzyme. This study has demonstrated that chemical manipulation of the functional groups within the active site of an enzyme can modify its catalytic activity and substrate specificity in a predictable way, suggesting that the incorporation of noncoded amino acids into proteins has great promise for the development of new enzymatic mechanisms and new binding interactions.     

Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of acetophenone derivatives: effect of charge density

In an effort to study the effect of substituent groups of the substrate on the alcohol dehydrogenase (ADH) reductions of aryl-alkyl ketones, several derivatives of acetophenone have been evaluated against ADHs from Lactobacillus brevis (LB) and Thermoanaerobacter sp. (T). Interestingly, ketones with non-demanding (neutral) para-substituents were reduced to secondary alcohols by these enzymes in enantiomerically pure form whereas those with demanding (ionizable) substituents could not be reduced. The effect of substrate size, their solubility in the reaction medium, electron donating and withdrawing properties of the ligand and also the electronic charge density distribution on the substrate molecules have been studied and discussed in detail. From the results, it is observed that the electronic charge distribution in the substrate molecules is influencing the orientation of the substrate in the active site of the enzyme and hence the ability to reduce the substrate.     

计算机硬盘基片CMP中表面膜特性的分析研究

目前,普遍采用化学机械抛光(Chemical-mechanical polishing,CMP)技术对计算机硬盘基片(盘片)表面进行原子级平整。CMP加工中,盘片表面膜及其特性对CMP过程及CMP性能具有关键作用。本文分别采用俄歇能谱(AES)、X射线光电子能谱(XPS)、扫描电镜(SEM)、纳米硬度计、电化学极化法等分析手段对盘片表面物理、化学及机械特性进行了研究,发现盘片CMP后表面发生了氧化,氧化膜在盘片的表层,厚度在纳米量级,氧化产物为Ni(OH)₂;氧化膜为较软的、疏松的、粗糙的多孔结构;氧化膜的存在加快了盘片表面的腐蚀磨损。结合盘片CMP试验结果,推测盘片的CMP机理为盘片表面氧化生成机械强度较低的Ni(OH)₂氧化膜及随后氧化膜的机械和化学去除,二者的不断循环实现表面的全局平面化。     

Effects of rhamnolipid on degradation of granular organic substrate from kitchen waste by a Pseudomonas aeruginosa strain

The effect of rhamnolipid produced by a Pseudomonas aeruginosa strain on the aerobic degradation of granular organic substrate from kitchen waste by the bacterium was studied and compared with that of two synthetic surfactants, SDS and Triton X-100. The adsorption of rhamnolipid on the substrate, the surfactant-interfered adhesion of bacteria on the substrate as well as physicochemical and microbial conditions of the substrate during degradation were investigated. The adsorption isotherm of rhamnolipid on the substrate fit Freundlich law and its interactions with the substrate and bacteria weakened the adsorption of the bacteria on the substrate. The two synthetic surfactants, however, did not have such microbial effects. During degradation, rhamnolipid slowed down water evaporation in the substrate and significantly strengthened the dispersion of organic matter into the substrate water phase. The number of cells in the rhamnolipid treatment was higher than that in control and the remaining organic matter content in the substrates also had faster decreasing. SEM examination showed the on-site degradation of the substrate organic matter without rhamnolipid and the transfer of the degradation site in the presence of rhamnolipid. The results indicated that interference of rhamnolipid in the substrate matrix plays a potential role, physicochemically or microbially, on the degradation of the granular organic substrate. SDS and Triton X-100 may have the above physicochemical effects, but not so significant.     

基板界面对PS/PMMA共混物薄膜相逆转组成比的影响

近年来高分子共混体系中的界面、表面效应逐渐引起了越来越多研究者的兴趣 .人们发现 ,当共混物薄膜厚度减至一定程度时 ,聚合物共混物薄膜中的相形态、相容性及相分离动力学与本体中有较大的不同[1～ 3] .基板界面作用对共混薄膜体系的热力学、动力学行为产生很大的影响 .我们以往的研究 [4 ,5]也发现 ,PP/EVAc(70 /30 )共混体系退火过程中 ,基板界面 (如玻璃 )作用可大大加速分散相(EVAc)粒子的粗化凝聚过程 .本研究用聚甲基丙烯酸甲酯和聚苯乙烯共混物的四氢呋喃溶液在不同基板介质 (如玻璃基板 ,PP基板 )上成膜 ,用相差显微镜观测了…     

Evidence of Substrate Melting of NiCr Particles on Stainless Steel Substrate by Experimental Observation and Simulations

Single NiCr splats were plasma-sprayed onto a polished stainless steel substrate held at room temperature. The splat-substrate interface was characterized by focused ion beam and transmission electron microscopy. The frequent observation of NiO particles, particularly in pores within the splat, and at the periphery of splat, suggests that the principal oxidation process occurs at the substrate surface, where the splats are exposed to a water vapor-rich environment. It was also observed that the splat adhered well in some locations where elemental-diffusion and jetting of the substrate occurred, suggestive of substrate melting. A three-dimensional numerical model was developed to simulate the impact of a splat onto a substrate. The simulation shows that the observation of the central pore in the splat and the phenomenon of substrate melting may occur. Based on these results, the effect of water release on oxide formation and splat morphology can be explained.     

Effect of replicated polymeric substrate with lotus surface structure on adipose-derived stem cell behaviors

We fabricated polystyrene substrates with lotus leaf surface structure (LLSS) and investigated cell behaviors, including attachment, morphology, proliferation, and differentiation of adipose-derived stem cells (ASCs) on them. Compared to the flat substrate, the LLSS substrate induced higher cell attachment rate, but did not significantly change the cell proliferation rate. In addition, ASCs on the LLSS substrate exhibited relatively narrower spreading morphology and less organized cytoskeleton, there by resulting in smaller sizes of cells than those on the flat substrate. According to histochemical staining and RT-PCR analysis, the LLSS substrate induced higher adipogenic differentiation of ASCs than the flat substrate, while chondrogenic and osteogenic differentiation were decreased.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

The effect of substrate concentration on biohydrogen production by using kinetic models

The effect of substrate concentration ranging from 0 to 300 g/L on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as substrate. The experimental results showed that, at 35℃ and initial pH 7.0, during the fermentative hydrogen production, the hydrogen production potential and hydrogen production rate increased with increasing substrate concentration from 0 to 25 g/L. The maximal hydrogen production potential of 426.8 mL and maximal hydrogen pro-duction rate of 15.1 mL/h were obtained at the substrate concentration of 25 g/L. The maximal hydrogen yield and the maximal substrate degradation efficiency were respectively 384.3 mL/g glucose and 97.6%, at the substrate concentration of 2 g/L. The modified Logistic model could be used to describe the progress of cumulative hydrogen production in this study successfully. The Han-Levenspiel model could be used to describe the effect of substrate concentration on fermentative hydrogen production rate.     

Macromolecules in a blend of poor and amphiphilic solvents

The globular state of the homopolymer macromolecule in a blend composed of a poor solvent and an amphiphilic solvent (substrate), whose molecules tend to be aligned with the solvent concentration gradient in the inhomogeneity region, was theoretically studied. The size of a homogeneous globule and the substrate concentration in its volume were calculated in terms of a bulk approximation. After the transition of the macromolecule from the coil to the globule state, its volume first decreases with a decrease in temperature and then begins to grow due to substrate molecules penetrating the globule. The substrate concentration in the globule insignificantly exceeds that outside the globule at identical second virial coefficients of interaction between monomer units and between substrate molecules. The expression for the free energy functional depending on the volume fractions of the components and on the orientation of substrate molecules was examined in the ground-state approximation. The orientation effect leads to narrowing of the surface layer and to a decrease in the surface tension of the homogeneous globule, thereby increasing its stability with respect to the transition to the unfolded-coil state.     

High turnover remote catalytic oxygenation of alkyl groups: how steric exclusion of unbound substrate contributes to high molecular recognition selectivity

H-bonding mediated molecular recognition between substrate and ligand -COOH groups orients the substrate so that remote, catalyzed oxygenation of an alkyl C-H bond by a Mn-oxo active site can occur with very high (>98%) regio- and stereoselectivity. This paper identifies steric exclusion-exclusion of non H-bonded substrate molecules from the active site-as one requirement for high selectivity, along with the entropic advantage of intramolecularity. If unbound substrate molecules were able to reach the active site, they would react unselectively, degrading the observed selectivity. Both of the faces of the catalyst are blocked by two ligand molecules each with a -COOH group. The acid p-(t)BuC6H4COOH binds to the ligand -COOH recognition site but is not oxidized and merely blocks approach of the substrate therefore acting as an effective inhibitor for ibuprofen oxidation in both free acid and ibuprofen ester form. Dixon plots show that inhibition is competitive for the free acid ibuprofen substrate, no doubt because this substrate can compete with the inhibitor for binding to the recognition site. In contrast, inhibition is uncompetitive for the ibuprofen-ester substrate, consistent with this ester substrate no longer being able to bind to the recognition site. Inhibition can be reversed with MeCOOH, an acid that can competitively bind to the recognition site but, being sterically small, no longer blocks access to the active site.     

固体基质室温磷光发光机理进展

本文从磷光体在基质中的分布、磷光体与基质中重原子的作用和磷光体基质中的刚性化三个方面对固体基质室温磷光发光机理进行了详细评述,着重介绍了磷光体与基质之间的作用 ,并对研究前景进行了讨论。     

Influence of substrate nature and growth conditions on the morphology of thin DMOAP films

We report some preliminary results on the morphology of thin N,N -dimethyl- n -octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) films. When deposited on a glass substrate, DMOAP forms a mono- or multi-layer structure parallel to the substrate. The surface topography of the film is probed by atomic force microscopy. In general, the free surface of such a film is not flat and smooth. Islands and holes are formed on the free surface of the films when a sufficiently flat substrate is used. The thin film surface topography depends strongly on the nature of the bare substrate, the curing conditions, and the immersion time of the substrate in the DMOAP solution. The film is always rougher than the bare substrate used. Annealing roughens the surface of the alkoxysilane thin films deposited on a glass substrate. For films on glass plates covered with an indium tin oxide layer, annealing has minor effects. The surface topography affects the microstructure of homeotropic smectic samples.     

衬底温度对强流脉冲离子束烧蚀沉积类金刚石薄膜化学结构的影响

采用强流脉冲离子束(High-intensitypulsedionbeam,HIPIB)烧蚀技术在Si(100)基体上沉积类金刚石(Diamond-likecarbon,DLC)薄膜,衬底温度的变化范围为298～673K.利用Raman光谱和X射线光电子谱(XPS)对DLC薄膜的化学结合状态与衬底温度之间关系进行研究.薄膜XPS的C1s谱的解谱分析得出薄膜中含有sp³C(结合能为285.5eV)和sp²C(结合能为284.7eV)成分,根据解谱结果评价薄膜中sp³C含量.根据XPS分析可知,衬底温度低于473K时,sp³C的含量大约为40%左右;随着沉积薄膜时衬底温度的提高,sp³C的含量降低,由298K时的42.5%降到673K时的8.1%,从573K开始发生sp³C向sp²C转变.Raman光谱表明,随着衬底温度的提高,Raman谱中G峰的峰位靠近石墨峰位,G峰的半峰宽降低,D峰与G峰的强度比I_D/I_G增大,说明薄膜中的sp³C的含量随衬底温度增加而减少.     

Kinetics of batch production of single-cell protein from cheese whey

A kinetic model for single-cell protein batch fermentation was developed using the numerical simultaneous integration approach of the fourth-order Runge-Kutta method. The model takes into account the effect of substrate inhibtion, maintenance energy, and cell death on the cell growth and substrate utilization during the fermentation process. The theoretical results obtained from the model compared well with the experimental data. The model was used to study the effect of the initial substrate concentration on the lag period, fermentation time, specific growth rate, population size, and cell productivity of batch fermentation. Increasing the initial substrate concentration increased the lag period and fermentation time and decreased the specific growth rate and cell yield. The growth limiting substrate concentration was 2.9 g/L, whereas the growth inhibiting substrate concentration was 69.0 g/L. Increasing the initial substrate concentration above 150 g/L significantly decreased the yeast population size.