羧甲基纤维素水凝胶生物降解动力学研究

用氯化铝对羧甲基纤维素进行交联,制得了水凝胶.考察了底物浓度、酶浓度以及降解温度对该水凝胶降解速率的影响,探讨了酶降解动力学及“表观”活化能对酶浓度的依赖关系.结果表明,该酶促反应最佳温度为37 ℃,降解反应对底物浓度和酶浓度的反应级数分别为1级和1.2级;得到了与传统的Michaelis-Menten动力学机制不同的非均相酶促反应动力学模型,确定了“表观”活化能与酶浓度之间的定量关系.     

PHBV/Sol-gel Bioglass多孔材料在模拟生理溶液中的化学反应研究

3-羟基丁酸-co-3-羟基戊酸共聚物(PHBV)/生物活性玻璃(SGBG)是一种用于骨和软骨组织工程支架的新型多孔复合材料,本文探讨了PHBV/SGBG在模拟生理溶液中的一系列化学反应,以及多孔材料在模拟生理溶液中浸泡后的成分和结构变化.研究结果表明,在SBF溶液中浸泡后,SGBG与SBF溶液的离子交换反应和PHBV的降解反应使SBF溶液的离子浓度发生变化,并在PHBV/SGBG表面形成了结晶态类骨碳酸羟基磷灰石.     

聚碳酸亚丙酯对聚(β-羟基丁酸酯-β-羟基戊酸酯)结晶行为的影响

用FTIR和DSC研究了PHBV及其与聚碳酸亚丙酯(PPC)熔融共混样的熔融结晶行为和等温结晶动力学.结果表明,PHBV与PPC熔融共混过程中发生了酯交换反应,两组分间存在一定的相互作用.PPC虽然能降低PHBV结晶折迭链的表面自由能,但同时也能够降低PHBV的结晶度、球晶径向生长速率、平衡熔点和结晶能力.     

利用色谱法研究α－淀粉酶变性动力学

提出用尺寸排阻色谱法研究酶的变性动力学。此法将高效色谱分离同灵敏的紫外检测结合起来,消除了紫外法测定蛋白质变性速度时变性体的影响。确定了个淀粉酶在盐酸胍溶液中变性时的反应级数,测定了不同浓度变性剂中酶的变性速度常数,并和失活速度常数进行了比较,讨论了影响酶变性速度的因素。     

聚(3-羟基丁酸酯-co-3-羟基戊酸酯)改性及纤维成形北大核心CSCD

聚(3-羟基丁酸酯-co-3-羟基戊酸酯)(PHBV)是一种微生物发酵生产的热塑性聚合物。从物理、化学改性及其纤维成形两个方面综述了PHBV的研究进展。PHBV的物理改性主要有无机纳米粒子共混体系(PHBV/iNPs)、有机纳米晶共混体系(PHBV/oNPs)、高聚物共混体系(PHBV/Polymer)和绿色全降解共混体系;化学结构构筑主要包括接枝共聚改性、嵌段共聚改性、端基扩链改性等。从改性的手段及介质,分析了改性方法的优缺点。PHBV纤维的成形方法主要有熔融纺丝法、干法纺丝法及静电纺丝法。从PHBV纤维应用领域看,熔融纺纤维应用目标在于替代现有石油基相关产品,而静电纺纤维主要应用于开拓组织工程再生医学领域。最后,对PHBV今后的研究及发展提出了展望。     

壳聚糖的酶法降解

用壳聚糖酶降解壳聚糖,探讨了壳聚糖降解过程中温度、pH值、底物浓度和金属离子对酶促反应的影响。结果表明:酶促反应进行到5 h左右时,即可得到聚合度小于10的壳寡糖。该酶促反应的最适温度为50℃,最适pH=5.5;最适底物浓度为0.02 g/mL;金属离子Ca2+和Mg2+对酶降解有一定的促进作用,而Zn2+、Cu2+对酶降解有较强的抑制作用。该酶促反应符合米氏动力学方程,米氏常数Km=7.80 g/L,最大反应速率Vmax=7.72 g/(min.L)。     

漆酶催化氧化水中有机污染物*

漆酶是一种含铜的多酚氧化酶, 属于铜蓝氧化酶蛋白, 能催化氧化多种难降解的有机污染物。漆酶催化氧化水中有机污染物具有底物广泛、能耗低、易操作、环境友好等优点,是一项前景广阔的生物处理技术。本文综述了漆酶对水中有机污染物的催化降解,主要从漆酶的化学组成、结构、催化降解机理、漆酶的固定化、影响因素（溶液pH值、温度、金属离子、营养因子、有机溶剂、漆酶的浓度、底物的初始浓度等）、降解的动力学以及漆酶在处理水中有机污染物的应用等五个方面介绍了相关的研究进展。漆酶催化氧化有机物的机理主要表现在底物自由基中间体的产生和氧气还原成水两个方面。本文提出了目前漆酶催化氧化水中有机污染物存在的主要问题,并展望了漆酶降解水体中有机污染物的发展方向。     

利用色谱法研究α—淀粉酶变性动力学

提出用尺寸排阻色谱法研究酶的变性动力学。此法将高效色谱分离同灵敏的紫外检测结合起来，消除了紫外法测定蛋白质变性速度时变性体的影响。确定了α－淀粉酶在盐酸胍溶液中变性时的反应级数。测定了不同浓度变性剂中酶的变性速度常数，并和答活速度常数进行了比较，讨论了影响酶变性速度的因素。     

碱性条件下L-抗坏血酸自降解的非酶褐变研究

在偏碱性溶液环境下,对L-抗坏血酸(ASA)自降解过程的非酶褐变反应进行了研究。考察了反应因素(温度、时间、p H值)对其非酶褐变过程的影响,主要考察了各反应因素对ASA自降解过程中的底物消耗及生成物(挥发性产物、无色中间体和褐色物质)生成的影响;利用经典动力学模型分析了ASA自降解过程中底物消耗、生成无色中间体和褐色物质的动力学特征。结果表明:较高温度、较长时间和较强碱性均会促进ASA的自降解,同时也有利于挥发性物质、无色中间本和褐色物质的生成。在碱性条件下挥发性产物主要为呋喃类化合物。ASA非酶褐变自降解过程的动力学表现为其自降解符合一级动力学特征,而无色中间体和褐色物质的生成符合零级动力学特征。     

PHBV纳米纤维的静电纺丝及在生物医用领域的研究进展

聚(3-羟基丁酸酯-3-羟基戊酸酯)(PHBV)纳米纤维具有高比表面积、高孔隙率、生物相容性、生物降解性等优点,可用作生物医用材料。本文综述了基于静电纺丝法制备的PHBV纳米纤维及其在生物医用领域的研究进展,讨论了PHBV纺丝溶液的溶剂、浓度、外加盐类以及聚合物等对PHBV纳米纤维形貌、结构及性能的影响规律,结合目前PHBV纳米纤维在组织工程和药物运输载体的研究现状,重点概述了其亲水改性、力学性能改善、功能化改性的研究进展。最后,对静电纺丝法制备PHBV纳米纤维存在的一些问题进行分析,并对其未来发展趋势和前景进行展望。     

Thermal and mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) plasticized by biodegradable soybean oils

The effects of soybean oil (SO) and epoxidized soybean oil(ESO) as biodegradable plasticizers for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were studied using thermal and mechanical analyses. PHBV/SO and PHBV/ESO blends were prepared by evaporating solvent from blend solutions. The levels of additive in the blend varied from 5% to 30%. As a plasticizer for PHBV, ESO was more effective than SO in depression of the glass transition temperature as well as in increasing the elongation at break and the impact strength of the films with increasing levels of additive. Biodegradation of the plasticized PHBV films was carried out by accelerated compost method. The degradation rates of the blend films with SO or ESO were found to be faster than that of PHBV film. From the thermogravimetric analysis, it was found that the thermal reaction between the epoxide groups of ESO and PHBV fragments with carboxylic chain ends, occurred during the degradation of PHBV/ESO blends.     

Real‐Time Monitoring of Mass‐Transport‐Related Enzymatic Reaction Kinetics in a Nanochannel‐Array Reactor

To understand the fundamentals of enzymatic reactions confined in micro‐/nanosystems, the construction of a small enzyme reactor coupled with an integrated real‐time detection system for monitoring the kinetic information is a significant challenge. Nano‐enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real‐time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass‐transport‐related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano‐enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 μL min^?1), the enzymatic reaction kinetics became the rate‐determining step. This change resulted in the decrease in the conversion efficiency of the nano‐enzyme reactor and the apparent Michaelis–Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.     

Kinetics of Enzymatic Synthesis of Cinnamyl Butyrate by Immobilized Lipase

This work illustrates the enzymatic synthesis of cinnamyl butyrate by esterification of butyric acid and cinnamyl alcohol. Experiments were performed to study the various operating parameters such as molar ratio, enzyme concentration, temperature, and speed of agitation. Also, the suitable kinetic model for esterification reaction was predicted and the various kinetic parameters were determined. It has been observed that the experimental results agree well with the simulated results obtained by following the ping-pong bi-bi mechanism with dead-end inhibition by both the substrate acid and alcohol. The highest 90% conversion of butyric acid was observed after 12 h at the following reaction conditions: substrate molar ratio 1:2 (butyric acid/cinnamyl alcohol), temperature 50 °C, enzyme loading 2% (with respect to the weight of the substrates), and agitation speed 250 rpm. Diffusional mass transfer limitations between substrate and enzyme surface do not show significant effect on reaction kinetics. Enzyme reusability study reveals that it retains 85% of its catalytic activity after five consecutive cycles.     

Effects of nonproductive binding on the kinetics of enzymatic reactions with patterned substrates

Existing models of ligand-receptor binding kinetics suggest that clustering surface-associated molecules tends to decrease the rates with which solution phase molecules associate and dissociate. Here, the authors use kinetic Monte Carlo simulations to study the case of an enzyme catalyzing the turnover of substrate molecules immobilized on a surface. The simulations reveal a crossover in the overall reaction rates for randomly distributed and clustered substrate molecules as the enzyme unbinding rate is varied. Approximate expressions for the effective kinetic parameters are introduced, and they show that the observed behavior derives from sequestration of the enzyme in the strong-sticking limit.     

Kinetics of bonded invertase: asymmetric polysulfone membranes

Asymmetric flat ultrafiltration membranes made from bromomethylated polysulfone were used to fix invertase chemically. The invertase reactivity of these membranes was compared with those where enzyme bonding was achieved by reacting bromomethylated polysulfone with ethylene diamine and glutaric dialdehyde which act as spacers. In both cases the invertase fixation was carried out such that its concentration at the surface facing the saccharose feed solution could be neglected The kinetic behavior of the immobilized invertase was modelled by investigating the influence of diffusive and of convective transport across the membranes. Considering axial back-mixing of the convective flow within the membrane, the reaction can be simulated at low substrate concentrations. The heterogeneous distribution of the enzyme within the membrane matrix prevents us from calculating the kinetic data of the enzymatic reaction over the entire range of parameters.     

Kinetics of adsorption and proteolytic cleavage of a multilayer ovalbumin film by subtilisin Carlsberg

Adsorption and proteolytic activity of the enzyme subtilisin Carlsberg have been studied on an immobilized, multilayer ovalbumin film. The cross-linked multilayer substrate permits protease adsorption to be examined unencumbered by the surface inhomogeneity typically observed in monolayer studies of protease surface kinetics. Decline of the protein film was measured over time using ellipsometry. Resulting kinetic data as a function of aqueous enzyme concentration and temperature were well fit by a Langmuir-Michaelis-Menten model for surface proteolysis. We observed that both the protein degradation kinetics and the in situ adsorption data were well described by the proposed model. The temperature dependence of the kinetic rate parameter yielded an activation energy of 12 kcal/mol. Further, the apparent Langmuir adsorption equilibrium constant of the enzyme at the protein/aqueous interface was 0.11 L/mg at 22 degrees C, 0.034 L/mg at 36 degrees C, and 0.011 L/mg at 50 degrees C. Although enzyme adsorption at a given aqueous enzyme concentration decreased at higher temperature, the enzyme cleaved the substrate more rapidly, leading to a net increase in the ovalbumin film degradation rate. We observed that the maximum enzyme coverage on the immobilized protein surface was approximately 40% of a close-packed monolayer at ambient temperature (22 degrees C).     

Preparation and Characterization of Polyhydroxyalkanoates Macroporous Scaffold Through Enzyme-Mediated Modifications

Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.     

Structural Effects on the Biodegradation of Aliphatic Polyesters

In advance of a discussion on structural effects on biodegradation, aliphatic polyesters as biodegradable structural materials were classified into four types regarding chemical structure, that is poly(ω-hydroxy acid), poly(β-hydroxyalkanoate), poly(ω-hydroxyalkanoate) and poly(alkylene dicarboxylate), and reviewed on synthesis route, thermal and physical properties, and biodegradability. The biodegradation mechanism of these aliphatic polyesters were discussed on the major mode of hydrolysis reaction in regard whether it was enzyme-catalyzed or not, and the substrate specificities of enzymes, such as lipases or PHA depolymerases, were discussed on the hydrolysis of the aliphatic polyesters in respect of primary structure. Moreover, the biodegradation behaviors were exceedingly influenced by solid-state morphology in addition to primary structure. The rate of enzymatic degradation of polycaprolactone fibers drawn with various draw ratios was dependent on draw ratios, suggesting that crystallinity and orientation of them affected biodegradability by lipase. In the study of enzymatic degradation of films made from butylene succinate – ethylene succinate copolymer, the dependence of degradation rate on polymeric compositions was ascribed to the degree of crystallinity rather than the primary structure. These studies revealed that the degree of crystallinity was the major rate-determining factor of biodegradation of solid polymers. © 1997 John Wiley & Sons, Ltd.     

Enzymatic determination of fenitrothion by cholinesterase and acetylcholinesterase on fluorogenic substrates

 The action of the enzymes acetylcholinesterase and cholinesterase on the substrates indoxylacetate and 2-naphthyl acetate was studied kinetically. Both enzymes convert the substrates to highly fluorescent products (3-hydroxy-indole and 2-naphthol, respectively). The kinetic curves present the initial rate as the variation of fluorescence for a unity of time (ΔF/Δt) against the substrate concentration. This enzymatic reaction was investigated in presence of the inhibitor organophosphate pesticide fenitrothion. From the kinetic curves the enzymatic parameters and enzyme and substrate concentrations used for the calibration curve can be obtained. Four simple spectrofluorimetric methods for the enzymatic determination of fenitrothion were developed showing detection limits between 5.5 to 19.5 μmol/l, depending on the enzyme and the substrate used. The precisions (R.S.D.) of the methods were between 1.6 and 9.6%. A comparative study of the kinetic enzymatic parameters and the detection limits was performed. A good correlation was obtained between inhibition constants and the detection limit. Received: 21 February 1996/Revised: 2 July 1996/Accepted: 9 July 1996