超声波在萃取中的应用

本文主要阐述了超声波强化萃取的原理－－机械作用和空化作用。综述了超声在萃取植物中酶、中草药和茶叶中特定成分、溶液中金属等所产生的影响。     

绿色木霉AS3．3711的葡聚糖内切酶V基因的克隆与表达

采用RT-PCR方法克隆到绿色木霉(Trichoderma viride)AS3．3711的葡聚糖内切酶V(EGV)的cDNA基因。测序后构建到酿酒酵母诱导型表达载体pYES2上，转化酿酒酵母，同时研究了超声波处理对酵母完整细胞转化的影响。转化子用2％的β-D-半乳糖进行诱导，用Northem杂交和CMC糖化力法分别对目的基因的转录和表达产物的葡聚糖内切酶活性进行检测。结果表明，EGV的cDNA基因开放阅读框长度为741bp，编码247个氨基酸，推测的蛋白质分子量为24．99kDa。超声波处理60s的酿酒酵母的转化率最高，在相同条件下是未处理组的2倍。酶活检测显示该基因能在酿酒酵母中表达并分泌到胞外。发酵液中的酶活在培养60小时达到最高0．046U／ml。最适酶解温度为60℃，最适pH值为5．4。     

声酶法合成生物柴油的比较研究

以玉米油、乙醇和Lipozyme TL IM脂肪酶为主要原料,研究了声酶法合成生物柴油不同因素的影响,并与静态、摇床作用下酶法合成生物柴油进行了比较,结果表明,随着乙醇量、反应温度或溶剂石油醚加入量的增大,生物柴油的产率均先增大后降低,随着酶添加量增大、反应时间的延长,生物柴油产率相应增大。适宜的反应条件为:油醇摩尔比1:1、反应温度60℃、酶添加量10%、溶剂加入量(mL)与玉米油质量(g)比为1:1、声酶法合成反应时间3h。超声波辐射显著提高了生物柴油的产率,在相同反应温度下,超声波辐射使生物柴油产率比静态条件下的产率提高了27%-33%,比摇床作用下的产率提高了6%-15%。超声波作用没有改变酶的最适反应温度。     

壳聚糖固定化L－天冬酰胺酶的制备及其性能研究

本文采用天然高分子化合物壳聚糖作为基体，制备了固定化Ｌ－天冬酰胺酶微球，研究了温度、酸碱性以及抗胰蛋白酶水解能力等条件对酶的性能影响。     

绿色木霉AS3.3711的葡聚糖内切酶Ⅴ基因的克隆与表达

采用RT PCR方法克隆到绿色木霉 (Trichodermaviride)AS3 371 1的葡聚糖内切酶Ⅴ (EGⅤ )的cDNA基因。测序后构建到酿酒酵母诱导型表达载体pYES2上 ,转化酿酒酵母 ,同时研究了超声波处理对酵母完整细胞转化的影响。转化子用 2 %的 β D 半乳糖进行诱导 ,用Northern杂交和CMC糖化力法分别对目的基因的转录和表达产物的葡聚糖内切酶活性进行检测。结果表明 ,EGⅤ的cDNA基因开放阅读框长度为 74 1bp ,编码 2 4 7个氨基酸 ,推测的蛋白质分子量为 2 4 99kDa。超声波处理 6 0s的酿酒酵母的转化率最高 ,在相同条件下是未处理组的 2倍。酶活检测显示该基因能在酿酒酵母中表达并分泌到胞外。发酵液中的酶活在培养 6 0小时达到最高 0 0 4 6U/ml。最适酶解温度为 6 0℃ ,最适pH值为 5 4。     

壳聚糖凝胶材料固定葡萄糖氧化酶制电极的研究

以壳聚糖为载体研究凝胶法固定葡萄糖氧化酶制电极。试验研究了载体壳聚糖的降解性；交联剂戊二醛的浓度、用量；电极的载酶量等固定化条件对所组建的传感器性能的影响。通过影响规律的分析、优化固定化条件的研究，找出了根据壳聚糖溶液粘度适当调整交联剂成二醛的用量和铂丝在酶膜母液中浸涂时间，克服壳聚糖的降解性对酶电极性能的影响，建立了制备性能相近的GOD传感器的方法。     

生物酶保鲜包装技术研究

酶是一种有高度催化活性的生物催化剂，它能大大降低反应的活化能。活化能越小，温度对反应速度常数的影响也就越小，所以许多由酶催化的反应在比较低的温度下，仍然能够以一定的速度进行。这也是农产品贮藏过程中许多酶促反应会发生的重要原因。     

酶解木质素改性沥青的制备及性能研究

采用酶解木质素对沥青进行改性,研究了酶解木质素及脲醛改性酶解木质素对改性沥青各项性能的影响。结果表明:添加酶解木质素及脲醛改性酶解木质素后提高了沥青的高温性能,略降低了沥青的低温性能,改善了沥青的抗老化性能;脲醛改性酶解木质素大大提高了沥青与石料之间的黏附性;酶解木质素的最佳掺量为12%。     

超声波处理对纤维素酶酶解竹粉效果影响的研究

为提高纤维素酶酶解竹粉效率,采用超声波处理竹粉。通过单因素试验分析确定超声波预处理竹粉的最优条件为:频率53kHz,功率180W,时间30min,浴比1∶10,温度30℃。用中性纤维素酶8000L酶解原竹粉和经超声波处理的竹粉,相同酶解条件下,超声波处理竹粉生成的还原糖量较原竹粉最大可提高18.05%。吸附性测试结果表明,超声波处理的竹粉对酶液的吸附性能略高于原竹粉。扫描电镜图片对比看出,经超声波处理的竹粉表层结构被破坏,整齐的纤维束结构变得杂乱松散,竹粉比表面积增加。而红外光谱测试结果表明,超声波处理并未引起竹粉官能团的明显变化。因此可初步认为超声波处理是一种温和而有效的预处理方式。     

聚对苯二胺的酶促合成及其结构性能初探

以过氧化物酶为催化剂，在有机溶剂－水混合溶剂中，合成了聚对苯二胺；研究了体系中有机溶剂的浓度对聚合产物分子量的影响，探讨了聚合产物的结构，并对其热稳定性进行了初步研究。     

Enzyme immobilization in latex dispersion coatings for active food packaging

Carboxylated styrene acrylate latex samples have been functionalized by the immobilization and entrapment of the enzyme glucose oxidase (GOx), which can be used as an oxygen scavenger in food packaging. GOx was covalently immobilized both on the surface of already formed films and on the latex particles in dispersion, as well as entrapped within the polymer matrix. In the latter two cases, polymer films were formed after the enzyme had been added to the latex dispersion. The storage stability of the enzyme and the influence of adding clay were also studied. For a given amount of enzyme, the enzyme immobilized on the film surface showed an enzyme activity about 10 times higher than that of the enzyme present within the polymer matrix. This is probably due to the diffusion limitations of the substrate in the polymer matrix. The films with the enzyme present within the polymer matrix, however, showed a higher total oxygen‐removal capacity than films with the enzyme immobilized on the surface. Entrapped enzyme showed a slightly higher activity than enzyme immobilized in the dispersion due to the negative effect of the activating chemicals used during the immobilization and on conformational constraints upon covalent bonding. Low amounts of clay added to the dispersion decreased the enzyme activity, but with higher amounts of clay the enzyme activity increased, probably because of the increased porosity and thus higher substrate accessibility. The most suitable storage condition for all the enzyme‐containing films was +8°C, which is just above the glass transition temperature of the polymer used. Copyright © 2007 John Wiley & Sons, Ltd.     

Upconversion Nanocarriers Encapsulated with Photoactivatable Ru Complexes for Near‐Infrared Light‐Regulated Enzyme Activity

Enzyme activity is important for metabolism, cell functions, and treating diseases. However, remote control of enzyme activity in deep tissue remains a challenge. This study demonstrates near‐infrared (NIR) light‐regulated enzyme activity in living cells based on upconverting nanoparticles (UCNPs) and a photoactivatable Ru complex. The Ru complex is a caged enzyme inhibitor that can be activated by blue light. To prepare a nanocarrier for NIR photoinhibition of enzyme activity, a UCNP and the caged enzyme inhibitors are encapsulated in a hollow mesoporous silica nanoparticle. In such a nanocarrier, the UCNP can harvest NIR light and convert it into blue light, which can activate the caged enzyme inhibitors. This photoactivation process is feasible in deep tissue because of the tissue penetration ability of NIR light. The nanocarrier is compatible to LNCaP, PC3, and SAOS‐2 cells, which show high enzyme expression. NIR irradiation induces release of the inhibitors and inhibition of enzyme activity in living cells. NIR light provides high spatiotemporal resolution to regulate enzyme activity in deep tissue.     

农残快速检测专用酶试剂的稳定性研究

本文报导通过添加2%正丁醇、5%蔗糖、1%动物血清白蛋白及自制高效保护剂,在保持所研制的农残检测胆碱酯酶高检测灵敏度的前提下,使该液态酶试剂的保存性得到很大提高,冷藏条件下可长期保存,室温保存15天酶活性无明显下降,37℃高温条件下放置二周仍能保持60%以上的酶活性。     

Preparation of biocatalytic nanofibres with high activity and stability via enzyme aggregate coating on polymer nanofibres

We have developed a unique approach for the fabrication of enzyme aggregate coatings on the surfaces of electrospun polymer nanofibres. This approach employs covalent attachment of seed enzymes onto nanofibres consisting of a mixture of polystyrene and poly(styrene-co-maleic anhydride), followed by a glutaraldehyde (GA) treatment that cross-links additional enzyme molecules and aggregates from the solution onto the covalently attached seed enzyme molecules. These cross-linked enzyme aggregates, covalently attached to the nanofibres via the linkers of seed enzyme molecules, are expected to improve the enzyme activity due to increased enzyme loading, and also the enzyme stability. To demonstrate the principle, we coated α-chymotrypsin (CT) on nanofibres electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The initial activity of CT-aggregate-coated nanofibres was nine times higher than nanofibres with just a layer of covalently attached CT molecules. The enzyme stability of CT-aggregate-coated nanofibres was greatly improved with essentially no measurable loss of activity over a month of observation under rigorous shaking conditions. This new approach of enzyme coating on nanofibres, yielding high activity and stability, creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, and biosensors.     

Enzyme inhibitor screening by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.     

Thionine covalently tethered to multilayer horseradish peroxidase in a self-assembled monolayer as an electron-transfer mediator

A new approach to construct a reagentless enzyme biosensor is described. Based on multilayer horseradish peroxidase in a self-assembled monolayer configuration, the biosensor was constructed using multilayer thionine covalently tethered to the enzyme as an electron-transfer mediator. The multilayer enzyme and the multilayer mediator were stepwisely synthesized onto an l-cysteine-assembled gold electrode using glutaraldehyde as a bifunctional reagent. The multilayer mediator tethered to the multilayer enzyme could effectively and stably shuttle electrons between the electrode and the multilayer enzyme linked onto the monolayer. The sensitivity of the resulting enzyme biosensor with eight layers of enzyme and three layers of mediator was more than 250 μA cm(-)(2) for 1.0 × 10(-)(4) mol/L hydrogen peroxide under optimal conditions, whereas such a modified electrode with one layer of enzyme and one layer of mediator did not yield a detectable response to 1.0 × 10(-)(4) mol/L hydrogen peroxide.     

A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.     

Potentiometric and voltammetric investigations of H2/H+ catalysis by periplasmic hydrogenase from Desulfovibrio gigas immobilized at the electrode surface in an amphiphilic bilayer assembly.

Interactions of an enzyme with an organized amphipilic bilayer are explored as a general means of enzyme immobilization in electroenzymatic systems. Immobilization of Desulfovibrio gigas hydrogenase at the electrode surface involves hydrophobic interactions of the enzyme with the bilayer assembly consisting of octadecyltrichlorosilane and octadecylviologen (C18MV2+) molecules. Due to a hydrophobic character of the enzyme, these interactions direct the enzyme to occupy a central position in the bilayer's hydrocarbon region and lead to immobilization of 3 pmol/cm2 of the enzyme in the plane of the bilayer. This corresponds to 50% surface coverage. The immobilized enzyme catalyzes H2 oxidation mediated by the C18MW2+/.+ couple. This electroenzymatic scheme functions under steady-state voltammetric as well as potentiometric conditions in the pH range 3.5-10. Coupling of enzymatic activity to the electrode surface is accomplished via lateral diffusion of the octadecylviologen molecules along the bilayer assembly.     

T4 DNA连接酶酶活力测定方法

工具酶的酶活力检测是工具酶产品质量控制的关键,研究和建立工具酶酶活力的测定方法是工具酶产品标准化制定的重点和难点。该文采用改进的粘性末端单位测定T4 DNA连接酶酶活力。经验证,方法重复性好,稳定性佳,为建立T4 DNA连接酶质量检测标准奠定了基础。     

Enzyme immobilization in porous silicon: quantitative analysis of the kinetic parameters for glutathione-S-transferases

Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (approximately 6.5 microm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is approximately 4x higher than the immobilized enzyme, for which we measured an apparent K'(m)(GSH) value of 1.0 +/- 0.3. The maximum velocity, V'(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is approximately 20 times lower than that in solution. Results suggest approximately 25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.