Enzymes immobilized in mesoporous silica: A physical–chemical perspective

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.     

Cold-Active Enzymes and Their Potential Industrial Applications—A Review

More than 70% of our planet is covered by extremely cold environments, nourishing a broad diversity of microbial life. Temperature is the most significant parameter that plays a key role in the distribution of microorganisms on our planet. Psychrophilic microorganisms are the most prominent inhabitants of the cold ecosystems, and they possess potential cold-active enzymes with diverse uses in the research and commercial sectors. Psychrophiles are modified to nurture, replicate, and retain their active metabolic activities in low temperatures. Their enzymes possess characteristics of maximal activity at low to adequate temperatures; this feature makes them more appealing and attractive in biotechnology. The high enzymatic activity of psychrozymes at low temperatures implies an important feature for energy saving. These enzymes have proven more advantageous than their mesophilic and thermophilic counterparts. Therefore, it is very important to explore the efficiency and utility of different psychrozymes in food processing, pharmaceuticals, brewing, bioremediation, and molecular biology. In this review, we focused on the properties of cold-active enzymes and their diverse uses in different industries and research areas. This review will provide insight into the areas and characteristics to be improved in cold-active enzymes so that potential and desired enzymes can be made available for commercial purposes.     

Taking Advantage of Hydrophobic Fluorine Interactions for Self‐Assembled Quantum Dots as a Delivery Platform for Enzymes

Self‐assembly of nanoparticles provides unique opportunities as nanoplatforms for controlled delivery. By exploiting the important role of noncovalent hydrophobic interactions in the engineering of stable assemblies, nanoassemblies were formed by the self‐assembly of fluorinated quantum dots in aqueous medium through fluorine–fluorine interactions. These nanoassemblies encapsulated different enzymes (laccase and α‐galactosidase) with encapsulation efficiencies of ≥74 %. Importantly, the encapsulated enzymes maintained their catalytic activity, following Michaelis–Menten kinetics. Under an acidic environment the nanoassemblies were slowly disassembled, thus allowing the release of encapsulated enzymes. The effective release of the assayed enzymes demonstrated the feasibility of this nanoplatform to be used in pH‐mediated enzyme delivery. In addition, the as‐synthesized nanoassemblies, having a diameter of about 50 nm, presented high colloidal stability and fluorescence emission, which make them a promising multifunctional nanoplatform.     

INACTIVATION OF CITRIC ACID CYCLE ENZYMES AS A RESULT OF PHOTODYNAMIC SENSITIZATION BY MITOCHONDRIAL INNER MEMBRANE

Abstract— Exposure to blue light of mitochondria under aerobic conditions resulted in inactivation of the regulatory enzymes of the citric acid cycle (CAC) contained in the mitochondrial matrix, except citrate synthase. When "soluble mitochondrial protein" was exposed to blue light under aerobic conditions, no significant loss of activity was observed for any CAC enzymes. However, the inclusion of submitochondria particles (SMP) in the photolysis system resulted in a substantial inactivation of the CAC enzymes. Of the CAC enzymes, NAD+-specific isocitrate dehydrogenase (ICDH) appeared to be most susceptible to the membrane dependent-photoinactivation. Imidazole protected the CAC enzymes against inactivation. In contrast, superoxide dismutase failed to protect them, except α-ketoglutarate dehydrogenase. The photoinhibition of ICDH activity was drastically depressed in the presence of SMP whose Fe-S centers were destroyed by the mersalyl acid treatment. The results obtained in this study suggest that the photoinactivation of the CAC enzymes in situ is mediated mainly by singlet oxygen, which is photoproduced primarily by the Fe-S centers of mitochondrial membranes.     

纳米通道内酶组装及其催化反应研究进展

研究酶的组装和催化反应不仅有利于探索生命活动的本质,同时对开发酶在工业合成、分析检测、疾病治疗等领域的实际应用价值具有重要的指导意义. 研究发现,酶的有效固定和有序组装是保持酶活性、酶促反应的稳定性和对酶催化过程进行控制的重要途径,而在纳米通道内进行单酶或多酶的有序组装,利用纳米通道的限域效应可有效保持酶的构型进而提高酶催化反应的选择性和催化效率,增强酶级联反应的动力学进程. 本文概述了近年来基于纳米通道的酶反应器在生物传感领域的研究进展,着重描述纳米通道限域空腔内酶的组装方法、酶催化反应及其动力学机制,并展望了基于纳米通道的酶反应器的应用前景.     

Enzyme mimics based upon supramolecular coordination chemistry

Recent advances in supramolecular coordination chemistry have allowed chemists to synthesize macromolecular complexes that exhibit various properties intrinsic to enzymes. This Review focuses on structures inspired by properties and functions observed in enzymes rather than precise models of enzyme active sites. These structures are synthesized using convergent, modular, and high-yielding coordination-chemistry-based methods, which allow one to tailor the size, shape, and properties of the resulting complexes. Many of the structures discussed exhibit reactivity and specificity reminiscent of natural systems, and some of them have functions that exceed the natural systems which provided the inspiration for initially making them.     

High-resolution X-ray structure of an acyl-enzyme species for the class D OXA-10 beta-lactamase

Beta-lactamases are resistance enzymes for beta-lactam antibiotics. These enzymes hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. Of the four classes of known beta-lactamases, the enzymes of class D are the least understood. We report herein the high-resolution (1.9 A) crystal structure of the class D OXA-10 beta-lactamase inhibited by a penicillanate derivative. The structure provides evidence that the carboxylated Lys-70 (a carbamate) is intimately involved in the mechanism of the enzyme.     

Dicarbonyl reduction by single enzyme for the preparation of chiral diols

Chiral diols are a group of key building blocks useful for preparing a variety of important chiral chemicals. While the preparation of optically pure diols is generally not an easy task in synthetic organic chemistry, three classes of enzymes, namely dicarbonyl reductase, dioxygenase and epoxide hydrolase, display remarkable ability to stereoselectively introduce two hydroxyl groups in a single-step enzymatic conversion. In this tutorial review, we pay special attention to dicarbonyl reductases that directly produce chiral diols through the bio-reduction of two carbonyl groups. The dicarbonyl reductases include diketoreductase, α-acetoxy ketone reductase and sepiapterin reductase. We present these exceptional enzymes in the context of source and properties, structure and catalytic mechanism as well as biocatalytic application. In addition to the broad substrate specificity, the excellent stereoselectivity and high catalytic efficiency of these enzymes have positioned them as valuable biocatalysts. With more sophisticated understanding of the structure-function relationship, the practical utilities of these enzymes associated with their interesting chemistry will be considerably appreciated over time. Moreover, rational redesign and molecular evolution of these unusual biocatalysts will truly enable their broader applications in the synthesis of chiral diols in the future.     

CALCIUM-REGULATED NUCLEAR ENZYMES: POTENTIAL MEDIATORS OF PHYTOCHROME-INDUCED CHANGES IN NUCLEAR METABOLISM?

Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.     

How critical is the use of commercially available enzymes for selenium speciation?

The aim of this work was to check whether commercially available enzymes are pure enough to be used for selenium speciation analysis and the contribution that impurities could make to Se determination in real samples. For this purpose, twelve commercially available enzymes with different origins and classifications (protease, amylase, cellulase, lipase) were analysed. After the dissolution of the enzyme in water, the Se species were separated by ion exchange chromatography, with inductively coupled plasma mass spectrometry used as the detection system. The results showed that the Se content was significant in several cases. The highest value was obtained for β-amylase from barley, 3100 ng Se per g of enzyme. Speciation analysis showed that Se-methionine, selenite, selenate and some unknown compounds were present in several enzymes. In general, the Se species identified represented a small fraction of the total Se. For instance, only 17% of the total Se was determined for β-amylase from barley. On the other hand, about 100% of the total Se was identified in protease from Streptomyces griseus. Upon comparing the results from different lots of the same enzyme, not all of them were found to be comparable. Thus, the presence of selenium species in commercially available enzymes could be due to the preparation procedure used for the enzyme; they could be present as degradation products. Therefore, when determining selenium species in samples with low Se contents, attention should be paid to enzyme purity in relation to selenium compounds when an enzyme is used for hydrolysis.     

二氧化硅纳米与微米颗粒作为固定化酶载体的生物效应

分别将二氧化硅纳米颗粒(SiNPs)与微米颗粒(SiMPs)作为固定化载体, 选择多聚酶牛肝过氧化氢酶(CAT)和单体酶辣根过氧化物酶(HRP)作为酶模型, 通过考察酶固定化后在酶活回收率、热稳定性、 酶促反应最适温度以及酶在水-有机溶剂混合体系中催化能力的变化, 对载体与酶所产生的生物效应差异进行了系统研究. 酶活回收率结果表明, SiNPs显示出比SiMPs优越的对酶无选择性的高生物亲和性, 而SiMPs则能使固定于其上的酶热稳定性大幅度提高, 且二者都能使固定化酶在有机相中的稳定性得到明显增强. 但酶促反应最适温度的变化结果表明, 对不同类型的酶所产生的生物效应则表现出无规律性.     

An efficient synthesis of cytidine monophospho-sialic acids with four immobilized enzymes.

Four CMP-sialic acids, among them the new derivative CMP-KDN, have been prepared straightforward from CMP, phosphoenolpyruvate and sialic acid by using four immobilized enzymes.     

镧对小鼠肝脏中几种酶活性的影响

分别从体内作用和体外作用两个方面研究了镧对鼠肝脏中的胆碱脂酶、异柠檬酸脱氢酶、谷丙转氨酶活性的影响 ,同时用电感耦合等离子质谱 (ICP -MS)研究了镧在肝脏中的累积情况 .结果表明 :经尾静脉注射后镧在肝脏中有明显的累积现象 ,其累积量随给药剂量的增大而增加 ;镧对上述三种酶的活性均表现出低剂量激活 ,高剂量抑制的Hormesis效应     

Biocatalytic Strategies towards [4+2] Cycloadditions

Long sought after [4+2] cyclases have sprouted up in numerous biosynthetic pathways in recent years, raising hopes for biocatalytic solutions to cycloaddition catalysis, an important problem in chemical synthesis. In a few cases, detailed pictures of the inner workings of these catalysts have emerged, but intense efforts to gain deeper understanding are underway by means of crystallography and computational modelling. This Minireview aims to shed light on the catalytic strategies that this highly diverse family of enzymes employs to accelerate and direct the course of [4+2] cycloadditions with reference to small-molecule catalysts and designer enzymes. These catalytic strategies include oxidative or reductive triggers and lid-like movements of enzyme domains. A precise understanding of natural cycloaddition catalysts will be instrumental for customizing them for various synthetic applications.     

Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Deubiquitinating enzymes are IN/(trinsic to proteasome function)

Covalent conjugation of the ubiquitin tag to cellular proteins plays a central role in a number of processes, the most notable among them being degradation by the 26S proteasome. A fundamental property of this process is that ubiquitination, in contrast to subsequent degradation, is reversible due to a number of deubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates. The uniqueness of ubiquitin as a reversible tag necessitates mechanisms to guarantee its efficiency. Interestingly, some deubiquitinating enzymes are associated with the 26S proteasome itself. We include a brief overview of the key proteasome-associated deubiquitinating enzymes such as Rpn11/POH1, UCH37/Uch2, Ubp6/Usp14 and Doa4/Ubp4. We go on to discuss how these enzymes may contribute to, or possibly counteract, proteolysis by the proteasome. For example, cumulative evidence points to a partitioning of proteasome action between proteolysis and deubiquitination. On the one hand, inhibition of proteolysis promotes deubiquitination, while on the other hand, inhibition of deubiquitination can promote proteolysis. The plethora of deubiquitinating enzymes may serve as proof reading devices altering the equilibrium between these two processes and allowing for reversal of fortune at various stages of the process. To promote degradation over deubiquitination, certain polyubiquitin conformations could be stabilized or protected from deubiquitinating enzymes in order that they can serve as efficient targeting signals leading to the proteasome. We hypothesize that polvubiquitin chains could also serve as "timers": by slowing down chain disassembly, longer chains allow ample time for unfolding and proteolysis of the substrate.     

Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺-dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

On-line-Bestimmung von Enzymen in der Bioproze?analytik unter Berücksichtigung von Probenahme und Flie?injektionstechnik

Summary The on-line determination of enzymes in biotechnical processes becomes an important factor with regard to process development and optimization. At present, most commonly enzymes are determined off-line in the laboratory after withdrawal of a separate sample. Wet chemical methods dominate in this respect, mainly because enzymes have to be measured according to the reaction schemes which are catalyzed by them. For an efficient process monitoring and control the time delay, the limited reliability and the man power needed for analysis of a large number of samples are crucial points. By using the technique of flow injection analysis (FIA) it should in general become easy to develop automatically operated enzyme determination procedures based on reaction schemes which can be used for fast and efficient process monitoring, providing the problems with the coupling of the analyzer at the bioreactor are solved. Continuous sampling in this respect plays a key role in developing on-line measuring techniques. This paper reviews the current status of on-line enzyme analysis, using flow-injection techniques. It is shown that the coupling problems can be solved by using a newly developed sampling module, which is based on membrane filtration. Some examples of on-line enzyme determinations in fermentation as well as in downstream processing illustrate the ease and reliability of the proposed concept for using FIA in connection with membrane separation.     

Towards practical biocatalytic Baeyer-Villiger reactions: applying a thermostable enzyme in the gram-scale synthesis of optically-active lactones in a two-liquid-phase system

Baeyer-Villiger monooxygenases (BVMOs) are extremely promising catalysts useful for enantioselective oxidation reactions of ketones, but organic chemists have not used them widely due to several reasons. These include instability of the enzymes in the case of in vitro and even in vivo systems, reactant/product inhibition, problems with upscaling and the necessity of using specialized equipment. The present study shows that the thermally stable phenylacetone monooxygenase (PAMO) and recently engineered mutants can be used as a practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions. For this purpose several parameters such as buffer composition, the nature of the solvent system and the co-factor regeneration system were optimized. Overall a fairly versatile and efficient catalytic system for enantioselective laboratory scale BV-oxidations of ketones was developed, which can easily be applied even by those organic chemists who are not well versed in the use of enzymes.     

General Ligand Affinity Chromatography in Enzyme Purification Ligands,Affinity Chromatography,Enzyme Purification

Recent developments in enzyme purification have revolutionized isolation techniques and at the same time provided a more profitable manner in which to obtain them. These developments are primarily concerned with the preparation and usability of imobilized ligands for the selective isolation and purification of biologically active substance^1–5, particularly enzymes. This technique is based on the unique principle that the biological macromolecule is able to be adsorbed to the immobilized ligand, specifically and reversibly, unlike the traditional methods by which proteins are separated by their dffferences in physicochemical properties. Numerous proteins have been purified since this technique was first applied by Campbell et al.⁶ for the purification and isolation of antibodies. From these preliminary efforts it is apparent that further development of thfs method would greatly facilitate our understanding and utilizattion of enzymes.