融合酶的设计和应用研究进展

酶的分子改造和重新设计是解决酶催化工业应用瓶颈的重要途径。基于融合蛋白设计的融合酶技术是分子酶工程的一个研究热点,已逐渐应用于多功能酶和酶靠近效应的构建与控制研究中,显示出重要的理论和应用研究价值。文中对近年来融合酶的分子设计策略和应用研究的进展进行了综述。首先介绍了融合酶的概念和特点,并对最近研究中出现的融合酶构建策略进行了归纳总结,重点阐述了不同种类连接肽对融合酶的影响及其可能机理。同时,对目前融合酶的应用研究进行了归纳和讨论。最后,结合本实验室的研究,指出了融合酶领域的关键问题并对其发展方向进行了探讨和展望。     

Deciphering the language of cells

Long distance cell-to-cell or organism-to-organism communications may be accomplished by transmission and reception of electromagnetic signals through membrane receptors or enzymes. Consistent with this idea is the observation that membrane ATPases are capable of absorbing energy from oscillating electric fields of defined frequency and amplitude and using it to perform chemical work. The concept of the 'electroconformational coupling' is used to explain how an electric signal can modulate the activity of a membrane protein, and conversely, how an energy-dissipating reaction can produce an electric signal.     

New algorithms and an in silico benchmark for computational enzyme design

The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design.     

Non-chromogenic peptide substrates for detection of enzymes by means of capacitance changes at metal electrodes

A new type of substrate for enzyme detection has been developed. The substrate is non-chromogenic and is used in an assay method based on electrode adsorption. The rate of change in the electric capacitance of the electrode is monitored and taken as a measure of the substrate adsorption. Substrate adsorption is in turn proportional to substrate bulk concentration and thus subject to changes by enzymes. The new substrate introduces a new concept in enzyme detection: as it is non-chromogenic it may contain appropriate amino acids on both sides of the bond subject to enzymatic cleavage.     

Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering

Measurement of enzyme activity and selectivity at in vivo concentrations is highly desirable in a range of fields including diagnostics, functional proteomics and directed evolution. Here we demonstrate how surface-enhanced resonance Raman scattering (SERRS), measured using silver nanoparticles, can be used to detect the activity of hydrolases at ultra-low levels. This approach was made possible by designing 'masked' enzyme substrates that are initially completely undetected by SERRS. Turnover of the substrate by the enzyme leads to the release of a surface targeting dye, and intense SERRS signals proportional to enzyme activity are generated. The method was used to rapidly screen the relative activities and enantioselectivities of fourteen enzymes including examples of lipases, esterases and proteases. In the current format the sensitivity of the technique is sufficient to detect 500 enzyme molecules, which offers the potential to detect multiple enzyme activities simultaneously and at levels found within single cells.     

Do electromagnetic fields interact with electrons in the Na,K‐ATPase?

The effects of low frequency electric and magnetic fields on several biochemical systems, including the Na,K-ATPase, indicate that electromagnetic (EM) fields interact with electrons. The frequency optima for two enzymes in response to EM fields are very close to their turnover numbers, suggesting that these interactions directly affect reaction rates. Nevertheless, generally accepted ideas about Na,K-ATPase function and ion transport mechanisms do not consider interactions with electrons. To resolve the clash of paradigms, we hypothesize interaction with transient electrons and protons that arise from flickering of H-bonds in the hydrated protein. These transient charges in the enzyme could provide a trigger for the sequence of conformation changes that are part of the ion transport mechanism. If the distributions of transient electrons and protons in the membrane are affected by their concentration and the membrane potential, as expected from electric double layer theory, this can account for the different effects of low frequency electric and magnetic fields, as well as for the observation that membrane hyperpolarization reverses the ATPase reaction to generate ATP.     

Size-modulated synergy of cellulase clustering for enhanced cellulose hydrolysis

Immobilization of enzymes onto nanoparticles for enhanced biocatalytic activity via enzyme clustering is a growing field. In this paper, the effect of nanoparticle size on the hydrolytic activity of artificial cellulosomes was investigated. A simple method based on metal affinity coordination was employed to directly conjugate two enzymes, an endoglucanase CelA and an exoglucanase CelE, onto CdSe–ZnS core–shell quantum dots (QDs) without the use of any chemical modification or linker molecules such as streptavidin. Artificial cellulosomes were created by clustering the enzymes onto two different QDs (5 and 10 nm) to systematically study the influence of particle size and QD to enzyme ratio on the enhancement in cellulose hydrolysis. Our results indicate that enzyme proximity is the most important factor for activity enhancement while the influence of particle size is relatively modest. This detailed understanding will provide insights for the design of other artificial cellulosomes based on nanoclustering of multiple catalytic domains with significantly enhanced activities, and may be applicable for designing improved nanobiocatalysts for biofuel production, bioremediation, and drug design.     

Understanding nature's catalytic toolkit

Enzymes catalyse numerous reactions in nature, often causing spectacular accelerations in the catalysis rate. One aspect of understanding how enzymes achieve these feats is to explore how they use the limited set of residue side chains that form their 'catalytic toolkit'. Combinations of different residues form 'catalytic units' that are found repeatedly in different unrelated enzymes. Most catalytic units facilitate rapid catalysis in the enzyme active site either by providing charged groups to polarize substrates and to stabilize transition states, or by modifying the pKa values of other residues to provide more effective acids and bases. Given recent efforts to design novel enzymes, the rise of structural genomics and subsequent efforts to predict the function of enzymes from their structure, these units provide a simple framework to describe how nature uses the tools at her disposal, and might help to improve techniques for designing and predicting enzyme function.     

工业蛋白质理性设计与应用

作为合成生物学与绿色生物制造等领域的底层核心技术,蛋白理性设计可有效解决天然功能元件性能不足等共性挑战,创制高性能人工酶元件。值此天津工业生物研究所(Tianjin Institute of Industrial Biotechnology, TIB)创立10周年之际,文中回顾了研究所在工业蛋白理性设计领域的系列重要工作进展。从酶设计方法学研究、新酶反应设计到生物催化应用等方面进行了分析讨论,并展望了本领域未来发展方向。望借此搭建学术界和产业界与酶理性设计的桥梁,促进新技术、新策略的开发应用,加速融合人工酶的基础研究与产业应用,推动我国生物制造领域的科技创新升级。     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

Constraint matching as a means of designing biochemical experiments in multi-enzyme systems

A method of qualitative analysis by constraint matching, where expectations derived from theory or from data bases are systematically compared against experimental findings, is described. This was originally developed as an artificial intelligence technique to analyze enzyme kinetic mechanism determinations. It is shown to have been used (without computer involvement) in designing experiments involving a few enzymes, and is suggested as a useful experimental design tool. The experiments in question validate the behavior of insulinoma extracts as models for pancreatic islet glycolysis, which conform to the expectations from the relevant enzyme literature. Possible generalization to other areas of biological research is suggested.     

Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes

Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.     

Resonance electroconformational coupling: A proposed mechanism for energy and signal transductions by membrane proteins

Recent experiments show that membrane ATPases are capable of absorbing free energy from an applied oscillating electric field and converting it to chemical bond energy of ATP or chemical potential energy of concentration gradients. Presumably these enzymes would also respond to endogenous transmembrane electric fields of similar intensity and waveform. A mechanism is proposed in which energy coupling is achieved via Coulombic interaction of an electric field and the conformational equilibria of an ATPase. Analysis indicates that only an oscillating or fluctuating electric field can be used by an enzyme to drive a chemical reaction away from equilibrium.In vivo, the stationary transmembrane potential of a cell must be modulated to become locally oscillatory if it is to derive energy and signal transduction processes.     

Tools and strategies for constructing cell-free enzyme pathways

Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.     

Biocatalysts: application and engineering for industrial purposes

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.     

Electromagnetic homeostasis and the role of low-amplitude electromagnetic fields on life organization

The appearance of endogenous electromagnetic fields in biological systems is a widely debated issue in modern science. The electrophysiological fields have very tiny intensities and it can be inferred that they are rapidly decreasing with the distance from the generating structure, vanishing at very short distances. This makes very hard their detection using standard experimental methods. However, the existence of fast-moving charged particles in the macromolecules inside both intracellular and extracellular fluids may envisage the generation of localized electric currents as well as the presence of closed loops, which implies the existence of magnetic fields. Moreover, the whole set of oscillatory frequencies of various substances, enzymes, cell membranes, nucleic acids, bioelectrical phenomena generated by the electrical rhythm of coherent groups of cells, cell-to-cell communication among population of host bacteria, forms the increasingly complex hierarchies of electromagnetic signals of different frequencies which cover the living being and represent a fundamental information network controlling the cell metabolism. From this approach emerges the concept of electromagnetic homeostasis: that is, the capability of the human body to maintain the balance of highly complex electromagnetic interactions within, in spite of the external electromagnetic noisy environment. This concept may have an important impact on the actual definitions of heal and disease.     

酶分子设计常用策略和进展

酶分子的生物学功能很大程度上是由其三维空间结构和所处溶剂环境共同决定的。因此,优化酶分子的结构性质以及探索其性质最优的溶剂环境是改善酶分子功能以及进行理性设计的一个可行途径。从实际应用的角度来看,分子设计方法可以为酶工程提供一种有效的解决方案。目前,酶分子设计有两个重要的研究方向,包括提高酶分子的催化活力和优化其稳定性。同时,对酶分子设计方法的研究也有助于对蛋白质生物学机理的探索。在近些年的学术界酶分子设计案例中,生物信息学方法得到广泛的应用。本文系统地总结基于生物信息学的酶分子设计方法的背景、策略和一些经典案例。     

Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree

We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.     

酶工程专刊序言

酶工程是酶学与工程科学融合的综合性科学技术,是现代生物技术的支柱之一。近年来我国在酶工程研究方面取得了较大进步,为促进国内酶工程研究的发展,本期"酶工程专刊"集中展现了我国酶工程专家学者在酶促生物转化、医药用酶、饲料用酶、环境修复用酶和生物能源用酶等领域所取得的最新进展。     

Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.