Protein engineering and de novo designing of a biocatalyst

Proteins as a biomolecule have been recognized as a “molecule with manifold biological functions”. The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by‐products and their non‐hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf‐life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter‐relationships. Copyright © 2016 John Wiley & Sons, Ltd.     

Enzymes with noncanonical amino acids

Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.     

Biocatalysis for pharmaceutical intermediates: the future is now

Biocatalysis is continuing to gain momentum and is now becoming a key component in the toolbox of the process chemist, with a place alongside chemocatalysis and chromatographic separations. The pharmaceutical industry demands a speed of development that must be on a parallel with conventional chemistry and high optical purity for complex compounds with multiple chiral centres. This review describes how these demands are being addressed to make biocatalysis successful, particularly by the use of micro-scale technology for high-speed catalyst screening and process development alongside discipline integration of biology and engineering with chemistry. Developments in recombinant technology will further expand the repertoire of biocatalysis in the coming years to new chemistries and enable catalyst design to fit the process. Further development of biocatalysis for green chemistry and high productivity processes can also be expected.     

Tools for Selective Enzyme Reaction Steps in the Synthesis of Laboratory Chemicals

The need for more selective reactions steps and the compatibility between process steps which follow on from each other has been a major driving force for organic synthesis. The synthesis of chiral compounds, metabolites, new chemical entities and natural products by a combination of chemical and enzyme reaction steps has become well established, due the existence of stable enzymes as selective catalysts which are inherently chiral by nature. Auxiliary tools such as suitable transfer reagents for reaching complete conversion, easy and robust reaction control as well as tools for straightforward workup and purification of the final product have been developed. Selective enzyme reaction steps in the area of hydrolyses, oxidation steps including hydroxylation and the Baeyer‐Villiger oxidation, carbon‐carbon bond formation and glycosylation reactions have compared favorably with existing methods of classical organic synthesis. The tools developed during optimization and scale‐up of these enzyme reaction steps have the potential to shorten development time. The introduction of selective enzyme reactions into an entire synthetic process has resulted in harmonization of improvements in economic efficiency with resultant solutions to health, safety and environment problems. This will become even more important in industrial synthetic chemistry in the future, for convenient solutions to certain intractable synthetic problems and for expanding the repertoire of chemistry by modular biocatalysts. Efficient isolation procedures for the final product are essential to take full advantage of the biocatalytic conversion to obtain high product yields.     

Substrate channeling and enzyme complexes for biotechnological applications

Substrate channeling is a process of transferring the product of one enzyme to an adjacent cascade enzyme or cell without complete mixing with the bulk phase. Such phenomena can occur in vivo, in vitro, or ex vivo. Enzyme–enzyme or enzyme–cell complexes may be static or transient. In addition to enhanced reaction rates through substrate channeling in complexes, numerous potential benefits of such complexes are protection of unstable substrates, circumvention of unfavorable equilibrium and kinetics imposed, forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of toxic metabolite inhibition, and so on. Here we review numerous examples of natural and synthetic complexes featuring substrate channeling. Constructing synthetic in vivo, in vitro or ex vivo complexes for substrate channeling would have great biotechnological potentials in metabolic engineering, multi-enzyme-mediated biocatalysis, and cell-free synthetic pathway biotransformation (SyPaB).     

New opportunities for biocatalysis: driving the synthesis of chiral chemicals

Various biocatalytic methods have been developed for the synthesis of chiral chemicals, which have made their synthesis more environmentally friendly and product-specific. New opportunities for biocatalysis, including new scientific developments in genomics and protein engineering technologies, novel process developments and the increased availability of useful enzymes, offer many possibilities for the manufacture of new chiral compounds and deliver greener and economically competitive processes. In this review, new opportunities for biocatalysis in the preparation of chiral molecules are outlined and highlighted.     

Advancing biocatalysis through enzyme, cellular, and platform engineering

Biocatalysis offers opportunities for highly selective chemical reactions with high turnover rates under relatively mild conditions. Use of whole-cell or multi-enzyme systems enables transformations of complexity unmatched by nonbiological routes. However, advantages of biocatalysis are frequently compromised by poor enzymatic performance under non-native reaction conditions, the absence of enzymes with desired substrate or reaction specificities, and low metabolic fluxes or competing pathways. During the 234th National Meeting of the American Chemical Society, these issues were addressed in the "Advances in Biocatalysis" sessions. Protein engineering and metabolic pathway engineering were used to develop efficient enzymes and whole-cell catalysts. Novel strategies for the use of enzymes at solid interfaces and in nonaqueous environments were discussed, and efficient biotransformation platforms were demonstrated. These advances broaden the applications of biocatalysis in biofuels, pharmaceuticals, fine chemicals, and human health.     

Entrapment of a Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl₂, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry.     

Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

Surface charge engineering has received considerable interest from the scientific and industrial community in the last few decades. Although it was previously hypothesized that the surface charge–charge interactions were not a fundamental force to determine protein folding and stability, many studies today show that surface charge plays a key role determining protein structure and activity. This review aims to (a) highlight the value of surface charged engineering of proteins to improve enzyme stability and activity in aqueous media and in the presence of ionic liquids (ILs) and organic solvents, (b) describe the existing approaches (genetic engineering or chemical modifications) for surface charged engineering, and (c) demonstrate the applicability of these surface charged enzymes in biocatalysis. The review provides a new foundation for the scientific and research community to exploit the surface engineering of protein concept for the development of new enzymes that are more active and stable in the presence of ILs and organic solvents, thereby offering new opportunities for industrial biocatalysis. Furthermore, this review is a useful tool for researchers to decide the best available technology to improve their enzyme system/process.     

Extremozymes

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.     

A window into biocatalysis and biotransformations

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium.     

From Enzyme Kinetics to Metabolic Network Modeling – Visualization Tool for Enhanced Kinetic Analysis of Biochemical Network Models

Model‐based analysis of enzyme kinetics allows the determination of optimal conditions for their use in biocatalysis. For biotransformations or fermentative approaches the modeling of metabolic pathways or complex metabolic networks is necessary to obtain model‐based predictions of steps which limit product formation within the network. To set up adequate kinetic models, relevant mechanistic information about enzyme properties is required and can be taken from in vitro studies with isolated enzymes or from in vivo investigations using stimulus‐response experiments which provide a lot of kinetic information about the metabolic network. But with increasing number of reaction steps and regulatory interdependencies in the network structure the amount of simulation data dramatically increases and the simulation results from the dynamic models become difficult to analyze and interpret. Demonstrated for an Escherichia coli model of the central carbon metabolism, methods for visualization and animation of simulation data were applied and extended to facilitate model analysis and biological interpretation. The dynamic metabolite pool and metabolic flux changes were visualized simultaneously by a software tool. In addition, a new quantification method for enzyme activation/inhibition was proposed, and this information was implemented in the metabolic visualization.     

工业生物催化技术

以蛋白质酶的工程应用为核心的工业生物催化技术,被认为是生物技术继生物医药和转基因植物之后的第三次浪潮。它的发展与应用将对人类的工业化学过程带来根本的变革。工业生物催化的兴起与以下的两个关键技术因素有密切的关系：(1)蛋白质定向进化技术的出现,(2)基因组学和蛋白质组学的发展。探讨了工业生物催化技术的现状和发展趋势,并对我国如何发展该领域的基础和应用研究提出一些见解。     

Levan and levansucrases: Polymer,enzyme, micro-organisms and biomedical applications

Abstract

Nowadays, levan synthesis and its biocatalysis is a step claiming attention due to its promising uses in biomedical field. Levansucrase, as the enzyme responsible of levan biocatalysis is studied in this work concerning sequence homology, signal peptide, metal binding, etc. More than 650 different levansucrase sequences were studied and many interesting results were obtained such as phylogenetic trees and conserved residues that could be essential for biocatalysis machinery. Moreover, a review of levan applications in biomedicine and health field was made. It is showed the advances in applications such as a film agent, a carrier for drug delivery systems, an anti-inflammatory compound or its potential use for functional food as prebiotic.     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Improving the production,activity, and stability of CLEAs with diepoxides

Among enzyme immobilization techniques, the preparation of cross‐linked enzyme aggregates has shown promising results in biocatalysis, because they are easy to prepare, versatile, and cheap. The method involves the precipitation of enzymes with ammonium sulfate or an organic solvent and subsequent cross‐linking with glutaraldehyde. However, the Schiff base produced with glutaraldehyde is reversible and can be broken with acids or bases, releasing proteins to the reaction medium. To solve this problem, we propose replacing glutaraldehyde with diepoxide compounds to obtain an irreversible secondary amine bond. Such a substitution avoids protein leakage during the biocatalytic process, contamination of the final products, and loss of enzyme. It also improves the synthesis of the biocatalyst, because, while the Schiff base is favored at mildly acidic pH, the epoxide reaction can be made at the optimal enzyme pH, assuring its structural stability and catalytic performance. The proposed method has been successfully used in the production and optimization of aldolase epoxy‐cross‐linked aggregates, which retain 98% activity. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1425–1429, 2017     

生物信息学在工业生物催化研究中的应用

随着石油等不可再生资源的日益减少以及环境污染问题的日益严重,应用工业生物催化技术改造或取代传统化工工艺已经成为新世纪化学工业可持续发展的研究热点。工业生物催化技术的研究对象是生物催化剂及其催化过程。近来,利用生物信息学技术进行工业生物催化研究已经越来越受到人们的重视。随着工业生物催化的发展,生物信息学将直接指导并加快新型高效生物催化剂的发现及功能改造进程。     

A Database to Facilitate a Process‐Oriented Approach to Urban Metabolism

In view of urbanization trends coupled with climate‐change challenges, it is increasingly important to establish less‐harmful means of urban living. To date, urban metabolism (UM) studies have quantified the aggregate material and energy flows into and out of cities and, further, have identified how consumer activity causes these flows. However, little attention has been paid to the networks of conversion processes that link consumer end‐use demands to aggregate metabolic flows. Here, we conduct a systematic literature search to assemble a database of 202 urban energy, water, and waste management processes. We show how the database can help planners and policy makers choose the preferred process to meet a specific resource management need; identify synergies between energy, water, and waste management processes; and compute optimal networks of processes to meet an area's consumer demand at minimum environmental cost. We make our database publicly available under an open‐source license and discuss the possibilities for how it might be used alongside other industrial ecology data sets to enhance research opportunities. This will encourage more holistic UM analyses, which appreciate how both consumer activity and the engineered urban system work together to influence aggregate metabolic flows and thus support efforts to make cities more sustainable.     

Chemical versus biochemical conversion: when and how to use biocatalysts

Developments in biotechnology have been extremely rapid the last two decades. This is particularly true for recombinant DNA technology, which has strongly stimulated the study of biocatalysis, and many examples of new applications of biocatalysts exist. Nevertheless, they have not yet been routinely applied in chemistry. In this study, the question is addressed as to when biocatalysts can be advantageously used in this field. If that is indeed the case, many decisions have to be made before they can be optimally utilized. In the present work, a decision tree is introduced which provides important questions to answer to come to such an optimal use. Examples are given to illustrate the decisionmaking. (c) 1996 John Wiley & Sons, Inc.