Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.     

Horseradish peroxidase: Modulation of properties by chemical modification of protein and heme

Horseradish peroxidase (HRP) is one of the most studied enzymes of the plant peroxidase superfamily. HRP is also widely used in different bioanalytical applications and diagnostic kits. The methods of genetic engineering and protein design are now widely used to study the catalytic mechanism and to improve properties of the enzyme. Here we review the results of another approach to HRP modification—through the chemical modification of amino acids or prosthetic group of the enzyme. Computer models of HRPs with modified hemes are in good agreement with the experimental data.     

结构修饰提高酶稳定性、活性

酶因其特异性和可持续性而成为广泛应用的绿色催化剂，其稳定性和催化活性是决定酶适用性的关键因素。为满足实际应用需求，通过蛋白质结构修饰赋予其所需的催化特性是当前的研究热点。提高热稳定性的策略有：引入非共价/共价相互作用（疏水相互作用、氢键、盐桥、芳香环相互作用、二硫键）、环截短、C端和N端工程，及增加脯氨酸/减少甘氨酸的数目等；获得具有高效性和多样性的生物催化剂的策略有：降低空间位阻、拓宽催化口袋、增加底物亲和力及调节活性位点灵活性等。然而，在稳定性或催化功能改造的过程中，新突变的引入会削弱其他功能，致使进化过程中稳定性和催化活性相互制约。因此，采用基于理性计算优选突变热点、基于多重蛋白质稳定性或活性改造策略的共进化，以及基于高度稳定的蛋白质骨架创造或/和优化蛋白质功能等多种策略克服酶稳定性-活性之间的权衡。本综述重点阐述了结构修饰方法在提高酶稳定性或/和催化活性方面的应用，并展望了该领域的未来发展前景。     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

Enzyme-mediated methodologies for protein modification and bioconjugate synthesis

Bioconjugates are valuable tools in many fields, including protein engineering and environmental and therapeutic research. Chemical methods are commonly used to synthesize protein-protein and protein-functional molecule bioconjugates because they permit easy tethering through covalent bonds. However, chemical methods often produce heterogeneous products and lead to degradation of protein activity due to random modifications. Recently, a number of techniques for modifying proteins or synthesizing bioconjugates have been reported, including more sophisticated chemical modification methods, utilization of noncovalent affinity, and protein splicing. Enzymatic methods in particular have attracted much attention due to the substrate specificity of enzymes, which enables site-specific tethering of proteins to other proteins or functional molecules. Here, we discuss newly developed methods for protein modification and bioconjugate synthesis that exploit the properties of acyltransferases, ligases, and other enzymes.     

Chemical modification of enzymes for enhanced functionality.

The explosion in commercial and synthetic applications of enzymes has stimulated much of the interest in enhancing enzyme functionality and stability. Covalent chemical modification, the original method available for altering protein properties, has now re-emerged as a powerful complementary approach to site-directed mutagenesis and directed evolution for tailoring proteins and enzymes. Glutaraldehyde crosslinking of enzyme crystals and polyethylene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability. Whereas crosslinking of enzyme crystals generates easily recoverable insoluble biocatalysts, PEGylation increases solubility in organic solvents. Chemical modification has been exploited for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality, such as the conversion of a hydrolase into a peroxidase. Despite the breadth of applicability of chemically modified enzymes, a difficulty that has previously impeded their implementation is the lack of chemo- or regio-specificity of chemical modifications, which can yield heterogeneous and irreproducible product mixtures. This challenge has recently been addressed by the introduction of a unique position for modification by a site-directed mutation that can subsequently be chemically modified to introduce an unnatural amino acid sidechain in a highly chemo- and regio-specific manner.     

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.     

Protein engineering of subtilisin

The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.     

Enzymes: An integrated view of structure, dynamics and function

Microbes utilize enzymes to perform a variety of functions. Enzymes are biocatalysts working as highly efficient machines at the molecular level. In the past, enzymes have been viewed as static entities and their function has been explained on the basis of direct structural interactions between the enzyme and the substrate. A variety of experimental and computational techniques, however, continue to reveal that proteins are dynamically active machines, with various parts exhibiting internal motions at a wide range of time-scales. Increasing evidence also indicates that these internal protein motions play a role in promoting protein function such as enzyme catalysis. Moreover, the thermodynamical fluctuations of the solvent, surrounding the protein, have an impact on internal protein motions and, therefore, on enzyme function. In this review, we describe recent biochemical and theoretical investigations of internal protein dynamics linked to enzyme catalysis. In the enzyme cyclophilin A, investigations have lead to the discovery of a network of protein vibrations promoting catalysis. Cyclophilin A catalyzes peptidyl-prolyl cis/trans isomerization in a variety of peptide and protein substrates. Recent studies of cyclophilin A are discussed in detail and other enzymes (dihydrofolate reductase and liver alcohol dehydrogenase) where similar discoveries have been reported are also briefly discussed. The detailed characterization of the discovered networks indicates that protein dynamics plays a role in rate-enhancement achieved by enzymes. An integrated view of enzyme structure, dynamics and function have wide implications in understanding allosteric and co-operative effects, as well as protein engineering of more efficient enzymes and novel drug design.     

酶类药物现状、问题及展望

随着生物制药的迅速发展,许多酶类药物应运而生,在治疗代谢疾病、心血管疾病、癌症等诸多疾病上发挥着越来越重要的作用。但是酶类药物也存在一些不足,如潜在的免疫原性、较短的体内半衰期,以及较差的组织靶向性,影响了酶类药物的疗效和应用。为克服这些缺点,人们已开发出多种技术,如通过糖基化、聚乙二醇修饰等分子工程技术提升酶蛋白药效,另一方面酶基因疗法也已成功用于多种酶缺陷疾病的治疗。基于酶类药物的迅速发展和广泛的应用前景,本文对酶类药物的现状进行较详细的阐述,并对酶类药物的优势、所存在的问题及未来发展趋势进行分析和评述。     

Design and engineering of allosteric communications in proteins

Allostery in proteins plays an important role in regulating protein activities and influencing many biological processes such as gene expression, enzyme catalysis, and cell signaling. The process of allostery takes place when a signal detected at a site on a protein is transmitted via a mechanical pathway to a functional site and, thus, influences its activity. The pathway of allosteric communication consists of amino acids that form a network with covalent and non-covalent bonds. By mutating residues in this allosteric network, protein engineers have successfully established novel allosteric pathways to achieve desired properties in the target protein. In this review, we highlight the most recent and state-of-the-art techniques for allosteric communication engineering. We also discuss the challenges that need to be overcome and future directions for engineering protein allostery.     

Incorporation of artificial receptors into a protein/peptide surface: a strategy for on/off type of switching of semisynthetic enzymes

Recent developments in new bioorganic methodologies have greatly facilitated the site-specific incorporation of non-natural amino acids into the protein framework. It is now desirable for chemists to explore promising concepts based on chemistry for regulation and extension of functions of naturally occurring enzymes using non-natural molecules, in order to promote the new trends in protein/enzyme engineering. This article demonstrates that the concepts of host-guest (or supramolecular) chemistry, which have been developed over the last few decades, provide powerful tools for the artificial control of the functions of native proteins and enzymes.     

Protein design for pathway engineering

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.     

l-Amino acid oxidase as biocatalyst: a dream too far?

l-Amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the “true” LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with “narrow” or “strict” substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.     

Vitamin K-dependent carboxylase. Control of enzyme activity by the "propeptide" region of factor X

A liver microsomal enzyme catalyzes the vitamin K-dependent posttranslational carboxylation of specific glutamyl residues of a limited number of plasma proteins to gamma-carboxyglutamyl residues. The intracellular precursor forms of these proteins are known to contain a homologous basic amino acid-rich propeptide region between the signal peptide region and the amino terminus of the mature protein. This region of the precursor protein has been implicated as a possible recognition site for the carboxylase enzyme. A 20-residue peptide containing the octadecapropeptide of human clotting factor X has now been shown to strongly stimulate the activity of the enzyme toward a noncovalently linked substrate. This stimulatory effect is seen at less than micromolar concentrations and is accompanied by a decrease in the Km of the glutamic acid substrate. These observations raise the possibility that the catalytic activity of other enzymes involved in protein processing may be regulated by a portion of their normal substrates.     

Small-molecule catalysts of oxidative protein folding

Oxidative protein folding occurs both in vivo and in vitro and involves the formation and rearrangement of protein disulfide bonds (SS bonds). In vivo these reactions are catalyzed by enzymes, including the eukaryotic enzyme protein disulfide isomerase (PDI). Using the physical properties of PDI as a guide, several small-molecule catalysts of oxidative protein folding have been designed, synthesized, and tested. These small molecules can improve the folding rate of the model substrate ribonuclease A by a factor of over 10 and improve the yield by up to a factor of 3 over traditional conditions. The molecules have also been demonstrated to significantly improve the in vivo folding of proteins as well.     

Directed evolution: an approach to engineer enzymes

Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.     

Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis

Despite their unparalleled catalytic prowess and environmental compatibility, enzymes have yet to see widespread application in synthetic chemistry. This lack of application and the resulting underuse of their enormous potential stems not only from a wariness about aqueous biological catalysis on the part of the typical synthetic chemist but also from limitations on enzyme applicability that arise from the high degree of substrate specificity possessed by most enzymes. This latter perceived limitation is being successfully challenged through rational protein engineering and directed evolution efforts to alter substrate specificity. However, such programs require considerable effort to establish. Here we report an alternative strategy for expanding the substrate specificity, and therefore the synthetic utility, of a given enzyme through a process of "substrate engineering". The attachment of a readily removable functional group to an alternative glycosyltransferase substrate induces a productive binding mode, facilitating rational control of substrate specificity and regioselectivity using wild-type enzymes.     

Structure-driven protein engineering for production of valuable natural products

Proteins are the most frequently used biocatalysts, and their structures determine their functions. Modifying the functions of proteins on the basis of their structures lies at the heart of protein engineering, opening a new horizon for metabolic engineering by efficiently generating stable enzymes. Many attempts at classical metabolic engineering have focused on improving specific metabolic fluxes and producing more valuable natural products by increasing gene expression levels and enzyme concentrations. However, most naturally occurring enzymes show limitations, and such limitations have hindered practical applications. Here we review recent advances in protein engineering in synthetic biology, chemoenzymatic synthesis, and plant metabolic engineering and describe opportunities for designing and constructing novel enzymes or proteins with desirable properties to obtain more active natural products.     

Incorporation of non-natural modules into proteins: structural features beyond the genetic code

The biotechnological application of enzymes necessitates a permanent quest for new biocatalysts. Among others, improvement of catalytic activity, modification of substrate specificity, or increase in stability of the enzymes are desirable goals. The exploration of homologous enzymes from various sources or DNA-based methods, like site-directed mutagenesis or directed evolution, yield an incredible variety of biocatalysts but they all rely on the restricted number of canonical amino acids. Chemistry offers an almost unlimited palette of additional modifications which can endow the proteins with improved or even completely new properties. Numerous techniques to furnish proteins with non-natural amino acids or non-proteinogenic modules have been introduced and are reviewed with special focus on expressed protein ligation, a method that combines the potential of protein biosynthesis and chemical synthesis. An erratum to this article can be found at