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.     

Enzyme engineering: Reshaping the biocatalytic functions

Enzyme engineering is a powerful tool to fine-tune the enzymes. It is a technique by which the stability, activity, and specificity of the enzymes can be altered. The characteristic properties of an enzyme can be amended by immobilization and protein engineering. Among them, protein engineering is the most promising, as in addition to amending the stability and activity, it is the only way to modulate the specificity and stereoselectivity of enzymes. The current review sheds light on protein engineering and the approaches applied for it on the basis of the degree of knowledge of structure and function of enzymes. Enzymes, which have been engineered are also discussed in detail and categorized on the basis of their respective applications. This will give a better insight into the revolutionary changes brought by protein engineering of enzymes in various industrial and environmental processes.     

Manipulation of enzyme properties by noncanonical amino acid incorporation

Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties through protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, noncanonical amino acid incorporation (NCAAI) has become a new and effective protein engineering technique. Recently, NCAAI has been used to improve intrinsic functions of proteins, such as enzymes and fluorescent proteins, beyond the capacities obtained with natural amino acids. Herein, recent progress on improving enzyme properties through NCAAI in vivo is reviewed and the challenges of current approaches and future directions are also discussed. To date, both NCAAI methods-residue- and site-specific incorporation-have been primarily used to improve the catalytic turnover number and substrate binding affinity of enzymes. Numerous strategies used to minimize structural perturbation and stability loss of a target enzyme upon NCAAI are also explored. Considering the generality of NCAAI incorporation, we expect its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.     

Protein engineering of oxygenases for biocatalysis

Oxygenase enzymes have seen limited practical applications because of their complexity, poor stabilities, and often low catalytic rates. However, their ability to perform difficult chemistry with high selectivity and specificity has kept oxygenases at the forefront of engineering efforts. Growing understanding of structure-function relationships and improved protein engineering methods are paving the way for applications of oxygenases in chemical synthesis and bioremediation.     

Altering protein specificity: techniques and applications

Protein engineering constitutes a powerful tool for generating novel proteins that serve as catalysts to induce selective chemical and biological transformations that would not otherwise be possible. Protocols that are commonly employed for altering the substrate specificity and selectivity profiles by mutating known enzymes include rational and random methods as well as techniques that entail evolution, selection and screening. Proteins identified by these techniques play important roles in a variety of industrial and medicinal applications and in the study of protein structure-function relationships. Herein we present a critical overview of methods for creating new functional proteins having altered specificity profiles and some practical case studies in which these techniques have been applied to solving problems in synthetic and medicinal chemistry and to elucidating enzyme function and biological pathways.     

Substrate tunnels in enzymes: Structure–function relationships and computational methodology

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. Proteins 2015; 83:599–611. © 2015 Wiley Periodicals, Inc.     

蛋白质工程：从定向进化到计算设计

定向进化通过建立突变体文库与高通量筛选方法,快速提升蛋白的特定性质,是目前蛋白质工程最为常用的蛋白质设计改造策略。近十年随着计算机运算能力大幅提升以及先进算法不断涌现,计算机辅助蛋白质设计改造得到了极大的重视和发展,成为蛋白质工程新开辟的重要方向。以结构模拟与能量计算为基础的蛋白质计算设计不但能改造酶的底物特异性与热稳定性,还可从头设计具有特定功能的人工酶。近年来机器学习等人工智能技术也被应用于计算机辅助蛋白质设计改造,并取得瞩目的成绩。文中介绍了蛋白质工程的发展历程,重点评述当前计算机辅助蛋白质设计改造方面的进展与应用,并展望其未来发展方向。     

Cytochrome P450 monooxygenases: an update on perspectives for synthetic application

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospeci?c oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.     

Protein design in metabolic engineering and synthetic biology

Starting from experimental data on sequence, structure or biochemical properties of enzymes, protein design seeks to construct enzymes with desired activity, stability, specificity and selectivity. Two strategies are widely used to investigate sequence-structure-function relationships: statistical methods to analyse protein families or mutant libraries, and molecular modelling methods to study proteins and their interaction with ligands or substrates. On the basis of these methods, protein design has been successfully applied to fine-tune bottleneck enzymes in metabolic engineering and to design enzymes with new substrate spectra and new functions. However, constructing efficient metabolic pathways by integrating individual enzymes into a complex system is challenging. The field of synthetic biology is still in its infancy, but promising results have demonstrated the feasibility and usefulness of the concept.     

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.     

Engineering of cellobiose phosphorylase for glycoside synthesis

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.     

Engineering of cellobiose phosphorylase for glycoside synthesis

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.     

Substrate-assisted catalysis: molecular basis and biological significance

Substrate-assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose-1-phosphate uridylyltransferase. Moreover, structure-based predictions of SAC have been made for many additional enzymes. Examples of SAC include both naturally occurring enzymes such as type II restriction endonucleases as well as engineered enzymes including serine proteases. In the latter case, a functional group from a substrate can substitute for a catalytic residue replaced by site-directed mutagenesis. From a protein engineering perspective, SAC provides a strategy for drastically changing enzyme substrate specificity or even the reaction catalyzed. From a biological viewpoint, SAC contributes significantly to the activity of some enzymes and may represent a functional intermediate in the evolution of catalysis. This review focuses on advances in engineering enzyme specificity and activity by SAC, together with the biological significance of this phenomenon.     

Molecular engineering of industrial enzymes: recent advances and future prospects

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.     

Bacterial detoxification of organophosphate nerve agents

Bacterial enzymes have been isolated that catalyze the hydrolysis of organophosphate nerve agents with high-rate enhancements and broad substrate specificity. Mutant forms of these enzymes have been constructed through rational redesign of the active-site binding pockets and random mutagenesis to create protein variants that are optimized for the detoxification of agricultural insecticides and chemical warfare agents. In this review, the catalytic properties of two bacterial enzymes, phosphotriesterase and organophosphorus anhdrolase, are examined for their ability to hydrolyze organophosphate nerve agents.     

Bacterial mandelic acid degradation pathway and its application in biotechnology

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.     

Transient protein expression systems in plants and their applications

The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools.     

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。     

疾病治疗及药物制备用酶的研究进展

生物技术的发展及对疾病机理的深入研究,使酶逐步应用于疾病治疗。与此同时,以酶作为催化剂制备非天然有机化合物具有反应条件温和、催化效率高、特异性高、选择性强、副反应少等优点。因而,酶也在药物制造方面展现了巨大潜力。此外,基因工程、酶化学修饰和固定化技术的应用进一步改善了酶的功能。基于此,文中结合本课题组的相关研究,综述了近年来酶作为药物用于疾病治疗及作为催化剂用于药物制造的研究进展,并对其存在的问题提出了相应的解决办法,最后对酶在医药领域的应用前景进行了展望。     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。