Recent Advances in Phospholipase C Applications in Food Industry: From Molecular Mechanisms to Green Processing Technologies.

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity.

Halogenated organic compounds (HOCs) are essential building blocks in pharmaceuticals, agrochemicals, and advanced materials. However, their conventional chemical synthesis often relies on hazardous reagents and generates significant environmental waste. Harnessing nature's solutions, halogenases and dehalogenases offer selective, eco-friendly alternatives for the biosynthesis and degradation of HOCs. Halogenases, including electrophilic (e.g., haloperoxidases, flavin-dependent), radical (α-ketoglutarate-dependent), and nucleophilic (S-adenosylmethionine (SAM)-dependent) types, facilitate precise C-X bond formation under mild conditions. Recent advances in protein engineering, such as the modification of tryptophan halogenases and fluorinases, have greatly expanded the repertoire and efficiency of biocatalytic halogenation, enabling the production of new-to-nature compounds for synthetic biology applications. In parallel, dehalogenases, ranging from reductive to hydrolytic and oxidative enzymes, play crucial roles in removing halogens from persistent pollutants, thereby supporting effective bioremediation and environmental detoxification. This review summarizes recent progress in enzyme discovery, mechanistic elucidation, protein engineering, and applied synthetic biology, with a focus on the integration of halogenases and dehalogenases into scalable platforms for both biosynthetic and remediation. Continued research aimed at improving enzyme stability, substrate scope, and operational robustness will be critical to fully realizing the industrial and environmental potential of these versatile biocatalysts.

Chapter 10 - Advances in protein engineering and its application in synthetic biology

Bispecific antibodies (BsAbs) are designed to recognize and bind to two different antigens or epitopes. In the last few decades, BsAbs have been developed within the context of cancer therapies and in particular for the treatment of hematologic B-cell malignancies. To date, more than one hundred different BsAb formats exist, including bispecific T-cell engagers (BiTEs), and new constructs are constantly emerging. Advances in protein engineering have enabled the creation of BsAbs with specific mechanisms of action and clinical applications. Moreover, a better understanding of resistance and evasion mechanisms, as well as advances in the protein engineering and in immunology, will help generating a greater variety of BsAbs to treat various cancer types. This review focuses on T-cell-engaging BsAbs and more precisely on the various BsAb formats currently being studied in the context of B-cell malignancies, on ongoing clinical trials and on the clinical concerns to be taken into account in the development of new BsAbs.

Polyarginine (poly-Arg) and arginine-rich peptides have been attracting enormous interest in chemical and cell biology as cell-penetrating peptides capable of direct intracellular penetration. Owing to advances in protein engineering, arginine-rich fragments are often incorporated into multifunctional bioorganic/inorganic core–shell nanoparticles, enabling them the novel unique ability to cross cells and deliver biopharmaceutical cargos. Therefore, understanding the molecular details of the adsorption, packing, and release of poly-Arg onto or from metal nanoparticles is one of the current challenges. In this work, we carry out atomistic molecular dynamics simulations to identify the most favorable location, orientation, and conformation of poly-Arg adsorbed onto a silver nanoparticle (AgNP). Herein, we utilize the constant protonation approach to identify the role of protonation of side chain arginine moieties in the adsorption of poly-Arg to AgNP as a function of pH. The strong adsorption of unprotonated poly-Arg30 onto the quasispherical surface of AgNP with an average diameter of 3.9 nm is primarily governed by multiple interactions of side chain guanidinium (Gdm) moieties, which get stacked and align flat onto the surface. The protonation of the arginine side chain enhances the protein–solvent interactions and promotes the weakening of the protein–nanoparticle binding. The formation of multiple H-bonds between the protonated Arg residues and water molecules favors exposing the charged Gdm+ moieties to the solvent. Protonated poly-Arg30 is found to be partially bound to AgNP due to some weak protein–nanoparticle contacts, maintained by binding of the amide oxygen atoms of the peptide bond. These results suggest that reversible acid–base switching between the arginine protonation states is able to drive the rearrangement of the polyarginine coating around AgNPs, which could be important for a rational design of “intelligent” multifunctional core–shell nanosystems.

Over the lifespan of therapeutic proteins, from the point of biosynthesis to the complete clearance from tested subjects, they undergo various biological modifications. Therapeutic influences and molecular mechanisms of these modifications have been well appreciated for some while remained less understood for many. This paper has classified these modifications into multiple categories, according to their processing locations and enzymatic involvement during the trafficking events. It also focuses on the underlying mechanisms and structural-functional relationship between modifications and therapeutic properties. In addition, recent advances in protein engineering, cell line engineering, and process engineering, by exploring these complex cellular processes, are discussed and summarized, for improving functional characteristics and attributes of protein-based biopharmaceutical products.

Amino acid transaminases (ATs) have garnered considerable attention in recent years as promising biocatalysts for the synthesis of high‐value chiral chemicals, including both natural and non‐canonical amino acids. These enzymes catalyze the transfer of amino groups from amino acids to keto acids, playing a pivotal role in various biological processes and industrial applications. Characterized by their high turnover rates, remarkable enantioselectivity, and broad substrate specificity, ATs exhibit exceptional versatility and potential. This review presents a comprehensive overview of the classification, reaction mechanisms, and activity assays of ATs. More crucially, we delve into the recent advancements in protein engineering of ATs through directed evolution and rational/semi‐rational design strategies, which have been instrumental in addressing limitations such as low catalytic efficiency and stability. Furthermore, we survey the recent synthetic applications of ATs in the production of aliphatic and aromatic amino acids, highlighting smart amino donors and coupling methods that effectively shift the equilibrium of transamination reactions, as well as enzyme cascades that further expand the scope of reactions. By bridging gaps in research on ATs, this review aims to provide valuable insights and guidance for future developments in the field of biocatalysis, ultimately fostering their continued utilization and advancement.

Yeast surface display (YSD) is a powerful tool in biotechnology that links genotype to phenotype. In this review, the latest advancements in protein engineering and high‐throughput screening based on YSD are covered. The focus is on innovative methods for overcoming challenges in YSD in the context of biotherapeutic drug discovery and diagnostics. Topics ranging from titrating avidity in YSD using transcriptional control to the development of serological diagnostic assays relying on serum biopanning and mitigation of unspecific binding are covered. Screening techniques against nontraditional cellular antigens, such as cell lysates, membrane proteins, and extracellular matrices are summarized and techniques are further delved into for expansion of the chemical repertoire, considering protein–small molecule hybrids and noncanonical amino acid incorporation. Additionally, in vivo gene diversification and continuous evolution in yeast is discussed. Collectively, these techniques enhance the diversity and functionality of engineered proteins isolated via YSD, broadening the scope of applications that can be addressed. The review concludes with future perspectives and potential impact of these advancements on protein engineering. The goal is to provide a focused summary of recent progress in the field.

Protein engineering has emerged as a powerful approach toward the development of novel therapeutics targeting the MYC/MAX/E-box network, an active driver of >70% of cancers. The MYC/MAX heterodimer regulates numerous genes in our cells by binding the Enhancer box (E-box) DNA site and activating the transcription of downstream genes. Traditional small molecules that inhibit MYC face significant limitations that include toxic effects, drug delivery challenges, and resistance. Recent advances in protein engineering offer promising alternatives by creating protein-based drugs that directly disrupt the MYC/MAX dimerization interface and/or MYC/MAX’s binding to specific DNA targets. Designed DNA binding proteins like Omomyc, DuoMyc, ME47, MEF, and Mad inhibit MYC activity through specific dimerization, sequestration, and DNA-binding mechanisms. Compared to small molecules, these engineered proteins can offer superior specificity and efficacy and provide a potential pathway for overcoming the limitations of traditional cancer therapies. The success of these protein therapeutics highlights the importance of protein engineering in developing cancer treatments.

Cytochrome P450 enzymes (CYPs) are versatile heme-containing monooxygenases involved in the metabolism of endogenous and exogenous compounds, as well as natural product biosynthesis. Their ability to catalyze regio- and stereoselective oxidation reactions makes them valuable in pharmaceuticals, fine chemicals, and biocatalysis. However, wild-type CYPs suffer from low catalytic efficiency, limited substrate specificity, and instability under industrial conditions. Recent advances in protein engineering—rational design, semi-rational design, and directed evolution—have enhanced their activity, stability, and substrate scope. These strategies have enabled CYPs to be engineered for applications like C–H functionalization, carbene transfer, and complex molecule biosynthesis. Despite progress, challenges remain in optimizing efficiency, expanding substrate ranges, and scaling production for industrial use. Future directions include integrating CYPs with other biocatalysts, improving high-throughput screening, and applying machine learning to enzyme design. This review highlights recent developments and the promising future of engineered CYPs in sustainable chemistry, drug development, and high-value chemical production.

A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives. These efforts are crucial in drug discovery and development, where there is a continuous quest for more potent agents. Both rational and random evolution techniques have been utilized in engineering these enzymes. This review will highlight some examples from several large families of natural products.

With the advent of green chemistry, the use of enzymes in industrial processes serves as an alternative to the conventional chemical catalysts. A high demand for sustainable processes for catalysis has brought a significant attention to hunt for novel enzymes. Among various hydrolases, the α-amylase has a gamut of biotechnological applications owing to its pivotal role in starch-hydrolysis. Industrial demand requires enzymes with thermostability and to ameliorate this crucial property, various methods such as protein engineering, directed evolution and enzyme immobilisation strategies are devised. Besides the traditional culture-dependent approach, metagenome from uncultured bacteria serves as a bountiful resource for novel genes/biocatalysts. Exploring the extreme-niches metagenome, advancements in protein engineering and biotechnology tools encourage the mining of novel α-amylase and its stable variants to tap its robust biotechnological and industrial potential. This review outlines α-amylase and its genetics, its catalytic domain architecture and mechanism of action, and various molecular methods to ameliorate its production. It aims to impart understanding on mechanisms involved in thermostability of α-amylase, cover strategies to screen novel genes from futile habitats and some molecular methods to ameliorate its properties.

Microbial amidases: Characterization, advances and biotechnological applications.

Introduction to Computer-Assisted Drug Design – Overview and Perspective for the Future

Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.