Enzymes in the Removal of Harmful Substances: The Potential of Biotechnology in Environmental Protection

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.

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.

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

Cellulosomes are multienzyme complexes originally found on the surface of certain anaerobic celluloytic bacteria and fungi that are specialized in the degradation of plant cell walls. Recently, the efficiency in lignocellulose conversion and architectural features of these intricate complexes inspired the construction of artificial chimeric complexes for targeted substrate degradation. The simultaneous advancements in synthetic biology, protein engineering, and the pursuit of greater sustainability across various industries have highlighted the immense potential of these artificially designed enzymatic complexes for diverse applications. Notably, they hold significant promise for industries specializing in valorization of plant biomass waste and the production of bio-based renewable energy. The article discusses the main architectural features, design and construction steps as well as various biotechnological applications of these intriguing nanomachines.

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.

Abstract: This chapter explores the transformative potential of microbial biotechnology in addressing some of the most pressing ecological challenges of our time. It delves into the diverse applications of microbes in environmental sustainability, agriculture, and industry, highlighting their role in processes such as bioremediation, biofuel production, and sustainable agriculture. The chapter also examines the latest advances in microbial genomics and synthetic biology, showcasing how these technologies are driving innovation and enabling the development of novel solutions for climate change mitigation, resource recovery, and pollution control. Ethical considerations and challenges associated with the use of microbial biotechnology are discussed, alongside a forward-looking vision for how these innovations can contribute to a more sustainable and resilient future. The chapter emphasizes the importance of interdisciplinary collaboration and global commitment in harnessing the power of microbes to achieve ecological sustainability and address global challenges. Keywords: Microbial Biotechnology, Environmental Sustainability, Bioremediation, Biofuels, Sustainable Agriculture, Microbial Genomics, Synthetic Biology, Carbon Sequestration, Circular Economy, Ecological Innovation.

Circular Urban Metabolism Framework

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.

Objective: This article aims to identify the main characteristics and trends in the development of the circular economy in EU countries and the key features of this process concerning the implementation of traditional linear economic systems. Theoretical framework: The industrial revolution and rapid economic development over a short time have changed the state of the environment. Excessive consumption, the basis of modern society, has caused climate change and intensified countless environmental and social problems. Method: Questionnaire survey, which was conducted by the authors of the study online to practically clarify the most critical issues related to the analysis of trends in the implementation of the circular economy. Results and conclusion: The study identified the top, most important features of the concept and the circular economy model, the main trends in the development of the circular economy in EU countries, and the key differences between the circular and linear economies. The research authors proposed classifying circular business models, which include four main types: closed loop, circular supply chain, resource recovery, and product life extension. The study also found that implementing circular economy principles in EU countries positively impacts economic growth, job creation, and environmental protection. Implications of the research: The authors concluded that the transition to a circular economy is a global trend that requires the joint efforts of all countries and stakeholders. Originality/value: The originality and value of this research lie in its specific focus on the circular economy as an alternative to the traditional linear economy, using the case study of the European Union (EU).

Immobilized Enzymes

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.

Food waste gives rise to many environmental problems. A large amount of food waste is produced by grocery retail stores. It is therefore important to apply efficient food waste treatment technologies with minimal environmental impact and investigate the optimal approach for food waste collection, transportation, and treatment. In the present study, a life cycle impact assessment (LCIA) was conducted to analyze different food waste disposal scenarios, including incineration, landfilling, composting, anaerobic digestion, and bioconversion. The impacts of the five scenarios on the environmental, economic, and social aspects were assessed. The results suggested that the landfilling scenario has the lowest net cost for the treatment of food waste, followed by the incineration scenario. The bioconversion treatment cost has the most significant positive effect on the net cost of the bioconversion scenario, and both the price and yield of compost have a significant negative effect on the net cost. The rankings of the five scenarios are the same under both weight determination methods, with the bioconversion scenario performing the best, followed by the composting scenario. The results of this study can help improve the disposal of food waste in grocery retail stores in the framework of sustainability and the circular economy.

Ecological and biotechnological aspects of Aplysina-associated microorganisms