Biocatalysis: modern problems and applications

The resource, energy, and environmental problems existing to date bring about the need to develop and scale up fundamentally new methods for matter and energy conversion. The material, engineering, and ecological development of society and the growth of population require more intense industrial production and, in some cases, a conceptual change in the fundamental engineering processes that form the basis of human existence. Global problems of relationship between humans and the environment are of prime importance. The assessment of trends in science and technology and the development of the basis for strategic national planning, taking into account these trends and the existing engineering experience, appear to be of particular importance.

In order to achieve the sustainable development declared by the UN in 2015, the international industrial and economic community is making significant efforts to transform and improve the existing production processes and technologies. Large amounts of data have been accumulated, and the search for solutions that can significantly change both the current technological structure of society and the systems of product consumption and service provision is in progress.

The intensity and speed of works aimed at the development and implementation of new, more advanced technologies are fairly high. The sustainable development technologies are based on several key criteria:

— renewable or virtually infinite resources;

— improvement of the environmental quality as a result of industrial implementation of a process;

— provision of the design and scaled implementation of new efficient processes of matter conversion as a result of science and engineering development;

— understanding and analysis of processes in nature and in the human body.

Chemical processes involving enzymes most fully meet the above criteria because these biomolecules have a high catalytic potential.

Comparison of enzymes with conventional chemical catalysts under similar conditions (temperature, concentration) demonstrates obvious advantages of enzymes for acceleration of chemical reactions.¹ For example, the known comparison of enzymes with the hydrogen ion (the hydroxonium ion is the most typical chemical catalyst for hydrolysis) indicates that the ratio of the reaction rate constants may reach 1.7 × 10¹²͘. This means that if an enzyme-catalyzed reaction proceeds within 1 s, the same reaction in the presence of a hydrogen ion will take ~  55 000 years.

Since the discovery of catalysis, including biocatalysis, by K.S.Kirchhoff (1812 – 1818), a Russian pharmacist and researcher, biocatalysis has attracted the attention of researchers for many decades as the main subject of biochemistry. However, in the mid-20th century, it became clear that biocatalysis could also be the basis of the most efficient chemical engineering processes. Owing to the catalytic activity, high specificity (selectivity), unique availability, and an enormous potential for the design of protein molecules, enzyme catalysis has become an object of intensive chemical and engineering studies. A fundamentally important contribution to elucidation of the nature of biocatalysis and to expansion of its practical application was made by Corresponding Member of the USSR Academy of Sciences, Ilya Vasilyevich Berezin, whose 100th anniversary of birth was celebrated in 2023. The decisive role in the further development of chemical enzymology in the USSR and then in Russia belongs to the scientific school of I.V. Berezin, Professor and Dean of the Faculty of Chemistry, Lomonosov Moscow State University. The I.V.Berezin’s scientific school formed under the influence of the famous school of chemical kinetics headed by Academician N.N.Semenov, Nobel Prize winner in Chemistry, and Academician N.M.Emanuel. I.V.Berezin’s achievements in science are well illustrated by the idea he stated: ‘Enzymes are chemical catalysts’. Before I.V.Berezin’s works, biological catalysis was considered as a complex phenomenon bearing some features of vitalism. Meanwhile, owing to the studies carried out by I.V.Berezin and his followers, it became clear that enzyme catalysis can be quite simply and reliably interpreted in terms of physical chemistry and has a huge potential for technical implementation of many chemical reactions. Data on enzyme structure and understanding of the mechanism of enzyme action provide fundamentally new prospects for medicine and ecology.

While discussing the trends in the development of chemical enzymology, one cannot but mention the factors that have had a significant impact on this field of science not only in Russia, but also all over the world. In the early stage, the attention was focused on the kinetics of enzymatic reactions and physicochemical mechanisms of enzyme catalysis, which subsequently promoted the application of enzymes in a wide range of human activities. The foundation of the Division of Chemical Enzymology at the Faculty of Chemistry of the Lomonosov Moscow State University in 1974 was an important organizational achievement. This made it possible to start training highly qualified specialists, who later became leaders of research groups in Russian and world universities and companies.^2, 3

Quite a few editions are to be mentioned among research and methodological materials.^4 – 12 These books and study guides written by the ‘first circle’ of I.V.Berezin’s students and followers summarized the most advanced experience in the study and application of biocatalysis known at that time and were useful for both the professional growth of the authors and the development of educational programs at the Division of Chemical Enzymology, which became popular among chemistry students.

The international conferences on biocatalysis regularly held in Russia have also become an important step towards popularization and development of domestic works in the field of biocatalysis. The active research into the chemical mechanisms of biocatalysis in the 20th century revealed the unique properties of enzymes and provided an understanding of how they can incredibly accelerate many reactions.^{13 – 17}

Along with numerous benefits of enzymes as catalysts, their most significant advantages over chemical catalysts were revealed, first of all, high chemo-, regio-, and stereoselectivity. Owing to appearance of immobilization methods, which enabled repeated use of enzymes that were expensive at that time, and to the accumulated fundamental knowledge about the kinetics, control, and mechanisms of biocatalytic reactions, the practical use of biocatalysis in industrial processes has markedly advanced since the 1960s. A fundamental issue that later promoted the large-scale use of enzymes in industrial production was the development of genetic engineering methods, which made it possible to express almost any enzyme using a suitable producer and accumulate the enzyme in large quantities. This made enzymes available catalysts and ultimately minimized the share of the biocatalyst in the cost of the target product. Currently, we are witnessing the active use of biocatalysis and enzymes in the production of a wide variety of products.

Other factors favourable for the use of biocatalysis should also be noted. First of all, mention should be made of the broad public and political discussion of environmental problems, which started in the 1980s and gave rise to the concepts of green chemistry, closed-loop processes, sustainable development, orientation towards renewable raw material and energy sources, and realization of the necessity of careful attitude to non-renewable resources.^{18 – 21} Raising of these issues was followed by formulation of particular tasks, the effective solution of which is largely associated with the use of biocatalysis.²² Solution of global problems requires application of a variety of methods, and in the wide range of available means, it is necessary to identify the areas in which the use of biocatalysis appears most promising. One hot spot for the practical application of the catalytic advantages of enzymes may be identified in terms of the E-factor (environmental factor reflecting the presence of pollutants) in the characteristics of wastes produced in various fields of industrial chemistry ²³ (Table 1). The need to reduce the amount of waste has become a key issue in the development of the green chemistry concept.

Table 1

\[ \]

Waste generation in various fields of industrial chemistry.²³

(1)

Enzymes are capable of performing chemical transformations at high rates and in high yields; however, it is necessary to find or create an enzyme catalyst that would have the required specificity and would be stable under the reaction conditions. Out of thousands of enzymes that perform the chemical reactions in living systems, only a few hundred are currently in use. This shows that the potential of biocatalysis in industry has not yet been sufficiently implemented.

Which of the set goals can be considered to be of the first priority? One such goal is active use of biocatalysis in fine organic synthesis and pharmaceutical industry (see Table 1). Analysis of the experience of large pharmaceutical companies in the synthesis ^24, 25 and a relevant discussion ²⁶ identified the following key problems of pharmaceutical industry that can be addressed using biocatalysis:

— the need to develop efficient methods for amide bond formation,

— decrease in the consumption of organic solvents, which make the largest contribution (up to 80%) to waste production,

— increase in the chemoselectivity of reactions,

— inadmissibility of genotoxic pollution in the final product (reduction of the number of catalysts based on heavy metal complexes).

Great prospects are also associated with implementation of distinctive features of enzyme catalysts such as chemo- and regioselectivity, which may eliminate the protection and deprotection steps in chemical synthesis and, due to decrease in the number of steps, may significantly increase the economic feasibility of the synthesis.

Apart from the chemo-, regio-, and stereoselectivity, biocatalysis can ensure mild reaction conditions, a simpler set of reactants, and less expensive and safer equipment design for the industrial process, because effective enzymatic reactions do not require high pressure or other drastic conditions (low or high pH or temperature) as well as explosion- and fire-hazardous organic solvents, since enzymatic reactions preferably proceed in aqueous solutions. This does not rule out the use of enzymes in organic solvents,²⁷ which attests to the possibility of complex processes that would combine the potential of biological and chemical catalysis. This idea is actively pursued in recent years.^{28 – 30}

The enzyme catalysis techniques and the conventional chemical catalysts are being developed in parallel and are complementary to each other. Comparison of chemical and biological catalysts has been made many times in various reviews and continues today in view of the dominance of certain trends in the development of the world economy and particular branches of economy. In the petroleum industry, processing of plastics, waste pyrolysis, Fischer – Tropsch synthesis, and other processes requiring high temperatures and pressures, inorganic catalysts are commonly obvious leaders. However, enzymes as basic biological catalysts are superior for processes that require low capital cost, highly specific action, enantioselectivity, high activity at low and moderate temperatures, and the highest possible yields of target compounds in catalytic reactions along with minimized amount of by-products.

Currently, enzymes (mainly hydrolases such as proteases, carbohydrases, lipases, polymerases, and nucleases) ^31, 32 are superior to almost any chemical catalyst and are used as components of low-temperature laundry detergents and feed supplements, in agriculture, in medicine for the treatment of various enzymatic disorders, in genetic analysis and diagnosis, and in the food industry (Table 2).

Table 2

\[ \]

Applications of enzymes as biocatalysts

(2)

Enzymes play an important role in the textile and pulp-and-paper industries and as components of biosensors. A classic example is glucose oxidase, which is widely used to monitor the blood glucose level.⁴⁰ The possibility of genetic modification of enzymes markedly expands their catalytic potential in relation to substrates, reaction temperature, and pH value.

In 2022, the cost of biocatalyst production was 25% of the total production cost of all catalysts in the world.⁴¹ In the next 10 years, the role of enzymes in the global economy, medicine, and ecology is expected to considerably increase.

Chemical and biological catalysis as the base of biotechnology forms the ground for modern methods of conversion of matter in a technology-based society. Large-scale production of enzymes for the needs of food industry, textile industry, and agriculture is an integral part of basic technological processes. Since microbiological industry is based on the knowledge of enzymes and methods for the control of enzyme expression, the practical significance of enzyme catalysis becomes obvious. There are known limitations on the industrial implementation of processes using protein catalysis:

— many practically important processes require high temperatures to meet the proper thermodynamic conditions; meanwhile, enzymes can operate at temperatures up to 90°C;

— enzymes are homogeneous catalysts and are used, in most cases, as expendable reagents if this is economically justified; although this problem can be solved by immobilization of enzymes on inorganic or polymeric supports, this procedure requires additional process stages and economic costs;

— the use of enzymes in large-scale production processes is often restricted by their relatively low stability on long-term operation; quite a few studies are aimed at increasing the stability of protein molecules through genetic modifications and protein engineering (see below);

— the industrial use of enzymes is complicated, in some cases, by their outstanding specificity and selectivity to the substrate (reactant); however, the unique capabilities of modern genetic engineering make it possible to modify the enzyme active sites and tune the active site towards new compounds (see below).

Since the general trend of development of chemical engineering processes is, to some extent, directed towards the use of natural organic resources (biofuels, biopolymers, biopharmaceuticals, etc.), the modern studies of enzymes fit well into this trend.

This review is devoted to analysis of the most important trends in the development of chemical and engineering enzymology, the outstanding results and promising applications of enzymes as catalysts, although detailed consideration of many industrial aspects of biocatalysis are beyond the scope of the review. The attention is concentrated on modern achievements in the physicochemical fundamentals of the molecular processes in enzyme active sites, the use of genetic engineering methods as a protein strategy to create enzymes with new properties, the role of enzymatic processes in molecular medicine, and development of new methods for analysis and diagnosis.

The accumulation of large amounts of information on amino acid sequences and protein structures in public databases and development of supercomputers made it possible to perform the bioinformatic analysis of large protein superfamilies ⁴² (Fig. 1). Hence, it became possible to identify the amino acid residues most important for the functioning of enzymes, reveal previously unknown binding sites for regulatory ligands,⁴³ and establish the relationships between various binding sites and the active site of the enzyme.⁴⁴ The use of bioinformatic and molecular modelling techniques provided a qualitatively new level in the study of the mechanism of enzyme action and structural organization of active sites.⁴⁵ For example, it was shown that hydrolases can be subdivided into four groups according to the type of active site organization and catalytic cycles, differing in the mechanism of water activation. The important role of Gly, Pro, and Cys residues in the structural organization of protein molecules and their active sites was established.⁴⁶

Fig. 1

Generalized strategy of bioinformatic approaches to study enzymes, in particular using supercomputers. There is spiral progression from simple to complex: from the primary amino acid (or nucleotide) sequence to a mature three-dimensional structure of the enzyme and then to the mechanism of its action; each subsequent level requires a higher amount of resources and gives more interesting results for science and practice.

Bioinformatic analysis is used to identify amino acid residues of two functional types: those in conserved positions of enzyme superfamily that directly participate in the catalytic mechanism and those in specific positions that are responsible for the functional diversity of superfamilies.⁴⁷ The subfamily-specific positions are identified using a scoring function based on genomic and structural information.^{46 – 49} An important feature of this analysis is that it is applicable not only to thoroughly characterized proteins, but also to little studied enzymes for which only the primary amino acid sequence is known.

The specificity and conservation of amino acid residues in structural cavities of enzymes serve as a criterion for evaluation of the functional significance of binding sites and can be used to identify and classify them.⁵⁰ The identification of the subfamily-specific positions (which are conserved within enzyme subfamilies of bacteria or mammals, but differ between subfamilies) helps to find the structural difference between the binding sites in the homologous enzymes of pathogenic bacteria and humans and between the involvements of the amino acid residues in the interaction with ligands.^50, 51 There are seven web servers for implementation of the above approaches, which are listed below (the web addresses are indicated in parentheses):

— Mustguseal (https://biokinet.belozersky.msu.ru/mustguseal);

— Zebra2 (https://biokinet.belozersky.msu.ru/zebra2);

— Zebra3d (https://biokinet.belozersky.msu.ru/zebra3d);

— pocketZebra (https://biokinet.belozersky.msu.ru/pocketzebra);

— visualCMAT (https://biokinet.belozersky.msu.ru/visualcmat);

— vsFilt (https://biokinet.belozersky.msu.ru/vsfilt);

— Yosshi (https://biokinet.belozersky.msu.ru/yosshi).

The key web server Mustguseal can automatically construct alignments for large protein superfamilies using data on the structure and sequences contained in public databases; a supercomputer protocol has been developed for high-throughput analysis of large data arrays. This web server possesses functions that are absent in the known world analogues and can not only align but also select structurally and functionally diverse related proteins from public databases and to construct large alignments of protein superfamilies involving thousands of structures and sequences of homologues. This is attained by using search algorithms based on structural similarity, in order to reveal evolutionarily distant proteins, to perform structural alignment, and to perform further similarity-based search for amino acid sequences for identification of evolutionarily related proteins in the families and subsequent alignment of amino acid sequences. These structure-based alignments using the developed approach can be performed in the automated online mode.

The related web servers Zebra2 and pocketZebra are able to analyze the obtained sequences in order to identify the conserved and specific positions in the superfamily, find unknown binding sites in the proteins (enzymes), determine their importance for functional properties, and identify distinctive features in a particular representative of the family. The visualCMAT web server can be used to reveal the relationship between different sites in the protein structure and to elucidate the network of interacting amino acid residues.⁴⁴

The Zebra3d web server finds specific 3D patterns in the protein superfamily that represent structural elements important for the enzyme action mechanism and are responsible for the difference of properties (such as substrate specificity, catalytic activity) between enzymes that belong to different functional subfamilies and between conformers of one enzyme, owing to the known spatial orientation of the key amino acid residues and parts of the backbone. The specific 3D patterns can be used for functional annotation and rational design of proteins.⁵² Determination of 3D motifs qualitatively supplements the information about specific and correlated positions in enzyme subfamily and expands the scope of protein design methods, e.g., by inserting 3D motifs of disulfide bridges into protein structures in order to generate biomolecules with altered functional properties (Yosshi web server).⁵³

The vsFilt web server is able to take into account the specific interactions between the protein target and a functional modulator while selecting the most promising compounds as drug prototypes.⁵⁴ The bioinformatic analysis of families of homologous enzymes identifies the differences between the active site structures and use the results to design selective inhibitors, for example, by means of the vsFilt structural filtration algorithm ⁵⁴ during virtual screening.

The data gained by bioinformatic analysis described above have been used to propose new ways to address some scientific and practical problems: new methods for regulating enzyme activity were substantiated, a search for selective inhibitors of pathogen enzymes was carried out, the amino acid substitutions for enzyme bioengineering were determined in order to deliberately change the functional properties of enzymes.^55, 56 The experience of application of bioinformatic techniques showed that they are efficient for studying the mechanism of action and for engineering of various enzymes.^{57 – 61}

A methodology for the search for new type of enzyme modulators proposed in recent years is based on identification of previously unknown binding sites other than the active site. Analysis indicates that proteins have numerous potential ligand binding sites the function of which is unknown. The functional significance of these sites might be indicated by the presence of specific or conserved amino acid residues, which are determined via bioinformatic analysis of protein superfamilies.^43, 62 The computational platform based on the Mustguseal web server is able to find such binding sites and identify complementary ligands, taking into account differences in the structural organization of the sites for pathogen, human, and animal enzymes.

The understanding of characteristic features and distinctions in the active site and other binding site structures in enzymes from various sources is important not only for the creation of selective inhibitors, but also for determining the influence of components of the reaction mixture on the efficiency of biocatalytic processes and for the search for enzymes with a desired specificity. The corresponding computer design procedure was tested by the search for bifunctional inhibitors of influenza virus neuraminidase,^63, 64 neuraminidase A from Streptococcus pneumoniae,⁶⁵ transketolase, glyceraldehyde 3-phosphate dehydrogenase, and L,D-transpeptidase-2 from Mycobacterium tuberculosis.^{66 – 69} This procedure identifies selective inhibitors that are effective against pathogens but safe for humans. Apart from computer screening, which precedes and accelerates the drug development, molecular modelling techniques are also actively used in drug design.^{16, 17, 50, 51} The introduction of bioinformatics and molecular modelling makes it possible to significantly accelerate the development of new drugs, including those with a fundamentally different mechanism of action.

The bioinformatic analysis of enzyme superfamilies and relevant web servers can also be used to identify the range of mutations in the key human proteins (enzymes) that are associated with a pathological condition. This may result in the creation of a unified database of human proteins (enzymes) that would list the pathological mutations; this may enable the development of a personalized medicine information service and an automated service based on the patient’s DNA sequencing data.

The fundamental causes for the high rate of action of protein catalysts are the subject of modern physical chemistry.¹ Revolutionary opportunities for the development of this research area are provided by supercomputer technologies,⁷⁰ which can trace the structural and kinetic changes in the protein molecule throughout the catalytic cycle on the basis of fundamental equations of quantum mechanics and methods of quantum chemistry.^71, 72

Molecular modelling has provided tools for elucidation of enzymatic reaction mechanisms, identification of metastable intermediates, and determination of transition state structures. The possibility of constructing the free energy change profile for transition from the reactant to the reaction product and identifying the extrema in the energy pathway proved to be highly important. Using this profile, it is possible to estimate the rate constants of all elementary steps of the catalytic cycle. Meanwhile, due to the instability of intermediates formed at high rates, identification of their structures and interconversion rates by modern experimental methods is virtually impossible. The supercomputer simulation opens up the way for solving this ‘unsolvable’ problem.^{73 – 79}

The following fundamental features of enzyme catalysis were clearly identified as a result of supercomputer simulation:

(1) all enzymatic reactions within the catalytic cycle of reactant transformation into products consist of numerous steps, including the formation of stable and unstable intermediates. The lifetimes of the intermediates are in the range of nano(micro, milli)seconds. Classic examples of enzymes that catalyze multistep reactions are serine hydrolases, ribonucleases, and aminotransferases. Modern molecular modelling techniques using high-performance computing through analysis of reaction mechanisms can identify all metastable intermediates;^{76 – 79}

(2) the catalytic cycle of enzymatic transformations is implemented within a conformationally flexible polymer matrix and involves functional groups that are identified as active site components. The structural and functional modelling of catalytic cycles by quantum mechanics/molecular mechanics (QM/MM) methods illustrates the important role of conformational changes in the active site during catalytic reactions. The conformational changes are determined by the structure and steric features of the active site and ensure positioning and migration of reactive groups into the required points of activation of particular bonds in the reactant or intermediate molecule.^{80 – 82} In a certain approximation, these changes during the catalytic cycle can be described as operation of a ‘molecular machine’ forming a sequence of necessary catalytic steps;

(3) numerous mechanisms of enzymatic reactions were analyzed by supercomputer simulation methods, e.g., the following reactions:

— synthesis of N-acetylaspartic acid in the presence of aspartate N-acetyltransferase encoded by the NAT8L gene;^{83 – 85}

— hydrolysis of the neuropeptide N-acetylaspartylglutamate, which plays an essential role in the functioning of glutamate synapses and mechanisms of acquiring and retrieving information that determine memory,⁸⁶ catalyzed by glutamate carboxypeptidase II;^{74, 75, 80 – 82}

— N-acetylaspartate hydrolysis with aspartoacylase, which is a key enzyme of neurovascular coupling participating in human brain metabolism in response to an external excitation signal.^{69, 87, 88}

Owing to the development of graphics accelerators and implementation of quantum chemistry software packages in them, it became possible to perform molecular dynamic calculations with QM/MM potentials. This substantially expanded the views on the mechanisms of enzymatic reactions and made it possible to consider the reaction pathways in hydrolases from the dynamic behaviour of enzyme – substrate complexes.^{63 – 65} Criteria were proposed for identification of electrophilic atoms in organic molecules containing carbonyl groups on the basis of analysis of three-dimensional electron density maps. The applicability of these procedures to analysis of various structures in enzyme – substrate complexes was demonstrated by Khrenova and co-workers.^{89 – 92} It was shown that QM/MM-based molecular modelling provides correct interpretation of experimental data,⁹² reveals the most probable chemical reaction mechanism, and gives explanation for the diversity of data that were obtained for a set of related systems, in particular enzymes, containing amino acid substitutions.⁹¹ The use of molecular modelling methods promotes the development of new enzyme inhibitors and elucidation of the mechanisms of their action.

Enzymes are unique highly specific catalysts. However, very often, this feature proves to be also a drawback, since during evolution, Nature optimized the enzyme specificity to natural substrates; however, many target compounds differ in their structure from natural molecules. As an example, consider penicillin acylase, which is fairly effective in the synthesis of ampicillin (Amp) and several times less active in the synthesis of amoxicillin, although the latter markedly surpasses Amp in the antibacterial properties and differ from Amp only by an additional hydroxyl group in the para-position of the benzene ring in D-phenylglycine. Particularly the enzyme specificity, together with relatively low stability and high cost of preparation from natural sources, substantially restricted the practical implementation of biocatalytic processes. In addition, the optimal conditions for enzyme functioning in the cell (e.g., pH required for enzyme activity) often differed from the optimal process parameters for the synthesis of the target product. As a result, conditions of the real process were a trade-off between the optimal conditions for functioning of the biocatalyst and for the chemical reaction. One more exceptionally important factor that affected the design and development of new biocatalytic processes was the absence of an enzyme with the required activity, with the known methods for the search for new biocatalysts being extensive and having a poorly predictable outcome. In addition, in most cases, microorganisms were used in practice as enzyme sources. However, it is well known that more than 99.9999% of microorganisms existing in nature cannot be obtained in a pure state in laboratory.

The situation started to change fundamentally (and the progress still continues) in the late 1980s to the early 1990s when the scientific community entered the so-called post-genomic era. In this period, new methods and approaches started to be used as research tools in life sciences.⁹³ Suffice it to mention here the Nobel Prizes in chemistry awarded for DNA sequencing (1980), development of various sorts of polymerase chain reaction (PCR) (1994), and development of methods and approaches of genetic engineering and protein design (2018). The methods such as X-ray diffraction analysis, electron microscopy, nuclear magnetic resonance, computer simulation, etc. gained completely new content and development. The term ‘post-genomic era’ should be conceived as not only technological progress in sequencing of various genomes, ranging from archaea or bacteria to humans, although this information is exceptionally important and useful, but also, for example, advances in the diagnosis of various hereditary diseases, discovery of new metabolic pathways and enzymes they involve, analysis of mechanisms of drug resistance, etc.

Modern high-throughput sequencing methods allow determination of DNA sequences in a sample and subsequent assembly of the resulting sequences into separate genomes. As a result, the problem of uncultivated organisms was quickly solved, and for enzymologists, the choice of potential enzymes immediately increased by millions of times. The sequencing of hundreds of thousands (and even millions) of genomes brought about the transition from quantity to quality. This gave rise to a giant information space; hence, appropriate bioinformatic methods and supercomputers were required to handle it. Data on the sequences and properties of known enzymes were combined into specialized databases, the use of which sharply narrows down the search for enzymes with specific properties. The large-scale analysis of amino acid sequences in proteins and enzymes is described in sufficient detail in the previous Section of the review, but the results of this analysis should be treated with great caution, as indicated by numerous errors in the annotation of new genomes both in the number of genes of a particular enzyme and in the functionality (as an example, see Section 3.1). The same is true for structural modelling. The errors are due to the fact that the calculation error is ± 1 kcal mol^–1, and, hence, the maximum and minimum protein – ligand interaction constants in the docking would differ by a factor of 50. For this reason, expert evaluation still plays a crucial role in the preliminary selection of enzyme candidates.

Nevertheless, the above-described methods are usually utilized in the initial stage of biocatalyst design tailored for a particular process. As a result, the ideology of development of new biocatalytic processes has completely changed. First, the optimal process conditions are selected; then the parent enzyme is chosen; and protein engineering techniques are applied to adjust the properties of the enzyme to the requirements of the process. Moreover, the biocatalyst itself can be changed. For example, reactions using α-D-amino acid ester hydrolase (HAA), instead of penicillin acylase (PA), are currently being developed for the preparation of aminopenicillins and aminocephalosporins. Higher efficiency of HAA is due to the fact that the acyl-containing enzyme is obtained from the methyl ester rather than from the much less reactive amide, as in the case of PA. In addition, since HAA is an esterase, hydrolysis of the final product (amide) with this enzyme proceeds much more slowly than that with PA, which is amidase. Consequentky, the yield of the final product is often much higher in the presence of HAA than with PA.

The post-genomic era is also characterized by the appearance of new biocatalytic processes. As an example, consider the CRISPR/Cas system for genome editing (Nobel Prize in Chemistry 2020). Currently, there are a few variants of this system, and new versions are being proposed, while the problem of its practical implementation refers to the ethical and legislative sphere.

Due to the restricted size of the review, below we consider the production of only classic biocatalysts. Tishkov et al.⁹⁴ addressed a variety of practically important enzymes; depending on the purpose, enzymes of the same type were obtained from different sources according to reported procedures (see examples below). As examples, we chose two enzymes, formate dehydrogenase (FDH) and D-amino acid oxidase (DAAO), because these biocatalysts represent two extreme cases of enzymes with high and very low homology. The milestones of this study are considered below.

The choice of enzyme is one of the most complicated issues. The differences between the properties of the same enzyme, but isolated from different sources, can be readily assessed by forming a reduced nicotinamidine adenine dinucleotide phosphate (NADPH) regeneration system based on formate dehydrogenase (EC 1.2.12) (Fig. 2). The FDH-catalyzed reaction in the general form in depicted in Scheme 1.

Fig. 2

Structures of FDH dimer from Pseudomonas sp.101 (a) and monomeric subunits of enzymes from Candida boidinii (b) and Arabidopsis thaliana (c). The structural parameters were retrieved from RCSB PDB (codes 2GUG, 8HTY, and 3NAQ, respectively) and are represented using the PyMOL program (version 1.7.6, Schrödinger, LLC)

Scheme 1

The specificity of dehydrogenases to NAD⁺/NADP⁺ is quantitatively expressed as coenzyme preference (CP) determined by formula (1) ⁶⁸

(1)

where k_cat is the catalytic constant, K_M is the Michaelis constant.

It was found that FDH from the bacterium Pseudomonas sp.101 (PseFDH) is characterized by CP of 2400, while the same enzyme from the yeast Candida methylica and C. boidinii (CboFDH) have CP of 250 000. It may seem that a 100-fold difference between CPs for bacterial and yeast enzymes is not high. However, in the case of PseFDH, the obtained mutants had a catalytic efficiency (k_cat/K_M) and specific activity for NADP⁺ equal to those for wild-type PseFDH for NAD⁺. In addition, the above values of the best yeast FDH mutants for NADP⁺ were 5 – 10 times lower.⁹⁵ While choosing the parent enzyme, one should also take into account characteristics that determine the production cost and application conditions. For the considered example, stability to the action of proteases is important, because the inactivation by protease impurity is one of the main reasons for the loss of activity during enzyme storage. The PseFDH enzyme is exceptionally stable to proteases; therefore, it can be stored at +4°C. The oxidation or modification of the SH groups of Cys residues also leads to enzyme inactivation. In the case of PseFDH, the mutants resulting from the replacement of two Cys residues did not lose activity for more than 10 years when stored in a phosphate buffer at +4°C, whereas CboFDH in which two Cys residues have been replaced should still be stored at –20°C in 50% glycerol.⁹⁶

Thus, the choice of the initial enzyme is highly important and can be performed using two main approaches.

1. Genome mining. The search for potential D-amino acid oxidases (DAAO) (Fig. 3) catalyzing the amino acid oxidation (Scheme 2) was performed in the thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaDAAO).^97, 98

Fig. 3

Structures of D-amino acid oxidase (a), D-aspartate oxidase from O. parapolymorpha (b), and human D-aspartate oxidase (c). The structural parameters were retrieved from the UniProt (W1QLN4 and W1Q8E7) and RCSB PDB (6RKF) data bases, respectively.

Scheme 2

The genome of this yeast was sequenced in 2013, and genome annotation revealed only two genes of potential DAAO, which had errors. Classic DAAO with a broad range of substrate specificity was annotated as specific D-aspartate oxidase (DASPO), while DASPO was interpreted as DAAO. Thorough analysis revealed five DAAO genes and one DASPO gene in the genome of O. parapolymorpha DL-1. It is noteworthy that formerly, it was considered ⁹⁷ that only one DAAO and one DASPO gene should be present in yeast genomes. The six genes were cloned and expressed in Escherichia coli cells. All enzymes turned out to be oxidases and differed in the substrate specificity and pH activity profile. Four DAAOs had no analogues in the literature. Analysis of the catalytic properties showed that various OpaDAAOs were superior in activity to previously described DAAOs and DASPOs from other sources.⁹⁸ Three-dimensional structures were determined for two OpaDAAOs. This gave a base with established structure – function relationships, which was used in the studies according to the second approach.

2. Genome screening. In this case, the labour intensity and the overall success of the work are determined by the degree of conservation of the primary structure. For example, in the case of FDH, the search for these enzymes in various genomes is quite simple, since the homology is > 50% even between distant organisms.⁹⁶ Formate dehydrogenase from Staphylococcus aureus (SauFDH) with a homology of only 40% was an exception.⁹⁶ According to evolutionary analysis, SauFDH belongs to a separate branch of evolution. The SauFDH gene encoding the FDH synthesis was cloned and expressed. The specific activity of this enzyme was found to be 2.5 times higher than that of other described formate dehydrogenases.⁹⁹

In the case of DAAO, the homology level is not more than 30 – 35%.¹⁰⁰ Therefore, for reliable identification of genes of potential DAAOs in the genomes of extremophile bacteria and archaea, one more stage of enzyme selection was added, that is, simulation of 3D structures and their comparison with experimental and model structures of known DAAOs and glycine oxidases. As a result, ten DAAOs were found in bacteria and one enzyme was found for the first time in archaeal cells. Analysis of the active site structure for DAAO from the bacterium Natronosporangium hydrolyticum ACPA39 (NhyDAAO) suggested that this enzyme could be highly specific to D-Phe (used to diagnose gestational diabetes in pregnant women); this was confirmed after NhyDAAO was cloned and expressed in E. coli cells.¹⁰¹

Analysis of the model active site structure of DAAO from the archaea Natrarchaeobius halalkaliphilus AArcht4 (NhaDAAO) showed that this enzyme can oxidize not only usual D-amino acids but also their N-substituted analogues. The produced recombinant NhaDAAO was found to actually exhibit this unusual specificity. Catalysis of the oxidation of mono- and trimethylated Gly (sarcosine and betaine, respectively) is of particular interest. The oxidation of betaine with enzymes has not been described in the literature.

The selected enzyme is optimized using both random mutagenesis (RM) and rational design methods. RM was most popular from the mid-1980s to the late 1990, but now rational design plays a major role in protein engineering, because it is much more efficient and cost effective. The studies are carried out along several lines: improvement of catalytic properties, creation of enzymes with new properties, and increase in the temperature and operational stability.

Protein engineering experiments were carried out with various enzymes such as FDH,¹⁰² DAAO,¹⁰³ peroxidase (PO),¹⁰⁴ penicillin acylase (PA),¹⁰⁵ α-D-amino acid ester hydrolase,¹⁰⁶ phenylacetone monooxygenase,¹⁰⁷ and so on. The FDH ⁷⁶ and DAAO mutants,¹⁰³ in which 5 – 7 substitutions resulted in enhanced catalytic properties and higher thermal and chemical stability, proved to be most interesting. Immobilized FDH and DAAO retained the activity at 80 and 90°C. The mutant peroxidase had an increased electron transfer efficiency within the protein globule.¹⁰⁴ The results of engineering of FDH from the yeast O. parapolymorpha DL-1 were unexpected. During cloning, the Gly or Ala residue was added to the N-terminus of the enzyme.¹⁰⁸ The Gly residue did not affect the properties of OpaFDH, while the addition of Ala induced a four-fold increase in the enzyme thermal stability.¹⁰⁸ The latest achievements and current trends of protein engineering can be found in the literature.¹⁰⁹

The approach that implies combining two or more enzymes into one polypeptide chain (creation of fusion proteins) proved to be efficient. Two types of hybrid biocatalysts were obtained: FDH with cytochrome P450 BM3 monooxygenase ¹¹⁰ or phenyl­acetone monooxygenase.¹¹¹ In the case of the FDH-P450 fusion construct, the efficiency of conversion of various substrates was 6- to 60-fold higher than that attained with a mixture of single enzymes.¹¹⁰

Thus, advances in bioinformatics, sequencing, and structural biology enabled the selection and engineering of effective enzymes for many areas of science and technology. A mere listing of recently developed processes involving biocatalysts would have taken several pages. To confirm the significant role of enzymes in the development and progress of modern technologies, it is sufficent to mention that enzymes are used as components of washing detergents and food supplements in agriculture (see also Section 1). Regarding the production output and cost, these two application areas alone account for more than a half of the market of enzyme products (see https://www.gminsights.com/industry-analysis/enzymes-market), which exceeded 20 billion dollars per year in 2022, according to various estimates. One more vivid example of application of enzyme engineering in modern technologies is the production of the Saphir Bst2.0 Turbo DNA polymerase mutant for the diagnosis of coronavirus SARS Cov-2 by isothermal polymerase chain reaction performed by Jena Bioscience within the shortest time possible (see https://www.jenabioscience.com/molecular-biology/isothermal-amplification-lamp/polymerases/pcr-390-saphir-bst-turbo-polymerase). Currently, these technologies are widely used to produce highly stable and active FDH and develop effective FDH-based biocatalysts for atmospheric CO₂ fixation.⁹⁴

One of the natural bioluminescent systems (Fig. 4, Scheme 3 and Scheme 4) is the luciferin–luciferase system of the Luciola fireflies (see Fig. 4a). The bioluminescence (emission of visible light in the 400 – 700 nm wavelength range) occurs during the oxidation of organic substrate (luciferin) catalyzed by the luciferase enzyme (EC 1.13.12.7).. In the enzyme molecule, two domains (large N-terminal domain and small C-terminal domain) are connected by a mobile non-structured polypeptide loop. Particularly the dynamic structure of the protein plays an important role in the function of firefly luciferase,¹¹² which can assume three different conformations: open conformation (in the absence of substrates) and two closed catalytic conformations corresponding to single reaction steps (adenylation and oxidation) (Scheme 3, see also Fig. 4).

Fig. 4

Structures of luciferase (Luc) from firefly Luciola mingrelica (a), bacterium Photobacterium leiognathi (b), and fungus Armillaria mellea (c). The structural parameters were retrieved from the RCSB PDB (codes 2D1R and 6FRI) and UniProt (A0A3G9JTR4) databases, respectively.

Scheme 3

Scheme 4

Upon binding of substrates, luciferin and adenosine triphosphate (ATP) (see Scheme 3), the free conformation of Luc is transformed into the adenylating conformation: the domains approach each other and are rotated through 90° relative to the orientation in the free conformation.^113, 114

Luciferin adenylation results in the formation of luciferyl adenylate, which is anhydride of a carboxylic and phosphoric acids. Unlike luciferin, it is highly reactive in the oxidation. The catalytically important C-domain residues (Lys529 and Thr527) are incorporated into the enzyme active site and promote the formation of luciferyl adenylate. In the oxidative conformation, C-domain is rotated around the N-domain by ~ 140°, and a part of the C-domain, Lys443 residue, enters the active site. The oxidative conformation is very unstable and is needed only to catalyze the oxidation of luciferyl adenylate. When light is emitted, the enzyme returns to the adenylating conformation.¹¹⁴ Owing to the dynamic structure of the protein, an active site configuration optimal for each step is implemented.

An important function of the Luc protein globule is the influence on bioluminescence spectra. The product of enzymatic reaction, oxyluciferin, can exist in solutions at various pH as six different species ^112, 113 (Fig. 5). Phenol forms I – III exist in non-polar solvents or at a very low pH; their fluorescence maximum (λ_max) occurs in the electronic spectrum at 450 nm. This blue luminescence was not observed for Luc. Phenolate forms IV–VI exhibit yellow-green (λ_max = 550 – 570 nm) or red (λ_max = ~ 620 nm) fluorescence, which is typical of the bioluminescence of this enzyme.

Fig. 5

Structures of oxyluciferin forms: I – III are phenol; IV–VI are phenolate; I, IV are enolate; II, V are enol; and III, VI are ketone forms. The values in parentheses are emission wavelengths. The Figure was created by the authors using published data.¹¹³

Studies of steady-state and subnanosecond time-resolved fluorescence of oxyluciferin and its structural analogues showed that bioluminescence in the firefly luciferin – luciferase system is generated by various tautomeric forms of electronically excited oxyluciferin, which are formed in the luciferase active site.^112, 115 The main factor determining the bioluminescence colour is the microenvironment of the emitter, which is located in the enzyme active site, where only one electronically excited product molecule is formed.¹¹⁶

Particularly the emitter molecule is a bioluminescent probe, which characterizes the state of its microenvironment at the instant of light emission.¹¹² A superposition of two or three forms of the emitters (E¹ – E³) detected in the bioluminescence spectra attests to the coexistence of various Luc conformers in a dynamic equilibrium in the reaction mixture. Each Luc conformer contains only one type of the emitter forms indicated in Fig. 5: ketone, enol, or enolate in Eqn (2), respectively.

(2)

By analysis of bioluminescence spectra, it is possible to qualitatively and quantitatively identify various enzyme conformers and trace the variation of their concentrations upon the change in the external conditions or luciferase mutations. For example, after a single substitution (Tyr35Asn or Tyr35His), the bioluminescence spectrum no longer depends on pH.^117, 118 The Tyr35 residue adjoins loop 233 – 237, the position of which is important to maintain the closed conformation of the luciferase active site needed for green bioluminescence to occur.

When the bulky aromatic residue Tyr35 is replaced by smaller Asn or His, the close packing near position 35 becomes more stable, and loop 233 – 237 is retained even at lower pH; hence, the closed conformation is not disrupted.¹¹⁷ Conversely, the His433Tyr mutation induces a shift of the bioluminescence λ_max at pH 7.8 (pH optimum of enzyme activity) from 566 to 606 nm, which is attributable to changes in the relative contents of various forms of the emitter.¹¹⁸

The His433 residue is located in the mobile loop formed by the Tyr427 – Phe435 amino acid sequence, which connects the N- and C-domains of Luc. This loop can be considered as a hinge linking the two luciferase domains. A temperature rise can also lead to increasing amplitude of the thermal vibrations of domains relative to each other and to increasing concentration of the conformer that generates red emission. It was shown for the luciferase of Luciola mingrelica and for some single mutants at Glu457 that the fraction of the red emitter at 42°C increases to 90% for native luciferase and to 100% for mutants.¹¹⁶

Directed evolution was used to obtain a mutant L. mingrelica firefly luciferase with eight amino acid substitutions (Ser118Cys, Cys146Ser, Lys156Arg, Arg211Leu, Thr213Ser, Ala217Val, Glu356Lys, and Ser364Cys), the thermal stability of which increased by a factor of 66 at 42°C (Fig. 6).^113, 119 The catalytic properties of the mutant were considerably enhanced compared to those of many native and other known mutants of this enzyme.

Fig. 6

Structure of the luciferase from Luciola mingrelica (Uniprot Q26304). The amino acid residues subjected to site-directed mutagenesis are shown by spheres. The Figure was created by the authors using published data.^{113, 119}

The Luc protein engineering design made it possible to develop a highly sensitive quantitative method for ATP analysis and a live cell detection method. Practical aspects of using this luciferase mutant for bioluminescent ATP assay, which is widely applied in many fields of science, industry, and medicine, are presented in detail in a review.¹²⁰

Organophosphate hydrolase (OPH, EC 3.1.8.1) (Fig. 7), which exists in nature as a homodimer, attracts considerable attention for both theory and practice, owing to its catalytic characteristics, which are manifested in the hydrolysis of various organophosphorus compounds (OPC),^{121 – 124} mycotoxins,^125, 126 and bacterial quorum sensing molecules (Scheme 5).^127, 128

Fig. 7

Structures of the organophosphate hydrolase dimer from Brevundimonas diminuta (a) and monomeric enzyme subunits from Agrobacterium radiobacter (b) and Mycobacterium tuberculosis (c). The structural parameters were retrieved from RCSB PDB (codes 1QW7, 2D2G, and 4IF2, respectively). The catalytically important Co²⁺ and Zn²⁺ ions in the enzyme active sites are shown as pink spheres.

Scheme 5

This enzyme proved to be efficient as a component of antidotes used in experiments with various animals ^{129 – 131} and as parts of antimicrobial compositions ^{132 – 134} tested on various microbial cells. The initial phase of investigation on this enzyme was focused on enhancement of its catalytic properties;¹³³ later, genetic modification of the enzyme was performed to simplify the isolation and purification procedures and thus to provide for active commercial use of OPH.

For this purpose, an amino acid sequence consisting of several, most often six (His₆), histidine residues was genetically introduced to the N- or C-terminus of the protein, which ensured the affinity binding of the His₆-OPH or OPH-His₆ enzyme, respectively, to various metal-chelating carriers.^135, 136 The intensity of interactions between the carrier and the genetically modified OPH is determined by the nature of the metal in the carrier and the polyhistidine (polyHis) sequence length and location at a particular terminus of the protein molecule.¹³⁷ As the length of polyHis sequence increases, the interaction of the carrier with the enzyme is enhanced, resulting in hindered elution of the protein and successful immobilization to give a stabilized biocatalyst. The increase in the polyHis length in OPH gives rise to enzyme oligomers (tetra- and octamers) and simultaneously markedly increases the enzyme stability.¹³⁶

Of considerable interest was the effect of the length and location (C- or N-terminus) of the genetically introduced polyHis sequence on the catalytic properties of the modified enzyme such as catalytic efficiency, substrate specificity, and the pH and temperature optimum for catalytic activity.^136, 137 It was found that genetic modification of OPH particularly at the N-terminus using polyHis sequences markedly enhances the catalytic performance of the enzyme toward substrates with more bulky substituents at phosphorus such as organophosphorus pesticides, warfare agents, and the products of their hydrolysis (Vx, diisopropyl fluorophosphate, methylphosphonic esters, etc.).^136, 137 The cause for these changes in the properties of enzyme derivatives was identified using computer simulation techniques. It was found that the polyHis-tagging of the OPH molecule involved in the dimer formation induces a conformational change, which results in pronounced opening of the hydrophobic pockets that form the substrate binding site. In the presence of the polyHis sequence, the enzyme conformation is slightly stretched at the outer edges of each OPH subunit forming the dimer (see Fig. 7a); this promotes a minor opening of the pockets, that is, the enzyme active sites that are located more closely to the centre of the dimer molecule. The fabrication of His₆-OPH-based recombinant fusion proteins ¹³⁶ combining the properties of OPH and other proteins and enzymes through their biosynthetic connection into a single molecule with a short amino acid linker further increased the tendency of enzyme – substrate binding pockets to expand and decreased the contact between the OPH subunits in the dimer to the extent of formation of stable monomeric structures with altered catalytic properties.

Thus, using the genetic modification of OPH with polyHis sequences, it is possible

(1) to simplify and accelerate the preparation of highly purified active enzyme by using supermacroporous carriers suitable for flow metal ion affinity chromatography;

(2) to combine enzyme immobilization on affinity carriers with purification to obtain various forms of catalytically active materials to degrade toxic compounds;

(3) to change physicochemical characteristics of OPH (the optimal temperature and pH for activity and stability in different media);

(4) to improve the catalytic properties of the enzyme towards various substrates, thus expanding the substrate specificity of His₆-OPH;

(5) to fabricate various fusion proteins containing increasing percentage of enzymes with combined properties in the soluble form upon expression in E. coli cells for the subsequent use to detect and degrade OPC in industry, agriculture, and ecology.

The post-translational modifications (PTMs) of proteins and enzymes underlie the control of protein–protein interactions that determine the vital activity of organisms (Fig. 8). For many proteins, no other functions are known except for the PTM-controlled ability to bind to other biomolecules. Currently, PTM types that determine the final protein function are being studied starting from the synthesis.¹³⁸ The biggest problems arise in the study of recombinant proteins, especially when they are expressed in foreign host cells. For example, the results obtained for recombinant eukaryotic proteins synthesized in bacteria are almost inapplicable for studying the functioning of these proteins in eukaryotic organisms. The post-translational modifications can be subdivided into three main types.

Fig. 8

Main approaches used in protein engineering and examples of protein PTMs: the introduced group is highlighted by a colour; hydrogen atoms are not shown for simplicity, and chemical elements are shown in blue (nitrogen), red (oxygen), white or purple (carbon), and orange (phosphorus).

(1) First type modifications: PTMs that control the interactions with other biomolecules and with structural elements of whole cells. There are quite a few such modifications, with protein phosphorylation and dephosphorylation catalyzed by special enzymes, protein kinases and protein phosphatases (Fig. 9), being most popular among them.¹⁰⁹ The introduction of additional negatively charged phosphate groups to serine, threonine, or tyrosine residue changes the protein interaction pathways with other biomolecules. The phosphatase-catalyzed hydrolysis reactions are depicted in the general form in Scheme 6.

Fig. 9

Structures of human protein phosphatase (a), human alkaline phosphatase (b), and phytase from Aspergillus niger (c). The structural parameters were retrieved from RCSB PDB (codes 6DNO, 1ZED, and 3K4Q, respectively). The catalytically significant Mg²⁺/Zn²⁺ ions in the enzyme active sites are shown as spheres.

Scheme 6

There are a lot of data on the key role of protein phosphorylation in the regulation of the vital activity in all organisms. A similar process involving sulfation and desulfation at tyrosine residues, which is also accomplished by special enzymes, has been less studied. The development of this research area implies gaining more in-depth knowledge about the role of well-known PTMs [non-enzymatic glycation, glycosylation, oxidation, ubiquitinylation, SUMOylation (SUMO is small ubiquitin-like modifier), and acetylation] and the search for new protein modifications to control their interaction with other biomolecules.

(2) The second type modifications are PTMs that affect the protein structure and change the conditions of protein functioning. These modifications not only alter the interactions of proteins with biomolecules, but also directly influence the catalytic function (change the substrate binding); they affect the amino acid residues of the active site and, as a rule, inactivate the enzymes. However, examples of increase in the enzyme activity upon modification of catalytically important amino acid residues are also known.^{139 – 141}

Modification of allosteric sites may cause both enzyme activation and inhibition. The reversible modification of amino acid residues of the active and regulatory sites can become a significant element in the regulation of catalytic activity of the enzyme and a particular metabolic pathway as a whole. In this field, promising trends are those related to the search for new ways of enzyme protection from irreversible inactivation, which is especially important for biotechnological applications. For example, the most vulnerable SH groups of the Cys residues involved in the catalysis are protected from irreversible oxidation using methods that exist in natural objects, e.g., additional Cys residues are introduced into the enzyme. Investigation of the role of PTMs in the regulation of protein properties allows task-oriented site-specific mutagenesis to produce enzymes with desired properties. For example, the replacement of Cys with serine residues may induce changes in the enzyme function that take place upon SH group oxidation to sulfenic acid (S – OH) group.¹⁴²

(3) The third type modifications are PTMs of amino acid residues in the active site that give rise to a new type of enzyme catalytic activity. This type of PTMs is most interesting, because there are few such examples. In addition, these modifications are closely connected with the effect of moonlighting proteins. These proteins are characterized by the presence of several functions in addition to the main function. Apart from their main catalytic activity, enzymes characterized as moonlighting proteins usually can react with other biological molecules being involved in a variety of processes. For example, they can bind to nucleic acids, thus regulating their transcription or translation, to various signalling proteins, thus inducing apoptosis, etc. Furthermore, while retaining the main catalytic activity, moonlighting proteins can perform other reactions. The additional reactions are usually not very complicated and are closely related to the main catalytic action, being due to the ability of enzymes to bind substrate analogues and to convert them as required. The existence of such moonlighting proteins with diverse functions, including a few types of catalytic activity, puts in question, to a certain extent, the well-known hypothesis of molecular biology, namely, one gene–one protein hypothesis. It should be noted that in most cases, moonlighting proteins fully retain their main function, i.e., they catalyze the reaction for which the enzyme is intended. However, simultaneously, these proteins participate in a few more processes. The post-translational modifications may enhance the ability of moonlighting proteins to perform additional functions. In some cases, after the modification of catalytically important amino acid residues in the active site, the enzyme starts to catalyze an absolutely new reaction to which it was previously inert. Certainly, the main catalytic activity fully disappears. Hence, owing to PTMs, the one gene–one enzyme hypothesis is transformed into one gene–two enzymes. Unfortunately, examples of this sort in which the mechanism has been convincingly proved are almost absent in the literature.

A well-studied enzyme of this type is glyceraldehyde 3-phosphate dehydrogenase (GAPD, EC 1.2.1.12), which oxidizes glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate by reducing NAD to NADH. Modification of the catalytically important Cys residue with GAPD completely inhibits the dehydrogenase reaction. However, GAPD can be easily converted to 1,3-diphosphoglycerate phosphatase upon oxidation of Cys to sulfenic acid residue with hydrogen peroxide, other reactive oxygen species, and even nitric oxide.¹⁴³ The appearance of a new type of activity in the enzyme intended for dehydrogenase reaction makes difference for the regulation of glycolysis and energetic processes in the cell. Owing to cleavage of 1,3-diphosphoglycerate, the oxidation and phosphorylation steps in the glycolysis are uncoupled, which is necessary for effective ATP synthesis in mitochondria under aerobic conditions.

The oxidation of Cys residues can also change the function of apurinic/apyrimidinic endonuclease-1 (APE1).^144, 145 The oxidation of SH groups of this enzyme to sulfenic acid groups is accompanied by a decrease in the endonuclease activity and increase in the G-quadruplex affinity. The oxidation results in a change in the function of APE1, which starts to participate in the modulation of transcription processes.

The above data indicate that PTM of one amino acid residue results in the conversion of the enzyme into a protein with a different catalytic activity, thus confirming the existence of one gene – two enzymes model. However, particular examples illustrating a change in the enzyme function upon PTMs are not numerous. Most often, they are discovered accidentally and are not always given due attention. These secondary types of catalytic activity can make an important contribution to the targeted regulation of metabolism and can be used to produce enzymes with new properties by genetic engineering.

Cereal grains (wheat, rye, oats, and barley) are widely used to produce animal feed. The feeds contain 13 – 15% non-starch polysaccharides (NSPs) such as cellulose, β-glucans, and xylans, which reduce the digestibility, as they hinder the access of digestive enzymes to nutrients (starch and proteins). Monogastric animals and poultry do not have their own enzymes capable of efficiently cleaving NSPs; therefore, enzyme preparations (EPs) composed of polysaccharide hydrolases (cellulases, β-glucanases, and xylanases) are used as additives in the feed production (Fig. 10). The enzymatic cleavage of NSPs (shown in Scheme 7 as a chain of monosaccharides) increases the uptake of nutrients.¹⁴⁶

Fig. 10

Structures of xylanase from Penicillium canescens (a), endoglucanase from P. verruculosum (b), and β-glucanase from Talaromyces funiculosus (c). The structural parameters were retrieved from RCSB PDB (codes 4F8X, 5I6S, and 6IMW, respectively)

Scheme 7

The economically feasible production of enzymes requires high productivity of cells. For this purpose, either mutagenesis and selection of enzyme producers are performed, or genetic engineering methods are used to modify the strains that secrete the target enzymes or complexes of the target enzymes. This genetic modification of producers makes it possible to deliberately change the properties of proteins, for example, to increase their operational stability and catalytic activity towards a number of substrates.¹¹⁷ Thus, using a few successive stages of induced mutagenesis, the wild type strain was converted to highly productive Penicillium verruculosum strain B221-151, which was further converted, also via mutagenesis, to the P. verruculosum B1-537 strain (ΔniaD), characterized by high secretion of extracellular protein (up to 50 – 60 g L^–1). This strain was an auxotroph with a defect in the niaD gene encoding nitrate reductase; this was used as a selection criterion for screening recombinant strains.¹⁴⁷

The P. verruculosum B1-537 strain produced a set of extracellular cellulases that were superior in activity to the analogues traditionally used for bioconversion of renewable plant raw materials. This strain was used to make recombinant producers of NSP enzymes,¹⁴⁸ out of which heterologous endoglucanase I from Trichoderma reesei (EGI), homologous EGII, and heterologous endoxylanase E from P. canescens (XylE) were chosen.

This gave cells that simultaneously produced EGI and EGII, EGII and XylE, and an XylE-producing strain. Using these strains, the plant Agroferment (https://agroferment.ru) currently produces a number of EPs as additives that improve feed digestibility (Table 3, Table 4). The specific endoglucanase (assessed using CMC) and xylanase activities of these EPs were 1.5 – 3.7 and 1.7 – 3.3 times, respectively, higher than these values for the control EP B1-537 produced with the P. verruculosum recipient strain B1-537 (ΔniaD). The total cellobiohydrolase (CBH) activity of feed EPs (estimated by MCC) decreased 1.3 – 1.8-fold. The control preparation B1-537 included a large amount of exodepolymerases CBHI and CBHII (58% of the total proteins), which is important for cellulose degradation to soluble sugars, but insignificant for a feed additive. In feed EPs, the CBH level decreased ~ 2.5-fold, while the content of endodepolymerases that degrade NSPs substantially increased.

Table 3

\[ \]

Specific activity of feed enzyme preparations on various substrates.¹⁴⁶

(3)

Table 4

\[ \]

Component composition of feed enzyme preparations.¹⁴⁶

(4)

Generally, the data of EP composition were correlated with their specific activity: an increase in the EG and XylE contents resulted in increasing specific CMCase and xylanase activities, respectively, while a decrease in the CBH content led to decreasing specific activity relative to MCC.

Phytates (D-myo-inositol-1,2,3,4,5,6-hexakis-phosphoric acid salts) are a stored form of phosphorus in the seeds of higher plants. The phytic phosphorus content is 60 – 88% of the total phosphorus in grains.^146, 147 Due to very low phytase activity in the digestive system, this form of phosphorus is inaccessible to monogastric animals, who do not digest 60 – 70% of phosphorus from plant-based feeds. In addition, phytic acid binds to Ca²⁺, Zn²⁺, Cu²⁺, Mg²⁺, Fe²⁺, and Fe³⁺ ions and to proteins, starch, and lipids, giving poorly soluble compounds, which substantially reduces the nutritional value of feed. Therefore, phytase (see Fig. 9), which hydrolyzes phytates to inorganic phosphate and myo-inositol, is widely used as a feed supplement.¹⁴⁶

A highly active recombinant producer of heterologous phytase A from Aspergillus niger (phytase-3) was obtained with the P. verruculosum expression system. Using this producer, the Agroferment plant currently manufactures enzyme preparations Argofit with phytase contents of up to 50% relative to the total protein content and Agrofit Pro (a mixture of phytase and NSP enzymes).

Thus, new high-capacity recombinant producing strains have been developed on the platform of the gene expression system of P. verruculosum microscopic fungus and are used for industrial-scale production of new-generation feed enzyme preparations in Russia.^148, 149