Structure and Intrinsic Disorder in Enzymology

By Vladimir N Uversky

Structure and Intrinsic Disorder in Enzymology offers a direct, yet comprehensive presentation of the fundamental concepts, characteristics and functions of intrinsically disordered enzymes, along with valuable notes and technical insights powering new research in this emerging field. Here, more than twenty international experts examine protein flexibility and cryo-enzymology, hierarchies of intrinsic disorder, methods for measurement of disorder in proteins, bioinformatics tools for predictions of structure, disorder and function, protein promiscuity, protein moonlighting, globular enzymes, intrinsic disorder and allosteric regulation, protein crowding, intrinsic disorder in post-translational, and much more.

Chapters also review methods for study, as well as evolving technology to support new research across academic, industrial and pharmaceutical labs.

• Unifies the roles of intrinsic disorder and structure in the functioning of enzymes and proteins
• Examines a range of enzyme and protein characteristics, their relationship to intrinsic disorder, and methods for study
• Features chapter contributions from international leaders in the field

• Language: English
• Publisher: Academic Press
• Release date: Nov 17, 2022
• ISBN: 9780323995344

Book preview

Preface

Munishwar Nath Gupta and Vladimir N. Uversky

In recent years, every time one of us had the occasion to mention intrinsic disorder to biochemists, invariably the response has been like—Is it higher flexibility? Many structural biologists (of the kind who use X-ray diffraction to probe protein structure) recognize that the lack of structure has been frequently seen in many cases of protein structures. Hence, it was felt that there is a need for a volume, which describes proteins not in terms of the binary of structured proteins and intrinsically disordered proteins but as a continuum of both structured regions and disordered regions seamlessly playing the respective functional roles. That way the message about the importance of intrinsic order lands softly without people feeling that the old (protein) world order has collapsed and has been replaced by a new one! So, it is not about structure vs disorder; it is about structure and disorder. There is a second perception which this volume tries to alter—that not just the other noncatalytic proteins but enzymes also utilize intrinsic disorder when it is more useful for a certain role.

In last two decades or so, our perceptions of the enzyme specificity as lock-and-key-type correspondence have changed gradually. The specificity is context-dependent, both in terms of space and time. The former relates to cellular location and the latter to the concentration of a metabolite in the real time. Often, there is no one-to-one relationship between the lock and the key. Moonlighting and promiscuity have emerged as fairly common protein traits. This volume tries to capture these paradigm shifts as well.

The early chapters emphasize that just like structure in proteins, disorder is also sequence-dependent; just like Lindstrom-Lang’s hierarchy in structured proteins, there is one in disordered proteins as well! Bioinformatics is hugely useful for finding and characterization of intrinsically disordered proteins and intrinsically disordered protein regions.

The last few chapters focus more on the consequences of disorder and how biology exploits it for creating ease of posttranslational modifications, curvatures, and condensates. Prions also seem to rely upon disorder.

We thank all the authors who took time to contribute. We thank Dr. Peter Linsley (Elsevier) for encouraging us in this task and Ms. Sara Pianawalla (Elsevier) for overseeing the production of this volume.

This volume is one of the early ones in the new series Foundations and Frontiers in Enzymology launched by Elsevier. Hopefully, this volume captures the snapshots of foundations (there is a chapter on history of enzymology!) as well as what is happening at the frontiers of enzymology as well in the world of proteins.

We hope that all those interested in enzymology will find this volume useful.

Chapter 1

Enzymology: early insights

Munishwar Nath Gupta¹ and Vladimir N. Uversky²,    ¹Former Emeritus Professor Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Delhi, India,    ²Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, United States

Abstract

The modern view of enzymes as complex biological machines with unique structures, complex catalytic mechanisms, and specific conformational dynamics and the wide spread of their industrial and pharmaceutical applications are the result of extensive efforts of a big army of scientists with very different backgrounds. In this chapter, we provide a brief admittedly subjective overview of the most important steps that led to the current understanding of the enzyme structure and function. We describe developments in applied enzymology and white biotechnology, talk about enzymes in neat solvents, and also describe some common myths about applications of enzymes.

Keywords

Enzymology; applied enzymology; white biotechnology; catalysis; protein purification; protein immobilization; structural biology

1.1 Introduction

The pioneers mostly have to fly blind. Early history of enzymes as biocatalysts illustrates this so well. In early days of biochemistry, most efforts were related to understanding basics of enzymology. For that reason the early history of enzymology is intertwined with how biochemistry evolved at that early phase. It has been pointed out that many recent disciplines/subdisciplines, such as biotechnology, bioinformatics, molecular biology, and biochemical engineering, have an umbilical cord with biocatalysis [1,2]. Let us backtrack and see how it all started with the chemical catalysts.

At present, it feels surreal to think that two very well-known chemists Justus Freiherr von Liebig (1803–73) and Jöns Jacob Berzelius (1779–1848) argued about the description of catalysis [3]. Liebig, while talking of the views of Berzelius about existence of catalysis, went as far as to say: The assumption of this new force is detrimental to the progress of science [3]. Furthermore, Liebig was one of the first scientists to recognize that there is no a rigid boundary between organic and inorganic and predicted that production of all organic substances no longer belongs just to living organisms. It must be seen as not only probable, but as certain, that we shall be able to produce them in our laboratories [4].

The critical feature about the nature of scientific inquiry is that in the long run it does not matter who said it; it is only important what was said. As new technologies/tools come into play, every generation gets a chance to reexamine what was said. One hallmark of a good scientist is that he/she is open to factoring in any new data. Leibig, by 1870, accepted the ideas of others, which by then included Jean Baptiste André Dumas (1800–84), Jacobus Henricus van’t Hoff (1852–1911), and Friedrich Wilhelm Ostwald (1853–1932), about catalysis. The nature of biocatalysis was first discussed in the context of fermentation. For quite some time, formed ferments (living yeast) and unformed ferments (invertase, diastase, pepsin, etc. as isolated from organisms) came to be viewed as very different catalysts. It was Wilhelm Kuhne (1837–1900) who in 1878 replaced the term unformed ferments with enzymes [5]. The cell free extract of yeast catalyzing fermentation of glucose was demonstrated by Eduard Buchner (1860–1917). The catalysts in his extract were recognized as enzymes in line with the earlier thoughts of Kuhne. As yeast invertase preparation showed very high catalytic activity but failed to show the presence of tryptophan by the known color reaction, Richard Martin Willstätter (1872–1942) (during 1920–30) questioned the prevalent view that these enzymes were proteins. Because of the stature of Willstätter, James B. Sumner’s (1887–1955) crystallization of urease in 1926 was overlooked as an evidence in support of enzymes being proteins. The following years saw the crystallization of several other enzymes by John Howard Northrop (1891–1987) and Moses Kunitz (1887–1978) [3]. Thus it came to pass that scientists accepted that enzymes are protein in nature. Chemists had not become familiar with the existence of polymers. So, till ultracentrifuge was designed; there was lots of skepticism about the views of some that proteins are much larger than the organic molecules known at that time. Ultracentrifugation also showed that proteins are not polydispersed like polysaccharides [6].

It is interesting to recall that in 1800, Academy of the First French Republic announced a prize of 1 kg of gold to anybody who could discover how proteins function as catalysts. We now know the answer to a very large extent, though the prize was never given [6].

The early biochemists focused on respiration, digestion, and fermentation [3]. Borrowing from the area of fermentation, Jan Baptist van Helmont (1580–1644) suggested the term ferment to describe the agent, which carried out the chemical transformations of food stuff during the process of digestion [3]. As 19th century ended, saliva and juices of stomach, pancreas and intestines were recognised as containing these ferments [3]. Earlier in 1752, René Antoine Ferchault de Réaumur (1683–1757) fed kites food packed inside metallic tubes with holes and found that the food inside the ejected tubes had undergone digestion [3]. The nature of digestion as a chemical process was confirmed after this during 1765–1900, by the independent observations of Lazzaro Spallanzani (1729–99), Theodor Schwann (1810–82), and Wilhelm Kuhne (1837–1900). In parallel, Louis Pasteur (1822–95), starting in 1857, began his seminal work on fermentation and established that fermentation as well involves chemical transformations and can be carried out even when cellular integrity is not preserved. Eventually, it was found that there are many common reactions between glycolysis, digestion of glucose, and fermentation [3].

In 1814 Gottlieb Sigismund Constantin Kirchhoff (1764–1833) reported the hydrolysis of starch to glucose by a wheat extract. This ferment, also found later in malt extract and saliva, was called diastase [3]. In 1830 Pierre Jean Robiquet (1780–1840) described an albuminoid, which hydrolyzes amygdalin; this substance was studied further by Liebig and Friedrich Wohler (1800–82), who named it emulsin in 1837. Around the same period (second half of 19th century), van’t Hoff and Ostwald put the concept of catalysis on a firm footing and provided a quantitative framework for further studies on catalysis [3]. Enzymes eventually came to be recognized as the catalysts, which facilitate biochemical reactions in the living cells. Arthur Kornberg has provided an entertaining account of his seminar at Washington University, St. Louis in 1953, where the chairman of the host department questioned his statement that all chemical reactions in a cell are catalyzed by enzymes: Do you mean to tell us that something as simple as the hydration of carbon dioxide [to form bicarbonate] needs an enzyme?… Yes, Joe, cells have an enzyme called carbonic anhydrase [7].

Christian M. Heckmann and Francesca Paradisi have recently written a good account of the timelines of the major milestones in the history of enzymes [8].

1.2 Isolation and purification of proteins

After the biocatalysts turned out to be protein molecules, organic chemists got into the act of isolating/purifying these molecules. Organic chemists typically use distillation, sublimation, extraction, and crystallization for isolation and purification. Distillation was obviously not useful; sublimation did prove useful down the line for concentration of aqueous extracts of proteins in the form of freeze-drying to sublimate ice from frozen solutions in vacuum [9]. We have already referred to early pioneering efforts on crystallization of proteins. Unlike small molecular weight organic compounds, those protein crystals were not a pure preparation. Polymer chemistry was still not developed, and these early enzymologists had very little insight into properties of macromolecules. So, they did not expect contaminating proteins to co-crystalize along with the target protein. Precipitation turned out to be one of the major techniques for protein purification. It really developed as a part of the war efforts (around 1930 and beyond) in the laboratory of Edwin Joseph Cohn (1892–1953) where John Tileston Edsall (1902–2002) was working with him. The project was fractionation of blood plasma proteins [10]. Organic solvents, salts, and metal ions were the precipitating agents [11]. Later polymers, such as dextrans and polyethylene glycol (PEG), were used [11,12]. Thus precipitation has occupied a dominant position among what are known as nonchromatographic methods of separation of proteins. An excellent overview of such methods, including precipitation, is recommended to capture why these were preferred by biochemical engineers who were focused on development of large-scale separation methods [13]. One such scalable precipitation technique that uses a synergy of precipitation by salt and organic solvent is three-phase partitioning (TPP) that uses t-butanol and ammonium sulfate to precipitate enzymes and proteins from aqueous solutions [14,15].

It was the work on sequencing of bovine pancreatic ribonuclease A by a group of the Rockefeller researchers, Derek George Smyth, William Howard Stein (1911–80), and Stanford Moore (1913–82) [16], which laid the foundation of chromatographic separations of proteins [17]. Not far from that group, Christian Boehmer Anfinsen (1916–95) developed affinity chromatography as a high-resolution method for protein purification [18]. Introduction of cyanogen bromide coupling method for designing affinity matrices by Jerker Porath (1921–2016) facilitated quick and wider adoption of affinity chromatography [19]. Meanwhile, biochemical engineers were busy developing other large-scale separation methods. Aqueous two-phase separations, membrane-based separations, and expanded-bed chromatography exemplify these efforts [20].

During the middle phase of development of this area, it was common to talk of upstream and downstream separation strategies. At that time, affinity chromatography was used at the end of the downstream protocols for getting high purity proteins. The advent of production of recombinant proteins changed this forever [20–22]. This was preceded by the adoption of metal ion chelates and dyes as the affinity ligands. These could replace the more expensive affinity ligands like coenzymes, antibodies, and lectins. Recombinant DNA (rDNA) techniques allowed polyhistidine fusion tags to be inserted in the recombinant protein which allowed proteins to be captured straightway on immobilized chelates of copper and nickel (immobilized metal ion affinity chromatography referred to as IMAC) [23]. Later, many such fusion tags or affinity tags have been described [20–22]. This made the distinction between upstream and downstream methods irrelevant as the shorter protocols of directly using these commercially available IMAC columns became widely adopted [20–22].

Chromatographic columns require a clear feed and centrifugation at the industrial scale is a very costly step. At that point, the other option was using membranes. Membranes were still expensive in those days and were liable to get clogged too (membrane fouling) with contaminating proteins and other contaminants. Using affinity interactions in free solution turned out to be a brilliant strategy. Affinity precipitation, aqueous phase affinity extractions, and macro-affinity ligand TPP were such hybrid techniques [15]. Expanded-bed chromatography anyway allowed direct loading of crude suspensions but expanded-bed affinity chromatography turned it into high-resolution technique [20].

One bump in the technological progress on production of recombinant proteins was that when bacterial systems were used for heterologous overexpression, the desired protein was generally found in the form of insoluble inclusion bodies, which contained misfolded protein molecules. Many protein refolding methods have been developed over the years. Many separation methods (both chromatographic and nonchromatographic) also turned out to be helpful in refolding the proteins from the solubilized inclusion bodies [24]. In a twist to the tale, later many inclusion bodies as such were found to be fairly catalytically active [24].

1.3 The dawn of structural biology

The early tools used for understanding protein structure came from chemists. It is remarkable how much was learnt by chemical modification of proteins/enzymes using various monofunctional and bifunctional reagents [25,26]. The contents of the volume on protein structure in the series methods in enzymology published in 1967 capture the dominating role, in which chemistry played in that early era [27]. Most of it describes chemical modification methods. One last section mentions pH titrations, hydrogen-deuterium (H–D) exchange, ultra violet (UV) and visible spectroscopy, and fluorescence methods [27]. That early era also saw the use of viscosity measurements, optical rotatory dispersion (before circular dichroism displaced it after commercial circular dichroism (CD) instruments became available), infrared (IR) spectroscopy (mostly in solid state), and proteolytic digestion [28]. An important milestone using the last method was discovery of RNase S by Frederic Middlebrook Richards (1925–2009) and Paul J. Vithayathil [29]. RNase S is obtained by limited proteolytic digestion and retains full activity of the parent enzyme RNase A. Richards started working on this as a postdoc in the laboratory of Kaj Ulrik Linderstrøm-Lang (1896–1959) who later generously agreed to Richards continuing to work on that problem when he moved to Yale [30]. The active RNase S contains the small fragment S-peptide noncovalently associated with the larger fragment S-protein. In a way, this was the harbinger of concepts like drug–receptor and hormone–receptor interactions wherein noncovalent associations form the basis of a biological function. Equally interesting is the comment of Christian B. Anfinsen who quoted Kaj Ulrik Linderstrøm-Lang and John Schellman (1924–2014) …so that the protein molecule will often consists of a number of regions of varying structures and stability. He referred to RNase S and active complex of the proteolytic fragments of staphylococcal nuclease as examples of proteins having regions of varying stability [31]. It should be added that the region, where S-peptide interacts with S-protein in RNase S, differs from RNase A in being disordered [32]. RNase A was among the early enzymes, the conformation of which based upon X-ray diffraction was available [32,33]. In an interesting review on bovine pancreatic ribonuclease A by two leading workers in their era, it is mentioned that as a part of their contribution to war efforts, an industry processing beef offered a preparation of the enzyme free to anybody wanting to work on it. This led to perhaps extensive work on this particular enzyme [33]. RNase A was also the first enzyme, the primary sequence of which was established by the Rockefeller group [16]. Around the same period, Christian B. Anfinsen published his famous experiment on refolding of reduced and denatured RNase A. His famous observation that primary sequence has enough information for the protein to fold into the correct native structure laid the foundation of what later developed into bioinformatics [34,35].

When early information about conformations of proteins started becoming available, biologists were skeptical about the relevance of these crystal structures to the biological function of the enzymes. The debate was settled by the results from Frederic M. Richard’s laboratory, in which crystals of RNase S and carboxypeptidase A were shown to be catalytically active [36,37]. This paved the way for X-ray diffraction emerging as an acceptable and a powerful tool to probe protein structure.

One of the excellent sources for knowing the history about the roles of X-ray diffraction analysis in understanding protein structure is a book dedicated to Linus Carl Pauling (1901–94) on his 65th birthday with contributions from his students, colleagues, and friends [38]. John Cowdery Kendrew (1917–97), writing in that book, underscores the fact that the term molecular biology was actually first used in the context of understanding protein structure [38]. As the term is now used more in the context of new biology in the postdouble helix era, we prefer the more current phrase structural biology to delve into the fascinating history of how it all happened. Ironically, lately we talk of phrases like earth is flat or globalization has shrunk the world; in reality, the understanding protein structure involved a free-wheeling exchange of ideas between scientists working in different parts of the world. The well-known book The eighth day of creation depicts that well [39].

The early history of protein X-ray crystallographic analysis has been described by Dorothy Crowfoot Hodgkin (1910–94) and Dennis Parker Riley in the aforementioned book by Rich and Davidson [38]. The large crystals of pepsin were accidentally obtained by John Philpot while working briefly at Uppsala. These were given to John Desmond Bernal (1901–71) at Cambridge (United Kingdom), where Dorothy Crawfoot Hodgkin looked at them by X-ray diffraction in 1934 [40]. She later went on to get the Nobel for solving the structure of cobalamin by X-ray diffraction. However, the X-ray structure of the first protein to be published was that of sperm-whale myoglobin, which was reported in March, 1958 [41] by John Cowdery Kendrew along with five collaborators [38,39]. Just to indicate the challenges involved in solving the structure of the tetrameric hemoglobin (the subunits of which resemble myoglobin which is a monomeric protein), it took two more years before its structure could be established [42]. It is interesting to note Perutz recalling Disbelief and indignation – I had expected naively that people would be delighted to see this problem solved. In fact, everybody was angry [39]. Hemoglobin had already been the system on which Jacques Lucien Monod’s (1910–76) group focused to explain the allosteric interaction of proteins. So, its structure was expected to provide complete molecular picture of it allosteric behavior as well. That was not immediately obvious from the structure.

By this time, Linus Carl Pauling (1901–94) had unveiled the α-helix model in 1953 and was delighted to see its presence in myoglobin structure [39]. A related significant development was calculations of the allowed ranges of conformations for a pair of peptide units by Gopalasamudram Narayanan Ramachandran’s (1922–2001) group at Madras (as it was then called) [43], which led to what are often called Ramachandran’s maps that are plots of the dihedral angles related to successive peptide units [38].

The first enzyme whose X-ray structure was solved was hen egg white lysozyme [44]. While X-ray crystallography approaches continue to dominate, other tools based upon fluorescence, cryo-electron microscopy, and nuclear magnetic resonance (NMR) have begun to extend our understanding of structural biology [45,46].

1.4 The early work on enzyme kinetics

It was in 1880 that Charles-Adolphe Wurtz (1817–84) based on insolubilization of papain in the presence of fibrin suggested that an enzyme binds to its substrate before the catalysis is initiated [46,47]. It is however in 1913 that Leonor Michaelis (1875–1949) and Maud Leonora Menten (1879–1960) published their paper Die kinetik der invertinwirkung [48], an English translation of the paper was published in 2013 to mark a century of what has come to be known as Michaelis–Menten kinetics [49]. It was a remarkable paper in several respects. First, it acknowledged the earlier works of C. F. R. S. O’Sullivan and Frederick W. Tompson [50], Émile Duclaux (1840–1904) [51], Adrian John Brown (1852–1919) [52], and Victor Henri (1872–1940) [53]. It took into account the effect of pH, a concept which Søren Peter Lauritz Sørensen (1868–1939) had introduced only few years back in 1909 [54]. It also recognized that the products inhibit the reaction and hence reaction should be followed only during the initial period, when sufficient product molecules are yet to form to get a clear picture of the kinetics.

The next phase was to be able to track the formation of enzyme-substrate complex. It so happens that Michaelis had spent some time (1922–23) at Nagoya University in Japan, which apparently helped the development of biochemistry in that country [55]. Kunio Yagi, who was quite influenced by Michaelis’s work and worked at the same University but few decades later, reported the data on Michaelis complexes of D-amino acid oxidase [56]. An interesting subsequent paper by Yagi reported that D-amino acid oxidase could act on L-proline [57]. This was an early lead showing that enzymes are not absolutely stereo-specific. Also, as the breakdown of the E–S complex with L-amino acids was much slower, the formation/existence of E–S complex could be established.

At this point, it is worthwhile to mention paper by Joseph Kraut (1926–2012), which gives an excellent account of how the idea of the transition state complex during the enzyme catalysis developed in 1930 [58]. He mentions how John Burdon Sanderson Haldane (1892–1964) was exceptionally prescient in suggesting that the key does not fit the lock quite properly but exercises a certain strain on it [58,59]. It was Henry Eyring (1901–1981) who developed the transition state theory soon thereafter [60] almost simultaneously with Meredith Gwynne Evans (1904–52) and Michael Polanyi (1891–1976) [61]. Linus Carl Pauling (1901–94) did suggest during 1946–48 that transition state formation powers the enzyme catalysis [62,63], but according to Kraut, his ideas did not get traction immediately in the context of how enzymes work [58]. Kurtz in 1963 developed the theoretical framework and William Platt Jencks (1927–2007) around 1966 advanced the idea of transition state-analog inhibitors [64–66]. It was Richard Vance Wolfenden [67] and Gustav E. Lienhard [68] who independently established the general importance of the transition state theory in enzyme catalysis during 1969–73 [58].

Among the earliest clear examples of covalent intermediates were acyl-enzymes formed during the catalysis by proteases and esterases wherein the acyl group is bound to either –OH of the serine side chain or to –SH of the cysteine side chains in the active sites of the enzymes [65]. In these very early studies, the formation of the acyl intermediate was found out to be the faster step (e.g., the initial burst of nitrophenol in the chymotrypsin-catalyzed hydrolysis of the nitrophenol esters accompanied the formation of the acyl-enzyme intermediate). The second, slower step of decomposition of the acyl intermediate was thus the rate-determining step. Jencks discussed why the overall catalytic rates for hydrolysis of different esters are different. Of the seven reasons described by him, two are more relevant for our discussion [65]. First, if the induced fit (involving the conformational change in the enzyme during binding of the substrate) is significant, then that step instead is rate-limiting. That may differ from substrate to substrate. Second, if the substrate is such that a poor leaving group is involved (e.g., in amides), then the rates of acylation and deacylation fall in the same range. The acylation can be partly or completely rate-determining [65].

Awareness of these different possibilities is important for understanding the concept of km. It is equal to Ks (dissociation constant of the E–S complex) only if acylation (formation of the E–S complex in general) is rate-determining. It is to be noted that this does not impact the text book definition of km as being equal to the substrate concentration, at which the initial velocity is half of the Vmax.

At this point in our discussion, it is worthwhile to add that for a given kcat/km (a measure of the specificity toward a substrate), maximization of kcat results from the high km (weak binding of the substrate) [69]. It is actually the strong binding at the transition state, which leads to the high catalytic rates. Nevertheless, as pointed out further by Alan Roy Fersht [69], a low km is useful for the first enzyme in a metabolic pathway, which is invariably an allosteric enzyme, to control the flow of the substrate into the metabolic pathway [69]. Fersht further discusses how the evolutionary pressures on km are dictated by the physiological concentrations of the substrates [69].

1.5 Assaying enzymes

Once the basics of enzyme kinetics were understood, the work on quantitative analysis of enzymes attracted the attention of biochemists. One of the early challenges was to quantify biological activity of enzymes. In early days, this was in the context of tissue slices, cell homogenates, etc. So, many of these early attempts at enzymes assays were in fact carried out while establishing metabolic pathways.

Joseph Barcroft (1872–1947) and J. S. Haldane had invented an apparatus for manometric measurements for evolution (or depletion) of gases [70,71]. About 10 years later, in 1902, Otto Heinrich Warburg (1883–1970) used it to determine consumption of oxygen and simultaneous evolution of carbon oxide. This enabled his group to measure the respiratory coefficients of metabolites [3]. Later, manometric method was used to assay enzyme activities of various enzymes in their preparations. This also resulted in facilitating early work on catalase [3,72]. Urease, carbonic anhydrase, and decarboxylases were other enzymes, which could be assayed by measurements of changes in amounts of a gas as a result of an enzymatic reaction. An innovative adaption of this approach was in assays of many other kinds of enzymes, which catalyze reactions with resultant changes in concentration of protons. Any acid liberated in the presence of bicarbonate could be quantified by measuring amount of carbon dioxide evolved manometrically. Thus it became possible to assay glyceraldehyde-3-phosphate dehydrogenase (an important enzyme of glycolytic pathway), enzymes catalyzing some other phosphate transfer reactions, esterases, and proteases manometrically. Many oxidases, which reduce ferricyanide with concomitant increase in the concentration of protons, could also be assayed in the presence of bicarbonate [3,72,73].

Another early method was to use electrodes to measure changes in concentration of protons directly [3,72–74]. The first commercial pH stat introduced by Radiometer was, hence, a great help in rapidly establishing standard assay protocols for esterases, proteases, and lipases. In many cases, these protocols are still used in spite of tremendous advances in enzymology [3,72–75].

Polarimetery was another existing method which could be easily used for measuring enzyme activities of enzymes like sucrase which result in the change of optical rotation. Fumarate hydratase was assayed by enhancing the specific optical rotation of malic acid by sampling the reaction mixture and adding the molybdate reagent to form a complex [72,73].

It is important to highlight the impact of commercial availability of colorimeters on practical enzymology [3,72–75]. Apart from making enzyme assays less tedious and faster, it facilitated a shift to use of synthetic substrates especially for hydrolases. The substrates for many hydrolases could be synthesized by introducing p-nitrophenyl group in the substrate structure. These were low-molecular-weight organic compounds that were soon available commercially in pure forms and were less expensive to use as compared to earlier substrates of biological origin. The characteristic absorption maximum of nicotinamide adenine dinucleotide (NADH) around 340 nm allowed the assay of many oxidoreductases that were dependent upon this coenzyme. Colorimetry also made it possible to use Thunberg method in which dehydrogenases were assayed by reduction of dyes (like methylene blue) [72].

Later as spectrophotometers (which covered ultraviolet region) became commercially available, assays based upon absorption spectroscopy began their dominance in enzymology. As many spectroscopy-based methods of protein estimation became available (to replace tedious methods like Kjeldahl method, which was based upon nitrogen estimation), parameters like specific activity or fold purification became popular for tracking isolation and purification of proteins/enzymes [72,73].

With the advent of fluorescence spectroscopy, umbelliferone-coupled substrates were employed (in place of p-nitrophenyl coupled substrates), wherever higher sensitivity is required during enzyme assays [73,74].

A new dimension to the development of enzyme assays appeared with the early studies on immobilized enzymes [76]. It paved the way for enzyme assays to be exploited in flow analyzers, the enzyme-linked immunosorbent assay (ELISA) reagents and numerous other forms of diagnostic tools and biosensors [46].

The use of enzymes in low-water media (organic solvents or ionic liquids containing small amounts of water or reverse micelles) resulted in use of high-performance liquid chromatography (HPLC) and high resolution gas chromatography (GC) for enzymes assays in a big way [76].

It may be added that there are a large number of biologically active proteins which are not enzymes. In almost all cases, principal approaches to their assays consist of adapting the assay methods developed for enzymes. Also, the phenomenon of moonlighting proteins indicates that a protein that may have a noncatalytic biological activity in a particular part of the cell may display a significant enzyme activity elsewhere. The distinction between an enzyme and noncatalytic proteins is getting blurred. Please see a later chapter in this book on moonlighting proteins.

Another important point to remember is that in classical enzymology, the assays are designed with the understanding that all enzyme activities fall under one of the six classes of the enzymes according to the enzyme commission [77]. The well-recognized phenomena of enzyme promiscuity means that this is strictly speaking not always true [78]. Lipases, though hydrolases according to the Enzyme Commission (EC) classification, are widely reported to catalyze formation of C–C bond formation. So, the choice of assay in those cases is not the assays conventionally used for lipases but based upon tracking its synthetic activity [78].

The design of an assay and interpretation of the data is not always a straightforward issue. There are some excellent sources that discuss these challenges [72–76].

One effort which is worth mentioning is setting up of the STRENDA (Standards for Reporting Enzyme Data) commission in 2003 [77]. Its first goal is to create a kind of checklist for ensuring that the kinetic data (which obviously involves assays) is obtained/reported in accordance with their guidelines. These include basic details like the molarity, pH, and nature of buffer which were used during the assay. The literature (notwithstanding the impact factor of the journal where the article is published.) is full of reports where even this minimum information is missing. The second laudable goal is to create tools for electronic submission of the data. This makes it possible to create reliable databases [77].

To a biochemist (especially ones which were directly or indirectly working with enzymes), there have been three invaluable sources for information about enzymes. The series of volumes called Enzymes and Methods in enzymology were (especially latter) go to sources for finding out protocols and some essential information about an enzyme in which one happens to be interested in. The Springer Handbook of Enzymes was more of a database. In 1987 BRENDA, the electronic version of these volumes of the handbook, became available [77]. In 2006 FRENDA (Full Reference Enzyme Data) came into existence. DRENDA (Disease Related Enzyme Information Database) was also released [77]. It is necessary to point out that all these electronic databases are based upon the classification of enzymes into the six classes. They mostly ignore the ramifications of the both promiscuous and moonlighting behavior of enzymes. That is a challenge for the future.

1.6 Enzyme immobilization

We have already briefly mentioned the impact of the emergence of the technique of protein/enzyme immobilization on enzyme assays. This was definitely a development, which had a dramatic effect on the growth of enzymology. Like in many other cases, the exact pinpointing of how it all began can be a matter of opinion. Klaus Mosbach, who edited those invaluable volumes of Methods in Enzymology on immobilization [79–82], referred the immobilized enzymes as insolubilized, matrix bound, carrier bound, entrapped, microencapsulated species in 1976 [79]. A book by Linqiu Cao [83] on the subject in 2005 mentions (besides adsorption, covalent coupling, entrapment, and encapsulation) another eight methods under unconventional enzyme immobilization. Enzymes constrained by membranes are now generally regarded as immobilized enzymes. There are enzymes bound/covalently linked to smart polymers, and these bioconjugates can catalyze as soluble biocatalysts and can be recovered by applying the appropriate stimulus [84,85]. Therefore the notion of a solid support as an essential component vanished quite some time back. Finally, moving away from even supports, cross-linked enzyme aggregates (CLEAs), cross-linked enzyme crystals (CLECs), and protein-coated microcrystals (PCMCs) are well-established examples of the support-free immobilization [26,86,87]. What about examples of lyophilized enzyme powders or enzyme precipitates as biocatalysts in low-water media like organic solvents or ionic liquids [86]? So, the list of what we call immobilized enzymes is quite elastic.

Somewhat underexploited approach is that of immobilizing enzymes in affinity layers [88,89]. This is an extension of affinity immobilization method described so well by Bo Mattiasson [90]. This enables one to deposit large amounts of activity on a small surface. With nanomaterials emerging as a favorite supports (it is sometimes wrongly stated that nanoparticles have large surfaces, an accurate statement is that these have large surface to volume ratios), this approach could be a winner. The challenge is that there is to keep the active sites of the enzymes accessible as one builds composite layers of the affinity material and the enzyme.

Broadly speaking, three major groups (with no intention to minimize the importance of work of others) can be identified as having given early impetus to this area. One was the group of Klaus Mosbach at Sweden who initiated the major work in this area along with Bo Mattiasson [80,82,90]. Second group was that of Ephraim Katchalski-Katzir (who later on became president of the country-Israel), who actually was most influential in nurturing the early development of this technology [91]. The third was the group of Ilya V. Berezin (1923–87) from Moscow State University in what used to be Union of Soviet Socialist Republics (USSR) [92]. Another distinct milestone was development of cyanogen bromide coupling method by Jerker Porath (1921–2016), Rolf Axén, and Sverker Ernback [19,93]. While the method was developed for preparing an affinity material, it could be easily (similar to almost all other methods for preparing affinity adsorbents) adopted to obtaining immobilized enzymes. Another game changer was perhaps the immobilization on nylon by P.V. Sundaram and William E. Hornby [94] as this paved the way for use of enzymes in flow analyzers. It had huge implication in clinical biochemistry, as large number of samples could be quickly analyzed in these flow analyzers [80,82].

Linqiu Cao [83] identifies distinct phases of the development of the area of protein immobilization as the early days (prior 1940s), the underdeveloped phase (1950s), the developing phase (1960s), the developed phase (1970s), the postdeveloped phase (1980s), and the rational design phase (1990s–present). While that may be a fair classification, every generation of the scientists used rational approaches based upon what was known at that time. Often, development of an area in science is a shift from unknown unknowns to known unknowns.

The approach of immobilization is obviously easy to extend to the areas beyond enzymology. Many drug-release formulations, extracorporeal shunts in medical treatments, and analytical devices are spin-offs of this approach [80,82]. It is also not sufficiently realized that much of the methodologies in protein immobilization and designing of affinity materials are common. After all, when it comes to chemical manipulation, the list of functional groups in the organic world is not so large [95].

Linqiu Cao [83] provides an extensive list of parameters, which can influence the behavior of the immobilized enzymes and were already identified by the 1970s. He also points out that by 1980s, 100 classes of supports for immobilization were commercially available. Commercial availability is not just the ready accessibility, it also means that a standardized preparation was available and so it was easy to reproduce the results in another laboratory. When it comes to supports, biochemical engineers made an immense contribution by identifying the external mass transfer constraints (related to diffusion of the substrates and products) and internal mass transfer constraints (due to the limited access to the enzyme sitting inside the pores of a porous support by the substrates and products) [96].

Generally, two main advantages of immobilizing enzymes are mentioned: stabilization and reusability. When it comes to stabilization, the term actually has different meaning in different contexts. For real-life applications, what really matters is operational stability—the survival of the activity during the catalytic cycle. A catalytic cycle can be arbitrarily defined as ending, when a certain prefixed % conversion has taken place. In an industry, that may be the point of conversion at which the process becomes profitable to the desired degree. That obviously involves a trade-off situation. For academic purposes, one may let the equilibrium be reached (in the case of equilibrium controlled reactions). The important point is the exposure of the biocatalyst to the substrate(s), which in industry is often a crude material rather than clear solution in a buffer. So, much of the data reported even in the high-impact journals with calculations of all sorts of numbers are either inadequate or not of direct utility in industrial enzymology. In that respect, it is better to trust biochemical engineers, whose work often does not find traction in biochemistry? In that respect, legendary former editor-in-chief of the journal Biotechnology and Bioengineering, Daniel I-Chyau Wang (1936–2020), deserves a credit for bringing in both biochemists and biochemical engineers closer by publishing their work in the same journal.

There is another common practice, which creates untrustworthy data on enzyme stability. In most of the publications, the biocatalyst is in far excess for reaching the maximum catalytic velocity. As a result, any deactivation process goes undetected. To sum up, the real stabilization for any real-life application is reflected in the reusability; that is, how much of the activity survived after how many cycles under the relevant conditions.

The second (and unfortunately very common) error in the literature has been to extrapolate the results of stability in the presence of some organic cosolvents to create low-water conditions. To start with, the enzyme (even in the free form) under low-water conditions is in solid phase. Therefore it is a case of heterogeneous catalyst. The outcome of spreading the enzyme (and hence catalytic centers) over the surface of the immobilization support is higher activity; this should not be termed stabilization. As pointed out by Halling et al., in most cases like lipases immobilized on celite by adsorption are just the breaking of the enzyme clusters and spreading it over a larger surface [97].

1.7 Applied enzymology and white biotechnology

The interest in enzymes (like with most of the things) originated in assessing their importance for living systems and in improving quality of our lives. Glycolysis is the first major metabolic pathway, which was investigated used both homogenates of animal tissues and yeast [3,6,8]. So, apart from understanding metabolism, we came to understand fermentative processes to obtain alcohol (and some other useful products) [98]. As chemical engineers became interested in working on biological systems, a new subdiscipline of biochemical engineering was born. These were closer to applied enzymologist. This area was called industrial enzymology with the term applied biocatalysis becoming more fashionable with time [98,99]. Later as white biotechnology took shape, much of the industrial enzymology was subsumed by that [100,101].

Not many enzymologists realize that one of the biggest markets for enzymes has been in their incorporation in detergents for cleaning clothes [98,102]. This application has an interesting history. Let us very briefly look at it. In prehistoric times, soft water to clean sheep fleeces (the material used by those ancients for garments) was used. With time, woolen garments came into use, and around 5000 BCE, river mud was found to be effective for their cleaning. By around 3500 BCE, wood ashes became popular for cleaning textile garments. By around 2100 BC, the Sumerians had discovered that a mixture of olive oil and wood ash upon boiling produced a material, which was very effective in cleaning their woolen garments. This was probably one of the oldest chemical reactions carried out by these early people. Egyptians, around 1000 BCE, were replicating it by mixing either animal fat/vegetable oil with sand from Wadi Natrum Desert, which was actually rich in sodium carbonate. To jump few centuries (and civilizations) in this story, synthetic detergents (alkyl sulfates) were commercially available by around 1930. It was Otto Karl Julius Röhm (1876–1939) who around the same period patented the use of pancreatic enzymes to remove stains before washing. Subsequently, Burnus, the very first enzyme-based detergent was introduced. It is instructive to analyze why this use of enzymes in cleaning detergents did not catch on for many years. First, these pancreatic enzyme preparations could be obtained only at small scale. Second, the serine proteases, which were responsible for removal of proteinaceous stains, were neither very active nor could their activities survive under the highly alkaline environments of detergency [102].

Both problems were solved by the use of the bacterial alkaline proteases in Bio-40 and Biotex [102]. It is also noteworthy that rather than using ill-defined pancreatic enzymes, these early commercial products incorporated industrial-grade enzyme preparations Alcalase from Novo Nordisk and Maxatase from Gist-Brocades, respectively. Industrial enzymology was on its way! With time, carbohydrases and lipases were incorporated in these detergents. These preparations were available from different companies in various parts of the world. As the local cuisines of different countries differ in amount of oil, etc. utilized in the food preparations, the percentages of different classes of enzymes in detergents sold in different countries may differ [102].

The ready availability of these detergent enzymes at economical prices had enormous impact on the development of enzymology in general. When enzymes started being used for synthesis of chemical/drug intermediates or agrochemicals, it is these very enzyme preparations, which were used as biocatalysts. There were some other spin-offs as well. It is not generally realized that most of the enzymes are allergens. The early enzyme-based detergents were in the powder form and became unpopular, when many cases of allergy were reported. This led to not only the development of formulation strategies (granular form or in liquid form), it also encouraged work on stabilization of enzymes in alkaline environments/solution forms [98,99].

In recent years, enzymes, which work well even at ambient temperatures (to save energy for heating water in washing machines), have been introduced. This has involved gaining important insights into structure-activity-temperature paradigm and provided further impetus to work on enzymes from psychrophilic/psychrotolerant organisms [103]. These enzymes provide us insight into adaption of microorganisms to low temperature [104–109]. They also found multiple industrial and biotechnological applications [110–122]. Overall, the application of enzymes in detergents is a good illustration of the meeting of minds of the twin tribes of enzymologists and applied enzymologists. Both classes of enzymologists have contributed to the development of the research areas of each other.

Let us look at the similar history of the other application domains of what was called industrial enzymology at one point. For the sake of continuity, let us discuss applications of enzymes in the textile sector before the cloth becomes garments, which require cleaning by detergents [98–100,123]. In case of cotton (cellulose) or its blends, before weaving, the threads have to be treated with starch (or its derivatives) to prevent breaking. Afterwards, desizing is done by amylases to remove such materials. The fabrics made from natural fibers (like cellulose) have fuzzy surface, and the engineered cellulases have been used to biopolish the fabrics.

In the case of denims, animal fat has been used as the size material sometimes (instead of starch-based materials). Hence, desizing of denims is carried out by combination of amylase and lipase [123]. The faded appearance (especially in jeans which has become so fashionable) can be achieved by biostoning using combination of cellulase and laccase activities. Interesting enough, the opposite process, that is, the process of dyeing, can also be carried out using laccases and peroxidases [98–100,123].

Jute is a natural fiber. The rough surface of jute-based fabrics can be improved by retting, which essentially softens the fiber. Bioretting involves pectinases and xylanases to yield an improved jute-based fibers/fabrics [98–100,123].

Hair and wool are proteinaceous fibers. Their functionalization by protein disulfide isomerase to assist dyeing and polishing is a promising approach. Proteases to achieve shrink resistance in both wool and silk have been described [98–100,123].

In fact, proteases have a very large footprint in the history of enzymology [99]. This has been pointed out earlier also in the contexts of isolation/purification and that of gaining structural and mechanistic insights into enzyme action. Proteases have been also used in brewing, baking, cheese making, and bating (softening) of leather. Haze formation in beer is inhibited by addition of papain or bromelain. Modification of the flours to alter the property of dough in a desirable way represents the use of fungal proteases in baking. Use of papain (and other proteases less frequently) in tenderization of meat is well-known. Alkaline proteases replace harsh chemicals in dehairing of the leather, whereas trypsin and some other microbial proteases are used to soften the leather [98,99].

The three proteases used as clot busters (thrombolytic agents), streptokinase, tissue plasminogen activator, and urokinases, are well-known examples of therapeutic applications of proteases [124].

Another major application of proteases has been in production of cheese since late 19th century. Historically, Arab nomads carried their milk in bags made of animal skins (the scourge of polythene bags was still many decades away.) [99]. It is believed that these bags essentially made from animal stomachs had rennet which curdled milk to produce an ancient version of cheese. Cheese making apparently caught on and led a Danish industry to market a commercial preparation of rennet. Rennet is actually a crude preparation containing only about 2%–3% of an aspartic proteinase called rennin (also contains chymosin). One of the finest and valuable resource for information related to industrial enzymology mentions many other protease preparations derived from animal, microbial, and plant sources which have been marketed/tried as an alternative to rennin [98]. In recent times, like in case of most of the other enzymes, industry has switched over to enzymes obtained by use of rDNA technology. Cheese production is a huge industry and few other enzymes are also used in addition to proteases. These include lipases (to improve its mouth feel by improving flavor) and lysozyme (to prevent butyric fermentation by destroying Clostridium tyrobutyricum cells). The flavors in cheeses are largely due to peptides produced by proteolytic action on milk proteins. The bitterness in some hard cheeses is also due to formation of bitter peptides [98,99].

Some other applications of common proteases are in digestive aids, debriding (cleaning of wounds by destroying dead cells in and around it) and as antiinflammatory agents [124]. An enzyme serratiopeptidase originally found in Enterobacteria serratia E 15 of silkworms has been used extensively as an antiinflammatory and analgesic agent [125]. Its major action mediating these therapeutic applications is on cyclooxygenase enzymes which in turn produce various molecules involved in signaling of pain and inflammation. It is now a widely used ingredient in many antiinflammatory medications in various forms and is also used during surgery, orthopedics, and dentistry [125].

Again, the list of the enzymes involved in therapeutics currently is a long one. The seminal work published by John S. Holcenberg and Joseph Roberts in 1981 captures the status till then very well [126]. The book mentions asparaginase, folate degrading enzymes (led by carboxypeptidase G), human ribonucleases, superoxide dismutases, enzymes which degrade both essential and nonessential amino acids, and those which showed early promises in the context of enzyme replacement in genetic diseases [126]. Some interesting early leads mentioned in that book are about the use of cell-surface antigenicity and control of the complement system. Evading immune surveillance by use of stealth polymers (the most promising one continues to be PEG) also finds mention in this early book. The use of other stealth strategies and drug release and targeting have been recently reviewed [127–130]. Enzymes can be used in various formats and their use in designing extracorporeal shunts had begun to be exploited fairly early; for example, degradation of folate by carboxypeptidase G [126].

Coming to the use of enzymes in food and feed processing sectors, their requirement of purity is not so high but it is necessary that these originate from a GRAS (generally regarded as safe) source [98,99]. Animal and poultry feed also constitute an important application sector for enzymes [98,99,124]. The soluble polysaccharide β-glucan is present in barley (a common poultry feed component) to a varying degree depending upon a variety of factors. This (depending upon its content) can lead to a viscous mass in the intestines of the birds. This not only reduces the nutritional uptake, but it also has multiple other deleterious consequences. Incorporation of microbial β-glucanase in the barley-based poultry feed has solved this pesky problem. Pentosan present in wheat presents a similar (though lot less troublesome) problem of enhancing the viscosity of the mass in intestine as digestion proceeds. Pentosanases added to the poultry feed again solve this problem. Substances, such as glucans and pentosan, are termed antinutritional factors, as they impede digestion and/or nutritional uptake. The classical examples of antinutritional factors are trypsin inhibitor and the lectin present in soybeans [131–133]. Phytin, though, is perhaps one of the most well-known examples of an antinutritional factor, which has been taken care of by incorporation of enzyme phytase in both animal and poultry feed. As high as (or even higher in some cases) 50% of the phosphorus in the cereals is present in the form of phytin (salt of phytic acid). Therefore phytase incorporation makes this phosphorous available to the birds/animals [124].

One of the major applications of enzymes is in oil and fat industries [98,99]. In fact, it starts with extraction of oils. An accepted method of olive oil extraction involves the treatment of the paste of milled olives with mixtures of cellulases, hemicellulases, and pectinases. Many industrial enzyme formulations tailored for this treatment are available. It has been pointed out that roughly extraction of oil from 1 t of olives requires 1 ton of water. One advantage of the enzyme treatment is that water requirement is easily reduced by about 10% [99]. The other advantages reported include better storage stability, improved flavor, and yield. Many oils contain high amounts of phospholipids and phosphatides. These cause gumminess, and the phospholipase A2 treatment is a good replacement for chemical degumming. The market size of the enzymes for this is significant enough for the enzyme manufacturing companies to continuously invest in development of better enzyme formulations [98–100].

For a long time, strong efforts have been put into the development of aqueous enzyme oil extraction processes, wherein enzymes are used in water to extract oil from seed powders [134]. In most of these cases, the concept is to disrupt the structure of oil bodies wherein oil is trapped. This has, however, never reached a point, where the approach can be deployed at the industrial scale.

Beyond extraction (and refining) steps, the use of lipases in fat splitting (hydrolysis of fats/oils to produce fatty acids and glycerol) has been known for a long time [98,99]. A relatively more recent subplot to this story has been transesterification of oils/fats by lipases to produce biodiesel and glycerol [135,136]. This actually led to the production of so much glycerol that it destroyed the industries which were producing glycerol chemically. On the positive note, a whole lot of technology has been developed for valorization of glycerol [137].

Coming back to biodiesel, chemically it is a mixture of esters of fatty acids present in the fat/oil with either methanol or ethanol. So, the transesterification is actually alcoholysis of the triglyceride (fat/oil) under the conditions of low water in the medium, which can be an organic solvent or ionic liquid. Solvent-free syntheses (where the mixture of the triglyceride and alcohol itself form the reaction medium) have also been reported. The esters of fatty acids (and other compounds like sugar) constitute a huge class of biotechnologically important compounds, such as biofuels, fragrances, flavors, and biosurfactants. These can be prepared by either esterification or transesterification under the low-water conditions [138].

There is a third type of reaction which can be catalyzed by lipases. Two esters of fatty acids can swap their alcohol/fatty acid components. This interesterification is also possible with one of the substrates being a triglyceride [138]. Discovery of interestrification many decades back led biochemists (those days the term biotechnologist/ biotechnology had still not caught on.) to work on fat engineering. The approach continued paying dividend when applied to enriching of oils with essential fatty acids (monounsaturated fatty acids and polyunsaturated fatty acids, etc.)