Biocatalysis: The Beginnings
Historically, cheese making has revealed valuable insights into enzymatic processes, with the earliest records indicating the use of fig tree extracts, which contain the proteolytic enzyme ficin Eventually, rennet, a source of the protease chymosin, gained popularity in cheese production Additionally, meat tenderizing has long utilized enzymes, with papaya (Carica papaya), known for its proteolytic enzyme papain, being an early and effective option for this purpose.
The pioneering research on gastric digestion, particularly regarding proteases by Rene Reaumur in 1751 and Lazzaro Spallanzani in 1780, established a scientific basis for enzyme catalysis Reaumur's systematic experiments on meat digestion marked the earliest documentation of enzyme activity, even before the term "enzyme" was introduced Theodor Schwann later coined the term "pepsin" to describe this digestive enzyme.
In 1836, Schwann investigated the proteolytic activity of gastric mucosa, conducting detailed quantitative experiments that demonstrated the necessity of acid for this reaction, although it was not the sole requirement Additionally, he is credited with coining the term "metabolism," highlighting his significant contributions to the field.
Parallel to the work on proteolytic enzymes, developments with fermentation and on starch hydrolysis have equally contributed to the initial growth of enzymology.
# Springer Nature Singapore Pte Ltd 2018
N S Punekar, ENZYMES: Catalysis, Kinetics and Mechanisms, https://doi.org/10.1007/978-981-13-0785-0_2
In 1815, Gottlieb Kirchhoff identified the activity of plant amylase, specifically α-amylase, while studying the hydrolysis of starch into sugar He demonstrated that the conversion of starch to sugar is facilitated by acid and recognized that this process during grain germination resembles chemical hydrolysis.
The work of Kirchhoff on starch hydrolysis was extended by Anselme Payen and
Jean Persoz (1833) They enriched (first attempts of enzyme purification!) the hydrolytic activity from malt gluten and termed it as diastase The name diastase
The term "diastasis," derived from Greek meaning "to make a breach," has played a crucial role in advancing enzymology In 1931, Erhard Leuchs discovered a starch-hydrolyzing activity in saliva, highlighting its potential practical applications.
Table 2.1 Landmarks in enzyme studies (enzymology classics)
R Reaumur 1751 Gastric digestion in birds
L Spallanzani 1780 Digestion of meat by gastric juice
A Payen & J Persoz 1833 Amylase (diastase) activity
J Takamine 1894 Patent on fungal diastase
E Fischer 1894 Lock and key concept
G Bertrand 1897 Co-ferment (coenzyme) conceived
P.E Duclaux 1898 Enzyme names to end with suffix “ase”
S.P.L Sorensen 1909 pH scale and buffers
L Michaelis & M Menten 1913 Equilibrium treatment for ES complex
R.M Willstatter 1922 Trager theory of enzyme action
G.E Briggs & J.B.S Haldane 1925 Steady-state treatment for ES complex
J.B Sumner 1926 Urease – Purification and crystallization
H Lineweaver & D Burk 1934 Double reciprocal plot (1/v versus 1/[S])
K Stern 1935 First ES complex observed
M Doudoroff 1947 Radioisotope use in enzyme mechanisms
A.G Ogston 1948 Asymmetric interaction with substrate
L Pauling 1948 Enzyme binds TS better than S
F Westheimer 1951 Enzymatic hydride transfer ( 2 H, 3 H used)
D.E Koshland Jr 1958 Induced fit hypothesis
C.H.W Hirs et al 1960 First enzyme sequenced – RNase A
Enzyme commission 1961 Enzyme classification and nomenclature
D.C Phillips et al 1962 First enzyme structure – lysozyme
W.W Cleland 1963 Systematization of enzyme kinetic study
J Monod et al 1965 Model for allosteric transitions
R.B Merrifield 1969 Chemical synthesis of RNase A
S Altman & T.R Cech 1981 Catalysis by RNA molecules
Early studies in the twentieth century identified two non-hydrolytic enzyme activities: peroxidase from horseradish and catalase Notably, Louis Thenard conducted the first quantitative analysis of catalase in 1819, suggesting that similar enzymatic activities could exist in various animal and plant secretions.
Enzymology, a vital intersection of chemistry and biology, has roots tracing back to prominent figures from the eighteenth century, including Reaumur, Spallanzani, Thenard, Schwann, Berzelius, Liebig, Berthelot, Pasteur, Buchner, and Fischer Notably, chemists like Berzelius, Liebig, and Berthelot made significant contributions to the field Jons Jacob Berzelius first introduced the term "catalyst" in 1836, describing it as a substance that awakens dormant energies through its mere presence, and he recognized parallels between catalysis in chemical reactions and within living cells However, Berzelius did not differentiate between catalytic processes in living and non-living systems During this period, the concept of a "vital force" was often linked to living cells, with biocatalysts playing a crucial role in this understanding It wasn't until later that the notion emerged that enzyme catalysis is governed by the same physical and chemical principles as other reactions.
Pierre Berthelot was the first to derive a second-order rate equation which influenced the publication by Guldberg and Waage on law of mass action leading to chemical kinetics.
As a part of their study on catalytic phenomena, Wohler and Liebig discovered
“emulsin”(aβ-glucosidase) from almonds in 1837 Indeed this enzyme was cleverly used by Fischer subsequently (almost 50 years later!) to define enzyme specificity.
In 1836, Swedish chemist Berzelius introduced the term "catalysis," derived from the Greek words meaning "to loosen wholly," without differentiating between chemical and biological catalysis, referring to catalysts as "contact substances." The term "enzyme," specifically for biological catalysts, has a complex history tied to debates over the existence of a "vital force" in living systems The name "diastase," introduced by Payen and Persoz in 1833 for starch hydrolysis, has since been commonly used to refer to biological catalysts.
1903 book on enzyme kinetics (an early classic on enzyme action) used diastase to mean an “enzyme.” Many other French scientists including Pierre Duclaux and
Gabriel Bertrand did use diastase to mean what we now call enzymes The suffix
“ase” – arising out of diastase– was subsequently recommended for all enzyme names (by Duclaux in 1898).
The term "ferment" originally referred to both living yeast and the actions of its cellular contents, with Berthelot's 1860 extraction of "ferment" from yeast cells marking a pivotal moment in enzymology by demonstrating enzyme activity outside living cells and challenging vitalistic biochemistry Key contributions from Kirchhoff, Payen, Persoz, and Berzelius highlighted the similarities between ferment-catalyzed and acid-catalyzed starch hydrolysis, while Schwann drew parallels for pepsin Willy Kuhne further advanced this understanding in 1867 by relating it to pancreatic protein digestion and introduced the term "trypsin" in 1877, solidifying the concept of "ferment." Kuhne also coined the term "enzyme" in 1877, designating trypsin as the first identified enzyme.
The term "enzyme" has evolved over time, with earlier descriptions such as "diastase," commonly found in French scientific literature, and "ferment" being used well into the early twentieth century.
The vitalistic theory wasfirmly laid to rest with Eduard Buchner’s conclusive demonstration that suitable extract from yeast cells could convert sucrose to alcohol.
In 1897, a groundbreaking discovery revealed that fermentation could occur "without living yeast," leading to the identification of a single substance called "zymase," later referred to as alcoholase by Emile Roux This substance encompasses the entire glycolytic sequence of reactions The debate surrounding the nature of alcoholic fermentation ultimately gave rise to the term "enzyme," highlighting the intersection of yeast activities with biology and chemistry, and paving the way for the field of enzymology.
Key Developments in Enzymology
The early study of enzymes faced challenges due to limited knowledge about the chemical nature of proteins and enzymes themselves To gain insights, researchers focused on purifying enzymes for detailed analysis Notably, Kuhne and Chittenden advanced the field by employing protein fractionation techniques using ammonium sulfate and introducing dialysis methods in 1883, which laid the groundwork for the development of more effective enzyme purification techniques.
Richard Willstätter introduced alumina Cγ gel, achieving such a high purity of peroxidase that it failed existing protein tests, leading him to mistakenly conclude that enzymes are not proteins in 1926 The pivotal discovery by James Sumner in 1933, which demonstrated that urease is indeed a protein, holds significant importance This perspective was further validated by Northrop and Kunitz through the purification and crystallization of three additional enzymes—pepsin, trypsin, and chymotrypsin—between 1930 and 1935.
8 2 Enzymes: Historical Aspects must impress anyone to note that all this was accomplished by just two simple purification techniques–fractional precipitation of proteins by ammonium sulfate and pH changes.
Laccase, identified by Bertrand in 1895 as an "oxidase," is one of the earliest known enzymes that is not classified as a hydrolase He proposed that this enzyme contains a divalent metal and introduced the term "co-ferment," referring to a nonprotein component of laccase, marking the first instance of an enzyme cofactor descriptor.
Advancements in protein purification and dialysis techniques significantly accelerated the field of enzymology, leading to the availability of numerous pure enzyme preparations By the late 19th century, there was a notable increase in enzyme reports, and by 1955, the sheer volume of identified enzymes necessitated a systematic classification To address this, the International Union of Biochemists established the International Commission on Enzymes, which developed guidelines for enzyme nomenclature, culminating in their recommendations published in 1961.
Enzyme kinetics can be effectively studied due to the exceptional catalytic properties of enzymes, even with limited understanding of their composition Although the protein nature of enzymes has been recognized, it has taken considerable time to connect their structural characteristics to their kinetic behavior.
In 1898, it was discovered that enzyme reactions can be reversible, exemplified by the yeast maltase synthesizing maltose from glucose This finding highlighted three important aspects: enzymes act as catalysts that accelerate reactions in both directions, certain metabolic steps can proceed in either direction, and enzymes play a crucial role in cellular biosynthetic processes.
The reversibility of enzyme catalysis has integrated it into thermodynamic analysis and physical chemistry, with J van’t Hoff establishing the thermodynamic constraints for both catalyzed and uncatalyzed reactions This foundation allowed JBS Haldane to connect enzyme kinetics with thermodynamics, leading to the well-known Haldane relationship Yeast invertase serves as a key example in early enzyme reaction kinetics and thermodynamics, with AJ Brown identifying the invertase-sucrose complex (the ES complex) from initial rate measurements in 1902 In 1903, V Henri derived the hyperbolic rate equation for single-substrate enzymatic reactions, emphasizing the importance of validating rate equations to confirm kinetic mechanisms The famous Michaelis–Menten equation, published in 1913, further advanced this field, while Briggs and Haldane introduced a more general form in 1925 using the steady-state approach Today, the Michaelis-Menten equation remains essential for describing substrate saturation in enzyme reactions, with the linear form of this relationship, known as the Lineweaver-Burk plot, widely utilized in enzyme kinetics studies.
A systematic investigation into enzyme reaction rates highlighted the necessity of using buffers to regulate hydrogen ion concentrations This research laid the groundwork for Sorenson's 1909 publication on the pH scale and the role of buffers in biological systems.
Michaelis and others emphasized the importance of pH on enzyme activity and routinely controlled it in all their studies.
The ES complex formation was a kinetic concept to begin with First direct observation of an enzyme substrate complex of catalase was made by KG Stern
(1935); he monitored the catalase–HOOEt complex using spectroscopy.
Emil Fischer was a pioneering organic chemist who significantly advanced the application of synthetic and analytical techniques to biological problems In 1894, he discovered that invertin (now known as invertase or sucrose hydrolase) does not act on the same substrates as emulsin (a β-glucosidase), highlighting the specificity of enzymes Fischer famously stated that “enzymes are fussy about the configuration of their object of attack,” emphasizing that the enzyme and its substrate must fit together like a “lock and key” for effective catalysis His insights laid the foundation for future developments in enzymology and biochemistry.
Fischer This laid the foundation for describing fundamental properties of enzyme like specificity, stereoselectivity, and the famous lock-and-key analogy for enzyme– substrate interactions.
In 1922, Willstätter proposed the "Trager" or carrier theory to explain enzyme function, suggesting that enzymes possess smaller reactive groups that specifically interact with certain substrate groups, thus ensuring enzyme specificity At that time, these reactive groups were believed to be attached to an inert colloidal carrier that constituted the enzyme, as the understanding that enzymes are proteins had not yet been established.
In 1948, AG Ogston proposed a hypothesis explaining how enzymes create chemical asymmetry through three-point contact with substrates, which led to further exploration of enzyme mechanisms This was soon followed by studies on redox reactions involving pyridine nucleotides and hydride transfer mechanisms Frank Westheimer and his team demonstrated that, in enzymes like alcohol dehydrogenase and lactate dehydrogenase, hydrogen from the substrate is selectively transferred to one side of the nicotinamide ring.
This pioneering research in 1953 made use of deuterium- and tritium-labeled substrates to establish the stereospecificity of these hydride transfers.
Michael Duodoroff’s group (1947) conducted pioneering research on bacterial disaccharide phosphorylases, utilizing radioisotopes like 32P phosphate to investigate enzyme mechanisms Their findings revealed that sucrose phosphorylase and maltose phosphorylase, both involved in disaccharide phosphorolysis, operate through fundamentally different mechanisms This significant discovery contributed to the understanding of single displacement versus double displacement reactions, ultimately leading to the identification of SN1 and SN2 reaction pathways.
The theory of kinetic criteria for distinguishing enzyme mechanisms, developed by WW Cleland in 1963, established a standardized language for presenting enzyme kinetic data, addressing the confusion caused by varying notations in the literature This systematization of enzyme kinetics achieved two key objectives: it created a unified kinetic notation for clearer presentation and summarized the criteria for correlating kinetic data with reaction mechanisms.
Over time, the rigid structure of enzyme active sites proved inadequate, leading to Linus Pauling's prophetic insight in 1946 that emphasized the importance of complementarity to the transition state rather than just the substrate or product This understanding highlighted the necessity of subtle protein motion at enzyme active sites, paving the way for the concept of conformational flexibility as essential for enzyme activity, which replaced the earlier lock-and-key model This evolution culminated in the induced fit hypothesis proposed by Koshland in 1958, where protein flexibility and ligand binding became crucial for allosteric transitions Consequently, the plasticity of protein structures for regulatory purposes emerged as a fundamental concept, exemplified by the Monod-Wyman-Changeux model that explains cooperative interactions in oligomeric proteins.
The recognition of enzymes' ability to accelerate reaction rates significantly sparked a quest to uncover the fundamental principles behind enzyme catalysis Researchers, including TC Bruice, WP Jencks, ML Bender, and DE Koshland Jr., have explored various physicochemical factors through model reactions and enzyme studies Key contributors to the remarkable rate enhancements observed in enzymatic reactions include intermolecular and conformational effects, general acid/base catalysis, and nucleophilic/electrophilic interactions It is now widely accepted that the extraordinary efficiency of enzymes arises from a combination of these factors Despite this understanding, the search for novel catalytic mechanisms developed by nature continues to thrive.
Exploiting Natural Diversity
Earth's rich biodiversity is closely linked to a wide variety of catalytic activities, where well-designed screens often yield enzymes with specific desirable properties For instance, thermostable proteases derived from Bacillus strains and DNA polymerases from Thermus aquaticus are notable examples of enzymes selected for their high-temperature stability The extensive natural diversity is evident in the numerous enzymes that serve specialized roles in the processing of carbohydrate polymers, proteins, and lipids.
Enzymes play a crucial role in the bioprocessing of polysaccharides, which are the primary biomolecules in biomass, serving functions in energy storage and structural integrity Enzyme technology originated from the need to hydrolyze sugar polymers, with microbes like bacteria and fungi being rich sources of amylases and cellulases The industry has effectively developed controlled hydrolysis of starch into sweeteners and sugar substitutes, utilizing various enzymes such as α-amylases, β-amylases, glucoamylases, pullulanases, and glucose isomerase Notably, the conversion of glucose to fructose via glucose isomerase is essential for producing many sucrose substitutes, offering significant economic and manufacturing benefits.
Despite the natural abundance of cellulose, converting cellulosic biomass into sugar remains challenging, requiring the coordinated action of multiple enzymes known as the "cellulase complex" (Payne et al 2015) Significant advancements in enzymatic processes for breaking down cellulose into fermentable sugars are underway Meanwhile, individual components of the cellulase complex have been successfully utilized in the textile and paper industries.
Proteases and lipases are crucial enzymes in various industries, particularly in food production, detergents, and leather tanning Historically significant enzymes like papain and digestive enzymes such as trypsin and chymotrypsin have paved the way for the extensive use of proteases Bacteria and fungi, especially Bacillus strains and Aspergillus species, are ideal for large-scale protease production Subtilisin, a well-known bacterial protease, is widely used in modern detergents due to its optimized pH and temperature stability Additionally, chymosin, a traditional milk coagulating enzyme sourced from calf stomach, has led to the exploration of microbial alternatives, with several Mucor strains identified as effective rennin substitutes for cheese making.
Many other enzymes including lipases and pectinases are also available in industrial scale A representative list of enzymes commonly used in industry is given in the table (Table3.2).
Enzymes play a vital role in the pharmaceutical industry, significantly benefiting drug discovery and development Both naturally sourced and genetically engineered enzymes are utilized, highlighting their importance in various medical applications The critical study of these enzymes enhances our understanding and effectiveness in developing new therapies.
• Active principles of many effective drugs are enzyme inhibitors (Robertson2005).
An enzyme situated in the intermediary metabolism presents a promising target for screening potential inhibitors Notably, several drugs have successfully emerged from these enzyme screening processes.
Cyclomaltodextrin-D -glucotransferase a–Amylase b–Amylase Pullulanase a–D-Glucosidase
Fig 3.1 Signi fi cant steps and enzymes employed in starch processing Glucose residues of starch are schematically represented as circles Filled circles indicate glucose residues whose
C 1 -OH remains free, indicating it has not formed a glycosidic linkage In addition to glucose isomerase, various enzymatic methods for breaking down starch are available, with some enzyme combinations offered as commercial industrial formulations.
The screening for enzyme inhibitors originated with Hamao Umezawa's research group in Japan in 1982, leading to the discovery of numerous enzyme inhibitors now in use One potential application of these inhibitors is in obesity management by preventing the absorption of dietary fats, specifically triglycerides Targeting a suitable lipase from digestive juices could facilitate this screening process.
Often the active chemical entity obtained from an enzyme screen may notfind direct application These lead compounds (inhibitors) are suitably altered/derivatized
Table 3.1 Component activities of cellulase complex and their applications
Cellulase component Substrate specificity Application β-Glucosidase
Cellulose! Cellobiose (exo – Nonreducing end)
Cellulose! Cellobiose(both exo and endo) Biomass conversion
Endoglucanase I (EG1) Cellulose (endo) Textile/fabric softening, Biopolishing Endoglucanase II (EG2) Cellulose (endo) Textile/fabric softening, Biopolishing
Xylanase Xylan Paper pulp deinking
All components Cellulose and Xylan Feed/fodder, biomass Conversion
Table 3.2 Large-scale use of enzymes in industry
Cellulase complex Biomass conversion, textile industry Pectinases, esterases Food industry, fruit juice, brewing
Glucose isomerase, invertase High fructose syrups, invert sugar
Papain, pepsin Meat and leather processing, treating dough
Subtilisin Detergents, leather and wool processing
Acting on lipids and esters
Lipases Food and detergent industry, cocoa butter
Penicillin acylase plays a crucial role in producing 6-aminopenicillanic acid (6-APA), which enhances bioavailability and minimizes toxicity A comprehensive kinetic analysis of enzyme inhibition, discussed in Chapter 28, is fundamental to contemporary drug discovery initiatives.
Many enzymes serve dual purposes as both targets for inhibitor screens and catalysts for synthesis, particularly in the production of β-lactam antibiotics A significant portion of these antibiotics consists of semisynthetic penicillins and cephalosporins While penicillin G is produced through fermentation, its key precursor, 6-aminopenicillanic acid, is derived from it Additionally, penicillin acylase plays a crucial role in the large-scale production of these antibiotics.
Enzymes, known for their catalytic potential and specificity, serve as valuable analytical tools in various applications Notably, alkaline phosphatase and peroxidase are widely used as reporter enzymes in enzyme-linked immunosorbent assays (ELISA), where their catalytic activity enhances signal amplification in antibody interactions Additionally, enzyme-antibody conjugates are routinely utilized for the detection of DNA, RNA, and proteins on blots Taq DNA polymerase plays a crucial role in DNA amplification through polymerase chain reaction (PCR), while numerous metabolites are analyzed in clinical settings.
Table 3.3 Examples of enzyme-targeted screens for active principles
Enzyme target Screening outcome End use
Angiotensin converting enzyme Captopril Hypertension
HMG CoA reductase Lovastatin Hypercholesteremia α-Amylase Acarbose Diabetes
Triacylglycerol lipase Orlistat (lipostatin) Obesity
Acetylcholine esterase Rivastigmine Alzheimer’s disease β-Lactamase Clavulanic acid Combination therapy
(involved in hydrolysis of triacylglycerols)
(natural or synthetic chemical libraries)
Fig 3.2 Flow chart outlining the design of a lipase inhibitor screen
Exploiting natural diversity through enzyme assays is crucial for analyzing various substances A comprehensive list of commonly used enzymes and their corresponding analytes can be found in Table 3.4 Additionally, several of these enzymes play a significant role as components in biosensors.
Enzymes play a crucial role in medicine, serving both diagnostic and therapeutic purposes Notable therapeutic enzymes include diastase (α-amylase) for digestive aid, asparaginase for leukemia treatment, rhodanese for cyanide poisoning, and streptokinase for dissolving blood clots These applications highlight the significant impact of enzymes in enhancing medical outcomes.
The penicillin (β-lactams) industry relies on specific enzymes and processes, with antibiotic resistance frequently attributed to β-lactamases To combat this, researchers are exploring novel antibiotic structures that remain effective against these enzymes Penicillin acylase plays a crucial role in producing 6-aminopenicillanic acid (6-APA), a key precursor for semisynthetic penicillins, which can be synthesized through both enzymatic and chemical methods Additionally, enzyme profiles from various bodily fluids such as serum, amniotic fluid, and urine are regularly monitored as clinical markers, with their levels often linked to specific disease conditions, aiding in accurate diagnosis.
The industrial preparation and use of enzymes involve significant safety and regulatory concerns, particularly regarding potential hazards such as allergenicity, functional toxicity, and chemical toxicity Large-scale enzyme production often results in partially pure preparations that may contain harmful contaminants, including mycotoxins from the source material It is crucial to address the risks associated with trace contamination from unsafe microorganisms in final enzyme products To ensure their safety in food applications, enzymes must be classified as GRAS (generally recognized as safe) Certain enzymes, especially proteases, pose risks to sensitive tissues when concentrated, and as proteins, enzymes can also serve as potent allergens.
Modifying Enzymes to Suit Requirements
Significant advancements in technology have enabled the alteration of enzyme properties, despite the extensive natural diversity of biological catalysts This modification process includes various techniques such as enzyme immobilization, chemical modification, genetic engineering, and the application of enzymes in nonaqueous solvents.
Immobilization of natural enzymes, which are typically water-soluble and unstable over time, significantly enhances their shelf life and facilitates easy recovery for repeated use, making it a cost-effective solution in cases where enzyme prices are high (Mateo et al 2007) The performance of immobilized enzyme systems is heavily influenced by the characteristics of the matrix used, which can be either inorganic or organic Key physical properties such as particle size, swelling behavior, and mechanical strength play a crucial role in determining the operational conditions of these systems Enzymes can be immobilized on the matrix through irreversible methods like covalent bonding and entrapment, or reversible methods such as adsorption and ionic binding Ultimately, the cost of the immobilization process is a critical factor in assessing its economic viability.
Enzyme immobilization employs various techniques, with several illustrated in Fig 3.4 Over the years, the technology and applications of enzyme immobilization have expanded significantly For comprehensive insights, numerous reference materials, including the Methods in Enzymology series, are available (Brena and Batista-Viera).
2006) This section covers very briefly on this applied aspect of enzymes, and the reader is encouraged to refer the more specialized literature for the purpose.
The selection of an enzyme immobilization method is influenced by the enzyme type and its intended application Non-covalent methods, such as physical entrapment and electrostatic adsorption, can result in enzyme leaching during use, while covalent anchoring necessitates bifunctional cross-linking reagents and compatible functional groups on the enzyme that do not impair its activity Advanced chemistry has been developed to prepare inert organic and inorganic polymers for enzyme immobilization For instance, carrier-bound penicillin acylase is effective in producing 6-APA and allows for the economical recycling of the catalyst, achieving over 99% conversion efficiency even after 1500 cycles Glutaraldehyde is utilized to cross-link glucose isomerase, enabling its repeated use in fructose syrup production Additionally, microencapsulation has mitigated the allergenic effects of detergent protease components, resulting in safer, dustless preparations Various successfully immobilized enzymes are detailed in Table 3.6.
Fig 3.4 Four different modes of enzyme immobilization
Table 3.6 Industrial uses of immobilized enzymes Enzyme Product
Aspartate ammonia lyase L-Aspartic acid
Lactase Lactose-free milk and whey
Glucose isomerase High-fructose syrup
Penicillin acylase 6-APA and penicillins
3.2 Modifying Enzymes to Suit Requirements 23
Immobilization of enzymes can influence their substrate specificity and catalytic potential, potentially leading to reduced activity due to changes in their microenvironment Factors such as heat, pH, and proteolytic enzymes can affect enzyme stability, and conformational changes may further decrease activity The pH optimum of immobilized enzymes can shift significantly, up to 2.0 pH units, depending on the type of carrier used; anionic carriers typically raise the pH, while cationic carriers lower it These shifts result from alterations in the ionization of amino acid residues, although this effect is diminished in high ionic strength environments where salt ions neutralize the carrier's charges.
The kinetic behavior of immobilized enzymes differs notably from that of free enzymes in solution, impacting both the maximal velocity (Vmax) and the Michaelis constant (KM), which reflects the enzyme's apparent affinity for the substrate When the carrier for the enzyme has an opposite charge to the substrate, the apparent KM decreases significantly due to electrostatic interactions that increase substrate concentration near the carrier Additionally, partition effects from ionic, electrostatic, or hydrophobic interactions alter the local concentration of ligands, further influencing kinetic constants Diffusion factors also play a crucial role, as the movement of substrate toward the enzyme and the diffusion of product away from it can significantly affect enzyme activity Restricted diffusion of the bulk substrate to the immobilized enzyme can lead to notably higher KM values.
Poor product diffusion away from the enzyme leads to inhibited enzyme activity When diffusion limits the maximum achievable substrate concentration, the immobilized enzyme may exhibit a lower apparent Vmax compared to the free soluble enzyme.
The primary goal of employing enzyme-catalyzed reactions in industrial settings is to maximize the conversion of substrates into products To effectively analyze these systems, the integrated form of the Michaelis-Menten rate equation is utilized, provided certain conditions are met: substrate concentrations must significantly exceed enzyme levels, the reaction should be irreversible, the enzyme must remain stable over time, and there should be no inhibition from products or substrates, with proper mixing maintained These factors can often be managed through strategic enzyme reactor configurations and process designs, such as using immobilized enzymes in various reactor types like stirred-tank, packed-bed, or fluidized-bed reactors Additionally, the flow of reaction mixtures can facilitate the introduction of fresh substrates, removal of products, and pH adjustments Notably, the use of membrane cassettes with penicillin acylase has significantly enhanced the efficiency of producing 6-APA, with pH control achieved through NaOH dosing and continuous product removal.
Enzyme biosensors are analytical devices that measure the concentration of biologically relevant substances, typically metabolites These biosensors utilize enzymes to transform biological responses into electrical signals, enabling precise monitoring and determination of various compounds.
Immobilized enzymes offer enhanced specificity and signal amplification, making them ideal for various applications Their ability to be reused and their elevated K M values allow for a proportional change in reaction rates across a significant linear range of substrate concentrations Additionally, these enzymes often demonstrate stability, with reaction rates remaining consistent regardless of pH, temperature, ionic strength, and inhibitors, which is particularly beneficial for metabolite measurements in real analytical samples.
The transducer is a vital element of biosensors, converting enzyme reaction outcomes into measurable signals Different types of biosensors utilize various transduction methods, including calorimetric biosensors for heat generation (e.g., measuring H2O2 with catalase), potentiometric biosensors for ion release or absorption (e.g., glucose with glucose oxidase), amperometric biosensors for current production (e.g., glucose, alcohol, and cholesterol detection), and optical biosensors for light absorption or emission (e.g., peroxides with horseradish peroxidase) Additionally, paper enzyme strips are employed for colorimetric detection of substances Currently, enzyme biosensors play a significant role in the analytical markets of healthcare, food safety, and environmental monitoring.
Nonaqueous enzymology explores the function of enzymes in organic solvents, highlighting their evolution as biological catalysts closely tied to aqueous environments While many enzymes are susceptible to denaturation by organic solvents, certain enzymes exhibit remarkable tolerance to high concentrations of water-miscible organic solvents, maintaining their activity in these unique conditions.
Enzymes require a monolayer of water molecules on their exposed surfaces and active sites, even in nonpolar solvents (Halling, 2004) This essential water layer allows them to maintain catalytic activity in organic solvents, as demonstrated by the pioneering research of Bourquelot and others.
Since 1913, research has indicated that only a limited number of enzymes can function effectively in environments with over 80% organic solvents, like ethanol or acetone This discovery is a significant precursor to the field of nonaqueous enzymology, which explores the use of enzymes in organic solvents for synthetic purposes Notably, the synthesis of a glucoside by maltase in 1898 demonstrated the early synthetic potential of enzymes.
The growing interest in biocatalysis within nonaqueous phases is driven by its advantages, including excellent enantioselectivity, the ability to reverse thermodynamic equilibrium, and the elimination of water-dependent side reactions Water is often not the optimal solvent for many organic reactions.
Genetic Engineering and Enzymes
Large-scale production of enzymes is often a prerequisite for most applications.
The extraction of enzymes from animal and plant sources, once common, has become increasingly challenging due to economic and ethical concerns Consequently, researchers are now focusing on the vast microbial biodiversity to identify enzymes with similar desirable properties A notable example is microbial rennin, derived from Mucor spp., which serves as an alternative to chymosin Additionally, recombinant DNA technology offers a promising method for producing these essential enzymes, ideally within microbial hosts.
The formidable tools of genetic engineering have allowed the expression and management of enzyme structures almost at will Detailed recipes of recombinant
DNA techniques are extensively documented in various texts and protocols, providing a foundational understanding of genetic engineering This process involves the precise manipulation of DNA sequences, allowing for the cutting and rejoining of DNA fragments or genes from different organisms The resulting recombinant DNA molecules can express either natural or modified enzyme proteins when introduced into a suitable host Once the genetic information is transcribed and translated in the new host, it can produce enzyme proteins that are foreign to that organism A general strategy for genetic engineering encompasses four essential stages, as illustrated in Fig 3.5.
Isolation of the Gene/ORF/cDNA for the Enzyme Protein Restriction endonucleases of different sequence specificities are employed to cut out the desired gene or the
An open reading frame (ORF) encodes an enzyme derived from a specific DNA source This essential DNA fragment can be amplified using polymerase chain reaction (PCR), or alternatively, the corresponding complementary DNA (cDNA) can be obtained through reverse transcriptase-PCR.
3.3 Genetic Engineering and Enzymes 27 relevant mRNA Alternatively, the required DNA may also be synthesized chemi- cally–if the desired nucleotide or amino acid sequence is known.
The insertion of a gene into an expression vector involves integrating the gene, ORF, or cDNA into a vector DNA to enhance its uptake and replication in a suitable host organism These DNA vectors are engineered with unique restriction endonuclease sites and carry selection markers to identify genetically modified host cells, known as transformants The resulting recombinant DNA is then introduced into an appropriate host for enzyme protein expression, typically using bacterial plasmid vectors for bacteria, while viral DNAs are often employed as vectors for animal and plant cells.
Transformation of the Host Cell The recombinant DNA vector is introduced into a host cell either directly (by the process of transformation) or by infecting it using a viral vector.
Fig 3.5 A general genetic engineering strategy for enzyme expression
The detection of inserted genes in host organisms is achieved using molecular techniques such as Southern blotting and PCR, which identify foreign DNA inserts Additionally, the functional expression of recombinant DNA can be assessed by monitoring enzyme activity and the presence of specific proteins.
Genetic engineering tools enable the production of any enzyme protein in the bacterium Escherichia coli, with options to use bacteria, yeast, plants, or mammalian cells for optimal expression and posttranslational processing Successful expression typically occurs in a homologous host Enzymologists manipulate the DNA blueprint of enzymes to modify existing features or introduce new ones, employing techniques such as site-directed mutagenesis (SDM) and directed evolution, which have become standard practices in enzyme design and redesign Further discussion on these genetic engineering strategies will be provided later in this book.
(Chap.39“Future of Enzymology–An Appraisal”) In addition, genetic engineer- ing has made optimal enzyme production possible in many different ways We will simply illustrate thefield with a few examples:
Engineered enzyme overproducing strains have been developed by overcoming regulatory mechanisms such as feedback inhibition, transcription, translation, and enzyme secretion The expression of amylases is frequently controlled by carbon catabolite repression, particularly glucose repression, while nitrogen metabolite regulation poses challenges for protease production Mutated bacterial and fungal strains that exhibit deregulation, constitutive expression, and hypersecretion are valuable for enzyme production Genetically stable mutant strains that integrate these characteristics are increasingly utilized in the enzyme industry.
Individual component activities of the cellulase complex and their specific combinations find industrial applications (Table 3.1) Producer strains (like
Trichoderma reesei strains that overexpress specific cellulase activities or have deletions in these activities are available These well-defined cellulase component cocktails are particularly effective for applications in the textile industry and biomass conversion processes.
Heterologous expression of mammalian or plant enzymes in microbial hosts offers a convenient method for producing recombinant proteins For instance, recombinant bovine chymosin was successfully generated in a fungus through a stable expression construct However, significant challenges remain, such as achieving economically viable secretion levels, ensuring protein stability, and obtaining appropriate posttranslational modifications, including glycosylation.
After cloning and expressing the structural gene for an enzyme, it becomes possible to create mutant variants of that enzyme This allows for modifications in properties such as stability, pH optimum, specificity, and regulatory features Site-directed mutagenesis is a key technique in protein and enzyme engineering, leading to practical applications in the enzyme industry, exemplified by the engineering of subtilisin for enhanced stability.
Glucose isomerase was improved for its metal preference, substrate specificity, and pH optimum DNA polymerase with high fidelity for PCR applications is another fruitful example.
The impact of genetic engineering on thefield of modern enzymology may be further gauged by examples presented in Chap.39 (“Future of Enzymology: AnAppraisal”).
Summing Up
Enzymes, remarkable catalysts of nature, have been utilized since the first enzyme application patent in 1894, well before their chemical properties were understood The industry has since flourished, particularly in starch processing, by leveraging enzymes from various life domains In clinical and pharmaceutical contexts, enzymes serve as disease markers, analytical tools, and targets for drug discovery Industrially, immobilized enzymes enhance cost-effectiveness by allowing reuse and scaling of reactions While most enzymes are optimized for aqueous environments, some can function in organic solvents, broadening their application for synthetic reactions With advancements in genetic engineering, enzymes are being tailored for specific features and large-scale production, significantly expanding their industrial applications This chapter provides a concise overview of these developments, with further details available in the cited literature.
Brena BM, Batista-Viera F (2006) Immobilization of enzymes In: Guisan JM (ed) Immobilization of enzymes and cells Humana Press, Totowa, pp 15–30
Halling PJ (2004) What we can learn by studying enzymes in non-aqueous media? Phil Trans R Soc Lond B 359:1287–1297
Li Q, Yi L, Marek P, Iverson BL (2013) Commercial proteases: present and future FEBS Lett 587:1155–1163
Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improve- ment of enzyme activity, stability and selectivity via immobilization techniques Enz Microb Technol 40:1451–1463
Payne CM, Knott BC, Mayes HB, Hansson H, Himmel ME, Sandgren M, Stồhlberg J, Beckham GT
Robertson JG (2005) Mechanistic basis of enzyme-targeted drugs Biochemistry 44:5561–5571
Umezawa H (1982) Low-molecular-weight enzyme inhibitors of microbial origin Ann Rev
Kirk O, Borchert TV, Fuglsang CC (2002) Industrial enzyme applications Curr Opin Biotechnol
Klibanov AM (2001) Improving enzymes by using them in organic solvents Nature 409:241 – 246
In recent studies, Sharma et al (2017) explored NAD(P)H-dependent dehydrogenases, highlighting their role in the asymmetric reductive amination of ketones, including insights into their structure, mechanism, evolution, and practical applications Additionally, van Beilen and Li (2002) provided a comprehensive overview of enzyme technology, discussing its significance and advancements within the biotechnology sector.
On Enzyme Nomenclature and Classification 4
What Is in the Name?
The term "wordenzyme," derived from the Greek word for yeast (ενζυμη), was first introduced by Kuhne in 1877 and is widely recognized today as a biological catalyst Most enzyme names currently use the suffix "-ase," a convention established by Duclaux for naming enzymes.
In 1898, proteolytic enzymes emerged as a notable exception to the typical naming conventions in biochemistry Many of these enzymes, such as trypsin, chymotrypsin, papain, and subtilisin, continue to follow the traditional practice of ending with the suffix "-in."
Enzymes are primarily proteins, although some RNA molecules, known as ribozymes, also serve as biological catalysts Most enzymes in biology are protein-based, constructed from 20 L-isomers of amino acids, which limits the types of reactive chemical groups available for catalysis While proteins contain many nucleophilic groups, they often lack sufficient electrophiles Consequently, nonprotein components called cofactors are essential for creating functional catalysts The inactive protein part without the cofactor is referred to as the apoenzyme, while the active enzyme, which includes the cofactor, is known as the holoenzyme Cofactors can include metal ions, enhancing the catalytic capabilities of enzymes.
Metal ions such as Mn[II], Ni[II], and Ca[II] serve as cofactors in enzymes like arginase, urease, and DNase I, respectively Additionally, organic molecules, including pyridine nucleotides (NAD+ and NADP+), flavin adenine dinucleotide (FAD), pyridoxal phosphate (PLP), thiamine pyrophosphate (TPP), biotin, cobamide, and heme, function as coenzymes The binding strength between a cofactor and its corresponding apoenzyme can vary, with tightly bound cofactors, known as prosthetic groups, being challenging to remove without damaging the enzyme Often, these prosthetic groups, such as lipoamide in transacylase, are covalently attached to the apoenzyme.
# Springer Nature Singapore Pte Ltd 2018
N S Punekar, ENZYMES: Catalysis, Kinetics and Mechanisms, https://doi.org/10.1007/978-981-13-0785-0_4
Non-covalent interactions between proteins, like apoenzymes, and cofactors can vary in strength, significantly impacting enzyme activity Enzymes requiring divalent metal ions exemplify this, with tightly bound metal ions classified as metalloenzymes, such as urease, and those with weakly bound metal ions categorized as metal-activated enzymes, like iron-dependent enzymes.
Enzymes are classified based on their metal ion dissociation constants (K D), with those having K D values of 10^8 M or higher categorized as metal-activated enzymes, while metalloenzymes possess K D values lower than 10^8 M This classification, however, is somewhat arbitrary, as there exists a continuum of binding strengths in nature Notably, non-covalent interactions can also exhibit significant strength, with some, like the avidin–biotin complex, being nearly irreversible.
(K Dẳ10 15 M;t 1/2 of 2.5 years) or for that matter the two strands of a double- stranded DNA!
Enzyme Diversity and Need for Systematics
In the mid-twentieth century, enzyme research surged dramatically, leading to an overwhelming number of newly identified enzymes This proliferation resulted in instances where similar enzyme activities were assigned different names, often lacking descriptive value, as seen with terms like "catalase." Consequently, systematic classification, cataloging, and nomenclature of enzymes became essential, though this task proved challenging Enzymes can be categorized based on various criteria.
Laccase is derived from the Japanese lacquer tree, while papain is sourced from papaya, and horseradish peroxidase is another notable plant enzyme Additionally, several digestive enzymes, including trypsin, chymotrypsin, carboxypeptidase, and lipase, are extracted from pancreatic juice Furthermore, lysozyme is commonly obtained from hen egg white.
(b) Nature of the substrate on which the enzyme acts: They could be classified into enzymes hydrolyzing (or acting on) proteins, carbohydrates, lipids, etc.
Enzymes can be categorized based on their cofactor requirements, with many being purely proteinaceous However, those that rely on cofactors can be classified into distinct groups, such as thiamine pyrophosphate (TPP) enzymes, pyridoxal phosphate (PLP) enzymes, and metalloenzymes.
Enzymes can be categorized based on their functional context, such as grouping glycolytic enzymes or those involved in histidine biosynthesis Additionally, they can be classified as soluble, membrane-bound, or associated with specific organelles like mitochondria.
34 4 On Enzyme Nomenclature and Classification
Enzymes can be categorized based on the specific type of reaction they facilitate, such as oxidation or hydrolysis, which defines their overall catalytic nature.
The reaction mechanism at the enzyme active site involves understanding the nature of intermediate complexes formed during the process For instance, proteases can be categorized based on the formation of an enzyme-bound acyl-enzyme intermediate, highlighting the significance of this interaction in enzymatic activity.
The classification and cataloging of enzymes has proven to be a complex challenge due to the vast diversity in their structures and functions For instance, RNA hydrolysis can occur through various means, including proteins like RNase A, protein-RNA complexes such as RNase P, or even RNA itself in the case of ribozymes Additionally, peptide bond hydrolysis can be facilitated by enzymes that operate efficiently under either acidic or alkaline conditions, often requiring divalent metal ions or specific functional groups like serine -OH or cysteine -SH Enzymatic decarboxylation of histidine may utilize pyridoxal phosphate or, in simpler forms, a bound pyruvate Interestingly, while pyridoxal phosphate is bound to glycogen phosphorylase, it primarily serves a structural role rather than acting as a cofactor Furthermore, alkyl-dihydroxyacetonephosphate synthase, which plays a role in ether phospholipid biosynthesis, employs FAD for non-redox reactions Given this complexity, no single classification criterion suffices, prompting the establishment of an international commission in 1955, which released its first report in 1961.
Enzyme Commission: Recommendations
Considering the diversity of enzyme sources, reactions, and mechanisms, it became apparent that a formal system of nomenclature and classification was required.
to each enzyme.
The Enzyme Commission has established a systematic approach to naming enzymes, allowing each to be assigned a trivial name and an EC catalog number (McDonald et al., 2009) Enzymes are classified into six general categories based on the overall reaction they catalyze, as outlined by a formal equation, without delving into the detailed mechanisms or intermediate complexes involved Each of these six classes encompasses a variety of unique enzymes, as documented in the BRENDA database.
In January 2009, a classification system for enzymes was established, highlighting six primary classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases, with redox and hydrolytic reaction enzymes being the most prevalent Each class is subdivided into various subclasses and sub-subclasses based on the nature of the catalyzed reactions Enzymes are assigned a unique four-number code, where the first digit indicates the main class, the second and third specify the subclass and sub-subclass, and the fourth number identifies the specific enzyme within its sub-subclass For instance, alcohol dehydrogenase is classified as "EC 1.1.1.1," with the first number signifying its membership in the oxidoreductase class (EC 1.x.x.x).
CH-OH group of donors bear the same subclass number (EC 1.1.x.x) Within this subclass, enzymes that use NAD or NADP as electron acceptor are given the number
Alcohol dehydrogenase is classified as EC 1.1.1.1, being the first enzyme in its category In contrast, all carboxylesterases share the same initial three digits in their EC code (EC 3.1.1.x), with the fourth digit differentiating them based on the specific carboxylic ester they hydrolyze.
Each enzyme is assigned a systematic name by the Commission, which includes the names of substrates and a reaction name ending in “-ase.” Due to the potential length of these systematic names, the Commission also recommends using simpler trivial names However, for common proteases such as pepsin, trypsin, and papain (group EC 3.3.3.x), acceptable systematic names have not yet been established Table 4.1 provides the enzyme Commission nomenclatures for representative enzymes of each class, including those frequently mentioned in this book.
The universally accepted EC classification and enzyme codes are finding place
(and direct utility) in a number of databases describing enzymes, genes, genomes, and metabolic pathways Some of these databases are listed in Table4.2.
Fig 4.1 Distribution of enzymes into six different classes according to EC classi fi cation Data from
36 4 On Enzyme Nomenclature and Classification
Enzyme commission nomenclatures categorize enzymes based on their functions and reactions Oxidoreductases facilitate the transfer of electrons, with examples including alcohol dehydrogenase, which converts alcohol and NAD+ into aldehyde, NADH, and H+, and lactate dehydrogenase, which transforms L-lactate and NAD+ into pyruvate, NADH, and H+ Transferases, such as serine hydroxymethyltransferase, transfer reactive groups between substrates, exemplified by the conversion of L-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate Hydrolases introduce water into substrates, illustrated by carboxylesterase, which hydrolyzes carboxylic esters into alcohol and carboxylate Each enzyme plays a crucial role in biochemical processes, highlighting the importance of understanding their classifications and reactions for applications in biochemistry and medicine.
Table 4.1 presents a comprehensive overview of various enzymes categorized by their reaction types It includes L-Arginine amidinohydrolase, which converts L-Arginine and water into L-Ornithine and urea, and Oxaloacetate acetylhydrolase, transforming oxaloacetate and water into oxalate and acetate The table also covers lyases, such as Pyruvate carboxy-lyase, which decarboxylates pyruvate into acetaldehyde and carbon dioxide Additionally, isomerases like Proline racemase facilitate the intramolecular rearrangement of L-Proline to D-Proline Ligases, such as L-Tyrosine:tRNA ligase, join molecules through covalent bond formation, exemplified by the reaction of ATP, L-Tyrosine, and tRNA to produce AMP and L-Tyrosyl-tRNA These enzymes play critical roles in various biochemical processes, highlighting their importance in metabolic pathways.
38 4 On Enzyme Nomenclature and Classification