The enzyme family of proteins catalyzes cellular reactions and thus makes them ideal drug targets. Adapted from Pediatric Critical Care, 4th Edition, 2011

Related terms:

Aminoglycoside Resistance–Modifying Enzymes

Among aerobic bacteria, aminoglycoside resistance is most commonly due to enzymatic inactivation through aminoglycoside-modifying enzymes. These may be coded by genes on plasmids or chromosomes. Several aminoglycoside-modifying enzymes have been shown to be carried on transposons.74

Aminoglycoside-modifying enzymes confer antibiotic resistance through three general reactions:N-acetylation,O-nucleotidylation, andO-phosphorylation. For each of these general reactions, there are several different enzymes that attack a specific amino or hydroxyl group. The nomenclature for these enzymes lists the molecular site where the modification occurs after the type of enzymatic activity. An aminoglycoside acetyltransferase (AAC) that acts at the 3′ site is designated AAC(3′) (Table 18.5). There may be more than one enzyme that catalyzes the same reaction, however, and Roman numerals may be necessary (e.g., AAC<3′>-IV).

Enzymatic aminoglycoside resistance is achieved by modification of the antibiotic in the process of transport across the cytoplasmic membrane.74 Resistance to a particular aminoglycoside is a function of two different rates—that of drug uptake versus that of drug inactivation. An important factor in determining the level of resistance is the affinity of the modifying enzyme for the antibiotic. If an enzyme has a high affinity for the specific aminoglycoside, drug inactivation can occur at very low concentrations of the enzyme.

The differences in the worldwide distribution of aminoglycoside-modifying enzymes may be partially a function of antibiotic selection pressures and may have had profound implications on the choice of antibiotics used at specific medical centers. Aminoglycoside phosphotransferase (APH)(3′) and APH(3″) are distributed widely among gram-positive and gram-negative species worldwide and have led to decreased use of kanamycin and streptomycin. The gene for aminoglycoside nucleotidyltransferase (ANT)(2″) has been associated with multiple nosocomial outbreaks in the 1990s across the United States. The gene for aminoglycoside acetyltransferase AAC(6′)-I has been found to be more prevalent in enteric bacteria and in staphylococci in East Asia.75 The AAC(3′) group of enzymes have been responsible for outbreaks of antibiotic resistance in South America, Western Europe, and the United States. Although each outbreak of aminoglycoside-resistant Enterobacteriaceae has its own pattern, the most typical manner of spread has been the appearance of a plasmid-carrying, aminoglycoside-resistant strain ofK. pneumoniae, usually carrying theANT(2″) gene, with subsequent dissemination to other strains of the species and further spread later to other species and genera of Enterobacteriaceae.76

Enzymes are catalysts that, within the mild conditions of temperature, pH, and pressure of the cells, carry out chemical reactions at amazing high rate. They are characterized by a remarkable efficiency and specificity.

Substrates are the substances on which enzymes act.

Enzymes are named by adding the suffix -ase to the name of the substrate that they modify (i.e., urease and tyrosinase), or the type of reaction they catalyze (dehydrogenase, decarboxylase). Some have arbitrary names (pepsin and trypsin). The International Union of Biochemistry and Molecular Biology assigns each enzyme a name and a number to identify them.

Enzymes are classified into six categories according to the type of reaction catalyzed:

Oxidoreductases, transferases, hydrolases, lyases, ligases, and isomerases.

Structurally, the vast majority of enzymes are proteins. Also RNA molecules have catalytic activity (ribozymes).

Coenzymes are small nonprotein molecules that are associated to some enzymes. Many coenzymes are related to vitamins. Coenzymes and the protein portion with catalytic activity or apoenzyme form the holoenzyme. The apoenzyme is responsible for the enzyme’s substrate specificity. Coenzymes undergo changes to compensate for the transformations occurring in the substrate.

Metalloenzymes are enzymes that contain metal ions.

The mechanism of action of enzymes depends on the ability of enzymes to accelerate the reaction rate by decreasing the activation energy. During the course of the reaction, the enzyme (E) binds to the substrate/s (S) and forms a transient enzyme–substrate complex (ES). At the end of the reaction, the product/s are formed, the enzyme remains unchanged, can bind another substrate and can be reused many times.

Active site or catalytic site is the specific place in the enzyme where the substrate binds. The structural complementarity between E and S allows an exact reciprocal fit. The enzyme adapts to the substrate via a conformational change known as induced fit. The presence in the active site of amino acids that bind functional groups in the substrate ensures adequate location of the substrate and formation of the transition intermediary, which will be subjected to catalysis.

Zymogens or proenzymes are inactive precursors of enzymes. They acquire activity after hydrolysis of a portion of their molecule.

Cellular location of enzymes varies, the majority being in different compartments of the cell, while others are extracellular.

Multienzyme systems are those composed of a series of enzymes or enzyme complexes. There are also multifunctional enzymes with several different catalytic sites in the same molecule.

Enzyme activity is determined by measuring the amount of product formed, or substrate consumed in a reaction in a given time. Initial velocity corresponds to the activity measured when the amount of consumed substrate is less than 20% of the total substrate originally present. One IU of enzyme catalyzes the conversion of 1 μmol of substrate per second under defined conditions of pH and temperature. Specific activity is the units of enzyme per milligram of protein present in the sample. Molar activity or turnover number are the substrate molecules converted into product per unit time per enzyme molecule, under conditions of substrate saturation.

The rate of the enzymatic reaction is directly proportional to the amount of enzyme rate present in the sample.

Also, at low and under constant conditions of the medium, enzyme activity rapidly increases with the raise in . At higher substrate levels, the activity increases slowly and tends to reach a maximum. The effect follows a hyperbolic function; at low the reaction is first order; at high the reaction is zero order with respect to the substrate.

Km or Michaelis constant is the at which the reaction rate reaches a value equal to half the maximum.

Under given conditions of pH and temperature, the Km value is distinctive for each enzyme and is used to characterize it. For most enzymes, the Km value is inversely related to the affinity of the enzyme for the substrate, the higher the affinity, the lower the Km.

Temperature affects enzyme activity, increasing it to reach a peak, which corresponds to the optimal enzyme activity. Beyond this maximum, enzyme activity rapidly drops. The optimal temperature for most mammalian enzymes is around 37°C. The inactivating effect of temperatures above 40°C is due to protein denaturation.

pH affects enzyme activity, by influencing the state of dissociation of functional groups involved in the ES complex. Enzymes have an optimum pH and extreme values of pH cause enzyme denaturation.

Enzyme inhibitors can be classified as:Irreversible, which permanently inactivate the enzyme, and

Reversible, which consist of the following inhibitors:Competitive: increase the Km but not the Vmax, its action is reversed by increasing . Some have structural similarity to the substrate and compete with it for the active site.

Noncompetitive: bind to the enzyme in a site different to the catalytic center. They decrease Vmax, leave Km unaffected, and are not influenced by .

Anticompetitive: reduce Km and Vmax.

Enzymes are subjected to regulation, to adapt to the requirements of different cells. When the in the cell is below the Km, changes in modify the activity. Allosteric enzymes are those modulated by agents that bind to them at a site different to the active center. The curve of initial velocity versus for allosteric enzymes is not hyperbolic, but sigmoid. Enzyme activity is also changed by covalent modification, such as phosphorylation.

Constitutive enzymes are those whose levels remain constant throughout the life of the cell. Inducible enzymes, are those whose synthesis is activated as required.

Isozymes are different proteins that have the same enzyme activity.

Robert M. Kliegman MD, in Nelson Textbook of Pediatrics, 2020

Serum Enzymes

Several lysosomal enzymes are released by damaged or degenerating muscle fibers and may be measured in serum. The most useful of these enzymes iscreatine kinase (CK), which is found in only three organs and may be separated into corresponding isozymes: MM for skeletal muscle, MB for cardiac muscle, and BB for brain. Serum CK determination is not a universal screening test for neuromuscular disease because many diseases of the motor unit are not associated with elevated enzymes. The CK level is characteristically elevated in certain diseases, such as Duchenne muscular dystrophy, and the magnitude of increase is characteristic for particular diseases. CK may also be elevated in certain nonneuromuscular disorders (Table 625.5).

Rhabdomyolysis is often a dramatic event associated with high plasma CK levels, myoglobinuria, and muscle pain or tenderness. It may be acquired (Table 625.6 andFig. 625.5), due to metabolic diseases (Table 625.7), or occur spontaneously or secondary to various triggers (Fig. 625.6).

VitaminCoenzymeReaction CatalyzedHuman Deficiency Disease
Water-Soluble Vitamins
Niacin (niacinate)NAD+, NADP'OxidationPellagra
NADH, NADPHReduction
Riboflavin (vitamin B2)FAD, FMNOxidationSkin inflammation
FADH2, FMNH2Reduction
Thiamine (vitamin B1)Thiamine pyrophosphate (TPP)Two-carbon transferBeriberi
Lipoic acid (lipoate)LipoateOxidation
Pantothenic acid (pantothenate)Coenzyme A (CoASH)Acyl transfer
Biotin (vitamin H)BiotinCarboxylation
Pyridoxine (vitamin B6)Pyridoxal phosphate (Pl.P)DecarboxylationAnemia
Cα–Cβ bond cleavage
α,β Elimination
Vitamin B12Coenzyme B12IsomerizationPernicious anemia
Folic acid (folate)Tetrahydrofolate (THF)One-carbon transferMegaloblastic anemia
Ascorbic acid (vitamin C)Scurvy
Water-Insoluble (Lipid-Soluble) Vitamins
Vitamin A
Vitamin DRickets
Vitamin E
Vitamin KVitamin KH3Carboxylation

This table is reproduced from

Reproduced from G. Litwack, Human Biochemistry and Disease, Academic Press/Elsevier, Table 3-1, page 119, 2008

Douglas P. Zipes MD, in Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, 2019

Lipoproteins, Apolipoproteins, Receptors, and Processing Enzymes

Lipoproteins are complex macromolecular structures coated by a water-compatible envelope of phospholipids, free cholesterol, and apolipoproteins covering a hydrophobic core of cholesteryl esters and triglycerides. Lipoproteins vary in size, density in the aqueous environment of plasma, and lipid and apolipoprotein content (Fig. 48.2andTable 48.1). The classification of lipoproteins reflects their density in plasma (the density of plasma is 1.006 g/mL) as gauged by flotation in an ultracentrifuge. The triglyceride-rich lipoproteins (TRLs) consist of chylomicrons, chylomicron remnants, and very-low-density lipoprotein (VLDL) and have a density of less than 1.006 g/mL. The rest (bottom fraction) of the ultracentrifuged plasma consists of low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein(a) (Lp).

Apolipoproteins have four major roles: (1) assembly and secretion of the lipoprotein (apo A-I, B100, and B48), (2) structural integrity of the lipoprotein (apo B, E, A-I, and A-II), (3) coactivators or inhibitors of enzymes (apo A-I, A-V, C-I, C-II, and C-III), and (4) binding or docking to specific receptors and proteins for cellular uptake of the entire particle or selective uptake of a lipid component (apo A-I, B100, and E) (Table 48.2). The role of several apolipoproteins (A-IV, A-V, D, H, J, L, and M) remains incompletely understood.

Many proteins regulate the synthesis, secretion, and metabolic fate of lipoproteins; their characterization has provided insight into molecular cellular physiology and targets for drug development (Table 48.3). Discovery of the LDL receptor (LDL-R) represented a landmark in understanding cholesterol metabolism and receptor-mediated endocytosis.12 The LDL-R regulates the entry of cholesterol into cells, and tight control mechanisms alter its expression on the cell surface, depending on intracellular cholesterol. The LDL-R belongs to a superfamily of membrane receptors that include LDL-R, VLDL-R, LDL-R–mediated peptide type 1 (LRP1; apo E receptor), LRP1B, LRP4 (MGEF7), LRP5 and LRP6 (involved in the process of bone formation), LRP8 (apo E receptor-2), and LRP9.13 LRP1, which mediates the uptake of chylomicron remnants and VLDL, preferentially recognizes apo E. LRP1 also interacts with hepatic lipase. The complex interaction between hepatocytes and the various lipoproteins containing apo E involves cell surface proteoglycans that provide scaffolding for lipolytic enzymes (lipoprotein lipase and hepatic lipase) involved in recognition of remnant lipoproteins. Macrophages express receptors that bind modified (especially oxidized) lipoproteins. These scavenger lipoprotein receptors mediate the uptake of oxidatively modified LDL into macrophages. In contrast to the exquisitely regulated LDL-R, high cellular cholesterol content does not suppress scavenger receptors, thereby enabling intimal macrophages to accumulate abundant cholesterol, become foam cells, and form fatty streaks. Sterol accumulation in the endoplasmic reticulum (ER) may lead to cell apoptosis via the unfolded protein response.14 Endothelial cells can also take up modified lipoproteins through specific receptors such as the oxidized LDL-R LOX-1.

Reinhard Renneberg, .. Vanya Loroch, in Biotechnology for Beginners (Second Edition), 2017

2.3 The Role of Cofactors in Complex Enzymes

Not all enzymes consist exclusively of protein, as does lysozyme. Many include additional chemical components or cofactors which serve as tools. Such enzymes are known as qualified enzymes and have more complicated reaction mechanisms.

Cofactors can consist of one or more inorganic ions (such as Fe3+, Mg2+, Mn2+, or Zn2+) or more complex organic molecules, known as coenzymes. Some enzymes require both types of cofactors.

Coenzymes are organic compounds that bind to the active site of enzymes or near it. They modify the structure of the substrate or move electrons, protons, and chemical groups back and forth between enzyme and substrate, negotiating considerable distances within the giant enzyme molecule. When used up, they separate from the molecule.

Many coenzymes are derived from vitamin precursors, which explains why we require a constant low-level supply of certain vitamins. One of the most essential coenzymes, NAD+ (nicotinamide adenine dinucleotide), is derived from niacin. Most water-soluble vitamins of the vitamin B group act as coenzyme precursors very much like niacin.

Otto Heinrich Warburg (1883–1970, Fig. 2.3) discovered the respiratory enzyme cytochrome oxidase (Fig. 1.14) and NAD. His discovery and subsequent structural analysis was one of the shining hours of modern biochemistry. In the absence of niacin in the diet, certain enzymes (e.g., dehydrogenases) cannot work effectively in the body. The affected human will develop pellagra, a disease caused by vitamin B (niacin) deficiency. Otto Warburg developed an optical test making it possible to quantify reduced NADH at a wavelength of 340 nm (the oxidized NAD+ does not absorb light at this wavelength). It was now possible to measure essential enzyme reactions, such as the detection of glucose using glucose dehydrogenase (see Chapter: Analytical Biotechnology and the Human Genome).

Nowadays, vitamins like B2 (riboflavin), B12 (cyanocobalamin), and C (ascorbic acid) are produced by the ton using biotechnological methods (see Chapter: White Biotechnology: Cells as Synthetic Factories).

Cofactors that are covalently bonded to the enzyme are called prosthetic groups. Flavin adenine dinucleotide acts as a prosthetic group for GOD. Peroxidase and cytochrome P-450 contain a heme group, as found in myoglobin and hemoglobin. The heme group itself consists of a porphyrin ring incorporating an iron ion in its center.

Coenzymes, by contrast, have only loose bonds, and, just like substrates, they undergo changes in the binding process and are used up. Unlike substrates, however, they bind to a whole host of enzymes (e.g., NADH and NADPH of nearly all dehydrogenases) and are regenerated and recycled inside the cells (see Section 2.13). Enzymes that bind to the same coenzyme usually resemble each other in their chemical mechanisms.

While we referred to the cofactors as “tools,” the protein section of the enzyme is the “craftsman” using these tools, who is responsible for their effectiveness. As always, craftsmen and tools rely on each other to achieve the best possible result.

Enzymes can be classified systematically according to the difference between reaction and substrate specificity, and the mechanism of action. The enzyme code (EC) shows such a classification. Notation according to EC number, i.e. EC X.X.X.X, is shown as follows. The first EC number classifies the enzyme reaction mechanism into six groups, namely oxidation–reduction, transition, hydrolysis, dissociation, isomerization and synthesis (creating new chemical bonds with the initial assistance of ATP). Examples of enzymes classified by EC number are:

EC 1.X.X.X-reductase

EC 2.X.X.X-transferase

EC 3.X.X.X-hydrolase

EC 4.X.X.X-lyase

EC 5.X.X.X-isomerase

EC 6.X.X.X-ligase.

Although standards of classification differ in each group, they are subdivided by the difference between enzyme reaction and substrate specificity. These numbers are assigned to the entire enzyme, and over 3000 enzyme reactions have been assigned an EC number. Moreover, enzymes have a variety of activities; for example, ATPase catalyzes the hydrolysis of both proteins and ATP. Sometimes, the substrate that the enzyme metabolizes is omitted from the systemic naming, based on the same rules as a systemic name. For example, the systemic name of EC is alcohol dehydrogenase. There are also many enzymes named in accordance with the naming convention, such as DNA polymerase.

Although an enzyme generally consists of protein, a few enzymes contain non-protein components such as nucleic acid. The ribozyme discovered by Thomas Cech and others in 1986 is a catalyst made of RNA, which acts on itself and cleaves RNA.

Some enzymes require other molecules to function and do not become active unless combined with cofactors (coenzyme, metal, etc.). An apoenzyme, a protein portion without a cofactor, does not have enzymatic activity, whereas a holoenzyme, a protein combined with a cofactor, has such activity.

The organic compound of the non-protein which assists an enzyme reaction in the active center is called a coenzyme. Since coenzymes are essential elements in the active form of the enzyme, they belong to a prosthetic group. Although they differ from typical prosthetic groups, they can easily separate from the enzyme and be consumed during an enzyme reaction with a substrate like NADH. For example, since the cytochrome P450 (CYP) enzyme is bound covalently with the heme iron, heme does not separate from the CYP enzyme. Therefore, this heme moiety is not called a coenzyme. Although lipoic acid is bound covalently to the enzyme, lipoic acid can be separated from the enzyme moiety, so lipoic acid is called a coenzyme. Therefore, the criteria defining coenzymes and prosthetic groups are not strict.

An enzyme may be comprised of two or more protein chains (peptide chain). When it consists of two or more peptide chains, each peptide chain is called a subunit.

P.D. Felgate, in Encyclopedia of Forensic Sciences (Second Edition), 2013

Cloned Enzyme Donor Immunoassay

CEDIA is a more recent homogeneous immunoassay that uses the binding of an antibody to change the activity of genetically engineered fragments of β-galactosidase from Escherichia coli as the enzyme label. The enzyme is present as two inactive fragments, the enzyme acceptor (EA) and the enzyme donor (ED). ED contains a small portion of enzyme missing from the larger EA fragment. Antibodies that bind to the hapten that is conjugated to the ED fragment prevent the reassociating of the enzyme and reduces the enzyme activity. As the amount of drug increases, the amount of bound antibody to the ED fragment decreases resulting in an increase in enzyme activity due to the reassociation of EA and ED. The enzyme hydrolyzes chlorophenol red-β-galactoside (CPGR) to chlorophenol red (CPR) and galactoside and the enzyme activity can be measured spectrophotometrically.

D.S. Stolzenberg, in Evolution of Nervous Systems (Second Edition), 2017 The Dynamic Interplay of Histone Acetyltransferase Enzymes and Histone Deacetylase Enzymes in Regulating Gene Expression

HAT and HDAC enzymes antagonize each other. To elucidate exactly how the interplay between these two enzymes affects gene expression, Wang et al. (2009) used a ChIP-sequencing technique to identify the location of several HAT and HDAC enzymes throughout chromatin. Not surprisingly they found that HATs were recruited to active gene promoters and positively correlated with both histone acetylation and gene transcription. Unexpectedly, however, they found that the distribution of HDAC enzymes frequently overlapped with HATS. Thus, HDACs were recruited to active gene promoters but typically absent in the promoters of silenced genes. These data suggest that most HDACs function to reset chromatin in active gene promoters by removing the acetylation that contributed to transcription initiation. The coincident recruitment of these stop-and-go signals confers tight temporal control on transcription in response to a cell-surface signal by a rapid repression of transcription when the signal has subsided. Therefore active gene promoters cycle between acetylated and deacetylated states.

Anil Kumar Patel, .. Ashok Pandey, in Biotechnology of Microbial Enzymes, 2017

2.1 Introduction

Enzymes are the active proteins (except RNAse) that can catalyze biochemical reactions. These are biomolecules required for both syntheses as well as breakdown reactions by living organisms. All living organisms are built and maintained by these enzymes, which are truly termed as biological catalysts having the capability to convert a specific compound (as substrate) into products at higher reaction rates. Like chemical catalysts, enzymes increase the reaction rate by lowering its activation energy (Ea), hence, products are formed faster and reactions reach their equilibrium state more rapidly. The rates of most enzymatic reactions are millions of times faster than those of the uncatalyzed reactions. They can perform conversions in minutes or even in seconds which otherwise may take hundreds of years (Dalby, 2003; Otten and Quax, 2005). Enzymes are known to catalyze about 4000 biochemical reactions in living beings (Bairoch, 2000). For example, lactase is a glycoside hydrolase that is able to hydrolyze lactose (milk sugar) into its constituent galactose and glucose monomers. It is produced by various microorganisms and also in the small intestine of humans and other mammals helping to digest milk completely. Enzymes are also enantioselective catalysts, which can be used either in the separation of enantiomers from a racemic mixture or in the synthesis of chiral compounds.

Humans recognized the importance of enzymes thousands of years ago; clarification and filtration of wines and beer being the earliest examples of the application of industrial enzymes. Enzymes have been used in brewing, baking, and alcohol production since prehistoric times; however, they did not call them enzymes. One of the earliest written references to enzymes is found in Homer’s Greek epic poems dating from about 800 BC, where it was mentioned that enzymes were used in the production of cheese. The Japanese have also used naturally-occurring enzymes in the production of fermented products like sake, Japanese schnapps brewed from rice, for more than a thousand years. Some enzymes have been designed by nature to form complex molecules from simpler ones while others have been designed for breaking up complex molecules into simpler ones, also a few modify molecules. These reactions involve the making and breaking of the chemical bonds in the components. Owing to their “specificity,” a property of an enzyme that allows it to recognize a particular substrate that they are designed to target, they are useful for industrial processes and are capable of catalyzing the reaction between particular chemicals even if they are present in mixtures with many chemicals. These enzymes are environmentally safe, natural, and are applied very safely in food and even pharmaceutical industries. Still, enzymes are proteins, which like any protein can cause and have caused in the past allergic reactions, hence, protective measures are necessary in their production and applications.

Enzyme technology is an ever evolving branch of “Science and Technology.” With the intervention and influence of Biotechnology and Bioinformatics, continuously novel or improved applications of enzymes are emerging. With novel applications, the need for enzymes with improved properties are also emerging simultaneously. Development of commercial enzymes is a specialized business which is usually undertaken by companies possessing high skills in:

Screening for new and improved enzymes

Selection of microorganisms and strain improvement for qualitative and quantitative improvement

Fermentation for enzyme production

Large-scale enzyme purifications

Formulation of enzymes for sale

Enzyme technology offers industries and consumers an opportunity to replace processes using aggressive chemicals with mild and environmentally friendly enzyme processes. About 3000 enzymes are known of which only 150–170 are being exploited industrially. At present only 5% of chemical products are produced through a biological route in this green era. However, economically feasible and eco-friendly enzymatic processes are emerging as alternatives to physico-chemical and mechanical processes. Based on the different application sectors, industrial enzymes can be classified as: (1) Enzymes in the food industry, (2) Enzymes for processing aids, (3) Enzymes as industrial biocatalyst, (4) Enzymes in genetic engineering, and (5) Enzymes in cosmetics.

Today, enzymes are envisaged as the bread and butter of biotechnology because they are the main tools for several biotechnological techniques (gene restriction, ligation, and cloning, etc.), bioprocesses (fermentation and cell culture), and in analytics in human and animal therapy as medicines or as drug targets. Furthermore they find applications in several other industries, such as food and feed, textiles, effluent and waste treatment, paper, tannery, baking, brewing, dairy, pharmaceuticals, confectionary, etc. (Pandey et al., 2006).

The enzymes utilized today are also found in animals (pepsin, trypsin, pancratin, and chimosin) and plants (papain, bromelain, and ficin), but most of them are of microbial origin, such as glucoamylase, α-amylase, pectinases, etc. The advantage of using microbes for enzyme production is their higher growing abilities, higher productivity, and their easier genetic manipulation for enhanced enzyme production, etc. Enzymes produced from microbial origins are termed as microbial enzymes. Microbes are mainly exploited in industries for enzyme production. Moreover, microbial enzymes are supplied, well standardized, and marketed by several competing companies worldwide. Depending on the type of process, enzymes can be used in soluble form (animal proteases and lipases in tannery) and in immobilized form (isomerization of glucose to fructose by glucose isomerase).