KERTÉSZETI ÉS ÉLELMISZERIPARI EGYETEM

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52

Bioreactors and immobilized enzymes

Judit Kosáry (2018-1)

The lecture deals with the characteristic properties of proteins especially enzymes in living organisms in order to understand the conditions of their use in biotechnological processes.

Biogenic elements

Building biomolecules: carbon (C), hydrogen (H), oxygen (O) and nitrogen (N) (and P és S). They are in the first and second periods of the periodic table (high charge concentration on their surface unit), their atoms are not susceptible to deformation and they form strong (-bonds with their own atoms and other biogenic elements.

Biomolecules

Building biomolecules: carbon (C), hydrogen (H), oxygen (O), nitrogen (N) (and P és S). They are in the first and second periods of the periodic table (high charge concentration on their surface unit), their atoms are not susceptible to deformation and they form with own atoms and other biogenic elements strong (-bonds.

Type of biomolecule

Units

Bonds between units

Proteins

(-Amino acids

Peptide bond (a special carboxylic acid amide, shortly carboxamide bond)

Carbohydrates

Simple sugars

O-Glycosidic bond (a special acetal bond)

Nucleic acids

______________________

Nucleotides

______________________

3’,5’-Phosphodiester bond

_____________________

Lipids (apolar biomolecules)

Simple lipids cannot be hydrolyzed by NaOH

Complex lipids can be hydrolyzed by NaOH

Characteristic data of the structure of proteins, carbohydrates and nucleic acids; characterization of lipids

Optical isomerism of biomolecules

When two molecules have some differences in their structure but their molecular formula (the composition of elements) is the same, they are isomers. When the atoms bond in different order in isomers they are structural (constitutional) isomers. There are different types of structural isomers. In biomolecules tautomerism (the difference between two isomers is in the position of one hydrogen atom and a double bond) can be found frequently (e.g. aldoses and ketoses in carbohydrate chemistry).

Stereoisomers have the same molecular formula and sequence of bonded atoms (constitution) but their atoms have differences in their three-dimensional orientation in space. There are different types of stereoisomers: optical isomers (enantiomers and diastereomers), geometrical isomers and conformers. Conformational isomers (conformers) differ by rotations around one or more single bonds (e.g. chair and sofa conformations of glucopyranoside).

In the case of optical isomerism the carbon skeleton is saturated. The geometry of saturated carbon atoms, due to hybridization (sp3), the angle of the bonds is 109.5° is tetrahedral. A carbon atom with four different substituents (marked by a star) is called a chiral carbon atom (on the basis of the Greek word kheir– hand). In the case of a single chiral atom two isomers, called enantiomers are possible. Enantiomers (antipodes) are related as mirror images. The chemical and physical properties of the enantiomers are the same because the microenvironment of the atoms is also the same. The only difference is in their optical rotation, which is the opposite. An enantiomer can be identified by the direction in which it rotates the plane of monochromatic and monopolarized light. If it rotates the light clockwise, that enantiomer is labeled (+), while its mirror image is labeled (−).

Many biologically active molecules are chiral, including the naturally occurring proteins, carbohydrates and nucleic acids. As enzymes are mostly proteins and proteins are chiral, they preferentially catalyze the transformation of only one of the enantiomers of a chiral substrate. Naturally, occurring proteins are made of L-(-amino acids, while carbohydrates, di-, oligo- and polysaccharides are all made of D-sugars. Nucleic acids contain also D-sugars: ribose or deoxyribose.

Rules of biochemistry at the molecular level

1. Bioaffinity – there is at least one biological surface to interact with a biomolecule.

2. Biocatalysis – in living organisms practically all reactions are catalyzed and the biocatalysts are called enzymes (mostly proteins).

3. Bioregulation – all biochemical processes are regulated.

Proteins

Proteins (polypeptides) are biopolymers made of (-L-amino acids connected by peptide bonds (a special type of carboxamide bond).

Units of the polypeptide chain, the L-(-amino acids

They are the building blocks of proteins connected by peptide bonds. Standard (protein, proteinogenous) amino acids build up proteins, non-standard (non-protein, non-proteinogenous) amino acids can be important metabolic intermediates. The name of standard amino acids is used generally in their abbreviated form. The modified Fischer conventions of the formulas of twenty standard amino acids and their abbreviations are presented in schemes. Ten amino acids (Val, Leu, Ile, Phe, Lys, Thr, Trp, Met, Arg, His) are called essential amino acids because the human body cannot synthesize them from other compounds at the level needed for normal growth, therefore they must be obtained from food. (Notice: while large quantities of the essential amino acids are needed, there are other essential compounds, e.g. vitamins, which we need only in small quantities). Often selenocysteine and taurine are also put on the list of standard amino acids, while Arg and His are classified as semi-essential amino acids by several authors.

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Essential polar and apolar characteristics

Polar character: H-bonds (hidrogénhíd or hidrogénkötés) with water molecules (polarized bonds, e.g. methanol: H3C–OH)

Apolar character: no H-bonds with water

a) non-polarized bonds (e.g. hydrocarbons)

b) polarized bond with a heteroatom of large ESP (e.g. methyl chloride: H3C–Cl)

In simple functional groups (the heteroatom directly connects to the carbon skeleton): amines – weak (gyenge) H-bonds, alcohols – strong (erős) H-bonds, ethers and chlorides – no H-bonds.

Essential polar and apolar characters of simple functional groups

Structural levels of proteins

Primary structure is the sequence of amino acids. On one end of every polypeptide chain, called the amino terminal or N-terminal, there is a free amino group. The other end, with its free carboxyl group, is called the carboxyl terminal or C-terminal.

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Primary structure of proteins with the N- and C-terminals of the chain

Peptide bonds are special carboxamide bonds with strong hydrogen bonds caused by a partial delocalization in the functional group. Due to this delocalization the peptide bond is planar and rigid. This partial delocalization is illustrated by the molecule acetamide.

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Partial delocalization and hindered rotation of acetamide illustrated by mesomeric structures

Secondary structures are established by hydrogen bonds between peptide bonds: righ-handed (-helix, (-sheet – between antiparallel chains, collagen structures – there are three left-handed extended helix structures rolled into a cable form of a right-handed helix in tropocollagen units containing Gly-Pro-Hyp triplets, hydroxyproline is synthesized by a direct oxidation of proline in peptide chain by means of L-ascorbate).

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Oxidation of proline to hydroxyproline in the peptide chain by L-ascorbate (vitamin C)

Tertiary structure: Connections between remote parts of the peptide chain by secondary bonds between the side chains of amino acids – globular structures (folded to three-dimensional structures, they contain all of the secondary structures) and fibrous structures (folded to fibers, they contain only one of the secondary structures). Interactions:

· hydrophobic interactions – glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), proline (Pro)

· ionic interactions – aspartic acid (Asp) glutamic acid (Glu), lysine (Lys), arginine (Arg)

· hydrogen bonds – serine (Ser), threonine (Thr), tyrosine (Tyr), asparagine (Asn), glutamine (Glu), tryptophan (Trp), histidine (His)

· disulphide bond – cysteine (Cys)

· dipole-dipole interactions methionine (Met).

Quaternery structure:– Connection between several polypeptide chains usually called protein subunits by secondary bonds between the side chains of amino acids.

Simple proteins contain only protein chains. Complex proteins contain other kinds of biomolecules or metal ions: glycoproteins (often in membranes), nucleoproteins (in ribosomes), lipoproteins (e.g. LDL – a cholesterol transferring lipoprotein), metalloproteins (e.g. some enzymes as lactate dehydrogenase contain zinc), chromoproteins (e.g. red hemoglobin), phosphoproteins (e.g. casein), etc.

Biological function of proteins

· Enzyme proteins – catalysts of biochemical reactions that are vital to metabolism

· Structural proteins – e.g. collage fibers as fibrin

· Contractile (mechanical) proteins – e.g. muscle proteins

· Transport proteins – e.g. hemoglobin transports oxygen

· Proteins for supply – e.g. myoglobin supplies oxygen

· Immune protection – etc. immunoglobulins

· Toxins (poisons) – e.g. snake poison.

1. Enzymes

Enzymes are globular proteins generally with a quaternary structure. As biocatalysts they give an alternative reaction for the product synthesis with lower activity energy than the original reaction of really high activity via forming a complex with substrate. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. Enzymes are known to catalyze about 4,000 biochemical reactions. Activity of enzyme is affected by temperature, chemical environment (e.g., pH and salt concentration) and the concentration of substrate.

Reaction diagram without and with enzyme

Enzyme reactions are reversible. The sum of the rate of the dissociation of the enzyme substrate complex (v-1) and the rate of the synthesis of product and regeneration of the enzyme (v2) from this complex can be equal to the rate of forming enzyme substrate complex (v1), this status is called ‘steady state’.

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A saturation curve can be found when the concentration of the product [P] is plotted against the reaction time. Additionally, a saturation curve can be found for the relation between the substrate concentration [S] and rate (v0). This rate (v0) is the rate of enzyme reaction at the first period of the reaction. The modified Michaelis-Menten plot that is called the equation of enzyme kinetics can characterize this. As the substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum rate (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis constant (KM), which is the substrate concentration required an enzyme to reach one-half its maximum rate.

Diagrams and equal of enzyme kinetics

Only the active site of an enzyme takes part in the catalytic reaction while other parts of the enzyme assure the active conformation of the active site that contains two important parts. The substrate-binding site can be characterized by KM for a given substrate, and this can show how tight the binding of the substrate is to the enzyme. The parameters and/or compounds decreasing the binding of the substrate can increase the value of KM. For a given substrate the catalytic site can be characterized by Vmax. The parameters and/or compounds decreasing the transformation of the substrate-enzyme complex to the product can decrease the value of Vmax.

The double reciprocal plot

The KM and Vmax values are the important kinetic constants of the kinetics of enzymes for a given substrate. The determination of these constants is given by a double reciprocal plot (Lineweaver-Burk plot) that yields a straight line with an intercept of 1/Vmax and a slope of KM/Vmax.

Certain compounds can alter the activity of enzymes. Enzyme activity can be decreased by various inhibitors or can be increased by activators. The effect of such compounds can be reversible or irreversible. Reversible inhibitors are classified according to their linkage to the active site. Compounds of similar structure to the substrate can bind to the substrate-binding site and are called competitive inhibitors. Compounds which disturb the function of the catalytic site are called non-competitive inhibitors. These are generally irreversible inhibitors because they create a covalent bond with the catalytic site. Compounds that can disturb the function of both the substrate-binding and catalytic sites are called mixed inhibitors.

The types of reversible inhibitions: competitive inhibition (kompetitív gátlás)), mixed inhibition (vegyestípusú gátlás), non-competitive inhibition (nonkompetitív gátlás), enzyme activity without inhibitors (gátlószer nélküli állapot))

The classification of enzymes

Enzymes can be identified by their number in Enzyme Nomenclature (Enzyme Catalogue EC). The EC number is a combination of four numbers. The first number of the combination shows the type of the reaction catalyzed.

1. Oxidoreductases – catalyze oxidation and reduction (dehydrogenases and oxygenases)

2. Transferases – catalyze substitutions

3. Hydrolases – catalyze hydrolysis (the reagent is a water molecule)

4. Lyases – catalyze addition and elimination

5. Isomerases – catalyze tautomerism

6. Ligases – catalyze reactions using the energy of macroerg bonds.

Oxidoreductases and transferases need reagents (compounds with coenzyme function) for the catalyzed reactions. Compounds with coenzyme function (henceforth they are called as coenzymes) are connected to enzymes either by secondary bonds (they are really coenzymes – they can be regenerated also in other reactions) or by covalent bonds (prosthetic groups – they can be regenerated only in their original place). Compounds with coenzyme function have two forms (unreacted and reacted) – only lipoic acid has three forms. The starting materials for coenzymes are water soluble vitamins and in a few cases essential amino acids).

Macroerg bonds

The phosphoric acid anhydride (pyrophosphate) derivatives of nucleotides are the nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP). Their anhydride bonds (one in NDP and two in NTP) are called macroerg bonds (they have a high phosphoryl-transfer potential) because their synthesis requires energy while their hydrolysis generates an energy of about 30,6 kJ/mol. The most important NTP is adenosine triphosphate.

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Compounds containing macroerg bonds: phosphoric acid anhydride, mixed anhydrides e.g. glycerate 1,3-bisphosphate, enolester e.g. phosphoenolpyruvate PEP, thiolester e.g. acetyl coenzyme A

There are different types of macroerg bonds. They are formed from an acid and a compound with acidic character. Anhydrates can be synthesized from two molecules of phosphates (phosphoric acid anhydrides e.g. ATP) or from a carboxylate and a phosphate (mixed anhydrides e.g. glycerate 1,3-bisphosphate). There are other compounds with acidic character that can form esters with an acid. An ester from phosphate and an enol (e.g. phosphoenolpyruvate – PEP) or a thiolester from a carboxylate and a thiol (e.g. acetyl coenzyme A – acetyl-CoA) (H3C–COSCoA) contain also macroerg bonds.

Influence of different parameters on the activity of enzymes

As the temperature rises, reacting molecules have more and more kinetic energy. An about 10°C rise in temperature can cause about 50 to 100% increase in the activity of most enzymes. The temperature at which an enzyme's catalytic activity is at its greatest is called optimum temperature. With further increase of the temperature the activity of enzymes abruptly declines because of protein denaturation. The animal enzymes are more sensitive to temperatures above 40°C than plant and microbial enzymes. Over a period of storage time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5°C or below is generally the most suitable. This fact can be important in immobilization processes.

This optimum instead of an exact date seems to be apparent because it is a combination of two opposite processes (activation and denaturation). The exact optimum temperature depends on the measuring method. The shorter the measuring process the higher is the optimum temperature. In the case of a long measuring process the effect of the denaturation can be detected at a lower temperature than in the case of a short measuring process.

The apparent optimum temperature of an enzyme reaction (t – time, T – temperature)

The influence of pH on enzyme activity is a complex. The pH point where the enzyme is most active is called the optimum pH. The structure of both sites (substrate-binding and catalytic sites) of active site of enzymes is important to understand the influence of pH on enzyme activity. There are two important functional groups in these sites. They can be easily protonated and deprotonated (mostly carboxylic groups, sometimes amino groups). The enzyme is active only in a special pH range when one of these groups is in protonated and the other is in deprotonated form in both sites. When the pH value is too acidic, both important functional groups are in protonated form and if it is too basic both are in deprotonated form, therefore the enzyme is inactive.

The connection of enzyme activity and pH is a logarithmic one. The active pH zone depends on the pK values of the mentioned functional groups. The situation is complicated in the case of substrate-binding site because of the presence of both enzyme and enzyme-substrate complex. The animal enzymes are more sensitive to pH than plant and microbial enzymes. Human enzymes are really sensitive: intracellular pH is 7.00(0.00, extracellular pH is 7.40(0.02. That is one of the reasons that generally microbial and sometimes plant enzymes are used for biotechnological procedures.

The effect of pH on the enzyme activity

The special acidic character of the hydroxyl group of serine in the active site of hydrolases

In the catalytic sites of hydrolases the hydroxyl group of serine can create a strong hydrogen bond with the imidazole ring of histidine. In this way the behavior of this hydroxyl group is similar to a carboxylic group. In this kind of hydrolases, this hydroxyl group of serine is the deprotonated one in the catalytic site. A good example is the function of choline esterase. Choline esterase is a hydrolase that produce choline (HO-(CH2)2-N((CH3) 3) and acetate (CH3COOH) from acetylcholine (CH3COO-(CH2)2-N((CH3)3), which is a neurotransmitter in nerve systems.

The function of the active site of cholinesterase

The reaction catalyzed:

CH3COO-(CH2) 2-N((CH3)3 + H2O ( CH3COOH + HO-(CH2)2-N((CH3) 3

The substrate-binding site contains a lot of carboxylate anion side chains, therefore its name is anionic site (anionos kötőhely). To this site the quaternary nitrogen with positive charge can be connected. The connection of acetylcholine starts the depolarization of the nervous cell. In the catalytic site (also known as esterase site), the ester group is attacked by the active hydroxyl group of the serine that is acetylated. That means that this catalytic reaction is a covalent one. This acetyl group is hydrolyzed by a water molecule. When after the hydrolysis choline leaves the anionic site that stops depolarization.

Solubility properties of enzymes based on their protein character

Inside the protein molecules different kinds of interactions are formed: in the secondary structures hydrogen bonds between peptide bonds and in the tertiary and quaternary structures secondary bonds between the side chains of amino acids (hydrophobic interactions, ionic interactions, hydrogen bonds, disulphide bond and dipole-dipole interactions. But for an active conformation of enzymes the presence of water molecules is also important. These water molecules create hydrogen bonds with the different parts of protein molecules. These hydrogen bonds not only help the dissolution of the protein molecule but they also provide the active conformation of the enzyme.

The changes in the solubility of albumins and globulins (fehérje oldékonyság) as a function of light salt content in aqueous solutions (salting in – besózás, salting out – salting out)

In most of the cases not only water molecules but the presence of neutral salts can increase the solubility of proteins in water. Albumins can be solved in water alone but globulins cannot be solved in this way. The salting-in process means that globulins can be solved only in the presence of neutral salts depending on the ionic strength of the salt solution. Divalent ions are more effective than monovalent ions. But at very high salt concentration the increased number of ion-water interactions decreases the possibilities of protein-water interactions therefore the solubility of protein molecules decreases. This process is salting out.

In the case of sodium, potassium and ammonium salts (light salts) salting in and salting out processes are reversible and they can be used for the separation of different enzymes. For the separation of protein mixtures different methods of chromatography are often used as well. There are other kinds of cations (heavy salts). They form insoluble complexes with protein anions. This is an irreversible denaturation (coagulation).

Extreme pH values cause denaturation as well. In very acidic media all basic sidechains and all carboxylic group containing side chains are protonated (poli-cation) and in really basic media anion all basic sidechains and all carboxylic group containing side chains are deprotonated (poli-anion). The isoelectric point is the proton concentration when the number of cations and anions of the proteins are the same. The stability of proteins is the lowest in isoelectric point.

The change in protein solubility (fehérje oldékonyság) as a function of pH

The presence of water-miscible solvents (e.g. ethanol) can disturb the secondary interactions between protein and water molecules therefore the solubility of the protein can be decreased.

Regulation of enzyme reactions

The regulation of biochemical processes are carried out by the regulation of enzyme reactions. There are different levels to regulate enzyme reactions.

In the case of direct regulation, the active site of the enzyme is influenced. Enzyme activity can be decreased by various inhibitors or can be increased by activators. The effect of such compounds can be reversible or irreversible. As it was mentioned earlier reversible inhibitors are classified according to their linkage to the active site. Compounds of similar structure to the substrate can bind to the substrate-binding site and are called competitive inhibitors. Compounds which disturb the function of the catalytic site are called non-competitive inhibitors. These are generally irreversible inhibitors because they create a covalent bond with the catalytic site. Compounds that can disturb the function of both the substrate-binding and catalytic sites are called mixed inhibitors. In most of the cases irreversible inhibitors create permanent, a covalent bond with the active site.

The concerned mechanism of allosteric regulation was described by Monod

In the case of feedback inhibition the activity of an enzyme that catalyzes the first step in a biosynthetic pathway is inhibited by a special molecule, in most of the cases the end-product of the whole biosynthetic pathway. In this way the too high concentration of that end-product the first step of the biosynthetic pathway can be prevented. A good example is the process of glycolysis. The high concentration of ATP (the end-product of terminal oxidation) can inhibit phosphofructokinase (PFK) that is the third enzyme of the glycolysis. This kind of regulation, which is called allosteric regulation, has an important role in the general regulation of the living organisms. The allosteric enzymes have at least two subunits. The catalytic subunit accomplishes the enzyme reaction. The activity of its substrate-binding site is influenced by the regulating subunit. The concerned mechanism of allosteric regulation was described by Monod (shared Nobel Price 1965). Both subunits can be in active (relaxed R) or inactive (tensed T) conformation but they also can be in the same conformation: RR or TT. The inhibitor connects to the regulating subunit causing a permanent T conformation. In this way the biosynthesis of this special end-product inhibitor is stopped, its concentration decreases. At low concentration of the inhibitor (the special end-product) leaves the binding site therefore the conformation of some of the regulating subunits turns to active (relaxed) form to start the biosynthesis.

Measurement of enzyme activity

There are two different methods to measure enzyme activity. In the case of the kinetic method the speed (velocity) of the enzyme reaction is measured at high substrate concentration (near to saturated concentration). In the case of the fixed time determination the change during a fixed period is measured. In most of the cases spectrophotometric methods are used to measure enzyme activity that is based on the quantity measurement of the concentration of different molecules having absorption in the visible or ultraviolet region of light. There are chemical methods to form that kind of molecules from molecules without absorption. The quantitative spectrophotometric methods are based on Lambert-Beer law.

A = log Io/I = SYMBOL 98 \f "Symbol"×L, A is absorption of the solution and SYMBOL 98 \f "Symbol"= SYMBOL 101 \f "Symbol"×c when L=1 cm

A = SYMBOL 101 \f "Symbol"×c, SYMBOL 101 \f "Symbol" is the molar absorption coefficient of the molecule

The Lambert-Beer law

The change of absorption as a function of the concentration of the molecule in the solution

The linearity of the Lambert-Beer law is valid only in the case of dilute solutions. In concentrated solutions every molecule can influence the behavior of the other molecules (e.g. the absorption).

The reaction catalyzed by lactate dehydrogenase

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The process of reduction of coenzymes containing a nicotinamide structure

Absorbance of coenzymes containing a nicotinamide structure

In the case of a direct measurement of enzyme activity the change of absorption can be directly followed because the substrate has an absorption. For example, lactate dehydrogenase (LDH) (EC 1.1.1.27) is the catalyst of reaction from pyruvate to lactate that is the last anaerobe step of glucose degradation. This enzyme is an oxidoreductase and the transformation of its coenzyme (NADH+H() ( NAD( can be followed at wavelength 340 nm. The NAD( molecule contains only aromatic ring systems (λmax = 260-280 nm). But one of the ring systems’ (NADH+H() molecule is not an aromatic one. Its structure is similar to the structure of quinone (λmax is about 340 nm).

Hydrolysis of sucrose by invertase

DINISA test

In the case of an indirect measurement of enzyme activity the change of absorption cannot be directly followed because the substrate does not have absorption. In this case, none of the participants of the reaction has absorption. For example, invertase (EC 3.2.1.26) is the catalyst of the hydrolysis of sucrose (saccharose). The official name of invertase is (-fructofuranosidase. It is a trehalose-type disaccharide as it is α-glucoside. From the reaction mixture from time to time samples are removed and injected into the measuring mixture in which the enzyme reaction is stopped and the concentration of glucose is measured after transforming to a derivative with a good absorption.

There are different possibilities to measure glucose concentration in a solution. Most of them are based on the reactivity of the aldehyde group of glucose. One of these possibilities is to use the 3,5-dinitro-salycilic acid (DINISA) test. Three molecules of glucose reduce one of the nitro group of DINISA to amino group that forms a Schiff base with another glucose that can be measured at 540 nm. This method measures the concentration of all kinds of reducing sugars, among them the concentration of fructose.

Glucose concentration measurement methods using enzymes

New methods use enzymes. They are popular. Nowadays measuring kits are available. For example glucose concentration can be measured by the combination of hexokinase (the first enzyme of glycolysis) and glucose-6-phosphate dehydrogenase (the first enzyme of pentose phosphate pathway)

The phosphorylation of glucose to glucose 6-phosphate by hexokinase is often called the ‘activation of glucose’. It is not a real activation step, because – as it was mentioned earlier – this ester does not contain a macroerg bond. But the phosphoric acid unit of sugars can help the formation of a connection between sugars and enzymes by ionic interactions. The pentose phosphate pathway is an alternative cytoplasmic oxidative degradation of glucose resulting in (NADPH+H() from NADP( as well as different pentose phosphate intermediates. The reduced coenzyme (NADPH+H() is the coenzyme of reductive biosynthesis for all kinds of living organisms. The concentration of (NADPH+H() can be measured at 340 nm.

A special type of measurement of enzyme activity

When there are no participants in the enzyme reaction with the possibility of producing measurable derivatives, consecutive enzyme reactions can be used. For example, the activity of phosphofructokinase (PFK) can be measured by means of an auxiliary enzyme system containing aldolase, triose phosphate isomerase (TPI) and 3-glycerolphosphate dehydrogenase (DH). Coenzyme ATP is used in saturated concentration. Only the concentration of (NADH+H() formed in the last reaction step can be measured at 340 nm. Its concentration is proportionate to the concentration of fructose-1,6-bisphosphate. In this way activity of PFK can be measured.

Measurement of activity of PFK by the help of an auxiliary enzyme system

Spectrophotometry is suitable not only for measuring enzyme activity but for the quality identification of different molecules. The place of absorption maximums can be characteristic for the molecule or the special parts of a molecule.

The change of absorption as a function of wavelength (λ)

Practical rules of the measurement of enzyme kinetics

Enzyme kinetic measurements, such as activity measurements, should always be performed at concentrations of the enzyme and substrate, so that the measurements provide the correct answer to our questions. For activity measurements we are wondering what the maximum performance of the enzyme is in ideal conditions. This requires a high substrate concentration, except in the case of substrate inhibition. Generally, the suitable substrate concentration is higher than the KM value of the enzyme that is literary data or can be measured by enzyme kinetics methods.

The double reciprocal plot

In the laboratory practice, the KM and Vmax values are not only the important kinetic constants of the kinetics of the enzyme examined for a substrate, but for the other laboratory parameters used. The determination of these constants is given by a double reciprocal plot (Lineweaver-Burk plot) that yields a straight line with an intercept of 1/Vmax and a slope of KM/Vmax. In representation of this data the best results are given using an inclination of the straight line about 45°. The data of not only the activity, but these kinetic constants of the native enzyme are essential to evaluate the success of the immobilization. These data are based on a lot of parallel determinations.

The protein content of the enzyme has to be measured not only before the immobilization but in all phases of the immobilization process. Both non-protein content and non-active protein content of the enzyme preparation have to be determined. It is also important to know the fate of the enzyme activity and the protein content of the enzyme during the immobilization process. There are different methods available for the measurement of protein content.

The absorption maximum of the aqueous solution is at 260-280 nm (UV light) because of their aromatic amino acid content that is individual for different peptides. Some measuring methods of protein concentration are based on the numbering of the peptide bonds. Consequently, they can be used for proteins containing different amino acids side chains.

The generally used assay is the biuret method. The peptide bonds are special amide groups and they can take part in an amide-imide tautomer isomerization in an alkaline solution. The difference in the structure of these constitution isomers is only in the position of a hydrogen and a double bond.

The biuret method is based on the complexation of cupric ion with the imide tautomer of two peptide bonds forming a violet-colored chelate in an alkaline solution, its absorption can be measured at 540 nm. The test is named biuret method because biuret (H2NCONHCONH2) also gives a positive reaction to the peptide-like bonds in the biuret molecule.

The amide-imide tautomer isomerization

Biuret method

Several variants on the test have been developed, such as the BCA test and the Modified Lowry test. The results of Biuret test are given in mg/ml. The calibration curve can be made by the concentration of the soluble crystalline bovine serum albumin (BSA) standard. There are also other colorimetric protein concentration assay methods e.g. Bradford protein assay.

2. Immobilization of enzymes

Biological catalysts can be used beyond biotechnology in different fields such as the textile, pharmaceutical and chemical industries. There are different kinds of biocatalysts (e.g. cells) but this part of the course primarily concerns enzymes. Nonetheless, occasionally the characteristic features of other biocatalysts are also discussed. Most of the enzymes are relatively unstable and their costs of isolation are still high. Moreover, when used in solution, it is technically very difficult to recover the active enzyme from the reaction mixture after use. Therefore, they are often used in immobilized form to different carriers. Immobilization means that the mobility of biocatalysts is restricted in a chemical or physical way. Immobilization can cause a decrease in the enzyme activity but the remaining catalytic activity (active conformation of the enzyme) is stabilized. Consequently, this immobilized form can be used repeatedly and continuously. Additionally, it is easily stored. The introduction of immobilized catalysts has greatly improved both in terms of the technical performance of the industrial processes and their economy. The further use of immobilized enzymes to other practical processes needs new methods and a development in the current techniques.

One of the first immobilization methods is an ancient one that is producing vinegar from diluted ethanol with acetic acid bacteria immobilized on wood. In the scientific world, immobilization of single enzymes (from 1960) followed by the creation of immobilized multiple enzyme systems (1985-1995) that tried to reproduce the biochemical processes. Practically, all human enzymes were immobilized with more or less success. The first industrial use of immobilization was the immobilization of aminoacylase (EC 3.5.1.14) from Aspergillus oryzae for the resolution of synthetic racemic (D-L) amino acids (1967, Chibata and coworkers). Resolution is the separation of optical isomers (D and L enantiomers).

Hydrolysis of acetyl-amino acids to amino acids and acetic acid by aminoacylase

In synthetic methods, only racemic mixture of amino acids can be synthesized. After acetylation and reaction with aminoacylase only L-amino acids were formed. In this way, D-acetyl-amino acids and L-amino acids could be separated easily.

Nowadays, some industrial processes are based on immobilized whole cells containing the desired enzyme. For example, immobilized oven yeast can be used for various biochemical processes depending on the applied substrate molecule.

Immobilized enzymes can be categorized in different ways: according to the supports or the nature of the bond between the enzyme and the support. Different types of supports (carriers) can be used. They can be polymer structural materials or membrane layers. Among natural polymers are polysaccharides (cellulose, dextran, agar, agarose, chitin, alginate, etc.), proteins (collagen and albumin) and different forms of carbon. Among synthetic polymers are polystyrene, polyacrylate, polyacrylamide, different varieties of polyamides, vinyl and allyl polymers, and so on. Among inorganic supports are bentonite, silica, glass (nonporous and porous), metals, metal oxides, etc. The porous carriers contain controlled pores. On the basis of the bond between the enzyme and the carrier, there are two categories: chemical bonds or physical interactions. Generally, enzymes without a quaternary structure can be immobilized without losing most of their activity.

There is a significant difference between the immobilization and consumption of enzymes and whole cells as biocatalysts. Enzymes can be used for only a special substrate or its analogues. They often need coenzymes and/or different salts. Sometimes the coenzyme can be immobilized together with the enzyme. The advantage of the use of enzymes is that they usually require simple equipment, it is easy to make up the reaction mixtures, and generally tolerate high concentrations and the presence of organic solvents. In the case of enzyme or whole cell immobilization the connection between the enzyme and carrier cannot disturb the functional groups taking part in the enzyme reaction.

There are living or lifeless whole cells with microbial, plant or animal origin. The whole cells contain all of auxiliaries for biochemical processes. However, whole cells often require expensive equipment and the extraction techniques can be complicated. Whole cells, particularly living cells, generally do not tolerate high concentrations and the presence of organic solvents.

There are reversible and irreversible immobilization methods. The reversible immobilization can be easily formed, but the bonds easily break down. Therefore, the stability of immobilized preparation is often not enough.

Reversible immobilizations (adsorption and ionic binding)

The adsorption interaction is the simplest and oldest type of enzyme immobilization by some kinds of secondary bonds, mostly van der Waals forces. The first example was immobilization invertase to active coal in 1916 (Nelson and Griffin). The first immobilized whole cells were living acetic acid bacteria to beech wood chips in the 19th century (Orleans production of acetic acid). The phases of immobilization are incubation (forming of adsorption interaction), separation technology (isolation of the insoluble immobilized preparation by filtering or centrifugation).

Immobilization by adsorption (after Hartmeier 1986)

During immobilization not only van der Waals forces but often hydrophobic interactions and hydrogen bonds can play a role as well. The advantage of this method is that it is easy and the conformation of the biocatalyst is hardly affected. Both inorganic and organic adsorbents are used e.g. alumina, calcium carbonate, cellulose, Sepharose gel (based on agarose) etc. There are modified carriers as well, e.g. treating yeast cells with aluminum ions binding them to glass. The first immobilized enzymes by this method were hydrolases and oxidoreductases (e.g. alkaline phosphatase, glucoamylase, alcohol dehydrogenase, etc.) but whole cells (e.g. yeast cells) were also immobilized.

The ionic binding is based on the electrostatic interaction between oppositely charged groups of carriers and the biocatalyst. There are anionic and cationic ion exchange carriers as well. Cationic exchange resins change all cations to protons and anionic exchange resins change all anions to hydroxide anions. The first immobilization by ionic binding was carried out by Mitz (1956). It was the immobilization of catalase on DEAE (diethylaminoethyl) cellulose.

Immobilization by ionic binding (after Hartmeier 1986)

The disadvantage of this method is that the ionic interaction between the enzyme and the carrier can often be disturbed by the experimental parameters (pH, ionic strength, temperature or the change of solvent). Generally, only strongly diluted electrolytes are used e.g. 0.01 M puffer solutions instead of 0.1 M puffer solutions.

A new variation of adsorption immobilization is affinity chromatography. Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and be bound within the column in order to be separated from the undesired material which will not interact and elute first. Therefore, this method can be used to purify and concentrate a substance from a mixture into a buffering solution, reduce the amount of unwanted substances in a mixture, identify the biological compounds binding to a particular substance and purify and concentrate an enzyme solution. The molecule of interest can be immobilized through covalent bonds. The carrier contains a molecule or a part of a molecule to which an enzyme can connect very well. This molecule can be some kind of pigment or a nucleotide unit (NDP or NMP). The last one is useful in order to bind enzymes using a NAD( and NADP( coenzymes (e.g. kinases).

Another variation is a carrier that can create chelate bonds (e.g. polysaccharides) with special metal ions (e.g. titanium) that can make complexes with the amino acid side chains of enzymes.

Irreversible immobilizations

Covalent immobilization

The covalent binding is the most widespread immobilization method. In this case, covalent bonds are created between the enzyme and the carrier. It is important to avoid the participation of the groups of the active site of the enzyme in the covalent bonds of immobilization. Therefore, the amino groups of enzymes are often used for covalent immobilization. In order to prevent the participation of the groups of the active sites, the immobilization reaction is carried out in the presence of substrate molecules. In most of the cases, the carriers must be provided with reactive groups. Theoretically, the groups of the enzyme can be activated as well but this can cause far more deactivation of the enzymatic function than the activating of the carrier. Often the amino groups (arginine and lysine), hydroxyl groups (tyrosine), thiol groups (cysteine) or carboxyl groups (aspartic acid, glutamic acid) of the side chains of the enzyme take part in the covalent immobilization.

Covalent binding is hardly used for the immobilization of whole cells because covalent bonds can cause structural changes that kill the cells. One of the rare examples of covalent immobilization of cells is the immobilization of Bacillus subtilis to agarose.

The possibilities of covalent bindings (after Hartmeier 1986)

The connection between the enzyme and the carrier can be formed directly or with the help of a spacer e.g. a covalent connection between amino groups of the enzyme and the carrier can be formed by glutaraldehyde.

Covalent immobilization by the help of the hydroxyl group of the carrier

There are several kinds of possibilities to activate the hydroxyl group of the carriers. A commonly used method is the activation by cyanogen bromide. For this method, carriers with vicinal (adjacent) hydroxyl groups are needed e.g. in the case of natural carbohydrate carriers. Cyanogen bromide reacts with both hydroxyl groups and forms such an imino derivative that forms imino derivatives with the amino group of the enzyme. In this reaction ammonia is also generated. These kinds of imino derivatives containing a C=N part are called Schiff base that is often mentioned as imidocarbonate.

Covalent immobilization of vicinal hydroxyl groups by cyanogen bromide (after Hartmeier 1986)

There is another possibility to form groups suitable for covalent immobilization in the case of carriers with vicinal (adjacent) hydroxyl groups, e.g. starch. The bond between this hydroxyl groups can be oxidized by metaperjodate ions (IO4-) to two aldehyde groups that can easily form imino groups with two amino groups of an enzyme. In this case no intermediary group is required. The imino groups can often be reduced by mixed metal hydrides (e.g. NaBH4, NaBH3CN) to amino derivatives.

Dialdehyde derivative from starch chain (after Hartmeier 1986)

Covalent immobilization by the help of aldehyde groups formed from polysaccharides (after Hartmeier 1986)

Other methods for using the hydroxyl groups of the carriers are also known, e.g. by using their silyl derivatives for the immobilization of enzymes or other proteins. This method can be used in the case of inorganic hydroxyl groups (e.g. porous glass).

Covalent immobilization with the help of the carboxyl group of the carrier

Among synthetic polymer carriers are polyacrylamides (acrylamide H2C=CH-CONH2). There are partly hydrolyzed derivatives of polyacrylamides in them. An appropriate proportion of amide groups are hydrolyzed to carboxylic groups e.g. BioGel C and CM preparations by Bio-Rad Laboratories. The carboxylic groups of these preparations can be used for different kinds of covalent immobilizations.

a) Immobilization by carbodiimide method

This method is often used earlier because carboxylic groups can react with the amino group of enzymes apparently in a direct way. Due to the fact that the total delocalization carboxylic groups cannot be attacked by nucleophilic reagents as amino group. Carbodiimides (R1-N=C=N-R2) are special reagents for carboxylic group forming a particular derivative electron distribution of which is similar to acid anhydrides. Therefore, this derivative can be attacked by the amine group of the enzyme producing a carboxamide group. Carbodiimides are synthesized from amines in three steps — the intermediates are isothiocyanate and thiocarbamide (thiourea) molecules — with the help of thiophosgene (CSCl2) and HgO as reagents. The by-product of this reaction is a carbamide (urea) derivative of the carbodiimide. Dicyclohexylcarbodiimide (DCC) was a successful reagent but its urea derivative was insoluble in water therefore the immobilized enzyme was contaminated. Nowadays other carbodiimides containing basic groups are used e.g. 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide and 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide. The salt of these urea derivatives is water-soluble. It can be washed out from the immobilized enzyme preparation.

Immobilization by carbodiimide method

Later on, it turned out that these carbodiimides can irritate the skin, causing unpleasant skin tingling and skin rashes. Consequently, this method is not as popular as it used to be.

b) Immobilization via forming hydroxymethyl groups

Immobilization with hydroxymethyl group formed from carboxylic group of the carrier

After esterification of the carboxylic group followed by the reducing of the ester by mixed hydrides a hydroxymethyl group can be formed. In this case, the carrier and the hydroxyl group are connected by a methylene group. The hydroxymethyl group reacts with thionyl chloride and the chloride reacts with the amine group of the enzyme.

Crosslinking

Special role of glutaraldehyde in the immobilization of enzymes

Glutaraldehyde (official name is glutar-dialdehyde) or other dialdehydes are suitable to make enzymes or other proteins insoluble without carriers. These dialdehydes can make connections between the amino groups of enzymes by forming double Schiff bases. The imino groups can be reduced by mixed metal hydrides to amino groups as well. With the help of these dialdehydes enzymes can be immobilized without using carriers.

Crosslinking of enzymes with glutaraldehyde (after Hartmeier 1986)

Using other groups for crosslinking

Crosslinking by urea groups

(after Hartmeier 1986)

For crosslinking other compounds with two functional groups may be suitable e.g. hexamethylene diisocyanate (O=C=N-(CH2)6-N=C=O) that can be synthesized from hexamethylene-diamine (H2N-(CH2)6-NH2) with phosgene (COCl2). From isocyanate groups and amino groups of the enzymes carbamide (urea) (-NHCONH-) groups can be formed.

Crosslinking can form disulfide bridges between cysteine CH2SH side chains as well, e.g. flocculent cells.

Matrix entrapment

In the case of matrix entrapment the enzymes or biocatalysts are embedded in some kinds of gel-like structures, e.g. in natural or synthetic polymers. The matrix can be formed in situ from the mixture of the biocatalyst and the monomer by polymerization or the mixture of biocatalyst and the solution of the polymer is treated by a precipitating agent. The biocatalyst cannot move in the mixture that is permeable for both substrate and product. In most of the cases the pores of the matrix are large enough to release the enzymes, therefore mostly whole cells are applied. There are matrix entrapped biocatalysts in spherical and fiber forms.

The forms of the matrix-entrapped biocatalysts, thermoreversible and ionotropic gelation (after Hartmeier 1986)

The generally used methods are:

Thermoreversible gelation (Agarose or gelatin can be liquified at 40 °C, gelation is in ice-cold water bath).

The structure of agarose (after Hartmeier 1986)

Ionotropic gelation (Alginate sodium salt is solved in water; this solution is dropped into calcium chloride solution because the calcium salt is insoluble in water).

The structure of alginate (after Hartmeier 1986)

Polymerization connected by copolymerization (most of the monomers are toxic for cells). Copolymerization of acrylamide and bis-(N,N)-methylenebis-acrylamide (BIS) are dense enough to form entrapped enzymes.

Enzyme entrapment in polyacrylamide (after Hartmeier 1986)

Membrane confinement

In the case of membrane confinement, the biocatalyst is not immobilized to the carrier but it is closed inside a membrane. Due to the fact that it can move inside the membrane, this immobilization method decreases the enzyme activity less than other methods. The enzyme is an aqueous solution and out of the membrane is aqueous solutions as well. In the membrane confinement methods, the enzyme is in retention within a defined space by a semipermeable membrane. The enzyme cannot pass over the membrane easily but the substrate(s) and the product(s) can.

The microencapsulation is a new method that is used temporarily only for enzymes and not for whole cells. The aqueous solution of the enzyme is surrounded by a semipermeable polymer membrane. This process can be carried out by a so-called boundary layer polymerization. The aqueous solution of the enzyme is surrounded by a semipermeable polymer membrane. This process can be carried out by a so-called boundary layer polymerization. The aqueous solution of the enzyme and a hydrophilic monomer is emulsified by a solvent immiscible with water (e.g. chloroform or cylohexane). The size of the droplets can be influenced by the intensity of the emulsification and by the addition of surface reactants. The hydrophobic monomer that only dissolves in the solvent phase is added. The polymerization is carried out on the surface of the droplets. After isolation, the capsules are repeatedly washed in order to eliminate the traces of monomers. The disadvantage of this method is that sometimes the hydrophilic monomers can partially inactivate the catalyst.

This problem can be avoided by the liquid drying method. An emulsion (1. emulsion) was made from the aqueous (polar, hydrophilic) enzyme solution and the polymer solution (e.g. ethyl cellulose or polystyrol) in organic solvent immiscible with water (apolar, hydrophobic) with a boiling point lower than that of water (e.g. chloroform or cyclohexane). From this emulsion a new emulsion (2. emulsion) is made by a large amount of polar, so-called protective colloid (e.g. gelatin or albumin solution). There are three phases in this emulsion, outside is the protective solution and around the enzyme solution is the apolar solvent phase solving the polymer. The organic phase was evaporated under vacuum. In this way, the solid polymer layer surrounds the enzyme solution as a microcapsule. In this method, the enzyme cannot be damaged by the monomer. The only disadvantage of this method is that the microcapsules are relatively large (about 20 µm in diameter).

The liposome technique produces soft, deformable and almost liquid membranes similar to the living cells. This bimolecular detergent layer can be formed from phospholipids or other surfactant molecules by sonification (ultrasonic treatment). Phospholipids (e.g. lecithines, the fatty acid parts can be different) are amphilic (detergent) molecules. This kind of molecules have a hydrophilic (polar) and a hydrophobic (apolar) part therefore they can form a lipid double layer with hydrophobic interior. Outward is the hydrophilic surface. This structure is similar to the liposomes in the cells. Nowadays in this way only very small (about a few nm in diameter) liposomes can be formed.

The hydrophobic (apolar) part and the hydrophilic (polar) part of the molecule

Chemical formula of phosphatidyl cholines (lecithines)

Membrane confinement techniques (after Hartmeier 1986)

In membrane reactors enzymes are often closed into hollow fiber membranes.

Hollow fiber reactors (after Hartmeier 1986)

Combined methods

There are different combinations of enzyme immobilization methods in order to avoid the disadvantages of the individual methods but maximize their advantages.

A simple combination type is the combination of adsorption and cross-linking. The enzymes are adsorbed onto a carrier (e.g. silica gel or polyamide), then the adsorbed molecules are connected by a bifunctional reagent (e.g. glutaraldehyde).

The combination of adsorption and cross-linking (after Hartmeier 1986)

In this way, the advantage is that the connection between enzyme molecules and the carrier is more stable than in the case of a simple adsorption. Similar techniques can be used in the case of porous carriers (e.g. porous glass) as well.

The prepolymer formation followed by entrapment (after Hartmeier 1986)

The prepolymer formation followed by entrapment is used in the case of the highly toxic monomers. At first water soluble so-called prepolymer is formed from two monomers. The toxic monomer is removed from the prepolymer. Then the prepolymer and the biocatalyst are connected by a coupling agent forming an insoluble polymer with the biocatalyst inside. In this way, the biocatalyst and the toxic monomer are always kept separate.

General aspects of immobilization experiments

The immobilization of enzymes is carried out by mostly organic chemical preparative methods but the rules of techniques of biochemical systems have to be taken also into account. This particular dichotomy (the combination of two different point of views) is also typical for preparative immobilization processes. The description of different immobilization methods sometimes seems to be complicated and it requires a wide variety of additives and processes. A part of these parameters and techniques is important (e.g. saturation of gels, binding of the enzymes by adsorption or covalent bonds, parameters necessary for the enzymes to function, etc.). But there are other prescriptions that can be safely omitted, as they are only there because of the inadequate biochemical or incomplete preparative organic chemical knowledge.

However, simplification can only be done with the utmost caution. There are a number of instances where a reaction step or component does not have the objective explanation but it is still a prerequisite for success. Therefore, it is strongly recommended that any recording attempts be initiated by the exact repetition of the literary description or analogy and only if this success is desirable to simplify the process.

During the immobilization, we need to know the degree of binding and deactivation of the enzyme. We need to know exactly how much protein and how much activity we want to apply. We need to know exactly how much protein and how much activity remained in solution, so it did not bind. For the same reason, we need to know how many proteins and how much of the activity of each washer fluid was removed. It is desirable to summarize the results in a tabular form.

In order to determine the relative amount of protein concentration, it is not necessarily a complexation-based photometric method, e.g. biuret reaction. After adequate calibration, the light absorption of proteins can also be utilized at 260 nm. This method is advantageous but only in this case because it does not result in loss of material, and the photometric solution can be reused without problem. Its disadvantage is that it can only be done with completely clear solutions. No amount of protein or activity can be detected, assuming it is bound.

If the ratio of protein and activity applied was not the calculated one, deactivation or activation occurred during immobilization. In the case of deactivation, some of the bound enzyme does not exhibit catalytic activity. Sometimes activation may also occur but it is very rare.

3Practical application of immobilized enzymes, bioreactors

Immobilized enzymes can be widely used. When only a small amount of immobilized biocatalysts is required, their preparation and use can be carried out with standard laboratory equipment and under normal laboratory conditions. However, for larger scale applications, especially for industrial applications, special equipment and reactors are required. The purpose of the various design reactors is to optimize the interaction between the immobilized enzyme and the appropriate substrate, in addition to the ideal parameters in the most economical way.

Types of bioreactors

The different reactor types are classified according to the implementation of the various parameters, in particular the immobilized enzyme and the substrate.

Stirred vessel reactors

In cylindrical mixed vessel reactors, the immobilized biocatalyst is mixed in the similar way as in the laboratory flasks. There is a mixer in the lower third of the reactor, which can be different, usually using two to six-bladed rigid mixers. When mixing, the solid particles rub against each other causing an attrition (abrasion).

Stirred vessel reactors (after Woodward 1985)

The position of the mixer should be optimized. If the mixing plate is too close to the bottom of the reactor, the interaction between the mixer and the reactor molecules reduces the energy utilization rate. If the mixer is in a position that is too high, there is insufficient mixing at the bottom of the reactor. If necessary, the introduction of air should also be provided. The air flow is introduced under the mixer. This type of reactor is intermittent, so at the end of the reaction the reactor must be emptied and recharged, this technical solution is called a batch reactor.

One type of this reactor is a fed-batch reactor in which the components of the reaction are filled intermittently but the reaction products are not released. By intermittent dosing, nearly constant substrate concentration is achieved. Batch feed reactors should also often be emptied.

Continuous-flow stirred-tank reactors, CSTR

This technical variation is very similar to the batch reactor but the introduction of the reaction components and the removal of the reaction products are constant. A steady state condition is formed inside the reactor for running the reaction. For the CSTR performance, choosing the parameters of the stationary state (the equilibrium point), in particular the planning of the average residence time of the reaction mixture, is crucial. CSTR works efficiently when the reaction medium is poor in the substrate and is rich in product. This means that CSTR should not be used for product inhibition or when the product is toxic but its use is particularly advantageous for substrate inhibition.

Continuous-flow stirred-tank reactors, CSTR (after Woodward 1985)

Due to their stationary state, CSTR generally works with lower efficiency than other continuous flow reactors. At the same time, CSTR is preferably used in the case of flexible (compressible) carriers (e.g. cellulose), since in these cases, packed-bed reactors are generally not usable due to deformation potential. Solutions for mixing and air intakes are often similar in this case to batch reactors. In this case, the damage of solid particles can also be found.

Packed-bed reactors

This bioreactor type resembles a column filled with column chromatography, the filling in this case being the immobilized biocatalyst. Similarly to the chromatographic column, the flowing liquid (which contains the dissolved substrate and any additives such as coenzyme) may be in the same direction (down flow) or in the opposite direction (up flow or upstream) of the gravity through the column by means a flow supporting energy source, usually a pump. In the case of upstream, the flow rate cannot exceed a given value because the gel bed is mixed and a fluidized bed solution is created. Contrary to the mixed systems, the bed of packed-bed reactors is stationary, only the fluid phase flows. Therefore no damage of solid particles can be found. The flow rate should be optimized because both in the case of too slow and too fast rate the conversion is insufficient.

Packed-bed reactors (after Woodward 1985)

Fluidized bed reactor (after Woodward 1985)

Fluidized-bed reactors

In the case where gravity is upstream of the packed bed reactor and the flow rate can exceed a given value, the gel bed will be mixed up, this is the fluidized-bed reactor. As a result of up flow f and downward direction of gravity, the particles of the immobilized biocatalyst float. In this case, therefore, the "immobilization" of the solid particles is a consequence of a stationary flow state. The floatability of the particles is a function of the flow rate. When the up flow is over, the grains settle down. There is virtually no interaction between the particles of the immobilized biocatalyst, so the abrasive effect of rubbing does not apply either. In case the up flow rate exceeds the force of gravity, the particles are washed out of the reactor. Ideally, background turbulence does not have to be counted. A great advantage of the fluidized bed reactor solution is that neither the fragments of the particles of the immobilized enzyme nor the gases generated during the reaction cause any problem in the flow. The fluidized bed solution is particularly advantageous for heavier particles.

Bubble-column reactors

In the case where the oxygen of the air is required for the catalyzed reaction, the blending of the biocatalyst particles can be carried out by means of a gas inlet through a tube. There is no fluid flow in the bubble column reactor, only mixing. This solution is rather close to mixed vessel reactors. The gas bubble is placed at the bottom of the reactor to avoid a too heterogeneous flow.

Bubble-column reactor (after Woodward 1985)

Internal-loop and external-loop variations of air lifted loop reactors (after Woodward 1985)

Air lifted loop reactors

In this technical solution, the flow of air results in a flow of fluid that moves in a way that the density of the fluid mixed with the air is less than that without air. Two tubes are used in air-driven loop reactors. One (rising up tube) introduces the air at the bottom of the reactor. As a result, the density of the gas mixing fluid in the reactor decreases and an upstream liquid flow is formed. The gas leaves the reactor at the roof.

The residual non-gas-containing liquid has a higher density, so the other tube (descending pipe, down comer) falls back to the bottom of the reactor. Both loops formed by the take-off and landing loops can be inside the reactor (internal loop) but the landing branch can be located outside the reactor (external loop). The air-driven loop solution is especially useful for high-volume bioreactors.

Liquid impelled loop reactors

This solution was developed for immobilized enzymatic reactions in biphasic fluid systems and it can be described by a hydrodynamic model. A typical example of such reactions is the catalytic synthesis of aspartame by thermolysin in a mixture of ethyl acetate and water. In this case, the advantages of the air-fed loop reactor are combined with the water-immiscible but with the reaction components and/or the product with well soluble organic solvents.

A type

B type

Liquid impelled loop reactors (after Woodward 1985)

A type – the density of the solvent is lower than the density of water

B type – the density of the solvent is higher than the density of water

The liquid-impelled loop reactor (LLR) is a reactor that consists of two parts: the main tube and the circulation tube (loop). Both parts are in open connection at the bottom and at the top. The reactor is filled with a liquid phase: the continuous phase of the main tube in our example the aqueous solution containing immobilized enzyme and other important components. Another liquid phase is injected in the main tube by means of pumping. This liquid phase is immiscible with the continuous phase and its density is significantly different. In our example the solution of substrate in organic solvent. If the density is lower than the density of the continuous phase injection takes place at the bottom (A type reactor). If the density is higher than the density of the continuous phase, injection takes places at the top of the main tube (B type reactor). Due to the density difference the dispersed-phase droplets that are formed will rise or fall, respectively. Due to the presence of dispersed phase in the main tube a pressure difference exists which causes circulation of the continuous phase in the reactor. This results in good mixing spontaneously. The collected organic phase (at the top in the case of type A and at the bottom in the case of type B) was recirculated repeatedly. Both types are known as external and internal loops.

Membrane reactors

The use of semipermeable membranes in bioreactors can have many advantages. Generally, ultrafiltration membranes and hollow fiber membranes are used. The pore size of the membranes should be chosen so that the substrate and the product are perfused and the biocatalyst is not allowed to pass. This solution is also advantageous in enzyme reactions in biphasic fluid systems. In addition to the enzyme reaction on the membrane, the phases of the organic solvent and water emulsion are also separated, thus avoiding energy-intensive separation of the phases.

The various types of membrane reactors

There are different types of membrane reactors. In variations (a) or (b) substrate and enzyme (either native or immobilized enzymes) are mixed and the product leaves the reactor through the membrane. The only difference is that in the case of variation (a) the mixture of enzyme and substrate is repeatedly recycled. In variation (c) a tube with entrapped enzyme is used. At a suitable flow rate, the substrate gives the product at the end of the passage.

Design of bioreactors

In addition to the knowledge required for the design of chemical reactors, the design of bioreactors requires special knowledge of biocatalysts. In addition to modeling bioreactors, external and internal material transfer effects, axial dispersion effect and heat transfer effects, it is necessary to model the Michaelis-Menten equation as well as to model the operational stability of the immobilized biocatalyst in the given system.

4Practical application of immobilized enzymes in analytical methods and chemical syntheses

Enzymatic analytical methods

The analytical methods that use an enzyme-catalyzed biochemical process is referred to as an enzymatic analytical method. Two major groups of enzymatic analytical methods are distinguished: analytical methods based on enzyme activity measurement and the determination of the concentration of organic molecules with the help of enzymes. The analytical use of both native and immobilized enzymes is presented.

Analytical methods based on enzyme activity measurement in biological systems

This group includes analytical methods based on the measurement of the activity of certain enzymes of different biological systems. This means that the activity of enzymes in the test medium is investigated. By measuring the activity of enzymes, their condition in the biological system can be deduced. The degree and extent of the activity of some enzymes depend heavily on the state of the biological system containing it. These include some medical diagnostic methods and food control tests.

These systems do not contain immobilized enzymes but necessarily belong to the investigation of enzymatic assays. It is known that the isoenzyme composition of the enzymes in different living tissues is different. Therefore, if an isoenzyme composition that is characteristic of another tissue appears in the blood, it is a sign of injury to that tissue. These isoenzyme composition assays can be measured by electrophoresis.

A particularly good example of this type of use is the study of glutamate-oxaloacetate transaminase (GOT) enzyme activity. The activity of this enzyme always increases out of the cells when the cells are injured. Such a case is a myocardial infarction in the human body that increases in the blood serum, so an infarct can be diagnosed by measuring the enzyme activity of the blood serum. Another enzyme, glutamate-pyruvate transaminase (GPT) has a similar indicator function.

A similar application can be found also in the food industry. The volume of ice is higher than that of water, and the cell is damaged by freezing. The enzyme activity in the extruded meat juice from fresh meat is lower than it is when the extrusion was after freezing. In this case, information on the antecedent of the meat can be obtained. It should be noted that lactate dehydrogenase activity is also appropriate for this purpose. The reaction catalyzed by lactate dehydrogenase was presented earlier.

The enzymatic examination of extracts made from food raw materials of plant origin can also provide useful information e.g. the propensity for rancidification in oily seeds can be estimated by a special examination of the activity of the lipid peroxidase enzyme.

The reactions catalyzed by GOT and GPT

Determination of the concentration of organic molecules by enzymatic methods

Another major group of enzymatic analyzes are analytical methods that can be used to determine the concentration of certain molecules, usually biomolecules, by enzymes. For such an analytical determination, almost all enzymes and biomolecules are suitable that allow for the change of an easily measurable physical or chemical parameter. Examples of this type of application abound. Increase in glucose concentration in the human blood indicates diabetes. In the food industry, the sugar composition of the various juices, such as glucose, is characteristic of the quality. Glucose concentration measurement methods using enzymes were presented earlier.

In such enzymatic analytical methods, the use of immobilized enzymes is very common. Especially in automated analyzers that are capable of serial measurement and which, after injecting the test solution, usually give the analysis results directly. Such a kind of instrument is called a flow injection analysis (FIA) system. These instruments generally work with membrane-encapsulated o