1

Enzyme Engineering & Technology

Lecturer Dr. Kamal E. M. ElkahloutAssistant Prof. of Biotechnology

2

CHAPTER 3Immobilized enzymes and their

uses

Enzyme reactors

• An enzyme reactor consists of a vessel, or series of vessels, used to perform a desired conversion by enzymatic means (Figure 5.1).

• Factors that affect choice of reactor for a process:• 1) Costs associated with substrate(s), downstream

processing, labor, depreciation, overheads and process development.

• 2) Costs concerned with building and running the enzyme reactor.

• 3) Form of the enzyme of choice (i.e. free or immobilized).• 4) Kinetics of the reaction.• 5) Chemical and physical properties of an immobilization

support (particulate, membranous or fibrous, density, compressibility, robustness, particle size and regenerability).

• 6) Scale of operation.• 7) Possible need for pH and temperature control.• 8) Supply and removal of gases .• 9) Stability of the enzyme, substrate and product.

(a) Stirred Tank Reactors

• Batch reactors generally consist of a tank containing a stirrer (stirred tank reactor, STR).

• The tank is normally fitted with fixed baffles that improve the stirring efficiency.

• In batch reactor all of the product is removed, as rapidly as is practically possible, after a fixed time.

• The enzyme and substrate molecules have identical residence times within the reactor.

• a) Stirred tank batch reactor (STR), which contains all of the enzyme and substrates) until the conversion is complete;

• b) batch membrane reactor (MR), where the enzyme is held within membrane tubes which allow the substrate to diffuse in and the product to diffuse out. This reactor may often be used in a semicontinuous manner, using the same enzyme solution for several batches;

• c) packed bed reactor (PBR), also called plug -flow reactor (PFR), containing a settled bed of immobilised enzyme particles;

• d)continuous flow stirred tank reactor (CSTR) which is a continuously operated version of (a);

• e) continuous flow membrane reactor (CMR) which is a continuously operated version of (b);

• f) fluidized bed reactor (FBR), where the flow of gas and/or substrate keeps the immobilised enzyme particles in a fluidized state.

• In some circumstances there may be a need for further additions of enzyme and/or substrate (i.e. fed -batch operation).

• Disadvantages:• The operating costs are higher than for continuous

processes due to the necessity for the reactors to be emptied and refilled both regularly and often.

• There are considerable periods when such reactors are not productive.

• It also makes uneven demands on both labor and services. • STRs can be used for processes involving non-immobilized

enzymes.• Batch reactors also suffer from pronounced batch-to-batch

variations.

• Reaction conditions change with time.• May be difficult to scale-up due to the changing power

requirements for efficient fixing. • Advantageous features:• Simplicity both in use and in process development. • For this reason they are preferred for small-scale production of

highly priced products, especially where the same equipment is to be used for a number of different conversions.

• They offer a closely controllable environment that is useful for slow reactions, where the composition may be accurately monitored, and conditions (e.g. temperature, pH, coenzyme concentrations) varied throughout the reaction.

• They are also of use when continuous operation of a process proves to be difficult due to the viscous or intractable nature of the reaction mix.

• All reactors would additionally have heating/cooling coils.

• (interior in reactors (a), and (d), and exterior, generally, in reactors (b), (c), (e) and (f)).

• Stirred reactors may contain baffles in order to increase (reactors (a), (b), (d) and (e) or decrease (reactor (f)) the stirring efficiency.

• The continuous reactors ((c) -(f)) may all be used in a recycle mode where some, or most, of the product stream is mixed with the incoming substrate stream.

• All reactors may use immobilized enzymes. • In addition, reactors (a), (b) and (e) (plus reactors (d)

and (f), if semipermeable membranes are used on their outlets) may be used with the soluble enzyme.

(b) Membrane reactors

• The main requirement for a membrane reactor (MR) is a semipermeable membrane which allows the free passage of the product molecules but contains the enzyme molecules.

• A cheap example of such a membrane is the dialysis membrane used for removing low molecular weight species from protein preparations.

• The usual choice for a membrane reactor is a hollow-fiber reactor consisting of a preformed module containing hundreds of thin tubular fibers each having a diameter of about 200 μm and a membrane thickness of about 50 μm.

• Membrane reactors may be used in either batch or continuous mode and allow the easy separation of the enzyme from the product.

• They are normally used with soluble enzymes, avoiding the costs and problems associated with other methods of immobilization and some of the diffusion limitations of immobilized enzymes.

• If the substrate is able to diffuse through the membrane, it may be introduced to either side of the membrane with respect to the enzyme.

• Otherwise it must be within the same compartment as the enzyme, a configuration that imposes a severe restriction on the flow rate through the reactor, if used in continuous mode.

• Due to the ease with which membrane reactor systems may be established, they are often used for production on a small scale (g to kg), especially where a multi-enzyme pathway or coenzyme regeneration is needed.

• They allow the easy replacement of the enzyme in processes involving particularly labile enzymes and can also be used for biphasic reactions.

• The major disadvantage of these reactors concerns the cost of the membranes and their need to be replaced at regular intervals.

• The kinetics of membrane reactors are similar to those of the batch STR, in batch mode, or the CSTR, in continuous mode.

• Deviations from these models occur primarily in configurations where the substrate stream is on the side of the membrane opposite to the enzyme and the reaction is severely limited by its diffusion through the membrane and the products' diffusion in the reverse direction.

• Under these circumstances the reaction may be even more severely affected by product inhibition or the limitations of reversibility than is indicated by these models.

Continuous flow reactors

• The advantages of immobilized enzymes as processing catalysts are most markedly appreciated in continuous flow reactors.

• In these, the average residence time of the substrate molecules within the reactor is far shorter than that of the immobilized-enzyme catalyst.

• This results in a far greater productivity from a fixed amount of enzyme than is achieved in batch processes.

• It also allows the reactor to handle substrates of low solubility by permitting the use of large volumes containing low concentrations of substrate.

• The constant reaction conditions may be expected to result in a purer and more reproducible product.

• There are two extremes of process kinetics in relation to continuous flow reactors;

• A) The ideal continuous flow stirred tank reactor (CSTR), in which the reacting stream is completely and rapidly mixed with the whole of the reactor contents and the enzyme contacts low substrate and high product concentrations;.

• B) The ideal continuously operated packed bed reactor (PBR), where no mixing takes place and the enzyme contacts high substrate and low product concentrations.

• The properties of the continuously operated fluidized bed reactor (FBR) lie, generally, somewhere between these extremes.

• An ordered series of CSTRs or FBRs may approximate, in use where the outlet of one reactor forms the inlet to the next reactor, to an equivalent PBR.

(c) Packed bed reactors

• The most important characteristic of a PBR is that material flows through the reactor as a plug; they are also called plug flow reactors (PFR).

• Ideally, all of the substrate stream flows at the same velocity, parallel to the reactor axis with no back -mixing.

• All material present at any given reactor cross -section has had an identical residence time.

• The longitudinal position within the PBR is, therefore, proportional to the time spent within the reactor.

• All product emerging with the same residence time and all substrate molecule having an equal opportunity for reaction.

• The conversion efficiency of a PBR, with respect to its length, behaves in a manner similar to that of a well -stirred batch reactor with respect to its reaction time.

• Each volume element behaves as a batch reactor as it passes through the PBR.

• Any required degree of reaction may be achieved by use of an idea PBR of suitable length.

• In order to produce ideal plug -flow within PBRs, a turbulent flow regime is preferred to laminar flow, as this causes improved mixing and heat transfer normal to the flow and reduced axial back-mixing.

• Consequent upon the plug -flow characteristic of the PBR is that the substrate concentration is maximized, and the product concentration minimized, relative to the final conversion at every point within the reactor; the effectiveness factor being high on entry to the reactor and low close to the exit.

• This means that PBRs are the preferred reactors, all other factors being equal, for processes involving product inhibition, substrate activation and reaction reversibility.

• They are easily fouled by colloidal or precipitating material.

• The design of PBRs does not allow for control of pH, by addition of acids or bases, or for easy temperature control where there is excessive heat output, a problem that may be particularly noticeable in wide reactors (> 15 cm diameter).

• Deviations from ideal plug-flow are due to back-mixing within the reactors, the resulting product streams having a distribution of residence times.

• In an extreme case, back-mixing may result in the kinetic behavior of the reactor approximating to that of the CSTR (see below), and the consequent difficulty in achieving a high degree of conversion.

• These deviations are caused by channeling, where some substrate passes through the reactor more rapidly, and hold-up, which involves stagnant areas with negligible flow rate.

• Channels may form in the reactor bed due to excessive pressure drop, irregular packing or uneven application of the substrate stream, causing flow rate differences across the bed.

• The use of a uniformly sized catalyst in a reactor with an upwardly flowing substrate stream reduces the chance and severity of non-ideal behaviour.

(d) & (e) Continuous flow stirred tank reactors

• This reactor consists of a well -stirred tank containing the enzyme, which is normally immobilized.

• The substrate stream is continuously pumped into the reactor at the same time as the product stream is removed.

• If the reactor is behaving in an ideal manner, there is total back-mixing and the product stream is identical with the liquid phase within the reactor and invariant with respect to time.

• Some molecules of substrate may be removed rapidly from the reactor, whereas others may remain for substantial periods.

• The reaction S>>>>>>>>>P is assumed, and substrate molecules that have long residence times are converted into product.

• The average residence time of the product being greater than that for the substrate.

• The composition of the product stream is identical with that of the liquid phase within the reactor.

• These reactors may be operated for considerably longer periods than that determined by the inactivation of their contained immobilized enzyme, particularly if they are capable of high conversion at low substrate concentrations.

• This is independent of any enzyme stabilization and is simply due to such reactors initially containing large amounts of redundant enzyme.

• In general, there is little or no back -pressure to increased flow rate through the CSTR.

• Such reactors may be started up as batch reactors until the required degree of conversion is reached, when the process may be made continuous.

• CSTRs are not generally used in processes involving high conversions but a train of CSTRs may approach the PBR performance.

• This train may be a number (greater than three) of reactors connected in series or a single vessel divided into compartments.

• In order to minimize back-mixing CSTRs may be used with soluble rather than immobilized enzyme if an ultrafiltration membrane is used to separate the reactor output stream from the reactor contents.

• This causes a number of process difficulties, including concentration polarization or inactivation of the enzyme on the membrane but may be preferable in order to achieve a combined reaction and separation process or where a suitable immobilized enzyme is not readily available.

(f) Fluidized bed reactors

• These reactors generally behave in a manner intermediate between CSTRs and PBRs.

• They consist of a bed of immobilized enzyme which is fluidized by the rapid upwards flow of the substrate stream alone or in combination with a gas or secondary liquid stream, either of which may be inert or contain material relevant to the reaction.

• A gas stream is usually preferred as it does not dilute the product stream.

• There is a minimum fluidization velocity needed to achieve bed expansion, which depends upon the size, shape, porosity and density of the particles and the density and viscosity of the liquid.

• This minimum fluidization velocity is generally fairly low (about 0.2 -I.0 cm s-1) as most immobilized-enzyme particles have densities close to that of the bulk liquid.

• In this case the relative bed expansion is proportional to the superficial gas velocity and inversely proportional to the square root of the reactor diameter. Fluidising the bed requires a large power input but, once fluidized, there is little further energetic input needed to increase the flow rate of the substrate stream through the reactor.

• At high flow rates and low reactor diameters almost ideal plug -flow characteristics may be achieved.

• However, the kinetic performance of the FBR normally lies between that of the PBR and the CSTR, as the small fluid linear velocities allowed by most biocatalytic particles causes a degree of back-mixing that is often substantial, although never total.

• The actual design of the FBR will determine whether it behaves in a manner that is closer to that of a PBR or CSTR.

• It can, for example, be made to behave in a manner very similar to that of a PBR, if it is baffled in such a way that substantial backmixing is avoided.

• FBRs are chosen when these intermediate characteristics are required, e.g. where a high conversion is needed but the substrate stream is colloidal or the reaction produces a substantial pH change or heat output. They are particularly useful if the reaction involves the utilization or release of gaseous material.

• The FBR is normally used with fairly small immobilized enzyme particles (20-40 mm diameter) in order to achieve a high catalytic surface area.

• These particles must be sufficiently dense, relative to the substrate stream, that they are not swept out of the reactor.

• Less-dense particles must be somewhat larger. • For efficient operation the particles should be of nearly

uniform size otherwise a non-uniform biocatalytic concentration gradient will be formed up the reactor.

• FBRs are usually tapered outwards at the exit to allow for a wide range of flow rates.

• Very high flow rates are avoided as they cause channeling and catalyst loss.

• The major disadvantage of development of FBR process is the difficulty in scaling-up these reactors.

• PBRs allow scale-up factors of greater than 50000 but, because of the markedly different fluidization characteristics of different sized reactors, FBRs can only be scaled-up by a factor of 10 -100 each time.

• In addition, changes in the flow rate of the substrate stream causes complex changes in the flow pattern within these reactors that may have consequent unexpected effects upon the conversion rate.

Immobilized Enzymes

• Immobilized enzymes are enzymes which are attached in or onto the surface of an insoluble support

• Immobilized enzymes have several advantages over the soluble enzyme: – Convenience: Miniscule amounts of protein dissolve in the

reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.

– Economical: easily removed from the reaction reusage– Stability: Immobilized enzymes typically have greater thermal and

operational stability than the soluble form of the enzyme

Immobilization criteria

• There are a number of requirements to achieve a successful immobilization:– The biological component must retain substantial

biological activity after attachment– It must have a long-term stability– The sensitivity of the enzyme must be preserved

after attachment – Overloading can block or inactivate the active site

of the immobilized biomaterial, therefore, must be avoided

Immobilization methods

a) adsorption b) covalent binding c) entrapment d) encapsulation

Adsorption and Ionic binding• Simplest immobilization method

– Mix the enzyme and support in suitable conditions• First immobilized enzyme model: invertase on the activated charcoal

(Nelson and Griffin, 1916)• Forces are weak so leakage is generally a problem• Supports such as alluminium hydroxide are often utilized • With a suitable charged matrix, ionic interactions may also be

promoted• This technique is technically undemanding and economically

attractive• Regeneration is easy• Best known industrial example: amino acylase immobilized on DEAE-

Sephadex in the production of amino acids

Gel-fibre entrapment and encapsulation

Entrapment• Enzymes may be entrapped within the matrix of a polymeric gel

– Incubate the enzyme together with the gel monomers– Promote gel polymerization

• Polyacrylamide and polymethacrylamide gels are examples• Gel pore size is a crucial factor

Encapsulation• Encapsulation involves entrapping the enzymes within a semipermeable

membrane such as cellulose nitrate and nylon-based membranes

Covalent immobilization

• The most widely used method for enzyme immobilization– It is technically more complex– It requires a variety of often expensive chemicals– It is time-consuming But immobilized enzyme preparations are stable and leaching is

minimal• Enzymes are immobilized by a suitable group in the surface:

– Hydroxyl groups in supports (e.g cellulose, dextran, agarose)– Amino, carboxyl and sulfhydryl groups in amino acids

Covalent immobilization

• The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding. Therefore, covalent binding may alter the conformational structure and active center of the enzyme, resulting in major loss of activity and/or changes of the substrate

• Covalent attachment to a support matrix must involve only functional groups of the enzyme that are not essential for catalytic action

• Higher activities result from prevention of inactivation reactions with amino acid residues of the active sites. A number of protective methods have been devised: – Covalent attachment of the enzyme in the presence of a competitive inhibitor

or substrate – A chemically modified soluble enzyme whose covalent linkage to the matrix is

achieved by newly incorporated residues

Site-specific immobilization

Three different approaches:(a) Gene fusion to incorporate a

peptidic affinity tag at the N- or C-terminus of the enzyme. The enzymes are then attached from this affinity tag to anti-tag antibodies on membranes

(b) Modification to incorporate a single biotin moiety on enzymes (see figure)

(c) Site-directed mutagenesis to introduce unique cysteines to enzymes. The enzymes are attached on thiol-reactive surfaces through the sulfhydryl group

Properties of support material

• The form, shape, density, porosity, pore size distribution, operational stability and particle size distribution of the supporting matrix will influence the result

• The ideal support is cheap, inert, physically strong and stable• Ideally, it should:

– increase the enzyme specificity (kcat/Km)– shift the pH optimum to the desired value for the process– discourage microbial growth and non-specific adsorption

• Some matrices may possess other properties which are useful for particular purposes such as – ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst

by means of magnetic fields)– a catalytic surface (e.g. manganese dioxide, which catalytically removes the

inactivating hydrogen peroxide produced by most oxidases)

Kinetic Properties• There is usually a decrease in specific activity of an enzyme upon

insolubilization: denaturation caused by the coupling process• Microenvironment after immobilization may be drastically different from

that existing in free solution: the physical and chemical character of the support matrix, or interactions of the matrix with substrates or products involved in the enzymatic reaction – The Michaelis constant may decrease by more than one order of magnitude

when substrate of opposite charge to the carrier matrix • The diffusion of substrate can limit the rate of the enzyme reaction: the

thickness of the diffusion film determines the concentration of substrate in the vicinity of the enzyme and hence the rate of reaction

• The effect of the molecular weight of the substrate can also be large. This may be an advantage in some cases, since the immobilized enzymes may be protected from attack by large inhibitor molecules

Effects of solute diffusion on the kinetics of immobilized enzymes

• external diffusion the transport of substrates towards the surface, and products away

• internal diffusion the transport of the substrates and products, within the pores of immobilised enzyme particles

Kinetics of immobilized enzymes

Partitioning effect

• The solution lying within a few molecular diameters (10 nm) from the surface of an immobilized enzyme will be influenced by both the charge and hydrophobicity of the surface

• The Km of an enzyme for a substrate is apparently reduced if [S] in the vicinity of the enzyme's active site is higher than that measured in the bulk of the solution

Kinetics of immobilized enzymes• A high concentration of ionising groups may cause a partitioning of gases

away from the microenvironment with consequent effects on their apparent kinetic parameters

• It is also a useful method for protecting oxygen-labile enzymes by 'salting out' the oxygen from the vicinity of the enzyme

• Partition of hydrogen ions The pH of the microenvironment may differ considerably from the pH of the bulk solution

• Enzyme immobilised on charged supports: free enzyme enzyme bound to a (+)ly charged support; a bulk pH of 5 is needed to produce a pH of 7 within the microenvironmentenzyme bound to a (-)ly charged support; a pH of 7 within the microenvironment is produced by a bulk pH of 9

Kinetics of immobilized enzymes

• If the surface is predominantly hydrophobic – Hydrophobic molecules will partition into the microenvironment of

the enzyme and hydrophilic molecules will be partitioned out into the bathing solution

– Partition will affect the apparent kinetic constants of the enzyme

• E.g. the reduction in the Km of immobilised alcohol dehydrogenase for butanol– If the support is polyacrylamide, the Km is 0.1 mM but if a more

hydrophobic copolymer is used as the support, the Km is reduced to 0.025 mM

Kinetics of immobilized enzymes

• A similar effect may be seen in the case of competitive inhibitors

Invertase

Ki, mM free Bound to PS (hydrophobic)

Aniline (hydrophobic) 0.94 0.39

Tris-(hydroxymethyl)-aminomethane (hydrophilic)

0.45 1.20

Kinetics of immobilized enzymes• Enzymatic depolymerisation (including hydrolysis) of

macromolecules may be affected by diffusional control

• Large molecules diffuse fairly slowly. • After reaction, the cleaved fragments normally retain

their ability to act as substrates for the enzyme• They are likely to be cleaved several times while they

are in the vicinity of the immobilised enzyme• This causes a significant difference in the molecular

weight profiles of the fragments produced by the use of free and immobilised enzymes

Kinetics of immobilized enzymes• Co-immobilization of the necessary enzymes for the pathway results in a

rapid conversion through the pathway due to the localized high concentrations of the intermediates

• The reduction in the apparent lag phase is most noticeable when there are more enzymes in the pathway

• It is least pronounced where the flux through the pathway is controlled by the first step

Mixture of free enzymesCo-immobilized enzymes

Application of immobilized enzymes

BioreactorsLarge scale production or conversion of various compounds

Application of immobilized enzymes

Biosensors An analytical device which can detect and quantify specific analytes in complex samples

BiologicalSample Detection TransducerSolution Element

Signal

Signal Processor

Readout

Enzyme biosensors

Electrodes detecting gases such as O2, CO2, NH3 and various ionic species are commercially available

Application of immobilized enzymes

Bioremediation• For the removal/detoxification of contaminants• E.g. Polyphenol oxidase immobilized on chitosan

coated membranes

Biosensors

a compact analytical device incorporating a biological sensing element with a transducer

Biosensors

Enzyme

Cell

Micro organism

Antibody

Nucleic acids

BIOELEMENT

Electroactive electrode substance

pH change pH electrode

Heat thermistor

Light photon counter

Mass piezoelectrical change device

READ

-OU

T

TRANSDUCER

PERFORMANCE FACTORS

Selectivity

Linear working range

Reproducibility

Response time

Lifetime

The biosensor should be cheap, small, portable and capable of being used by semi-skilled operators

Applications of Biosensors

• Health care and life sciences research applications – Glucose and urea sensors– Proteomics – Genomics – Toxicology – Oncology – Drug discovery

• Process industries – Monitoring of active component or pollutants

• Food and drink– Measuring Ripeness – Contaminant/Pathogen Detection – Process/Quality Control – Detection of Genetically Modified Organisms in Food

• Environmental monitoring– BOD, Pesticide

• Defence and security– Military; Nerve gases and explosives– Forensics; DNA identification

Beetle/Chip Sensor Schroth P. et al., Sensors and Actuators B, 78: 1-5, 2001

Whole-beetle

Antenna

Receptor Molecules

Detection of a single, damaged potato plant within a field of a thousand undamaged plant …

Bioelectronic sniffer for nicotineMitsubayashi K. et al, Analytica Chimica Acta

A bioelectronic sniffer for nicotine in the gas phase was developed with enzyme inhibition principle to butyrylcholinesterase activity

Micromachined sensor for lactate monitoringC.G.J. Schabmueller et al, Biosensors & Bioelectronics 21:1770-1776, 2006

Sport Medicinelocation independent, permanent real-time measurement of the lactate concentration during exercise

The size of the chip is 5.5 × 6.4 × 0.7 mm

What else?

• Engineers for the Japanese company Toto have designed a toilet that analyses urine for glucose concentrations, registers weight and other basic readings, and automatically sends a daily report by modem to the user's doctor...

• One glucose sensor that looks like a watch sits on the skin and produces small electric shocks, which open up pores so that fluid can be extracted to monitor tissue glucose concentrations

Cyclodextrin as a dehydrogenase mimic R. Kataky and E. Morgan, Biosensors & Bioelectronics, 18:1407-1417, 2003

• Cyclodextrins are very attractive as biomimetic materials; a suitably modified cyclodextrin may bind the substrate and then catalyse a reaction, mimicking an enzyme-catalysed reaction

• The model compound: a simple β-cyclodextrin derivative with a nicotinamide group attached to the secondary face of a β-CD

• The nicotinamide group would act as the electron transfer agent and the cyclodextrin would provide a suitable cavity for the reaction to take place in

• Only small, unbranched alcohols such as ethanol are small enough to fit into the β-CD cavity along with the reacting groups...

Dendrimers as synthetic enzyme mimics

C. Liang and J.M.J. Fréchet, Progress in Polymer Science, 30: 385-402, 2005

• Substrate migration into the dendrimer interior: The nanoenvironment is greatly influenced by the branching units. These favorable conditions encourage transition state stabilization. The resulting product is released from the nanoreactor into the solvent

(a) Substrate drawn from water into the hydrophobic region in close proximity to amines

(b) Charged tetrahedral transition state (TS) intermediate is stabilized by the amide group

(c) TS intermediate collapses and p-nitrophenolate is released into water

Membrane Proteins

Membrane proteins account for 20–25 % of all open reading frames

Wide range of central functions

At least 50 % of all drug targets are membrane proteins PROBLEMS… Insoluble in aqueous solution so hard to work

with

Model Membrane Systemsto offer a native environment for the membrane proteins

Tethered Bilayer Membranes (tBLM)

Artificial lipid bilayers attached to solid surfaces allow the opportunity to use several surface sensitive techniques

Atomic force microscopy Surface plasmon spectroscopy Impedance spectroscopy Quartz crystal microbalance etc