ENZYME IMMOBILIZATION

Enzyme immobilization can be defined as the confinement of enzyme molecules onto a matrix physically or chemically or both, in such a way that it retains its full activity or most of its activity. Enzyme immobilization is becoming a powerful tool to reduce the production costs or to develop novel industrial processes based on biotransformation ^[1]. Strategies that are carrier-bound and carrier-free offer novel alternatives for intensive and extensive enzyme use at a large scale.

A typical example is chitosan and its derivatives for enzyme immobilization. Numerous strategies have been conducted to produce several chemical modifications on the chitosan molecule before, during, and after its coagulation to form carrier beads, which resulted in a protective effect of the matrix. The use of chitosan for matrix synthesis and further modification turned out to be a viable low-cost strategy for achieving highly active and stable insolubilized derivatives in comparison with high-cost commercial supports. In addition, nano technological devices are offering novel and robust alternatives for enzyme encapsulation from nano-particles, nano-sheets, lipid vesicles, and nanotube. But this story is just at the beginning, and in the future, will bring outstanding contributions to the bio-transformation process.

It is possible to visualize four steps in the development of immobilized bio catalysts. In the first step at the beginning of the nineteenth century, immobilized microorganisms were being employed industrially on an empirical basis. This was the case of the microbial production of vinegar by letting alcohol-containing solutions trickle over wood shavings overgrown with bacteria, and that of the trickling filter or percolating process for waste water clarify cation.The modern history of enzyme immobilization goes back to the late 1940s, but much of the early work was largely ignored for biochemists since it was published in Journals of other disciplines. Since the pioneering work on immobilized enzymes in the early 1960s, when the basis of the present technologies was developed, more than 10,000 papers and patents have been published on this subject, indicating the considerable interest of the scientific community and industry in this field ^[2].

In the second step, only immobilized single enzymes were used but by the 1970s more complex systems, including two-enzyme reactions with co-factor regeneration and living cells were developed. As an example of the latter we can mention the production L-amino acids from α-keto acids by stereo selective reductive amination with L-amino acid dehydrogenase. The process involves the consumption of NADH and regeneration of the coenzyme by coupling the amination with the enzymatic oxidation of formic acid to carbon dioxide with concomitant reduction of NAD+ to NADH, in the reaction catalyzed by the second enzyme, formate dehydrogenase. More recently, in the last few decades, immobilized enzyme technology has become a multidisciplinary field of research with applications to clinical, industrial and environmental samples. The major components of an immobilized enzyme system are: the enzyme, the support and the mode of attachment of the enzyme to the matrix. The term solid-phase, solid support, support, carrier, and matrix are used synonymously.

During physical methods of immobilization there is no covalent bond formation, rather dependent on physical forces (like, Electrostatic, Protein-Protein etc). In principle, completely reversible i.e., original enzyme can be regenerated. Details of various physical methods are given below.

This technique is also called lattice entrapment, inclusion, occlusion etc. Method involves the formation of a highly cross-linked network of a polymer in the presence of an enzyme. Enzyme molecules are physically entrapped within the polymer lattice and cannot permeate out of gel matrix. Appropriately sized substrate and product molecules can transfer across to insure a continuous transformation. Thus, this method can be applied to selective enzyme systems. Usually with this method no change in the intrinsic properties of the enzyme are anticipated. Method has several advantages: simplicity, different physical shapes, no chemical modification and small amounts of enzymes required. However, this method suffers from some disadvantages e.g., leakage or leaching of small sized substrate/products, operationally good immobilization requires delicate balance of experimental factors (e.g., good mechanical properties versus activity). Important examples include urease immobilization onto polyacrylamide and alginate.

The method consists of addition of enzyme to a surface-active adsorbent, and removal of any non-adsorbed enzyme by washing. The absorbents usually require special pre-treatment in order to insure good adsorption ^[3]. The adsorbents may be organic or inorganic in nature. It is dependent on experimental variables like, pH, temperature, nature of the solvent, ionic strength, concentration of enzyme and adsorbent. In principle, it should be a completely reversible process; a change in pH, ionic strength should lead to desorption. In practice, however, this does not happen. Irreversible binding is not a problem, if the immobilized enzyme is still active and is to be used in a continuous process. Commonly employed adsorbents include alumina, anion and cation-exchange resins, celluloses, collagen etc. Method has several advantages: simplicity, large choice of carriers, mild process, simultaneous purification and immobilization (e.g. Asparginase on CM-cellulose). Method suffers from some disadvantages e.g. attaining optimum conditions is often a matter of trial and error; If strong binding does not occur, desorption will occur and this can be a nuisance, especially when operating at high substrate concentrations.

Microenapsulated enzymes are formed by enclosing enzyme within spherical semipermeable polymer membranes, having diameter of 1-100 µm. It can be done using permanent or non-permanent membranes ^[4]. Permanent membranes are made of chemicals like cellulose nitrate, polystyrene while non-permanent membranes are made of liquid surfactant. Main advantage of this method is extremely large surface area for contact of substrate and enzyme within a relatively small volume. Some disadvantages include that the size of substrate/product should be sufficiently small and high concentrations of enzyme are required.

Chemical methods of immobilization involve formation of at least one covalent bond (or partially covalent). In reality, more than one covalent bond is involved. Such a method is usually irreversible. There are basically two types of chemical methods which are as follows:

Method involves formation of covalent bonds between enzyme molecules by means of bi- or multi-functional reagents, leading to formation of multiple covalent bonds. Though many multifunctional reagents are involved only glutaraldehyde has found extensive use ^[5].

Figure shows attachment of α-amylase using glutaraldehyde onto Amberlite MB 150 beads leading to its colour change on immobilization. Multifunctional reagents can be used not only to link enzyme molecules to polymer but to link enzyme molecules to each other. This is probably because it reacts with proteins readily under mild conditions. Major disadvantages of this method: difficulty in controlling the reaction, the need for large quantities of enzyme, much of which is lost by involvement of the active site in bond formation, gelatinous nature of the final product.

Covalent attachment takes place via non-essential amino acid (other than active site groups) to a water-insoluble, functionalized supports (carrier, matrix, polymer), which are either organic or inorganic polymers ^[6]. Bond formation is also termed as fixation, linkage, binding, bonding, coupling, grafting etc. Supports are either synthetic: acrylamide-based, methacrylic acid-based, styrene based etc; or natural: glass, Sephadex, Agarose, Sepharose etc. Majority of support requires activation (as they do not possess reactive groups but have only hydroxyl, amide, amino, carboxyl groups) before they can be used for immobilization. Some of the reactions used are diazotization, schiff base (imine bond) formation etc.

Enzyme immobilization leads to change in its microenvironment that may be drastically different from that existing in free solution. The new microenvironment may be a result of the physical and chemical nature of the support matrix alone, or it may result from interactions of the matrix with substrates or products involved in the enzymatic reaction. Following are the various parameters which are affected by enzyme immobilization due to change in its microenvironment:

Optimum pH of the enzyme is determined by the pKa of amino acids present at its active site ^[7]. There is change in optimum pH either the enzyme is present on the surface of the matrix or entrapped inside it. It is either broadened or shifts to acidic or basic side with respect to soluble enzyme depending on type of immobilization chosen, charge and surface area of the matrix. In case of charged matrices, change in optimum pH profile is defined by following equation:

∆pH = pHi - pHa = 0.43εϕ/kT

Where, pHi represents pH in vicinity of the immobilized enzyme and pHo represents pH of the bulk solution, ε represents the positive charge on the proton and φ represents the average electrostatic potential, k is Boltzmann’s constant and T is the temperature. When the support is cationic (positively charged), the protons in the vicinity of the support are repelled. Hence, the local (i.e., in the vicinity of the enzyme) pH is higher that that of the bulk. Thus, there would be shift in optimum pH towards acidic side while vice versa in case of anionic support.

An immobilized enzyme preparation having high loading leads to substrate diffusion limitation.

Figure shows scanning electron micrograph of immobilized β galactosidase from Pisum sativum (PsBGAL) onto Amberlite MB150 beads having diameter of 5 µm leading to broadening of optimum pH when lactose was the substrate. As the enzyme has high intrinsic specific activity (occurs during immobilization), the substrate concentration gradient through the particle becomes steep and consequently the substrate may not penetrate to the centre of immobilized enzyme particle (enzyme present on the surface or inside the matrix). With the constraint of pH (below or above optimum pH), the substrate concentration gradient becomes less steep. Thus, allow the substrate to penetrate further into the immobilized enzyme particle having high intrinsic specific activity, due to increased enzyme concentration during immobilization. Two factors therefore work antagonistically on the reaction rate, the change in pH reducing the rate while the rise in effective enzyme concentration tends to increase the rate, thereby moderating the effect of the pH change. Therefore, due to high local enzyme concentration and substrate limitation leads to broadening of optimum pH.

Immobilization of enzyme to the matrix led to a barrier towards free movement of enzyme ^[8]. Therefore, gain in kinetic energy by the immobilized enzyme due to an increase in temperature is limited to much extent leading to an increase in optimum temperature. Displacement of optimum temperature for immobilized enzymes has been observed in many cases, but the extent of displacement would differ with type of matrix and interactions between the enzyme and the matrix. Further broadening of optimum temperature has been observed in various cases due to high intrinsic specific activity (Zulu effect) as observed in optimum pH.

1) Enzyme must be attached to the matrix in correct conformation (most appropriately via residues away from active site).

2) Diffusion barrier (internal and external): Internal diffusion barrier is present in case of immobilized enzyme system where enzyme is present inside the matrix. Here, substrate has to diffuse inside the matrix to get hydrolyzed to its products. On the other hand, external diffusion barrier (when enzyme is present on the surface of the\ matrix) is a result of the thin, unstirred layer of solvent that surrounds the polymer n particle, called the ‘Nernst layer ^[9]’. Solutes diffuse in this layer by a combination of passive molecular diffusion and convection. Higher the concentration of solute greater would be the external diffusion barrier. The thickness of this layer is affected(within limits) by the speed at which the solvent around the immobilized enzyme particle stirred. Increasing the stirring rate will reduce this external diffusion layer as represented by following equation:

α = Vmax/Km

where α is the diffusion constant.

3) Partitioning effects arise due to polyionic matrices leading to interaction between matrix and the ionic solute. If partition coefficient p = Si/So, where Si is the concentration of ions around enzyme and So is the concentration of ions in bulk phase. Thus, Michaelis-Menten equation becomes:

v = Vmax x So x p/Km x So x p

and thus,

Km(app.) = Km/p

1) Simple and cheap, High catalytic activity, No conformational change of the biocatalyst, No need to use reagents, Reuse of expensive material ^[10].

2) Protection of biocatalyst, Allows the transport of low molecular weight compounds, Enables continuous operation due to maintained cell density, Facilitates cell separation and simplified downstream process.

3) Allows controlled release of product, strong biocatalyst binding, prevents leakage, decreases desorption, increases the stability of biocatalyst.

4) Strong binding, High heat stability, Facilitates the enzyme contacts with its substrate, Prevents elution of biocatalysts, Flexibility in design of support material and method.

1) Low stability, Possible loss of biomolecules, weak bonds might cause desorption of biocatalyst ^[11].

2) Limitations on mass transfer, low enzyme loading.

3) Might cause alternation in active site, Diffusion limitations, loss of enzyme activity.

4) Limited enzyme mobility causes decreased enzyme activity, less effective for immobilization of cells, support materials are not renewable.

Immobilized enzymes are used in medicine from 1990, immobilized enzymes are used for diagnosis and treatment of diseases in the medical field. The inborn metabolic deficiency can be overcome by replacing the encapsulated enzymes (i.e, enzymes encapsulated by erythrocytes) instead of waste metabolites, the RBC acts as a carrier for the exogenous enzyme drugs and the enzymes are biocompatable in nature, hence there is no immune response.The enzyme encapsulation through the electroporation is a easiest way of immobilization in the biomedical field and it is a reversible process for which enzyme can be regenerated.The enzymes when combined with the biomaterials it provides biological and functional systems ^[12].

In food industry, the purified enzymes are used but during the purification the enzymes will denature. Hence the immobilization technique makes the enzymes stable ^[13]. The immobilized enzymes are used for the production of syrups. Immobilized beta-galactosidase used for lactose hydrolysis in whey for the production of bakers yeast. The enzyme is linked to porous silica matrix through covalent linkage. This method is not preferably used due to its cost and the other technique developed by Valio in 1980, the enzyme galactosidase was linked to resin (food grade) through cross linking. This method was used for the various purposes such as confectionaries and icecreams.

Biodiesel is monoalkyl esters of long chain fatty acids. Biodiesel is produced through triglycerides (vegetable oil, animal fat) with esterification of alcohol (methanol, ethanol) in the presence of the catalyst. The production of catalyst is a drawback of high energy requirements, recovery of glycerol and side reaction which may affect the pollution. Hence the biological production of liquid fuel with lipases nowadays has a great consideration with a rapid improvement. Lipase catalyses the reaction with less energy requirements and mild conditions required. But the production of lipase is of high cost, hence the immobilization of lipase which results in repeated use and stability. The immobilization of lipase includes several methods entrapment, encapsulation, cross linking, adsorption and covalent bonding ^[14]. Adsortion method of immobilization is widely used in recent years when compared to covalent bond, entrapment and cross linking. In the biological production of biodiesel the methanol inactivates the the lipase, hence the immobilization method is an advantage for the biodiesel production. The low cost of lipase, candida sp as origin is of more industrial use.

The enzymes derived from microbial origin are of great interest in textile industry. The enzymes such as cellulase, amylase, liccase, pectinase, cutinase etc and these are used for various textile applications such asscouring, biopolishing, desizing, denim finishing, treating wools etc. Among these enzymes cellulase has been widely used from the older period to till now. The textile industries now turned to enzyme process instead of using harsh chemical which affects the pollution and cause damage to the fabrics. The processing of fabrics with enzymes requires high temperatures and increased pH, the free enzymes does not able to withstand the extreme conditions ^[15]. Hence, enzyme immobilization for this process able to withstand at extreme and able to maintains its activity for more than 5-6 cycles. PolyMethyl Methacrylateis linked with cellulose covalently. In this method the nanoparticle is synthesized with cellulase as core particle endoglucanase is a component of cellulase enzyme, Endoglucanase is microencapsulated with Arabic Gum is a natural polymer with the biodegradable property is used as a matrix for encapsulation of endoglucanase. Encapsulation of endoglucanase prevented it to retain its activity in the presence of detergents.

Enzyme immobilization has been largely a trial and error approach and progress is being made towards targeted immobilization of enzymes. Recent advances in the design of materials with specified pore sizes and surface functionality has enabled more precise control of the immobilization process with retention of catalytic activity and stability. Simulation of the surface characteristics of the target enzyme can be used to aid in the design of appropriate support materials still this approach has not found much awareness. As the structure and mechanism of more enzymes become available, more controlled immobilization methods will be generated. It should be noted that, even in those cases where a three dimensional structure of the enzyme is unavailable, structural models can be built up by using homology modelling methods. In particular, successful industrial applications of biocatalysts require systems that are not only stable and active, but are low in cost and can undergo repeated re-use. Enzyme immobilization is slowly turning into a well understood science part of it still remains an art.