Enzyme Immobilization Dr Bhanu Kakrani, Purvi Kakrani & Dr.

Harish Kakrani Vallabvidyanagar

What is an Immobilized Enzyme? Enzymes are protein molecules which serve to accelerate the chemical reactions of living cells (often by several orders of magnitude). Without enzymes, most biochemical reactions would be too slow to even carry out life processes. Enzymes display great specificity and are not permanently modified by their participation in reactions. Since they are not changed during the reactions, it is cost-effective to use them more than once. However, if the enzymes are in solution with the reactants and/or products it is difficult to separate them. Therefore, if they can be attached to the reactor in some way, they can be used again after the products have been removed. The term "immobilized" means unable to move or stationary. And that is exactly what an immobilized enzyme is: an enzyme that is physically attached to a solid support over which a substrate is passed and converted to product. It is important to understand the changes in physical and chemical properties which an enzyme would be expecte d to undergo upon immobilization (sometimes referred to as insolubilization). There are a number of factors that affect the rate of the enzyme's catalytic activities. Changes have been observed in the stability of enzymes and in their kinetic properties due to the micro environment and the product's characteristics. Methods of Immobilization When immobilizing an enzyme to a surface, it is most important to choose a method of attachment that will prevent loss of enzyme activity by not changing the chemic al nature or reactive groups in the binding site of the enzyme. In other words, attach the enzyme but do as little damage as possible. Considerable knowledge of the active site of the enzyme will prove helpful in achieving this task. It is desired to avoid reaction with the essential binding site group of the enzyme. Alternatively, an active site can be protected during attachment as long as the protective groups can be removed later on without loss of enzyme activity. In some cases, this protective function can be fulfilled by a substrate or a competitive inhibitor of the enzyme. The surface on which the enzyme is immobilized is responsible for retaining the structure in the enzyme through hydrogen bonding or the formation of electron transitio n complexes. These links will prevent vibration of the enzyme and thus increase thermal stability. The micro environment of surface and enzyme has a charged nature that can

cause a shift in the optimum pH of the enzyme of up to 2 pH units. This may be accompanied by a general broadening of the pH region in which the enzyme can work effectively, allowing enzymes that normally do not have similar pH regions to work together. Carrier-Binding : the binding of enzymes to water-insoluble carriers y Cross-Linking : intermolecular cross-linking of enzymes by bi-functional or multifunctional reagents. y Entrapping : incorporating enzymes into the lattices of a semi -permeable gel or enclosing the enzymes in a semi-permeable polymer membrane Carrier-Binding The carrier-binding method is the oldest immobilization technique for enzymes. In this method, the amount of enzyme bound to the carrier and the activity after immobilization depend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier:
y

The selection of the carrier depends on the nature of the enzyme itself, as well as the:
y y y y

Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition

In general, an increase in the ratio of hydrophilic groups and in the concentration of bound enzymes, results in a higher activity of the immobilized enzymes. Some of the most commonly used carriers for enzyme immobilization are polysaccharide derivatives such as cellulose, dextran, agarose, and polyacrylamide gel. According to the binding mode of the enzyme, the carrier -binding method can be further sub-classified into: y Physical Adsorption y Ionic Binding y Covalent Binding Cross-Linking

Immobilization of enzymes has been achieved by intermolecular cross -linking of the protein, either to other protein molecules or to functional groups on an insoluble support matrix.. Cross-linking an enzyme to itself is both expensive and insufficient, as some of the protein material will inevitably be acting mainly as a support. This will result in relatively low enzymatic activity. Generally, cross-linking is best used in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage from polyacrylamide gels.

Since the enzyme is covalently linked to the support matrix, very little desorption is likely using this method. Marshall (1973), for example, reported that carbamy phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only 16% of its activity after continuous use in a column at room temperature for fourteen days. The most common reagent used for cross -linking is glutaraldehyde. Cross -linking reactions are carried out under relatively severe conditions. These harsh conditions can change the conformation of active center of the enzyme; and so may lead to significant loss of activity. Entrapping Enzymes The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types.

This method differs from the covalent binding and cross linking in that the enzyme itself does not bind to the gel matrix or membrane. This results in a wide applicability. The conditions used in the chemical po lymerization reaction are relatively severe and

result in the loss of enzyme activity. Therefore, careful selection of the most suitable conditions for the immobilization of various enzymes is required. Lattice-Type entrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and natural polymer (starch) have been used to immobilize enzymes using this technique. Microcapsule-Type entrapping involves enclosing the enzymes within semi permeable polymer membranes. The preparation of enzyme micro capsules requires extremely well-controlled conditions and the procedures for micro capsulation of enzymes can be classified as: y Interfacial Polymerization Method: In this procedure, enzymes are enclosed in semi permeable membranes of polymers. An aqueous mixture of the enzyme and hydrophilic monomer are emulsified in a water -immiscible organic solvent. Then the same hydrophilic monomer is added to the organic solvent by stirring. Polymerization of the monomers then occurs at the interface between the aqueous and organic solvent phases in the emulsion. The result is that the enzyme in the aqueous phase is enclosed in a membrane of polymer.
y

Liquid Drying: In this process, a polymer is dissolved in a water-immiscible organic solvent which has a boiling point lower than that of water. An aqueous solution of enzyme is dispersed in the organic phase to form a first emulsio n of water-in-oil type. The first emulsion containing aqueous micro droplets is then dispersed in an aqueous phase containing protective colloidal substances such as gelatin, and surfactants, and a secondary emulsion is prepared. The organic solvent in the n removed by warming in vacuum. A polymer membrane is thus produced to give enzyme micro capsules. Phase Separation: One purification method for polymers involves dissolving the polymer in an organic solvent and re -precipitating it. This is accomplished by adding another organic solvent which is miscible with the first, but which does not dissolve the polymer.

The form an of immobilized enzyme can be classified into four types: particles, membranes, tubes, and filters. Most immobilized enzymes are in particle form for ease of handling and ease of application.
y y

Particles - The particle form is described in the above section. Membranes - Enzyme membranes can be prepared by attaching enzymes to membrane-type carriers, or by molding into membrane form. The molding is done after the enzymes have been enclosed within semi -permeate membranes of polymer by entrapment. Tubes - Enzyme tubes are produced using Nylon and polyacrylamide tubes as carriers. The polymer tube is first treated in a series of chemical reactions and the enzyme is bound by diazo coupling to give a tube in a final step.

Fibers - Enzymes that have been immobilized by entrapment in fibers to form enzyme fibers. The solid supports used for enzyme immobilization can be inorganic or organic . Some

organic

supports

include:

Polysaccharides,

Proteins,

Carbon,

Polystyrenes,

Polyacrylates, Maleic Anhydride based Copolymers, Polypeptides, Vinyl and Allyl Polymers, and Polyamides. Applications of immobilized biocatalysts Isomerization of glucose to fructose (High fructose syrup) Lactose hydrolysis (dairy products conv erted to avoid lactose intolerance) Penicillin acylase Amino acid synthesis Additional applications in new term project Isomerization of glucose to fructose First, an understanding of the forms D-glucose looks like:

of

glucose

and

fructose

is

needed.

while D-fructose looks like:

This isomerization converts glucose which is not very sweet to fructose, the most sweet of the natural sugars. Syrups from this process compete with sucrose (cane sugar) in many food applications. Almost all manufacturers of soft drinks use high fructose syrups

because they are less expensive than sucrose. This was devastating to world price s of cane sugar and crippled the economies of some countries. The isomerization of glucose to fructose is part of the glycolysis cycle that converts glucose to pyruvate. The way this is done is to isomerize the aldehyde (hemiacetal) glucose to the ketone (as a hemiacetal) fructose,and make another phosphate ester. The isomerization takes advantage of the ease of breakage of a C-H bond which involves a carbon next to a carbonyl carbon. This is important in the next step which cleaves the bond between carbon s three and four of fructose. It is noted that this bond involves the carbon next to the carbonyl carbon of fructose. This cleavage would not have been possible without the isomerization of glucose to fructose, because the carbonyl group of glucose is too far from carbons three and four to make that bond breakable. Glucose isomerase (D-glucose ketoisomerase) causes the isomerization of glucose to fructose. Glucose has 70-75% the sweetening strength of beet sugar (sucrose), but fructose is twice as sweet as sucrose. Thus, processes for the manufacture of fructose are of considerable value. Novo Industries has developed glucose isomerase from B. coagulans for commercial use. In this immobilized enzyme process, the microorganism carries out a direct isomerization of the glucose. This glucose isomerase is primarily a xylose isomerase, so xylose, or a xylose-containing compound must be added for the induction of the enzyme. In batch processes, B. coagulans does not form the enzyme during the log phase. So, as soon as the glucose concentration in the nutrient solution approaches zero, growth ceases. Additional carbon sources present in the medium are then metabolized, and enzyme production begins. Enzyme activity is at a maximum about 24 hours after incubation. In wild strains of B. coagulans, both cobalt and magnesium are required for maximum enzyme production, but mutants have been isolated that don't require cobalt. Either yeast extract or corn steep liquor can be used as the nitrogen source; this choice is vital for the yield, and the concentration must be optimized for the specific process at hand. Because it is often difficult to standardize the nitrogen source, the fermentation yield may vary considerably from b atch to batch. A table illustrating this dramatic effect is shown below

In a continuous fermentation process, the growth-limiting substrate must be glucose. For optimal enzyme yields, oxygen limitation is also necessary since microaerophillic conditions inside the cells stabilize the system. The commercial process for production of fructose from glucose became feasible only when procedures for immobilization of the enzyme were developed, so that the same batch of enzymes could be used repeatedly. Since glucose isomerase is formed intracellularly in most strains, many commercial processes are carried out with immobilized cells or by the addition of partly broken cells. Procedure for carrying out the conversion Lactose hydrolysis An important application of immobilized enzymes The main purpose of using immobilized enzymes here is to convert the disaccharide lactose via hydrolysis into its monosaccharide components , glucose and galactose. Lactose is a disaccharide that occurs naturally in both human and cow's milk. It is widely used in baking and in commercial infant -milk formulas. One large problem with lactose is that many people are lactose intolerant - meaning that their body is incapable of digesting lactose. So it must be hydrolyzed into its monosaccharide components, allowing digestion which is the purpose of products today such as LACTAID. Like cellobiose and maltose, lactose is a reducing sugar. It exhibits muta - rotation and is a 1,4'-beta-linked glycoside. Unlike cellobiose and maltose, however, lactose contains two dif~erent monosaccharide units. Acidic hydrolysis of lactose yields 1 equiv of D -glucose and 1 equiv of D-galactose; the two are joined by a beta-glycoside bond between C1 of galactose and C4 of glucose. In other words, 100 g of lactose will produce 50g each of galactose and glucose.

Lactose, a [4-0 -(beta-D-Galactopyranosyl)-beta-D-glucopyranose] You may prefer the Haworth formula:

1,4'-beta-glycoside

Throughout the processing of milk, the disaccharide lactose accumulates in estimated 1.2 million tons annually from the dairy by -product, cheese whey. The hydrolytic conversion of lactose to glucose and galactose repr esents one way ofadding value to whey and whey derived products. For the enzymatic lactose hydrolysis various mesophilic -glycosidases have been described, some of which have already made it to the market. The application of known glycosidases is however partly hampered because of the moderate thermal stability and narrow pH profile of enzyme activity as well as due to the significant inhibition by galactose. The use of hydrolases from thermophilic microorganisms could help to overcome at least some of these problems. USE OF IMMOBILIZED ENZYMES IN BIOREMEDIATION There are two main categories of bioreactors used in bioremediation, suspended growth and fixed film. Suspended growth bioreactors consist of batch, plug flow, and completely mixed reactors. Microorganisms here are suspended in the medium in the reactor. Fixed film bioreactors consist of fixed bed, fluidized beds, air -sparged, or rotating media reactors. Here, microorganisms grow on or within a solid medium in the reactor. This is where immobilized enzymes come in. USE OF IMMOBILIZED ENZYMES IN HAZARDOUS WASTE TREATMENT METABOLISM IN IMMOBILIZED ENZYMES TYPES OF FIXED-FILM BIOREACTORS PROPERTIES OF IMMOBILIZED BIOMASS ADVANTAGES OF IMMOBILIZED BIOMASS REACTORS

HAZARDOUS WASTE TREATMENT USING IMMOBILIZED ENZYMES

METABOLISM IN IMMOBILIZED ENZYMES

In fixed-film bioreactors, microbial cells continue to grow by metabolizing the toxic components in the system. Three forms of metabolism that can exist are aerobic, anoxic, and anaerobic. Aerobic systems are limited in their degredation rate (rate of re moval of toxic substances) of high organic concentrated wastes due to the rate of oxygen transfer. Heavy biomass buildup creates high oxygen demand. Anoxic metabolism consists of placing the microorganisms into a particular growth substrate (ex. acetate) a nd placing the combination into an aquifer containing nitrate and sulfate. The system will first use up the nitrate first, which must then be removed in order for the sulfate to perform its required task, dehalogenation. Anaerobic metabolism occurs when th e microorganism used toxic compounds as its main energy source and form carbon dioxide and water. Rate of removal of toxic compounds is also limited by mass transfer and diffusion within the biomass. PROPERTIES OF IMMOBILIZED BIOMASS Microorganisms attach to the exposed support media in an aqueous environment These immobilized cells grow, divide, and produce a gel -like material (a polysacharide) called a biofilm The activity of the biofilm depends on local surface conditions to transport nutrients, substrate, electron acceptors, and donors to the immobilized cells This biofilm adsorbs the necessary components for metabolism. Microorganisms for the system are selected on the basis of the material (hazardous substances they metabolize The biofilm collects other microorganisms during the process which changes the nature of the biofilm The solution of hazardous wastes must be held in the reactor for long periods of time for metabolism of all toxic substances to take place The depth and composition are influenced by many factors: Microbial diversity changes due to concentration gradients of electron donors and acceptors and other biological transformation factors including biological transformation products Both anaerobic and aerobic envi ronments can exist within the same biofilm The biomass adsorbs organic compounds from the bulk solution yielding an enriched energy source at the biomass film surface Oxygen transport through the biofilm is diffusion limited and the concentration gra dient for diffusion is significantly reduced by the biofilm's respiration activity. This results in anaerobic conditions within the biofilm. The nature and concentration of the organic substrate and the nature of the microrganisms The superficial velocity (the velocity through the reactor if there were nothing inside it) The pH and eletrolyte concentrations of the water Characteristics of the support media which include surface area, surface roughness, pore volume, pore size, surface charge, ad sorption properties, density, and specific gravity It is important to maintain large microbial diversity in stable form to create high mean cell residence time, reduce sludge handling to maintain biomass, and create better response to toxicity ADVANTAGES OF IMMOBILIZED BIOMASS REACTORS

 PREVENTS WASHOUT OF BIOMASS IT IS EASIER TO OPERATE IMMOBILIZED REACTORS (IN SUSPENDED GROWTH REACTORS, YOU MUST SEPARATE THE BIOMASS FROM THE WATER WHEN THE PROCESS IS FINISHED. THIS IS NOT NECESSARY IN IMMOBILIZED BIOREACTORS.) MORE BIOMASS CAN BE PRODUCED PER VOLUME OF REACTOR CELLS LIVE FOR A MUCH GREATER PERIOD OF TIME IS MORE RESISTANT TO TOXIC LOADING SINCE MOST OF THE BIOLOGICAL ACTIVITY TAKES PLACE ON THE SURFACE, WHICH BUFFERS MOST OF THE MICROORGANISMS FROM THE SHOCK OF THE TOXIC SUBSTANCES MANY USEFUL MICROORGANISMS WILL GROW WELL WHEN IMMOBILIZED, BUT NOT IN SUSPENDED REACTORS. HIGHER RATES OF DEGRADATION (REDUCTION OF TOXIC COMPONENTS IN THE SYSTEM) ARE POSSIBLE ALLOWS FOR LARGER MICROBIAL DIVERSITY IT IS POSSIBLE TO CONTINUALLY REMOVE REACTION INHIBITORS FOR A SPECIFIC BIOMASS COULD PROVIDE A MORE STABLE GENE POOL AND ENHANCED RATES OF GENETIC TRANSFER MOST IMMOBILIZED REACTORS ARE CLOSED SYSTEMS. THEREFORE, THEY MINIMIZE EMMISIONS OF TOXIC VAPORS.