ERT 317 Biochemical Engineering Sem 1, 2015/2016 CHAPTER 1: ENZYME KINETICS AND APPLICATIONS (Part 2 : Applied Enzyme Catalysis Immobilized enzyme systems ) ERT 317 Biochemical Engineering Sem 1, 2015/2016

Outline Immobilized enzymes Advantages of immobilization Methods Large scale enzyme production Industrial utilization of enzymes Medical utilization of enzymes

Immobilized Enzyme System Immobilized Enzyme System - The containment of enzyme solution within a confined space for the purpose of retaining and re-using enzyme in processing equipment. Enzyme Immobilization – restriction of enzyme mobility in a fixed space

Advantages of Immobilized System Multiple/repetitive use of a single batch of enzymes The ability to stop the reaction rapidly by removing the enzyme from the reaction solution (or vice versa) Product is not contaminated with the enzyme (useful in the food and pharmaceutical industries) Analytical purposes: long half-life, predictable decay rates, elimination of reagent preparation, etc Reduce cost of operation compared to free enzyme systems where additional separation and purification steps are needed. Some immobilization can increase enzyme activity

Disadvantages of Immobilized System Many immobilized enzymes exhibit lower activity compared to free enzymes. More expensive to prepare than free enzymes Mass transfer limitations due to immobilization methods.

Methods of Immobilization 1. Entrapment a)Matrix-entrapped b)Membrane-entrapped 2. Surface Immobilization/Bound a) Adsorption b) Covalently binding It is most important to choose a method of attachment that will not change the chemical nature or reactive groups in the active site of the enzyme (will lead to inactivate the enzyme)

Surface immobilization

Method of enzyme immobilization

Entrapment : Matrix-entrapment Normally used polymeric materials (Ca-alginate, agar and collagen) Solid matrices like activated carbon and porous ceramic also can be used Matrix : can be particle, membrane or fiber. When immobilizing in a polymer matrix, enzyme solution is mixed with polymer solution before polymerization (formation of gel) takes place.

Example of Matrix Entrapment: Your Experiment 2 Effect of Immobilization Method On Enzymatic Activity

EXPERIMENT 2 EFFECT OF IMMOBILIZATION METHOD ON ENZYMATIC ACTIVITY OBJECTIVES To understand the principles of enzyme immobilization by using gel entrapment method in alginate gel To analyze the effect of immobilization on the activity of enzyme COURSE OUTCOMES CO1 – Ability to develop enzyme reactions based on its kinetics study and applied catalysis INTRODUCTION An immobilized enzyme is an enzyme that is attached to an inert, insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride). This can provide increased resistance to changes in conditions such as pH or temperature. The immobilization of enzymes has proven particularly valuable, because it has allowed enzymes to be easily reused multiple times for the same reaction with longer half-lives and less degradation and has provided a straightforward method of controlling reaction rate as well as reaction start and stop time. It has also helped to prevent the contamination of the substrate with enzyme/protein or other compounds, which decreases purification costs. These benefits of immobilized enzymes have made them highly applicable to a range of evolving biotechnologies.

OBJECTIVE 1 : To understand the principles of enzyme immobilization by using gel entrapment method in alginate gel Enzymes such as ·-amylase play a key role in the starch liquefaction process leading to refined syrups and sweeteners widely used in the food industry. Physical entrapment of alpha-amylase in calcium alginate beads has shown to be a relatively easy, rapid and safe technique (Dey et. al, 2003). It is widely used matrix for entrapping enzymes because the gel formation occurs at mild conditions and poses no risk of harm to humans. Entrapment requires that the immobilized enzyme should be a large enough molecule to be kept inside the gel matrix but the substrate and product should be small enough to pass through the pores of the gel. Calcium alginate beads made with 2% (w/v) solution have an average pore diameter of 80 to 100 Å (Stewart and Swaisgood, 1993). Starch molecules are very large, often reaching a molecular weight of 80 million Daltons (Gerhartz 1990). It is expected that starch hydrolysis reaction could occur more effectively if enzyme bound to surface. Enzymes (alpha-amylase) are well mixed with monomers/ polymers and cross-linking agents (calcium ions) in a solution. Immobilization allows for easy separation of the enzyme from the starch hydrolysis products which can save the enzyme, labor, and overhead costs (Gerhartz, 1990)

The preparation of alginate beads involves mixing of sodium alginate and alpha-amylase solutions in buffer, followed by dripping of the solution into calcium chloride solution to form a gel (Sadhukhan et al., 1993) Preparation of immobilized enzyme beads Mix 0.5 ml of α-amylase enzyme with 5 ml of 3% (w/v) sodium alginate solution Drip the polymer solution prepared in step 1 from a height of approximately 20 cm into the stirred calcium chloride (CaCl2) solution with a syringe at room temperature. Leave the beads in the calcium solution to cure for 15 minutes. Filter the formed beads and wash thoroughly with distilled water. Dry the beads using filter paper (Whatman no.1) followed by exposure to the open air for 15 minutes before use.

OBJECTIVE 2 : To analyze the effect of immobilization on the activity of enzyme This was determined through enzyme activity analysis Kinetic behavior of an immobilized enzyme may differ significantly from that of its free molecules. Different enzymes respond differently to the same immobilization protocol (a suitable immobilization protocol has to be worked out for a given enzyme). The effects on enzyme kinetics (i.e. activity) may be due to the influence of matrix or due to conformational changes in the enzyme molecules induced by the procedure of immobilization. Immobilization protocol may increase or decrease enzyme stability.

When immobilization produces a strain in the enzyme molecules, they become more prone to inactivation by higher temperatures, pH, etc. (simultaneous hydrolysis and fermentation process) In contrast, binding of an enzyme molecule at several points without creating any strain in the enzyme molecule may lead to substantial stabilization. This is primarily due to the physical prevention (due to multipoint binding) of large changes in the conformation of enzyme molecules, which is essential for their inactivation. However, enzymes whether free or immobilized, loose activity with time due to denaturation. Higher enzyme activity normally is observed with small beads indicating the larger surface/mass ratio allows a greater amount of immobilized enzyme to have access to substrate

Entrapment : Membrane-entrapment Membrane entrapment of enzyme is possible. Eg: hollow fiber unit have been used to entrap an enzyme solution between thin, semi-permeable membrane. Membrane like nylon, cellulose, polysulfone and polyacrylate are commently used.

Allowing small MW compound access to enzyme (substrate/product) Retain high MW compound (enzyme) Semi-permeable membrane is used to retained high-molecular weight compound (enzyme) while allowing small molecular weight compounds (substrate/product) access to the enzyme. Special form of membrane-entrapment is microencapsulation (microscopic hollow spheres are formed). The sphere contain the enzyme solution, while the sphere is enclosed within a porous membrane.

Surface Immobilization/Bound : Adsorption Attachment enzyme on the surfaces of support particles by week physical forces (Van Der Waals or dispersion forces) Active site of the adsorbed enzyme is usually unaffected and nearly full activity is retained upon adsorption. Problems : Desorption of enzyme (in the presence of strong hydrodynamic forces since binding forces are week. Adsorption of enzyme may be stabilizied by cross-linking glutaraldehyde. Support materials used for enzyme adsorption can be inorganic materials (alumina,silica, porous glass, ceramics etc) or organic materials (cellulose starch, activated carbon and ion exchange rasin) Effective immobilization if support materials are been pe-treat (chemically or physically)

Surface Immobilization/Bound : Covalent Binding Covalent binding : retention of enzymes on support surfaces by covalent bond formation. Enzyme molecules bind to support material via certain functional groups (amino, carboxyl, hydroxyl and sulfhydryl groups) which must not be at active site. One common trick is to block the active site by flooding the enzyme solution with a competitive inhibitor prior to covalent binding. Functional group on support material are usually activated by using chemical reagents (cynogen bromide).

Diffusional Limitation in Immobilized Enzyme Systems Diffusional limitations in immobilized enzyme systems vary depending on the nature of the support (porous, non-porous), bulk phase hydrodynamic conditions, and distribution of the enzyme inside or on the surface of the support Whether resistance to diffusion has an effect on the rate of the enzymatic reaction depends on the relative rates of diffusion and reaction, as characterized by the Damköhlernumber (Da): Where [Sb] is the substrate concentration in bulk liquid, and kL is the mass transfer coefficient When Da>>1, diffusion rate is limiting; Da<<1, reaction rate is limiting; Da≈1, the diffusion and reaction resistances are comparable

Diffusion Effects in Surface-bound Enzymes on Nonporous Support Materials Assume the enzyme catalyzed reaction rate follows Michaelis-Menten type kinetics. E+S Ss: substrate concentration at the surface; Sb: substrate concentration in bulk solution.

Assume: Enzyme are evenly distributed on the surface of a nonporous support material. All enzyme molecules are equally active. Substrate diffuses through a thin liquid film surrounding the support surface to reach the reactive surface. The process of immobilization has not altered the enzyme structure and the intrinsic parameters (Vm, Km).

At steady state, mass transfer rate = the reaction rate

The mass transfer rate (g/cm2-s): is the liquid mass transfer coefficient (cm/s) or stirring rate. Since the product formation rate is : the maximum reaction rate per unit surface area. (g/cm2-s or mol/cm2-s)

Diffusion Effects in Enzymes Immobilized in a Porous Matrix Substrate diffuses through the tortuous* pathway within the porous support to reach the enzyme. Substrate reacts with enzyme on the pore surface. Diffusion and reaction are simultaneous at steady state Example: Spherical support particles tortuous = full of twist and turn, lengthy and complex

Large Scale Enzyme Production Enzymes are manufactured at large-scale using overproducing strains of certain organisms (in particular Bacillus, Aspergillus,Flavobacterium) Cell are cultivated in large fermentation vessels, and then the enzyme is harvested using a product-specific purification process, intended to maximize product yield and purity, while minimizing costs. Enzymes are either produced extracellularly (secreted) or intracellularly Extracellular enzymes can be purified directly from the fermentation broth Intracellular enzymes must be purified by breaking open the cells, removing the resulting cellular debris, and then processing the cell-free suspension/solution

Flowsheet for the Production of an Extracellular Enzyme

U.S. Industrial Enzyme Market

Industrially Important Enzymes

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