Applications of Immobilized Enzymes

1. Applications of Immobilized Enzymes Shipin School of Food Science & Technology Jiangnan University June 24, 2010

2. Benefits of immobilization Protection from degradation and deactivation Retention of enzyme, enzyme-free products Recycling, repetitive use Cost efficiency Enhanced stability Use as controlled release agents Better process control Long half-lives and decay rates Stabilization of tertiary structureBetter process control Long half-lives and decay rates Stabilization of tertiary structure

3. Background Nomads stored milk in containers made from stomachs of animals for cheesemaking Rennin present in stomach lining, coagulates milk into curd and whey First use of immobilized enzymes in industry: invertase for production of golden syrup. Increased use of immobilized enzymes in 1960s

5. Applications of Immobilized Enzymes Films and Coatings (biosensors) Selective adsorbents Controlled release (liposomes, polymeric matrix, gels)

6. Biosensors (1) Coating of immobilized enzymes encapsulated in biomaterial Species to be analyzed must diffuse towards encapsulated enzyme (diffusion path must be short as possible to minimize response time) Chemical response of enzymatic reaction is then transformed into measurable signal (electrochemical or optical) An enzyme specific for the substrate of interest is immobilized between two membrane layers, polycarbonate and cellulose acetate. The substrate is oxidized as it enters the enzyme layer, producing hydrogen peroxide, which passes through cellulose acetate to a platinum electrode where the hydrogen peroxide is oxidized. The resulting current is proportional to the concentration of the substrate. YSI membranes contain three layers. The first layer, porous polycarbonate, limits the diffusion of the substrate into the second layer (enzyme), preventing the reaction from becoming enzyme-limited. The third layer, cellulose acetate, permits only small molecules, such as hydrogen peroxide, to reach the electrode, eliminating many electrochemically-active compounds that could interfere with the measurement. An enzyme specific for the substrate of interest is immobilized between two membrane layers, polycarbonate and cellulose acetate. The substrate is oxidized as it enters the enzyme layer, producing hydrogen peroxide, which passes through cellulose acetate to a platinum electrode where the hydrogen peroxide is oxidized. The resulting current is proportional to the concentration of the substrate. YSI membranes contain three layers. The first layer, porous polycarbonate, limits the diffusion of the substrate into the second layer (enzyme), preventing the reaction from becoming enzyme-limited. The third layer, cellulose acetate, permits only small molecules, such as hydrogen peroxide, to reach the electrode, eliminating many electrochemically-active compounds that could interfere with the measurement.

7. Glucose Biosensor Use in diabetes monitoring H2O2 is oxidized at the platinum anode, producing electrons. The electron flow is proportional to the H2O2 concentration and, therefore, to the concentration of the substrate. it places the sensor (pictured above, right, sitting on a finger) inside the body, in continuous contact with the environment to be measured. It is powered by an external reader (pictured above, left) which receives information and provides it to the patient through a harmless, noninvasive, electromagnetic transmission. By eliminating sampling The device, which measures about a centimeter by half a centimeter and is about the depth of a thin hair, is composed of an I-shaped backbone crossed by a series of 10 slightly decreasing bars. The shape vaguely resembles a miniature harp. The backbone and crossbars are made of a magnetorestrictive metallic glass � a material that changes shape with application of a magnetic field and generates a magnetic field when it changes shape � coated with a polymer that reacts to changes in acidity. This coating is then topped with glucose oxidase, the enzyme that reacts with glucose. The acid sensitive coating makes the device swell or shrink, changing mass depending on the surrounding acidity. The acid that causes the changes comes from the reaction of glucose with glucose oxidase, which produces gluconic acid. When a magnetic field from the outside is placed near the sensor, the sensor vibrates at a frequency dependent on the mass of the sensor. A magnetic coil can read the magnetic flux of the sensor and determine the amount of glucose in the blood. H2O2 is oxidized at the platinum anode, producing electrons. The electron flow is proportional to the H2O2 concentration and, therefore, to the concentration of the substrate. it places the sensor (pictured above, right, sitting on a finger) inside the body, in continuous contact with the environment to be measured. It is powered by an external reader (pictured above, left) which receives information and provides it to the patient through a harmless, noninvasive, electromagnetic transmission. By eliminating sampling The device, which measures about a centimeter by half a centimeter and is about the depth of a thin hair, is composed of an I-shaped backbone crossed by a series of 10 slightly decreasing bars. The shape vaguely resembles a miniature harp. The backbone and crossbars are made of a magnetorestrictive metallic glass � a material that changes shape with application of a magnetic field and generates a magnetic field when it changes shape � coated with a polymer that reacts to changes in acidity. This coating is then topped with glucose oxidase, the enzyme that reacts with glucose. The acid sensitive coating makes the device swell or shrink, changing mass depending on the surrounding acidity. The acid that causes the changes comes from the reaction of glucose with glucose oxidase, which produces gluconic acid. When a magnetic field from the outside is placed near the sensor, the sensor vibrates at a frequency dependent on the mass of the sensor. A magnetic coil can read the magnetic flux of the sensor and determine the amount of glucose in the blood.

8. Coatings: require only small amounts of embedded functional molecules, exhibit fast response times with external reagents, potential for miniaturization, multi-layer configurations. Coatings: require only small amounts of embedded functional molecules, exhibit fast response times with external reagents, potential for miniaturization, multi-layer configurations.

9. Selective adsorbents (2) Applications in food industry: Lactose removal in milk Milk contains 5% lactose which must be removed before feeding babies deficient in �-galactosidase (lactose hydrolyzing enzyme). A column packed with �-galactosidase immobilized in polyacrylamide gel allows continuous lactose removal. Hydrogen peroxide removal in milk after sterilization Immobilized catalase by covalent binding with carboxychloride resin Bitterness removal in orange juice Naringinase immobilized on copolymer, used in both batch and column systems

10. Selective adsorbents continued Applications in waste treatment: Reduction of phenol content in wastewater using immobilized polyphenol oxidase Removal of cyanide: Improve use of microorganisms to decompose cyanide by entrapping enzyme in polyacrylamide gel and packed into column Does not allow simultaneous removal of several different substances but allows removal of specific substances *blood detoxification using heparinase, phospholipase A2*blood detoxification using heparinase, phospholipase A2

11. Uses in medicine Blood detoxification: immobilization of phospholipase A2 and heparinase in bioreactors Urease-containing microcapsules injected intraveneously decrease urea content in blood Wound dressings: Immobilization of proteolytic enzymes (trypsin) onto textiles used as wound dressings to accelerate cleaning and healing of infected wounds and reduces amount of enzyme used as compared with use of enzyme solutions Low Molecular Weight Protamine (LMWP) Reverse haparin after surgery Some patients are sensitive to protamine Intramuscular implantation of enzyme-containing polymeric material. Enzyme releases from pellet and creates a high local concentrationIntramuscular implantation of enzyme-containing polymeric material. Enzyme releases from pellet and creates a high local concentration

12. Benefits of Controlled Release (3) localization prolongation of the desired action with minimal side effects one-time application

13. Mechanisms of Controlled Release diffusion controlled release pressure (rupture) activated release (carbonless paper, scratch and sniff products) solvent activated release (chewing gum) pH sensitive release (protein solubility) melting activated release membrane controlled release tearing or peeling release (magazine perfume samples) osmotically controlled release temperature sensitive release (microwave food packaging)

15. Drug delivery from environmentally sensitive release systems Drug delivery from environmentally sensitive release systems

16. Immobilized enzymes for controlled release Two major applications: Pharmaceutical (enzymes as drugs) Prolonged circulation in blood Local deposition in particular tissue, organ Food (release of enzymes from liposomes) Higher stability against denaturing actions and prolonged circulation life-time without loss of activity due to degradation, inhibition or capture by cells of reticuloendothelial system Where enzyme presence in other organs and tissues is undesired Cheeses with higher fat content promote lipid breakdown as lipid exchange between milk fat globule membranes and liposomes occur. *ability of targetingHigher stability against denaturing actions and prolonged circulation life-time without loss of activity due to degradation, inhibition or capture by cells of reticuloendothelial system Where enzyme presence in other organs and tissues is undesired Cheeses with higher fat content promote lipid breakdown as lipid exchange between milk fat globule membranes and liposomes occur. *ability of targeting

17. Liposomes Carriers for immobilized enzymes

19. Examples in Food Industry Food additives: encapsulated �-galactosidase added to milk. The enzyme is only active during digestion process, when the enzyme is released from the lipid vesicles Acceleration of cheese ripening: hard cheeses take up to 2 yrs to reach optimum flavor and texture; need to reduce ripening costs. Immobilize ripening enzymes into liposomes. Method of release: many parameters in cheese can induce a destabilization of lipid bilayers. Sensitivity to pH, temperature, and interactions with other lipids could induce release.

20. Targeted release Ligand-receptor binding: Cell surface receptors Antibodies Magnetic carriers -ligand-receptor affinity -carriers with ferromagnetic properties-ligand-receptor affinity -carriers with ferromagnetic properties

21. Summary Immobilized enzymes are used in a variety of fields in industry Method of immobilization is crucial in determining how the enzyme will function More work is required to optimize usage of immobilized enzymes for improved use Intramuscular implantation of enzyme-containing polymeric material. Enzyme releases from pellet and creates a high local concentrationIntramuscular implantation of enzyme-containing polymeric material. Enzyme releases from pellet and creates a high local concentration