CHAPTER 1: ENZYME KINETICS AND APPLICATIONS

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1 CHAPTER 1: ENZYME KINETICS AND APPLICATIONS EM /13 ERT 317 BIOCHEMICAL ENGINEERING

2 Course details Credit hours/units : 4 Contact hours : 3 hr (L), 3 hr (P) and 1 hr (T) per week Evaluations Final Exam 50% Midterm Tests 20% Course works 30% Laboratories 15% Assignments 15% CARRY MARKS 50%

3 Course details Course Outcome (COs) will be covered: CO1 Ability to develop enzyme reactions based on its kinetics study and applied catalysis Course works Assignments 1 (Due 19 Sept) Quizzes 1 (19 Sept) Midterm test 1 Class participations Max. of 3 points

4 Important reminder Attendance should not less than 80%, or else you will be barred from taking final examination. Plagiarism and copying other students work is strictly prohibited especially in doing assignments and lab reports, or else both parties will get zero. Cheating in quizzes and examinations is also prohibited, or else both parties will get zero. Therefore, study hard and smart. Take note of the important chapters or things that will be highlighted throughout lectures.

5 C1.2 Applied Enzyme Catalysis Week 3 (24-28 Sept 2012) Reading assignment: 1. Chapter 3, Bioprocess Engineering basic Concepts. Shuler and Kargi (Main)

6 Outline Immobilized enzymes Advantages of immobilization Methods Electrostatic and Steric Effects Large scale enzyme production Industrial utilization of enzymes Medical utilization of enzymes Enzyme biosensors

7 Immobilized Enzymes Advantages 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) Enzymes are usually stabilized by bonding Product is not contaminated with the enzyme (especially useful in the food and pharmaceutical industries) Analytical purposes: long half-life, predictable decay rates, elimination of reagent preparation, etc.

8 Methods of Immobilization Entrapment Matrix-entrapped Membrane-entrapped Micro-encapsulation Immobilization between macroscopic membranes Surface Immobilization Adsorbed Covalently bound 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)

9 Immobilization Methods

Carrier Binding The carrier-binding method is the oldest immobilization techniques for enzymes In this method, both the amount of enzyme bound to the carrier, and

10 Carrier Binding The carrier-binding method is the oldest immobilization techniques for enzymes In this method, both the amount of enzyme bound to the carrier, and the enzyme activity after immobilization, depend on the nature of the carrier Three types of carrier binding: physical adsorption, ionic binding, covalent binding

11 Physical Adsorption A major advantage of the physical adsorption method no requirement for use of any reagents, just involved minimum number of activation steps Adsorption tends to be less disruptive to enzymatic proteins than chemical means of attachment due to weaker binding (what type of bindings?) As such, the enzyme is likely to retain its original 3D conformation, as well as its normal activity If a suitable carrier can be identified, this method can be both simple and inexpensive

12 Disadvantages of the Physical Adsoption Method Due to the weak bonds involved, desorption of the protein is often observed (Factors involved?) Other methods of stabilization? The carriers used in physical adsorption can also permit additional, non-specific adsorption of other proteins or non-protein substances The consequences of this disadvantage?

13 Ionic Binding This method relies on the ionic binding of the enzyme protein to water-insoluble carriers containing ion-exchange residues The binding of an enzyme to the carrier is easily carried out, and the conditions are much milder than those needed for the covalent binding method Result of this binding on 3D conformation? Leakage of enzymes from the carrier may occur in substrate solutions of high ionic strength or upon variation of ph (How?) Enzyme-to-carrier linkages are much stronger for ionic binding than for physical adsorption, but are much weaker than for covalent binding

14 Covalent Binding The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding Result of this binding on 3D conformation? However, the binding force between enzyme and carrier is so strong that no leakage of the enzymes occurs, even in the presence of substrate or solution of high ionic strength Covalent attachment to the support matrix must involve only functional groups of the enzyme that are not essential for catalytic action

Cross-Linking Cross-linking: the formation of intermolecular linkages, either between the protein molecules themselves, or from the protein molecules to functional groups on an insoluble support

15 Cross-Linking Cross-linking: the formation of intermolecular linkages, either between the protein molecules themselves, or from the protein molecules to functional groups on an insoluble support matrix Cross-linking an enzyme to itself is both expensive and inefficient (WHY?) Generally, cross-linking is best used in conjunction with another immobilization method (carrier-binding, entrapment) Roles of cross-linking?

Entrapment Matrix-or lattice-type entrapment: involves entrapping enzymes within the interstitial spaces of a cross-linked, waterinsoluble polymer Microcapsule-type

16 Entrapment Matrix-or lattice-type entrapment: involves entrapping enzymes within the interstitial spaces of a cross-linked, waterinsoluble polymer Microcapsule-type entrapment: involves enclosing the enzymes within semi permeable polymer membranes. The preparation of enzyme microcapsules requires extremely well-controlled conditions.

17 Mass Transfer Effects of Enzyme Immobilization 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): Da maximum rate of reaction maximum rate of diffusion ' Vm k S 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 L b

18 Diffusion Effects for Surface-Bound Enzymes on Non-Porous Supports For surface-bound enzyme reactions, at steady-state the rate of reaction is equal to the rate of mass transfer (eq. 3.53): J s k L S S b S V K m ' m SS S S

19 Diffusion Effects for Enzymes Immobilized within a Porous Supports Substrate diffuses through the tortuous pathways among pores, and reacts with enzyme immobilized on pore surfaces Diffusion and reaction are simultaneous at steady state: D e 2 d dr " S 2 d S V m SS r dr 2 K S m S

20 Electrostatic and Steric Effect in Immobilized Enzyme (IE) System Bulk ph of immobilized enzyme will shift from its soluble form when the IE are in charged matrix Charged matrix repel/attract substrates, product, cofactors and H+ depending on the type and quantity of surface charge The shift in ph-activity profile is given by: ph ph ph i e zf RT Where z = charge on the substrate, F = Faraday constant,ψ is electrostatic potential and R is gas constant

21 Electrostatic and Steric Effect in Immobilized Enzyme (IE) System The expression is also valid for other nonreactive charged medium components Intrinsic enzyme activity is altered by local changes in local changes in ph and ionic constituents Further alteration are due to repulsion or attraction of substrate or inhibitors Enzyme activity is reduced towards HMW substrate compared to LMW due to the steric hindrance by the support For example, starch have MW comparable to enzymes, thus not be able to penetrate to active site of IE Immobilization also effects thermal stability of enzymes. HOW?

22 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

23 Flowsheet for the Production of an Extracellular Enzyme

24 Industrial Utilization of Enzymes Enzyme preparations have been used in the manufacture of foods and in industrial processes for many years The enzyme rennet (found in calf stomachs) has been used in cheese manufacturing since biblical times Enzyme preparations have a long history of being used in food production processes in the dairy, wine, brewing and distilling, starch, and baking industries Since the early 1900s, some enzyme preparations have been known to enhance cleaning action; they were first widely introduced into detergent formulations in the 1960s for this purpose Enzyme preparations are also used as diagnostic reagents and in the pharmaceutical, tanning (leather goods), textiles and paper industries

25 Industrial Utilization of Enzymes Industrial enzymes are typically produced in microbial expression systems vs. plant and animal sources because: Microbes are generally cheaper to grow The enzyme contents of microorganisms are more predictable and controllable (HOW?) Reliable supplies of raw materials of constant composition are more easily arranged Plant and animal tissues contain more potentially harmful materials than microbes (HOW?)

26 U.S. Industrial Enzyme Market

27 Industrially Important Enzymes

28 Medical Applications for Enzymes Since enzymes are specific biological catalysts, they should make desirable therapeutic agents for the treatment of metabolic diseases Unfortunately a number of factors severely reduce the potential clinical utility of enzymes: Size - enzymes are too large to be distributed simply within the body's cells Immunogenicity (HOW?) Stability - their effective lifetime within the circulation may be only a matter of minutes

29 Medical Utilization of Enzymes

30 Enzyme-Based Biosensors Biosensor: a compact analytical device incorporating a biological sensing element either integrated within, or intimately associated with, a physicochemical transducer For example: (Important!!) 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

31 Enzyme-Based Biosensors

Development of Enzyme Immobilization Technique

Development of Enzyme Immobilization Technique Professor SEUNG-WOOK KIM Laboratory of Bioprocess Department of Chemical and Biological In this presentation Enzymes Enzymes are are biological biological