Enzyme Inhibition & Immobilization PDF

ENZYME INHIBITION Engr. Vera Marie L. Lanaria TYPES OF INHIBITION  COMPETITIVE INHIBITION inhibitor binds to the active site and prevents binding of the substrate  RATE EQUATION Steps involved: 𝑣𝑝 = 𝑘5 [𝐸𝑆] 1) [E] + [S] ⇄ [ES] [E0]...

ENZYME INHIBITION Engr. Vera Marie L. Lanaria TYPES OF INHIBITION  COMPETITIVE INHIBITION inhibitor binds to the active site and prevents binding of the substrate  RATE EQUATION Steps involved: 𝑣𝑝 = 𝑘5 [𝐸𝑆] 1) [E] + [S] ⇄ [ES] [E0] = [ES] + [EI] + [E] 2) [E] + [I] ⇄ [EI] k1[E][S] = k2[ES] 3) [ES] → [P] + [E] k3[E][I] = k4[EI] 𝑘5 𝐸0 𝑆 𝑣= 𝐼 𝑘𝑠 1+ + 𝑆 𝑘𝐼  UNCOMPETITIVE INHIBITION inhibitor binds to the ES complex and prevents conversion to product  RATE EQUATION Steps involved: ▪ 𝑣𝑝 = 𝑘5 𝐸𝑆 1) [E] + [S] ⇄ [ES] ▪ [E0] = [ES] + [ESI] + [E] 2) [ES] + [I] ⇄ [ESI] ▪ k1[E][S] = k2[ES] 3) [ES] → [P] + [E] ▪ k3[ES][I] = k4[ESI] 𝑣𝑚𝑎𝑥 𝑆 𝐼 𝑎𝑝𝑝 1+ 𝑣𝑚𝑎𝑥 𝑆 𝑘𝐼 𝑣𝑝 = 𝑘𝑠 = 𝑎𝑝𝑝 +𝑆 𝐾𝑀 + 𝑆 𝐼 1+ 𝑘𝐼  NON-COMPETITIVE INHIBITION inhibitor can bind to either free enzyme or enzyme-substrate complex, and likewise, the substrate can bind to free enzyme or the enzyme-inhibitor complex  RATE EQUATION Steps involved: 𝑣𝑝 = 𝑘9 𝐸𝑆 1) [E] +[S] ⇄ [ES] [E0] = [ES] + [EI] + [EIS] + 2) [E] + [I] ⇄ [EI] [ESI] + [E] 3) [EI] +[S] ⇄ [EIS] k1[E][S] = k2[ES] 4) [ES] + [I] ⇄ [ESI] k3[E][I] = k4[EI] 5) [ES] → [P] + [E] k5[EI][S] = k6[EIS] k7[ES][I] = k8[ESI]  𝑣𝑚𝑎𝑥 𝐼 𝑆 1+ 𝑎𝑝𝑝 𝑘𝐼 𝑣𝑚𝑎𝑥 𝑆 𝑣𝑝 = = 𝑘𝑠 + 𝑆 𝑘𝑀 + 𝑆 SUMMARY ENZYME IMMOBILIZATION By: Engr. VERA MARIE L. LANARIA Chemical Engineering Department CIT University The movement of the enzyme in a fixed location is restricted by attaching them to an insoluble support medium or enclosed by the support medium which is also known as the carrier. DEFINITION Reusable Continuous operations are possible Easier enzyme/product recovery More stable enzyme Facilitates process control (optimization of product yield and quality) WHY IMMOBILIZED? Methods of Immobilizing Enzymes Inert Cheap Physically strong and stable Reduces product inhibition Discourages microbial growth and nonspecific adsorption, etc. IDEAL CARRIER/SUPPORT SPECIFICATIONS Oftendone in gels or fibers (with polyacrylamide, calcium alginate, gelatin) A convenient method for use in processes involving low molecular weight substrates and products ENTRAPMENT TECHNIQUE Enzymes are not chemically modified. Enzyme properties are not altered. ADVANTAGES (ENTRAPMENT) The deactivation of enzyme may take place during gel formation. Enzymeleakage may take place continuously depending upon the pore size of the gel. Diffusional limitations may pose reduced accessibility for the substrate. DISADVANTAGES (ENTRAPMENT) Enzymes are entrapped within semi-permeable membrane in the form of microscopic hollow spheres. This method is not suitable for proteolytic enzymes, or for macro molecular substrates. MICRO ENCAPSULATION The enzyme is attached to the surface by covalent bond formation via certain functional groups. COVALENT BINDING Amino groups Carboxyl groups Hydroxyl groups Sulphydryl groups MOST COMMONLY USED FUNCTIONAL GROUPINGS Very strong bonding (less enzyme leakage) Small amounts of enzymes are immobilized Providesmore permanent linkage between the enzyme and the support material ADVANTAGES Synthetic support materials - acrylamide-based polymers - maleic anhydride polymers - styrene-based polymers Natural support materials - agarose, cellulose, dextran, glass, & starch SOME WATER-INSOLUBLE SUPPORT MATERIALS The oldest immobilization method Enzymes can be adsorbed physically on a surface- active adsorbent by weak physical forces such as van der Waals forces or dispersion forces. ADSORPTION Silica gel Metal oxides (hydrous) Glass Organic polymers Porous carbon Clay, etc. MATERIALS FOR ADSORPTION Immobilization procedure is easy and simple Adsorption process is reversible Enzymes are not deactivated by adsorption It is possible to separate and purify the enzymes while being immobilized. ADVANTAGES Since adsorption is a non-specific process, many other substances may also be attached to the carrier in addition to the immobilized enzyme. The loading of enzyme on a unit amount of surface is always very low, and the bonding strength is very weak. DISADVANTAGES Thank You for Your Attention! Physical and Chemical Controls of Microbes Engr. Vera Marie L. Lanaria ChE Department CIT University Why is microbial control necessary? It is mainly to inhibit the growth of pathogens.  Sterilization it refers to the removal or destruction of all microbes, including viruses and bacterial endospores, in or on an object is an absolute term that implies the complete and total removal of all living things  Aseptic it describes an environment or procedure that is free of contamination by pathogens Ex. vegetables & fruit juices are available in aseptic packaging; surgeons & lab technicians use aseptic techniques to avoid contamination while doing their job  Disinfection it refers to the use of physical or chemical agents known as disinfectants this term is used only for treatment of inanimate objects Antisepsis refers to a process of using chemical or antimicrobial agent on skin or other tissue the chemical agent is called antiseptic  Degerming is the removal of microbes from a surface by scrubbing  Sanitizing is the process of disinfecting places and utensils used by the public to reduce the number of pathogenic microbes to meet accepted public health standards  Pasteurization is the use of heat to kill pathogens and reduce the number of spoilage microorganisms in food and beverages  What is microbial death? Death – is a phenomenon that involves the permanent termination of an organism’s vital processes  Factors that affect Death Rate 1) Number of microorganisms. 2) Nature of microorganisms. 3) Temperature & pH of the environment. 4) Concentration of the agent. 5) Mode of action of the agent. 6) Presence of solvents, interfering organic matter, and inhibitors.  How Antimicrobial Agents Work? The cellular targets of physical & chemical agents fall into 4 general categories: 1. the cell wall 2. the cell membrane 3. cellular synthetic processes (DNA, RNA) 4. proteins  Methods of Physical Control Heat Radiation Filtration Ultrasonic waves Cold  HEAT Moist Heat: - hot water, boiling water, steam (vaporized water) - temperature ranges from 60-135 0C Dry Heat: - that has been heated by a flame or electric heating coil - temperature ranges from 1600C up  What is the effect on microorganisms when moist heat is used as compared to dry heat? Moist heat generally coagulates and causes denaturation in microbes. In denaturation, proteins separate as an insoluble mass as they revert from their 3-dimensional structure to a 2-dimensional structure. In dry heat, the primary effect is due to oxidation of large molecules; is a less efficient process which requires a longer period of process time  COLD “Cold merely retards the activities of most microbes.” Lyophilization – a combination of freezing and drying; a common method of preserving microorganisms and other cells  Radiation Types of radiation that can be used as anti- microbial control agents: 1) Ionizing radiation – include x-rays and gamma rays, which form free radicals in cytoplasm and the free radicals destroy microbial proteins and DNA 2) Ultraviolet radiation – will effect nucleic acids by binding together adjacent thymine bases; microbes will die because DNA cannot function or replicate itself  Ultrasonic waves These high-frequency sound waves causes vibrations that coagulate cellular proteins and disintegrate cellular components. Ultrasonic vibrations are commonly used as cleaning agent for lab materials and as a cell disrupters.  Filtration Modern microbial filters: cellulose acetate; polycarbonate; plastic materials (teflon & nylon) where pores size can vary from coarse (8 microns) to ultrafine (0.02 micron)  Applications of Filtration use to prepare liquids that cannot withstand heat, including serum & other blood products, vaccines, drugs, IV fluids, enzymes, and media use for decontaminating milk & beer without altering their flavor used in water purification used in removing airborne contaminants that are common source of infection & spoilage  Chemical Agents Gases that can perform sterilization: ethylene oxide (ETO) is used to sterile plastics (such as petri dishes) beta propiolactone (BPL) is used to sterile liquids formaldehyde can be used for various materials  Qualities in choosing antimicrobial chemical agents: ▪ rapid action even in low concentration ▪ solubility in water or alcohol and long-term stability ▪ broad-spectrum microbicidal action without being toxic to human and animal tissues ▪ penetration of inanimate surfaces to sustain a cumulative or persistent action ▪ resistance to becoming inactivated by organic matter ▪ noncorrosive or nonstaining properties ▪ sanitizing and deodorizing properties ▪ affordability and ready availability Thank you ! STERILIZATION ENGR. VERA MARIE L. LANARIA CHE DEPARTMENT CIT UNIVERSITY  FERMENTATION A BIOCHEMICAL PROCESS OF PRODUCING METABOLIC PRODUCTS BY THE ACTION OF MICROORGANISMS OR A GROUP OF MICROORGANISMS ON A SUBSTRATE, IN THE PRESENCE OF NUTRIENTS IN THE MEDIUM FERMENTATION INVOLVES THE FOLLOWING: MICROORGANISM MEDIUM FERMENTER NUTRIENTS & OTHER ADDITIVES AIR (AEROBIC PROCESS) ILLNESSES OF FERMENTATION: ▪ THE FOREIGN ORGANISM MAY CONTAMINATE THE FINAL PRODUCT. ▪ THE MEDIUM WOULD BE CONSUMED UNNECESSARILY TO SUPPORT THE GROWTH OF CONTAMINATING ORGANISMS. ▪ THE CONTAMINATED PRODUCT MAY OUTWEIGH THE DESIRED PRODUCT; PARTICULARLY, IT IS MORE SO IN THE CASE OF CONTINUOUS FERMENTATION PROCESS. ▪ THE CONTAMINATED PRODUCT MAY INTERFERE WITH THE RECOVERY OF THE DESIRED PRODUCT, AND MAY RENDER IT INACCESSIBLE. ▪ UNSTERILE AIR IN AEROBIC FERMENTATIONS MAY RESULT IN THE SPOILAGE OF THE FERMENTATION PRODUCT.  STERILIZATION OF THE MEDIUM EMPLOYING AS PURE AN INOCULUM AS POSSIBLE STERILIZATION OF THE FERMENTER STERILIZING THE PIPES, VALVES, BENDS, ETC. WHICH COME IN CONTACT WITH THE FERMENTATION PROCESS STERILIZATION OF ALL MATERIALS TO BE ADDED TO THE FERMENTER STERILIZATION OF AIR  DISINFECTING THE FERMENTER AND CONTACT PARTS WITH A NON-TOXIC DISINFECTANT MAINTAINING ASEPTIC CONDITIONS IN THE FERMENTERS DURING FERMENTATION MAINTAINING OPTIMUM OR DESIRED PH WHICH DISCOURAGES THE GROWTH OF CERTAIN CONTAMINANTS OR UNDESIRED ORGANISMS OR PRODUCTS  STERILIZATION OF MEDIUM STERILIZATION IS NORMALLY DONE BY: BOILING IN WATER PASSING STEAM AUTOCLAVING (SUBJECT THE MEDIUM TO STEAM UNDER PRESSURE IN A PRESSURE COOKER) FOR LARGE SCALE PROCESSES: ▪ADJUSTING PH ▪USING CONTAMINATION INHIBITORS (SUCH AS LACTIC ACID) THINGS TO CONSIDER: SYNTHETIC MEDIA MAY REQUIRE A SMALL AMOUNT OF HEATING FOR STERILIZATION AS COMPARED TO CRUDE MEDIA. IF PH IS A CRITICAL FACTOR DURING STERILIZATION, IT IS ADVISABLE TO ADJUST THE PH TO NEUTRALITY. AFTER STERILIZATION, BRING BACK THE PH TO THE ORIGINAL VALUE BY ADDITION OF PRE-STERILIZED ACID OR ALKALI.  IF SOME OF THE ENZYMES AND NUTRIENTS LIKE VITAMINS ARE SENSITIVE TO HEAT STERILIZATION, THEY ARE SEPARATED INITIALLY BY PASSING THROUGH A BACTERIOLOGICAL FILTER; STERILIZATION IS CARRIED OUT, AND THE SEPARATED ENZYMES/NUTRIENTS ARE ADDED BACK TO THE MEDIUM.  STERILIZATION OF AIR METHODS OF AIR STERILIZATION: ❖BY HEATING ❖USE OF UV RAYS & OTHER ELECTROMAGNETIC WAVES ❖USE OF GERMICIDAL SPRAYS ❖BY FILTRATION NORMALLY AIR IS STERILIZED IN THE PROCESS INDUSTRIES BY PASSING IT THROUGH A FILTER BED. 2 TYPES OF AIR FILTERS: PORES ARE SMALLER THAN THE SIZE OF THE MICROORGANISMS TO BE REMOVED PORE SIZE IS BIGGER THAN THE SIZE OF THE MICROORGANISMS  STERILIZATION OF FERMENTERS STEAM AT 15 PSIG IS ADMITTED AND KEPT INSIDE FOR APPROXIMATELY 20 MINUTES FOR STERILIZATION TO COMPLETE. AFTER STERILIZATION, THE FERMENTER SHOULD BE FLUSHED WITH STERILE AIR KEPT UNDER POSITIVE PRESSURE.  Introduction to Biochemical Engineering Course By: Engr. VERA MARIE L. LANARIA Broad Definition: BIOTECHNOLOGY “Commercial techniques that uses living organisms, or substances from those organisms, to make or modify a product, including techniques used for the improvement of the characteristics of economically important plants & animals and for the development of microorganisms to act on the environment.”… (Congress of United States, 1984) ….. the integrated use of biochemistry, microbiology, and chemical engineering in order to achieve the technological and industrial application of the capacities of micro-organisms and cultured tissue cells. Biochemical Engineering … the extension of chemical engineering principles to systems using a biological catalyst to bring about desired chemical transformation … is concerned with conducting biological processes on an industrial scale In ancient times….  fermentation of beverage and food  cross-breeding of plants and animals for better yields Applications of Biotechnology  Pharmaceuticals: Antibiotics, antigens, insulin, interferon, vaccines  Animal Agriculture: Products similar to those being developed in the pharmaceutical industry; development of disease-free seed stocks & healthier, higher-yielding food animals  Application to Biotechnology  Plant Agriculture: Transfer of stress-, herbicide-, and pest-resistance traits to important crop species; development of plants with the increased abilities of photosynthesis or nitrogen fixation; development of biological insecticides and non-ice nucleating bacterium  Specialty Chemicals: Amino acids, enzymes, vitamins, lipids, hydroxylated aromatics, and biopolymers  Application to Biotechnology  Agricultural Chemicals: Pesticides, fungicides, herbicides  Environmental Applications: Mineral leaching, metal concentration, pollution control, toxic waste degradation, and enhanced oil recovery  Foods and Beverages: Alcoholic beverages, sweeteners, single-cell protein Application to Biotechnology  Commodity Chemicals: Acetic acid, acetone, butanol, ethanol, and many other products from biomass conversion processes  Bioelectronics: Biosensors, biochips https://www.ddw-online.com/the-protein-biochip-content- problem-1226-200308/ What’s the role of a biochemical engineer??? To carry out a bioprocess on a large scale, biochemical engineers need to work together with biological scientists:  to obtain the best biological catalyst for a desired process  to create the best possible environment for the catalyst to perform by designing the bioreactor and operating it in the most efficient way  to separate the desired products from the reaction mixture in the most economical way Major advantages of biological process…  Mild reaction condition  Specificity  Effectiveness  Renewable resources  Recombinant DNA technology Disadvantages….  Complex product mixtures  Dilute aqueous environments  Contamination  Variability https://www.labcompare.com/General-Laboratory-Equipment/25007-Laboratory-Bioreactors-and-Fermentors/ BIOLOGICAL BASICS  CELLS  Basic unit of living organism.  Different types but the same essential properties. … are living entities, surrounded by a membrane, that are capable of growing, reproducing, responding, and metabolizing. Microbial Nomenclature  Microbiologists use the binomial system. Ex. Bacillus subtilis; Escherichia coli  Proper names are always italicized.  First word is the name of the genus and starts with a capital letter. Ex. Bacillus – a small rod Clostridium – a small spindle  Second word is the species. Note: When the same genus name is repeated several times, it is abbreviated.  Chemical Composition of the Cells  Water: 75-85% of the cell mass (upper limit for viruses  6 irreplaceable elements: C, H, O, N, S and P  Trace elements: Cu, Co, Cr, Ni, Se, Mn, Mo, W, V, Zn, etc. → enzymatic reactions  Major + trace elements = nutrients  Proteins: about 50% of dry weight of cells is protein, largely enzymes  Nucleic acids (which contains the genetic code and machinery to make proteins): 10-20% of dry weight  Lipids: 5-15% of dry weight  In general, the intracellular composition of cells varies depending on the type and age of the cells and the composition of the nutrient media Microbial Diversity…  Psychrophile: optimum temp.  20 0C  Mesophile: 20 0C  optimum temp.  50 0C  Thermophile: 50 0C  optimum temp.  Aerobic: growth in the presence of O2  Anaerobic: growth without O2  Facultative: growth under either circumstances  Coccus: spherical or elliptical  Bacillus: cylindrical or rod  Spirillum: spiral What does life really look like?????  Father of Microbiology a Dutch tailor, merchant, and lens grinder the man who discovered the microbial world Developed the theory that all living things are composed of CELLS. Two types of cells….  Prokaryotes  Eukaryotes Prokaryotes are simple cells Eukaryotes are complex cells Some organelles and its functions… Classifications of Living Things…  DERIVATION OF MICHAELIS-MENTEN MODEL ENGR. VERA MARIE L. LANARIA CHE DEPARTMENT CIT UNIVERSITY  USING MICHAELIS-MENTEN APPROACH Recall: This approach assumed that the product-releasing step is much slower than the reversible reaction and which determines the rate of the whole reaction. Starting from the mechanism postulated by Adrian Brown (1902): 𝑆 + 𝐸 ⇄ 𝐸𝑆 𝐸𝑆 → 𝑃 + 𝐸 If the slower reaction determines the overall rate of reaction, the rate of product formation and substrate consumption is proportional to the concentration of the 𝑑𝐶𝑝 −𝑑𝐶𝑆 enzyme-substrate complex as: 𝑟 = = = 𝑘3 𝐶𝐸𝑆 → 𝐸𝑞𝑛. (1) 𝑑𝑡 𝑑𝑡  From the equilibrium step, the forward reaction is equal to the reverse reaction so that 𝑘1 𝐶𝑆 𝐶𝐸 = 𝑘2 𝐶𝐸𝑆 → 𝐸𝑞𝑛. (2) From the assumption that the total enzyme contents are conserved, the free-enzyme concentration 𝐶𝐸 can be related to the initial enzyme concentration 𝐶𝐸𝑜. 𝐶𝐸𝑜 = 𝐶𝐸𝑆 + 𝐶𝐸 → 𝐸𝑞𝑛. (3) 𝑘2 𝐶𝐸𝑆 Substitute Eqn. (2) to Eqn. (3) for 𝐶𝐸 , then rearranging for 𝐶𝐸𝑆. 𝐶𝐸 = 𝑘 1 𝐶𝑆 𝐶 𝐶𝑆 𝐶𝐸𝑆 = 𝑘2𝐸𝑜 𝑘1 +𝐶𝑆  𝐶𝐸𝑜 𝐶𝑆 𝒓𝒎𝒂𝒙 𝑪𝑺 Then substitute to Eqn. (1): 𝑟 = 𝑘3 𝑘2 ≈ +𝐶𝑆 𝑲𝑴 + 𝑪𝑺 𝑘1 𝑘2 where: = can be replaced by KM 𝑘1 𝑘3 𝐶𝐸𝑜 = can be replaced by 𝑟𝑚𝑎𝑥 (𝑜𝑟 𝑣𝑚𝑎𝑥 ) KM can be equal to the dissociation constant Kdiss , or the reciprocal of equilibrium constant 𝐶𝑆 𝐶𝐸 1 Keq : 𝐾𝑀 = 𝐾𝑑𝑖𝑠𝑠 = = 𝐶𝐸𝑆 𝐾𝑒𝑞  USING BRIGGS-HALDENE APPROACH Recall: The change of the intermediate concentration with respect to time is assumed to 𝑑𝐶𝐸𝑆 be negligible, that is, = 0. 𝑑𝑡 Again, starting from the mechanism postulated by Adrian Brown (1902) 𝑆 + 𝐸 ⇄ 𝐸𝑆 𝐸𝑆 → 𝑃 + 𝐸 Then the rates of product formation and substrate consumption are: 𝑑𝐶𝑃 = 𝑘3 𝐶𝐸𝑆 → 𝐸𝑞𝑛. (1) 𝑑𝑡 𝑑𝐶𝑆 − = 𝑘1 𝐶𝑆 𝐶𝐸 − 𝑘2 𝐶𝐸𝑆 → 𝐸𝑞𝑛. (2) 𝑑𝑡  𝑑𝐶𝐸𝑆 Assume that the change of 𝐶𝐸𝑆 with time, = 0, is negligible compared to that of 𝐶𝑃 𝑑𝑡 𝑑𝐶𝐸𝑆 or 𝐶𝑆. = 𝑘1 𝐶𝑆 𝐶𝐸 − 𝑘2 𝐶𝐸𝑆 − 𝑘3 𝐶𝐸𝑆 = 0 → 𝐸𝑞𝑛. (3) 𝑑𝑡 If this is substituted to Eqn. (2), it will confirm that the rate of product formation and that of the substrate consumption are the same, that is, 𝑑𝐶𝑃 𝑑𝐶𝑆 𝑟= =− = 𝑘3 𝐶𝐸𝑆 𝑑𝑡 𝑑𝑡 Again, if it is assume that the total enzyme contents are conserved, 𝐶𝐸𝑜 = 𝐶𝐸𝑆 + 𝐶𝐸 → 𝐸𝑞𝑛. (4)  Substituting Eqn. (4) into Eqn. (3) for 𝐶𝐸 , and rearranging for 𝐶𝐸𝑆 𝐶 𝐶𝑆 𝐶𝐸𝑆 = 𝑘2+𝐸𝑜 𝑘3 → 𝐸𝑞𝑛. (5) 𝑘1 + 𝐶𝑆 𝑑𝐶𝑃 𝐶𝐸𝑜 𝐶𝑆 𝒓𝒎𝒂𝒙 𝑪𝑺 Substituting Eqn. (5) into Eqn. (1): 𝑟 = = 𝑘3 𝑘2 + 𝑘3 ≈ 𝑑𝑡 + 𝐶𝑆 𝑲𝑴 + 𝑪𝑺 𝑘1 𝑘 2 + 𝑘3 Take note that KM is equal to … but 𝑘3 can be eliminated if 𝑘2 ≫ 𝑘3 which means 𝑘1 that the product-releasing step is much slower than the enzyme-substrate complex dissociation step. EXERCISE PROBLEM: When glucose is converted to fructose by glucose isomerase, the slow product formation step is also reversible as: 𝑆 + 𝐸 ⇄ 𝐸𝑆 𝐸𝑆 ⇄ 𝑃 + 𝐸 Derive the rate equation by employing: (a) the Michaelis-Menten approach; (b) the Briggs- Haldene approach. Explain when the rate equation derived by the Briggs-Haldene approach can be simplified to that derived by the Michaelis-Menten approach.