Biochemical Engineering Introduction And Enzyme Kinetics PDF

Biochemical Engineering INTRODUCTION OF BIOCHEMICAL ENGINEERING ENZYME KINETICS Engr. A. Corpuz 09/2021 Chemical Engineering Cagayan State University – Carig Campus, Tuguegarao City 9/11/2022...

Biochemical Engineering INTRODUCTION OF BIOCHEMICAL ENGINEERING ENZYME KINETICS Engr. A. Corpuz 09/2021 Chemical Engineering Cagayan State University – Carig Campus, Tuguegarao City 9/11/2022 ARC Aug2021 1 1. Introduction 2. Bioprocessing Applications 3. Biotechnology 4. Scaling up of Bioprocessing Technologies 5. Biological Process 6. Fermentation 9/11/2022 ARC Aug2021 2 Introduction Biochemical Engineering concerned with conducting biological processes on an industrial scale, providing the link between biological sciences and chemical engineering Increasing relevance due to dramatic developments of biotechnology in the recent years An interdisciplinary field 9/11/2022 ARC Aug2021 Netherlands (2020): Algae unit for Algae production as sustainable alternative 3 biomass to produce fuel, oil and protein (https://www.alamy.com/)  Bioprocessing Essential part of many foods, chemicals, and pharmaceutical industries The operations may make use of microbial, animal, and plant cells and their components (proteins, enzymes, metabolites) to manufacture new useful products and destroy harmful wastes. Also signifies the utilization of waste (production of alcohol, biofuels, therapeutics chemicals such as antibacterial, vaccines, etc) ARC Aug2021 9/11/2022 4  Bioprocessing Applications From: Advances in bioreactors for lung bioengineering: From scalable cell culture to tissue growth monitoring (Mahfouzi et al, 2021). Pharmaceuticals and medicines (Insulin, Penicillin, other Antibacterials, Proteins) Organ growth in reactors (Pancreas for transplanting islet cells for Type I Diabetes; Lung transplantation) Foods (Fermented foods, Beverages; Functional foods) Computers based on biological molecules rather than silicon chips (DNA vs silicon: biochips for new-age computers) Other industrial processes (superorganisms for removal of pollutants; biofuels) 9/11/2022 ARC Aug2021 5 Insulin story Slow- Type 1 release Biosynthetic diabetes insulin 95% of insulin global users (poor (added a prefer chance of protein found (rat insulin gene human normal in fish sperm, spliced into a protamine) bacterium) E. coli insulin life) 1921 1936 1970’s 1977 1982 2001 Human insulin insulin insulin first approved from a from genetically dog's pancreas engineered pancreas of cattle, pharmaceutical F. G. Banting, pigs product Risk: C. H. Best animal Eli Lilly Corporation 9/11/2022 diseases ARC Aug2021 6  Biotechnology Traditional biotechnology (Congress of the United States, 1984) Commercial techniques that use 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 and animals, and for the development of microorganisms to act on the environment Modern biotechnology refers to 1. recombinant DNA - allows the direct manipulation of genetic material of individual cells, which may be used to develop microorganisms that produce new products as well as useful organisms ❖ Genetic Engineering - the genetic manipulation within living cells Genetic engineering 2. cell fusion - a process to form a single hybrid cell (hybridoma) with nuclei and cytoplasm from two different types of cells in order to combine the desirable characteristics of the two ❖specialized cells of the immune system can produce useful antibodies - for disease diagnosis, treatment, and protein purification Hybridoma technology (use of tumor cells: immortality and rapid proliferation) 9/11/2022 ARC Aug2021 8  Bioprocessing Technologies Successful commercialization requires the development of a large-scale process that is technologically viable and economically efficient. Components: Raw materials – biomass (nutrients, energy source) Medium – liquid mixture (growth factors) Cell culture – pure strain (microorganism, animal or plant cell, or enzyme catalyst) Bioreactor or fermenter – with multiple components to control fermentation (optimal design, operation and control) Product recovery and purification (max purity, minimal cost) process development and design ✓ Basic sciences for the process (microbiology, biochemistry, molecular biology, genetics) ✓ Reaction kinetics ✓ Process control 9 9/11/2022 ✓ Separation processes ARC Aug2021  Biological Process o Biological processes have advantages and disadvantages over traditional chemical processes. Advantages Disadvantages Mild reaction condition Complex product mixtures (room temperature, atmospheric (multiple enzyme reactions, various cell components and pressure, and fairly neutral pH) metabolic by-products) Specificity Dilute aqueous environments (enzyme catalyst to chemical reaction/s) (small amounts of and heat sensitive products) Effectiveness Contamination (faster enzyme-catalyzed reaction) (molds, bacteria) Renewable resources Variability (biomass) (changing environment, sensitive enzymes) 9/11/2022 Recombinant DNA technology ARC Aug2021 10  Fermentation the process for production of alcohol or lactic acid from glucose (traditional) Fermentation originally referred to the metabolism of an organic compound under anaerobic conditions an enzymatically controlled transformation of an organic compound (A Merriam-Webster, 1977) modern industrial fermentation includes both aerobic and anaerobic large-scale culture of organisms 9/11/2022 ARC Aug2021 11 HW1: due 1 Oct Submit a 2-page report on a chosen biotechnology application discussing the rationale/historical background of the technology, the methodology, sample points of applications. Cite references used. ❖Biotechnology has applications in four major industrial areas, including health care (medical), crop production and agriculture, non-food (industrial) uses of crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and environmental uses. Examples: golden rice, penicillin, gene therapy 1. Enzymes as catalysts 2. Enzyme specificity 3. Classification and Applications of Enzymes 4. Catalytic effect 5. Factors affecting enzyme activity 6. Enzyme Kinetics 9/11/2022 ARC Aug2021 13  Enzymes (E) as catalysts proteins produced by living cells (animal, plant, and microorganisms: pure culture) act as catalysts (in small amounts) in biological reactions by decreasing activation energy, AE catalytic ability is based on protein structure specific chemical reaction is catalyzed at the active site, small portion of the surface of the enzyme rate of an enzyme-catalyzed reaction is usually much faster than that of nonbiological catalysts Grooves as active sites 14 9/11/2022 ARC Aug2021 (flipper.diff.org)  Enzyme Specificity An enzyme catalyst is highly specific, and catalyzes only one or a small number of chemical reactions E S P α-amylase starch glucose + maltose + oligosaccharides lactase lactose glucose + galactose lipase fat fatty acids + glycerol maltase maltose glucose urease urea + H2O 2NH3 + CO2 cellobiase cellobiose glucose rennin milk Curd (cheesemaking) pepsin proteins at acidic pH Hydrolyzed protein trypsin proteins at mild alkaline pH Hydrolyzed protein glucose isomerase glucose fructose glucose oxidase D-glucose + O2 + H2O gluconic acid alcohol dehydrogenase Ethanol + NAD + acetaldehyde + NADH2 9/11/2022 ARC Aug2021 15  Classification of Enzymes (Crueger and Crueger, 1984) Industrial enzymes: amylases, proteases, glucose isomerase, lipase, catalases, and penicillin acylases -tons usage Analytical enzymes: glucose oxidase, galactose oxidase, alcohol dehydrogenase, hexokinase, muramidase, and cholesterol oxidase e.g., in biosensors -milligrams to grams usage in their pure forms; high production costs Medical enzymes: such as asparaginase, proteases, lipases, and streptokinase for drug manufacture, therapeutics, cleaning wounds, diagnosis -milligrams to grams usage in their pure forms; high production costs 9/11/2022 ARC Aug2021 16  Alkaline protease: cleaning aid in laundry detergents 9/11/2022 Protease: meat tenderizer, cheese making ARC Aug2021 17 9/11/2022 ARC Aug2021 18 9/11/2022 ARC Aug2021 19 9/11/2022 ARC Aug2021 20 9/11/2022 ARC Aug2021 21  Modulators – substances which can combine with enzymes to alter their catalytic activities Others: shear, product concentration 9/11/2022 ARC Aug2021 22  Shear Enzymes had been believed to be susceptible to mechanical force, which disturbs the elaborate shape of an enzyme molecule to such a degree that denaturation occurs. The mechanical force that an enzyme solution normally encounters is fluid shear, generated either by flowing fluid, the shaking of a vessel, or stirring with an agitator. 9/11/2022 ARC Aug2021 23  ENZYME KINETICS Enzyme kinetics deals with the RATE of enzyme reaction and how it is affected by the various chemical and physical reactions. Kinetic studies of enzymatic reactions provide information about the basic mechanism of the enzyme reaction and other parameters that characterize the properties of the enzyme. The rate equations developed from the kinetic studies can be applied in calculating reaction time, yields and optimum economic condition, which are important in the design of an effective bioreactor. 1. Simple Enzyme Kinetics A. Michaelis Menten-approach 1. Batch PFR 2. CSTR 2. Complex Enzyme Kinetics A. Competitive B. Noncompetitive Inhibition 9/11/2022 ARC Aug2021 24  Simple Enzyme Kinetics Rate or reaction, rs Change of product and substrate conc. wrt time 9/11/2022 ARC Aug2021 25  (Brown, 1902) k3 max. reaction rate rmax or vmax k2 9/11/2022 rmax is proportional to Enzyme conc. within the range of the enzyme tested. 26 ARC Aug2021  Simple Enzyme Kinetics The effect of substrate conc. on the initial reaction rate Empirical expression (Henri, 1902) Kinetic parameters rmax and KM to be experimentally determined. 9/11/2022 ARC Aug2021 27  Simple enzyme kinetics substrate or product inhibition not accounted for 1 To derive rate equation: 2 Measurable entities: CS, Cp, CE0* Michaelis-Menten approach: assumed that the product-releasing step (2) is much slower than the reversible reaction (1) – justified by weak interaction within E-S complex; employed in heterogeneous catalytic reactions in chemical kinetics Briggs-Haldane approach: d(CES)/dt = 0, pseudo steady-state (or quasi-steady-state) assumption in chemical kinetics; applied in homogeneous catalytic reactions Numerical solution: solution of DE 9/11/2022 ARC Aug2021 28  Michael-Menten Kinetics Rate-limiting step 2 1 Equilibrium rxn 1 2 Enzyme conservation 3 Kinetic parameters: (2&3) eliminate Ce KM, Michaelis constant Michaelis-Menten rmax, max. rxn rate equation when KM is equal to CS, r =1/2 rmax (4&1) KI, dissociation constant 9/11/2022 ARC Aug2021 Keq, equilibrium constant 29 Exercise 2.1 Rate expression for numerical solution: expressed in terms of rate constant(s) and reactant concentration(s) 𝑑𝐶𝑝 = 𝑑𝑡 𝑑𝐶𝐸𝑆 = 𝑑𝑡 𝑑𝐶𝑆 = 𝑑𝑡 Conservation equation: simultaneous differential equations can be solved numerically by using a computer; requires the knowledge of elementary rate constants, k1, k2, and k3 and initial molar concentration of enzyme. The elementary rate constants can be measured by the experimental techniques such as pre-steady-state kinetics and relaxation methods 9/11/2022 ARC Aug2021 30  Example 2.1: Derive the rate equation (2.23 into 2.21) (2.19 & 2.20) eliminate Ce (2.20 & 2.22) eliminate Ce 9/11/2022 ARC Aug2021 31  Evaluation of Michaelis-Menten Parameters Conduct a series of batch runs of different S concentrations at constant CE0 Get initial reactions rates, r, measured at CS,0 By regression 𝑟𝑚𝑎𝑥 𝐶𝑠 Linearization: 𝑟= 𝑘𝑀 + 𝐶𝑠 Langmuir plot 𝐶𝑠 𝑘𝑀 𝐶𝑠 = + 𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥 1 𝑘𝑀 1 Lineweaver-Burk plot = + 𝑟 𝑟𝑚𝑎𝑥 𝐶𝑠 𝑟𝑚𝑎𝑥 Eadie-Hofstee plot 𝑟𝑘𝑀 𝑟 = 𝑟𝑚𝑎𝑥 − 𝐶𝑆 Nonlinear regression: Solver excel, see example (2.3) 9/11/2022 ARC Aug2021 32  Evaluation of Michaelis-Menten Parameters Conduct a series of batch runs of different S concentrations at constant CE0 Get initial reactions rates, r, measured at CS,0 By regression 𝑟𝑚𝑎𝑥 𝐶𝑠 Linearization: 𝑟= 𝑘𝑀 + 𝐶𝑠 1 𝑘𝑀 1 Lineweaver-Burk plot = + 𝑟 𝑟𝑚𝑎𝑥 𝐶𝑠 𝑟𝑚𝑎𝑥 9/11/2022 ARC Aug2021 33  Evaluation of Michaelis-Menten Parameters Conduct a series of batch runs of different S concentrations at constant CE0 Get initial reactions rates, r, measured at CS,0 By regression 𝑟𝑚𝑎𝑥 𝐶𝑠 Linearization: 𝑟= 𝑘𝑀 + 𝐶𝑠 Langmuir plot 𝐶𝑠 𝑘𝑀 𝐶𝑠 = + 𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥 9/11/2022 ARC Aug2021 34  Evaluation of Michaelis-Menten Parameters Conduct a series of batch runs of different S concentrations at constant CE0 Get initial reactions rates, r, measured at CS,0 By regression 𝑟𝑚𝑎𝑥 𝐶𝑠 Linearization: 𝑟= 𝑘𝑀 + 𝐶𝑠 𝑟𝑘𝑀 Eadie-Hofstee plot 𝑟 = 𝑟𝑚𝑎𝑥 − 𝐶𝑆 9/11/2022 ARC Aug2021 35 Exercise 2.3 Linear and Non-linear regression to estimate kinetic parameters 9/11/2022 ARC Aug2021 36 9/11/2022 ARC Aug2021 37 HW 2 due 19Sept 5PM 9/11/2022 ARC Aug2021 38  Estimate the values of the kinetic parameters using linear (3 techniques) and nonlinear HW2: regression. Compare the results. You may use kinetic parameters evaluation calculator and/or Excel, write complete solutions including calculated x,y data points for each plot and generated equations. Upload plots in MS teams. ARC Aug2021 9/11/2022 39 HW2: Deriving rate equation Hints: Set up rate equation based on rate limiting step Additional 2 relations from equilibrium rxns Setup enzyme conservation One by one, aim to express rate equation in terms of CS, CP, kinetic parameters and rate constants noting that kxE0 ~ Vmax ARC Aug2021 9/11/2022 40 HW2: mumol = µmol 1 unit activity = 1µmol product/min Hints: Initial rate is the slope To compute activity, how much enzyme was used? Relate to initial rxn rate ARC Aug2021 9/11/2022 41  Enzyme Reactor with Simple Kinetics pH control packed with immobilized enzyme Residence time Assumptions 1. Batch reactor contents are well mixed. 2. At steady state PFR, the properties will be constant wrt time. Change of CS with time in a batch reactor can be predicted. 1. Batch Reactor 2. Steady-State Plug-Flow Reactor (PFR) or Tubular flow plot of (CS0-CS)/ ln (CS0/CS) vs t / ln(CS0/CS) ideal reactor can approximate the long tube, packed-bed, and hollow fiber, or multi-staged reactor ARC Aug2021 42 9/11/2022  3. Continuous Stirred-Tank Reactor substrate balance of a CSTR:. At steady-state, dCs/dt=0, substrate concentration of the reactor should be constant. Using M-M equation for rS, Dilution Residence rate time Assumption: reactor contents are well mixed. Easy to automate Continuous operation eliminates downtime plot of CS vs CS τ /(CS0 - CS) in a series of steady- F, flow rate state CSTR runs with various flow rates V, volume of reactor contents rS is equal to dCS /dt when F is zero [batch operation] rS, the rate of substrate consumption for the enzymatic reaction 9/11/2022 ARC Aug2021 dCS /dt, the change of the substrate concentration in43the reactor Models for More Complex Enzyme Kinetics Inhibited ▪ Inhibitor – decreases enzyme activity either competitively, noncompetitively, or partially competitively Competitive competitive inhibitor has a strong structural resemblance to the substrate, both the inhibitor and substrate compete for the active site of an enzyme; binds reversibly the reaction rate decreases due to the presence of inhibitor, but a larger amount of substrate is required to reach the maximum rate Non-competitive NC inhibitors can bind to the enzymes reversibly or irreversibly at the active site or at some other region. maximum reaction rate will be decreased by the presence of a noncompetitive inhibitor while KM will not be affected resultant complex is inactive Other factors concentration of various components (substrate, product, enzyme, cofactor, and so on), pH, T, shear 9/11/2022 ARC Aug2021 44 Competitive Inhibition The mechanism of competitive inhibition can be expressed as follows: where, KS and KI are dissociation constants slow reaction Employing M-M approach and eliminating CE, CES, and CEI yields where, 9/11/2022 ARC Aug2021 45  Competitive vs non-competitive rMax not affected KM not affected KM increased rMax decreased competitive noncompetitive 9/11/2022 46 ARC Aug2021  Noncompetitive Inhibition Inhibitor can bind to the enzymes reversibly or irreversibly at the active site or at some other region. The mechanism of noncompetitive inhibition can be expressed as: Since S and I do not compete for a same site for the formation of E-S or E-I complex, we can assume that dissociation constant for the 1st equilibrium rxn is the same as that of the 3rd equilibrium rxn; similarly to 2nd and 4th rxns Employing M-M approach Case 2. When enzyme-inhibitor- substrate complex can be decomposed to produce a product and the enzyme-inhibitor complex. Partially Competitive Inhibition 9/11/2022 slow reaction ARC Aug2021 47 References Lee, J. M. (2009). Biochemical engineering. Englewood Cliffs, NJ: Prentice Hall Doran, P. M. (2013). Bioprocess engineering principles. Elsevier. 9/11/2022 ARC Aug2021 48