BC Lecture 2 - Biocatalysis, Enzymes, Chemical Kinetics - PDF

Welcome to Lecture 2 Last time: Advantages of biocatalysis High selectivity (stereo-, regio- and chemo-) Mild reaction conditions e.g. temp, pressure, pH (very energy efficient) Minimal formation of side products...

Welcome to Lecture 2 Last time: Advantages of biocatalysis High selectivity (stereo-, regio- and chemo-) Mild reaction conditions e.g. temp, pressure, pH (very energy efficient) Minimal formation of side products Green appeal Green chemistry: what it means and the 12 principles Sustainability: ~1 trillion single- use plastic bags are used annually across the globe. That's nearly 2 million every minute (!) U.S. consumption of plastic per capita is >200lb/year, but only ~8% of this is recycled as compared to 66% for paper and 27% for glass  Much more plastic is landfilled and burned than recycled! Must recycle! Many other problems We consume crazy amount of pharmaceuticals In 2020, the volume of medicines used globally reached 4.5 trillion doses The life cycle of pharmaceutical drugs Science, 2022 These are molecules designed to trigger biological response at very low concentrations! 1  Wild life disappearing… Risk of extinction in different taxonomic groups Science, 2019 ~20%! Today vs 2050 Moving towards greener, bio-based economy The refinery of the future Electricity generation capacity by source Realistic? Who is going to barrels pay for it? per day Nature, 2024 The ultimate goal: Carbon neutrality refers to achieving a balance between emitting carbon and absorbing carbon from the atmosphere in “carbon sinks”, such as forests, soils, and oceans 2  Industrial applications And we briefly talked about industrial applications A great diversity of products Any questions on the last class? Plan for today: Biocatalysis: market and products Introduction to catalysis (mostly a reminder) But first a quick look at your HW Industrial processes Industry sector analysis* Points to note: No Bioethanol; polymers got The proportions shown reflect bigger, but… the number of processes in each sector; NOT the scale No pharmaceuticals among “bulk” products, which are manufactured on a scale of tens of thousand tons per annum Thus, if the scale was taken into account, industries other *based on 134 industrial processes than pharma would be far from an industry survey carried out more important about 20 years ago Current Opinion in Biotechnology, 2002 3  Product characteristics “Chemical” distribution Points to note: Dominated by natural products and their derivatives Most products are chiral! Fat and carbohydrates are predominantly in the food sector The rest are fine chemicals, agri-chemicals and pharma *based on 134 industrial processes Why is that? from an industry survey carried out about 20 years ago Current Opinion in Biotechnology, 2002 Enzyme producers Novozymes: almost 50% The industry is of sales worldwide dominated by one Novozymes market company share in enzymes for industrial use Novozymes: Small and mature market (sales of ~$2.5B) Hence, worldwide sales of enzymes, COVID-related dip excluding pharma is only about ~$5-6B Novo-Nordisk, their pharmaceuticals arm span off in 2000, had sales of $32B in 2023 From: Novozymes annual report 2023 4  Novozymes’ business Sales by business areas and by geography Sales increase 2023: 5% organic Sales in Latin America increased sales growth overall by 13% after 17% increase in the prior year; North America did well too for the 2nd year in a row, but practically no growth in Europe and Asia Pacific In 2020 double-digit growth was in three out of five business areas: (i) Bioenergy, (ii) Food, Beverages Human/Health and (iii) Grain & Tech Processing, but on 2023 only in Bioenergy From: Novozymes annual report 2023 Recent product launches Biofresh™ - a tablet for oral use, which helps reduce bad breath and Biofresh CleanTM - unique combo of enzymes that enables consumers to protect teeth and maintain a healthy oral microbiome HiPhorius™ – a fourth generation phytase from the DSM-Novozymes Feed Alliance that releases phos- Alterion® – a probiotic phorus from animal feed (2022) solution that helps poultry Prefur Odorelief enables bio- farmers to lower costs by logical cat litter control with improving feed conversion an easy spray application (2022) and gut health. Fiberlife® – enzymatic treatment enabling It also provides textile manufacturers to produce higher a natural quality, and longer-lasting fabrics (2022) alternative to Vertera® Probite for plant-based meat growth industry to improve the texture and mouth- promoters feel of plant-based meat products (2023) From: Novozymes Protide® L – a protease that helps to raise annual reports the nutritional value of animal feed (2023) 5  Enabling technology In $$$ terms the biocatalyst market is pretty small Projected β-lactam antibiotics $65Bln ~$30Bln globally by 2025 Polyketide-derived Biocatalysts Bioethanol ~ 115 mln pharmaceuticals sale: ~$6Bln metric tons in 2018 in ~$30Bln excl. enzyme the U.S. alone drugs (Pharma) Many others natural and semi-synthetic drugs tens of $Bln Bioplastic And plenty of other products… ~$10Bln About $10Bln now, but Any questions? Remember ? expected to grow rapidly Disadvantages of biocatalysis Any ideas? Too narrow a range of suitable reaction conditions e.g. temperature, solvents, pH Too specific i.e. industry likes generic catalysts: once developed – good for multiple use Availability – a limited number of enzymes exists and relatively few are commercially available Other [$$$] considerations: (i) Often [more] expensive to develop and produce (ii) Proteins/cells are generally less stable than chemical catalysts – higher overall costs (iii) Often lower productivity i.e. higher production costs (iv) Different processing requirements/equipment… 6  Let’s compare Advantages vs Disadvantages Very selective Too selective Catalyze chemically difficult Low availability reactions, enable shortcuts Too narrow a range of Mild reaction conditions suitable reaction conditions Green appeal, marketing Economics: often too expensive Biocatalysis: the drivers Make sense? In what kind of products and/or processes the use of biocatalysts is likely to be advantageous?  Products that are  Environmental  Energy savings difficult or expensive impact to produce otherwise  Raw materials  Regulatory  Simplification of overall pressures  Marketing! manufacturing process But the bottom line: favorable economics! What is a catalyst? Catalyst is a chemical substance that increases the rate of reaction without being changed or consumed itself Enzyme is a protein-based catalyst What are catalysts? What are enzymes? Why are enzymes such remarkable catalysts? but to begin with… A quick Gen Chem reminder: Chemical kinetics NOTE: Questions related to this material will be included in your quiz and the final exam! 7  Chemical kinetics The study of the rate of chemical reactions Chemical reaction is a process in which one or more substances (reactants or substrates) are transformed into one or more new substances (products) Energy may be released or absorbed, but no loss of mass or individual atoms occurs, i.e. total molecular weight remains the same The reaction rate for a given reactant or product is defined as the amount of this reactant/product consumed/formed per unit of time Why rates for different reactions are different? What does the rate of a chemical reaction depend upon? Collision theory Collision theory (Max Trautz and William Lewis, 1916) qualitatively explains how chemical reactions occur and why reaction rates differ for different reactions For reaction to occur the reactants must collide However, only a certain fraction of these collisions would lead to the formation of products (the effective collisions) This is because only some of the reacting molecules have sufficient energy and the right orientation at the moment of impact to break the existing bond(s) and/or form the new one(s) Collision theory provide a simple framework for mathematical analysis of reaction rates and their dependence on reactants’ concentration, temp, etc - chemical kinetics 8  Collisions and kinetics Implications: dependence of reaction rates on Concentrations Molecular orientation Temperature Concentration dependence  To react the molecules must collide with each other, i.e. higher reaction rate at higher concentration of reactants Orientation dependence CH2=CH2 + HCl CH3CH2Cl Chloroethane Ethylene Would all of No these collisions work? The reaction occurs only Yes when the hydrogen of H-Cl attacks the double bond; HCl other collisions are ineffective HCl HCl The bottom line: molecules must collide in No the correct orientation to allow for specific re- arrangement of atoms And others… 9  Temperature dependence As temperature increases, the number of effective collisions increases – hence, the reaction rate should increase too Maxwell Boltzmann Distribution and activation energy And a familiar Activation diagram energy, Ea Good visualization The higher the temperature, the larger the proportion of The minimal amount of energy needed for the molecules that can jump molecule to react is called activation energy high enough to get over Quantitatively The effect of T on the reaction rate is described by the Arrhenius equation: Pre-exponential “frequency” Activation energy Ea is the factor that describes collision minimum (threshold) energy frequency and takes into necessary for a chemical account orientation reaction to occur No need to k = rate constant k=Axe –Ea/RT memorize A = frequency factor the equation R = 8.314J/mol K (the gas Graphical representation: but… constant) Ea = Jmol-1 Plot of lnk vs. 1/T gives a linear T = reaction temperature graph with the slope of -Ea/R in Kelvin -Ea 1 A rule of thumb: the rate increases lnk = + ln[A] R T 2-3 times for every 10oC rise in temp 10  First order reactions Let’s take a simple model reaction: A P Examples? and work out what happens with A and P over time As the reaction progresses, [A] decreases and [P] increases but how? Reaction rate equation At any given time the reaction rate is proportional to the number of reactant molecules A present, hence v = k [A], where k is the rate constant specific to this particular reaction and reaction conditions The decrease in the concentration of A over [At] = [A0] e-kt time can be expressed as: d[A] v=- = k [A] dt that can be rearranged as: d[A] - = k dt (1) [A] and integration of (1) gives us: The concentration of A decreases ln [At] = -kt + ln [A0] (2) exponentially as a function of time Note that a plot of –LnA vs time is a straight line with a slope of k 11  Half Life ln [At] = -kt + ln [A0] ln ([A0]/[At]) = kt “Half life” is the time for the concentration of a reactant to drop to 1/2 of its original value If At = ½ A0, then k x t1/2 =ln2 or t1/2 = ln2/k = 0.693/k i.e. it is constant Half-life for the 1st order reaction does not depend on the A0 Does it make sense? Pseudo 1st order reactions Pseudo-first order: concentration of one reactant remains essentially constant over time e.g. because it is present in large excess as compared to the other reagent Examples? Hydrolytic reactions A+B C+D but [B] practically (unless carried out at a -d[A] does not change VERY HIGH concentration v= k [A] [B] of substrate/reactant) are dt = often pseudo-first order -d[A] reactions because [H2O] v= k/ [A] where k/ =K[B] in H2O is ~55M! dt = and as before ln [A0] = ln [At] – k/t and t1/2 = ln2/k The treatment of these reactions is essentially the same as for the 1st order reaction. However, what you actually measure is an “apparent” value of k. To get to the real value it must divided by [B] 12  Second order reactions A+B P (or more products) -d[A] -d[B] Examples? v = k [A] [B] = = dt dt if x is the decrease in concentration of A and B in time t, then d[x] dt = k [A0-x] [B0-x] This equation can be solved analytically, but the math is horrendous  assuming for simplicity after integration this gives us: A0 = B0 , we get: 1 1 -d[A] = kt + [At] [A0] v= k [A]2 = dt Much more user-friendly  t1/2 for the 2nd order 1 1 and At = ½ A0, then the half if [At] = kt + [A0] life can be calculated as follows: And the visual Note the difference! 1 t1/2 = k[A0] The good news : We are not going to talk about the 3rd order! 13  Zero order reactions Zero order reactions are rare, but they exist e.g. light- and solubility-limited, and some electrode reactions Insoluble Dependence on surface area rather than concentration particle diffusion reaction A (fast) (fast) Slow dissolution This integrates to give [A] = -kt + [A0] Because the rate is Plotting [A] [A] independent of the See next versus t gives reactant [A] slide  a straight line -d[A] with slope -k v = dt = k How about t1/2? Time, t Summary at a glance Note that units for rate constants are different Required 14  Comparison 12 Let’s compare nd order Which Note the rate line is the acceleration for 1st order? the blue line! 0 order And this  How long does it take to complete? 0 order 2nd order Zero order: the rate is constant as the reaction progresses; linear decrease [A] First order: the rate is proportional to the concentration. As the reactant is consumed, the concentration drops and so does the rate of reaction Second order: the rate increases with the square of the concentration, producing an upward curving plot. Also, the rate of reaction decreases rapidly as the concentration of reactants decreases; note the shape of [A] vs t curve Reaction rate comparison Reaction Change in rate of reaction when order reactant concentration is doubled 0 No change; 20 1 Doubles; 21 2 Quadruples; 22 3 Increases eight times; 23 What would happen if the concentration of reactants is halved (decreased two times)? Thank you  Any questions? 15  Reversible reactions Let us now consider a reversible reaction K1 rate constant for k1 K-1 rate constant for the forward reaction A P the reverse reaction k-1 at equilibrium the forward and reverse reactions have the same rate hence, k1[A] = k-1[P] and k1/k-1 =[P]/[A] = K (eq constant) or for a more general case: aA + bB = cC + dD k1 [A]a [B]b = k-1 [C]c [D]d where k1 and k-1 are rate constants for the forward and reverse reactions or [C]c [D]d K is independent of the k1 K= = initial concentrations and K-1 [A]a [B]b the reaction pathway Enzyme kinetics The kinetics of most enzymatic reactions is described by Michaelis-Menten equation Vmax[S] v= Km + S Michaelis-Menten equation provides a relationship between the reaction rate and substrate concentration I assume you all know the meaning and derivation of the MM equation, but still a quick reminder Questions in this material will be included in quizzes and the final exam 16  Understanding the MM Consider an enzymatic reaction: k1 kcat E+S ES E+P where [S]>>[E] k-1 In a steady state the rate of formation of the ES complex and the rate of its decomposition (both way – towards P and S) are the same: What does this mean? k1 What does the Bath tub How quickly water level of water in comes in and out the tub depend on? ( i.e. the rate) Just like ES complex k-1+kcat S>>E V = k1 [E] [S] = k-1 [ES] + kcat [ES] (1) Formation of ES (water in) Decomposition of ES (water out) Derivation of the MM In addition to this: V = k1 [E] [S] = k-1 [ES] + kcat [ES] (1) We also need [E] + [ES] = [Eo], where Eo is total enzyme (2) The rest is trivial After numerous substitutions/ Km is different re-arrangements*, we arrive to from Ks!!! [Eo][S] *Full derivation will be added [ES] = (k-1+kcat)/k1+[S] to your handouts at the end Because (k-1 + kcat)/k1 = KM is a constant, called Michaelis- Menten constant or “KM”, we can write the following equation: [Eo][S] Because Vmax[S] [ES] = v= kcat x [Eo] Km + S Km + S = Vmax All the E d[P] kcat [Eo] [S] The rate: v= = kcat [ES] = is in ES - dt Km + S max rate 17  Partial solutions (again) What if Km>>S? What if S>>Km? Thanks  Vmax[S] Vmax[S] Vmax[S] then v= ~ v= Km+[S] ~ = Vmax Km+[S] [S] ~ Vmax[S] = K [S] Km app This is effectively a zero order reaction i.e. the reaction rate does NOT depend on the concentration of reactants (S in this case) this is a constant Effectively a first Why does the initial rate order reaction i.e. increase, when [S] increase? the reaction rate is proportional to the What can we learn from concentration of comparing enzymatic reactant’s (S) and chemical kinetics? A quick comparison How can the rate of chemical reaction be increased to catch up with the enzyme? Ramp up the temp (or increase [S])! Rate as a function of temp Oops… Enzymatic reaction (schematic) Same conditions Chemical Note: exponential! reaction Vchem