Process Kinetics Lecture 1 Mahesh Bule 4/27/2017
Template D Plain-white-dark Introduction to Processes in Biofuel
4/27/2017 Introduction to Processes in Biofuel Heterotrophic and phototrophic pathway forbiofuel production Template D Plain-white-dark Important Process and Its Kinetics
Enzymatic hydrolysis Fermentation of soluble sugar Downstream e.g. extraction, purification etc. Enzyme Kinetics Enzymes are the agents of saccharification process
What we want to be able to determine: Maximum velocity Substrate affinity Inhibitor affinity What it can tell us: Utilization of substrates What can we do with the information: Control and manipulate process Enzyme Kinetics Basics
Enzyme kinetics studies the reaction rates ofenzyme-catalyzed reactions and how therates are affected by changes inexperimental conditions An essential feature of enzyme-catalyzedreactions is saturation: at increasingconcentrations of substrates the rateincreases and approaches a limit wherethere is no dependence of rate onconcentration Consideration of Enzyme Kinetics
Conformation of proteins and positions of side chains areimportant for enzyme-substrateinteractions and catalysis. Forces involved in protein folding and structure are also involvedin catalysis- enzyme-substrate specificity To use enzymes in biotechnology NEED TO KNOW KINETICPARAMETERS OF THE ENZYME REACTION. We may want enzymes that WORK FAST- convert more substratein a fixed unit of time. To do this optimization we have toperform and analyze the enzyme catalyzed reaction. You can adjust pH, temperature and add co-factors to optimizeenzyme activity. You cannot adjust substrate selectivity. Just like chemical reactions, enzyme catalyzed reactions havekinetics and rates Reaction kinetics is Michaelis-Menten kinetics. Important things to study
Michaelis-Menten kinetics Interpretations and uses of the Michaelis- Menten equation Enzyme inhibitors: types and kinetics Enzyme-substrate cycle Enzyme Kinetics Equation Michaelis-Menten Equation Initial Velocity (vo) and [S]
The concentration of substrate [S] present will greatly influence the rate of product formation, termed the velocity (v) of a reaction.Studying the effects of [S] on the velocity of a reaction is complicated by the reversibility of enzyme reactions, e.g. conversion of product back to substrate. To overcome this problem, the use of initial velocity (vo) measurements are used.At the start of a reaction, [S] is in large excess of [P], thus the initial velocity of the reaction will be dependent on substrate concentration Michaelis-Menten Curve Substrate Saturation of an Enzyme
A. Low [S]B. 50% [S] or Km C. High, saturating [S] Steady State Assumption
The M-M equation was derived in part by making several assumptions.An important one was:the concentration of substrate must be much greater than the enzyme concentration. In the situation where [S] >> [E] and at initial velocity rates, it is assumed that the changes in the concentration of the intermediate ES complex are very small over time (vo).This condition is termed a steady-state rate, and is referred to as steady-state kinetics.Therefore, it follows that the rate of ES formation will be equal to the rate ES breakdown. Michaelis-Menten Equation Derivation
Rate of ES formation = k1([ET] - [ES])[S] (where [ET] is total concentration of enzyme E and k-2 is considered neglible) Rate of ES breakdown to product = k-1[ES] + k2[ES] Michaelis-Menten Equation Derivation (cont)
Thus for the steady state assumption: k1([ET] - [ES])[S] = k-1[ES] + k2[ES] This equation is the basis for the final Michaelis-Menten following algebraic rearrangement and substitution of Km and Vmax terms. [S] V0 = Vmax [S]+Km k-1 + k2 Km= k1 When V0=Vmax, Km= [S]
Vmax and Km [S] V0 = Vmax [S]+Km k-1 + k2 Km= k1 When V0=Vmax,Km= [S] Km is unique to each Enzyme and Substrate.It describes properties of enzyme-substrate interactions Independent of enzyme conc. Dependent on temp, pH etc. Vmax is maximal velocity POSSIBLE. It is directly dependent on enzyme conc. It is attained when all of the enzyme binds the substrate. (Since these are equilibrium reactions enzymes tend towards Vmax at high substrate conc but Vmax is never achieved. So it is difficult to measure). When an enzyme is operating at Vmax, all enzyme is bound to substrate and adding more substrate will not change rate of reaction (enzyme is saturated). (adding more enzyme will change the reaction). Important Conclusions of Michaels - Menten Kinetics
when [S]= KM, the equation reduces to when [S] >> KM, the equation reduces to when [S]