Production Kinetics of Alkaline Protease Production

Dr. Apurba Dey Professor Department of Biotechnology, National Institute of Technology, Durgapur.

ENZYMES
Enzymes are Biocatalyst that increase the rates of chemical
reactions and they are protein in nature .
Properties of Enzymes
Have enormous catalytic power Highly specific Activities of some enzymes are regulated Transform different kinds of energy Do not alter reaction equilibria Decrease the activation energy of reactions catalysed by them

Class 1. Oxidoreductases catalyze redox Oxidoreductasesprocesses Example: RCH2-OH p RCH=O

Class 2. Transferases transfer chemical groups Transferasesfrom one molecule to another or to another part of the same molecule. O O Example: CH3-C-SCoA + XR p CH3-C-XR + HSCoA acetyl CoA acetyl group transferred

Class 3. Hydrolases cleave a bond using water Hydrolasesto produce two molecules from one. O H2O O example: --CNH-R p --C-OH + H2N-R cleavage of a peptide bond Class 4. Lyases remove a group from or add a Lyasesgroup to double bonds. H-X ---C=C--- p H X ---C--C---

Class 5. Isomerases interconvert isomeric Isomerasesstructures by molecular rearrangements. CH3 CH3 HC-OH HO-CH COOH COOH Class 6. Ligases -- join two separate molecules by the formation of a new chemical bond usually with energy supplied by the cleavage of an ATP. example: O ATP ADP+Pi O -OOC-C-CH -COO-OOC-C-CH + CO 2 3 2 pyruvate oxaloacetate enzyme = pyruvate carboxylase

Enzymes from Microbial sources

Enzymes Sources Applications

Enzymes from Animal and Plat sources

Enzymes Sources Applications

Enzyme kinetics
Study of the rates of enzyme-catalyzed reactions Provides information on enzyme specificities and mechanisms
Why study enzyme kinetics? a) the precise scheduling of reactions in a cell is important to the cell and our understanding of its workings b) enzyme mechanisms, e.g., the number of kinetic steps and the detailed chemistry can be learned (enzymology). c) understanding enzyme function leads to better drugs.

Steps for a simple enzyme-catalyzed reaction

k2 ES E  P p E  S n p k1 k1

E = free enzyme S = substrate ES = enzyme-substrate complex P = product

MODELS FOR ENZYME SUBSTRATE COMPLEX

LOCK and KEY MODEL
Active site of enzyme by itself is complementary in shape to that of the substrate.

INDUCED FIT MODEL

The enzyme changes shape upon binding substrate. The Active site has a shape complementary to that of the substrate only after the substrate is bound.

Enzyme-catalyzed reactions exhibit saturation kinetics

k2 ES E  P p E  S n p k1

k1

At high [S], the enzyme is said to be saturated with respect to substrate

Steady State
The more ES present, the faster ES will dissociate into E + P or E + S. Therefore, when the reaction is started by mixing enzymes and substrates, the [ES] builds up at first, but quickly reaches a STEADY STATE, in which [ES] remains constant. This steady state will persist until almost all of the substrate has been consumed.

MichaelisMichaelis-Menten Equation Vmax[S] V= [S] + Km

Measuring Km and Vmax

Curve-fitting algorithms can be used to
determine K m and V max

from v vs. [S] plots Michaelis-Menton equation can be rearranged to the "double reciprocal" plot and K m and V max can graphically determined be

Km
Km is the [S] at 1/2 Vmax Km is a constant for a given enzyme Km is an estimate of the equilibrium constant for S binding to E Small Km means tight binding; High Km means weak binding

Vmax
The theoretical maximal velocity

Vmax is a constant for a given enzyme Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved To reach Vmax would require that ALL enzyme molecules have tightly bound substrate

Enzyme Activity
Amount of reaction that a certain amount of enzyme will produce in a specified period of time Activity determined by measuring the amount of product formed or substrate that disappeared
IU of enzyme activity is

The amount of enzyme necessary to produce 1 mole of product (or the loss of 1 mol of substrate) per minute under specified conditions of substrate concentration, pH and Temperature

The Turnover Number

A measure of catalytic activity

The kcat is a direct measure of the catalytic production of product under saturating substrate conditions. kcat, the turnover number, is the maximum number of substrate molecules converted to product per enzyme molecule per unit of time. According to M-M model, kcat = Vmax/Et Values of kcat range from less than 1/sec to many millions per sec

Enzyme Inhibition Many different kinds of molecules inhibit enzymes and act in a variety of ways. One major distinction is whether the inhibition is
1. Competitive
k1 k2 ES E  P p E  S np k 1

 I c KI EI
Competitive

Non- Competitive

Non-competitive

Effect of inhibitors on kinetic parameters for enzyme activity

Antipain (Serine Protease Competitive Inhibitor) Kinetic parameter 0.025mM 0.05mM 0.1mM

Aprotinine (Serine Protease Competitive Inhibitor) 0.025mM 0.05mM 0.1mM

Maximum enzyme activity (vm) 286.9 Saturation constant (KIamp) 0.006953 Inhibition factor (YI) 0.01348 0.02703 0.006073 0.01144 0.02288 286.6 286.2 285.3 285.3 285.4

14.81

28.71

57.56

12.93

24.36

48.73

NB. When no inhibitor then Vmax is 286.9 and Km is 0.0047mM

Process Kinetics
Cell Growth Kinetics Substrate Utilization Kinetics Production Kinetics

PROTEASES
Proteases are enzyme that hydrolytically cleave the peptide pond of proteins. For enzyme digestive enzyme and Blood clotting enzymes.

Classification of proteases

Serine proteases Cysteine proteases Aspartate proteases Metallo proteases

Application of Proteases

1.In beverage industry for stabilizing Beer 2.In cheese Industry for coagulation of casein and cheese ripening 3.In leather Industry de hearing of hides and softening the lathers 4.In food industry as meat tenderizer 5.As ingredient in detergent industry for removing the stain 6.For cleaning contact lenses

Cell Growth Kinetics

A. Monod Model
dX ! QX dt
Gabriel Monod (1844 - 1912)
0.08 0.06 0.04 0.02 0.00 0 10 20 30 40 50

X (t ) ! X 0 e

Qt

Specific growth rate (h

Qmax S Q! Ks  S

-1

Starch Concentration (gl -1)

Table 2. Substrate inhibition kinetic models used in this study.

Names of Model Andrew Aiba Competitive substrate inhibition model Non-competitive substrate inhibition model Edward
Q!

Substrate Inhibition model

Q !
s

R2 value 0.9908

Q m ax S  S  IS2
i

Qmax S  S Q! .e Ks  S
Q! Qmax S S K s (1  )  S Ki

0.9906 0.9691

Q!

Q max S K S (1  S )(1  ) S Ki

0.9691

Qmax S 2 S  K S  (S )(1  S ) Ki KS

0.9177

Andrews Model
Specific growth rate (h
-1

0.08 0.06 0.04 0.02 0.00 0 10 20 30 40 50

Q m ax S Q ! Ks  S  KIS2
max = 0.109 h-1 KS = 11.1 gl-1 Ki =0.012 l/g R2 =0.9908
Starch Concentration (gl -1)

Where KI is the inhibition constant in Andrew s model

Substrate Utilization Kinetics

Where YX/S and YP/S are yields of cell mass and product with respect to substrate and m is the maintenance coefficient for cells.

dS 1 dX 1 dP !   mX dt YX dt YP dt
S S

dS 1 !  dt YX
S

dX  mX dt

Assumption: Amount of carbon substrate used for the product formation is assumed to be negligible.

Maintenance Calculation

dS 1 !  dt YX
S

dX  mX dt

Assumption: At the stationary phase where dX/dt is zero and X is Xm. Therefore, m can be obtained using the following equation:

m= 0.0035

h-1

m!

[(dS

dt

)]st

Xm

YX/S Calculation

dS 1 !  dt YX
S

dX  mX dt

for
Monod Model Andrews Model YX/S = 1.003 m= 0.0035 h-1

starch used for cell growth was computed after deduction of starch used for maintenance of the cell from the experimental residual starch.

YX / S

dX ! dS

YX / S

X  Xo ! So  S

Production kinetics
Alkaline protease production kinetics was done using Leudeking-Piret model (Luedeking & Piret, 2000). According to this model, the product formation rate depends on both the instantaneous biomass concentration, X and growth rate, in a linear manner.

d dX !E FX dt dt
Where and are the product formation constants, which may vary with fermentation conditions. Dividing both sides by X, we get the following equation
is specific production rate is specific growth rate

1 d 1 dX !E F X dt X dt

R ! EQ  F

Note: Regression analysis was used for best fit of straight line on plot of and for finding out the parameters , .

..

Fig. Plot of specific alkaline protease production rate vs specific growth Rate using Leudeking-Piret model.

References
A. Anwar, M. Saleemuddin, Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations,Biotechnol. Appl. Biochem, 31 (2000) 85-89. J.F. Andrews, A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates, Biotechnol Bioeng, 10 (1968) 702-723. R. Luedeking, E.L. Piret, A kinetic study of the lactic acid fermentation. Batch process at controlled pH, Biotechnol Bioeng, 67 (2000) 636-644. M.L. Shuler, F. Kargi, Bioprocess Engineering: Basic Concepts, Practice Hall of India Private Limited, New Delhi, 2008 S.D. Yuwono, T. Kokugan, Study of the effects of temperature and pH on lactic acid production from fresh cassava roots in tofu liquid waste by Streptococcus bovis, Biochem Eng J, 40 (2008) 175183. M. Phisalaphong, N. Srirattana, W. Tanthapanichakoon, Mathematical modeling to investigate temperature effect on kinetic parameters of ethanol fermentation, Biochem Eng J, 28 (2006) 3643.

The End