Chapter 6 and 7 EPF3111

CHAPTER 6
ENZYMATIC REACTION
45
• Technology Oriented • Business Driven • Sustainable Development • Environmental Friendly
Enzymatic reaction
• Enzyme classification
• Enzyme Kinetics
2
Enzyme classifications
• Enzyme Commission number; a system for classification
of enzymes that also serves as a basis for assigning code
numbers to them
• The first number shows to which of six main divisions
(classes) the enzyme belongs,
• The second indicates the subclass,
• The third gives the sub‐subclass,
• The fourth is the serial number of the enzyme in its
subclass
3
Enzyme classifications
Group ReacVon catalyzed Typical reacVon Enzyme
example(s) with
trival name
EC 1 To catalyze oxidation/reduction reactions; transfer of H AH + B ➔ A + BH (reduced) Dehydrogenas,
Oxidoreductases and O atoms or electrons from one substance to another A + O ➔AO (oxidized) oxidase
EC 2 Transfer of a functional group from one substance to AB + C ➔ A+ BC Transaminase,

Transferases another. The group may be methyl‐, acyl‐, amino‐ or kinase
phosphate group
EC 3 Formation of two products from a substrate by hydrolysis AB + H2O ➔ AOH + BH Lipase, amylase,
Hydrolases pepAdase
EC 4 Non‐hydrolytic addition or removal of groups from RCOCOOH ➔ RCOH + CO2 Decarboxylase
Lyases substrates. C‐C, C‐N, C‐O or C‐S bonds may be cleaved or [X‐A‐B‐Y] ➔ [A‐B + X‐Y]
EC 5 Intramolecule rearrangement, i.e. isomerization changes AB ➔ BA Isomerase,
Isomerases within a single molecule mutase
EC 6 Join together two molecules by synthesis of new C‐O, C‐ X + Y + ATP ➔ XY + ADP + Pi Synthetase
Ligases S, C‐N or C‐C bonds with simultaneous breakdown of ATP
4
Please refer: http://www.chem.qmul.ac.uk/iubmb/enzyme/rules.html
Oxidoreductase (subclasses)
Oxidoreductases are classified as EC 1 in the EC number classificaAon of enzymes. Oxidoreductases can be further classified into 22 subclasses:
• EC 1.1 includes oxidoreductases that act on the CH‐OH group of donors (alcohol oxidoreductases)
• EC 1.2 includes oxidoreductases that act on the aldehyde or oxo group of donors
• EC 1.3 includes oxidoreductases that act on the CH‐CH group of donors (CH‐CH oxidoreductases)
• EC 1.4 includes oxidoreductases that act on the CH‐NH2 group of donors (Amino acid oxidoreductases, Monoamine oxidase)
• EC 1.5 includes oxidoreductases that act on CH‐NH group of donors
• EC 1.6 includes oxidoreductases that act on NADH or NADPH
• EC 1.7 includes oxidoreductases that act on other nitrogenous compounds as donors
• EC 1.8 includes oxidoreductases that act on a sulfur group of donors
• EC 1.9 includes oxidoreductases that act on a heme group of donors
• EC 1.10 includes oxidoreductases that act on diphenols and related substances as donors
• EC 1.11 includes oxidoreductases that act on peroxide as an acceptor (peroxidases)
• EC 1.12 includes oxidoreductases that act on hydrogen as donors
• EC 1.13 includes oxidoreductases that act on single donors with incorporaAon of molecular oxygen (oxygenases)
• EC 1.14 includes oxidoreductases that act on paired donors with incorporaAon of molecular oxygen
• EC 1.15 includes oxidoreductases that act on superoxide radicals as acceptors
• EC 1.16 includes oxidoreductases that oxidize metal ions
• EC 1.17 includes oxidoreductases that act on CH or CH2 groups
• EC 1.18 includes oxidoreductases that act on iron‐sulfur proteins as donors EC
• 1.19 includes oxidoreductases that act on reduced flavodoxin as a donor EC
• 1.20 includes oxidoreductases that act on phosphorus or arsenic in donors EC
• 1.21 includes oxidoreductases that act on X‐H and Y‐H to form an X‐Y bond
• EC 1.97 includes other oxidoreductases 5
Transferase (subclasses)
Transferases are classified as EC 2 in the EC number classification. Transferases can be further
classified into nine subclasses:
• EC 2.1 includes enzymes that transfer one‐carbon groups (methyltransferase)
• EC 2.2 includes enzymes that transfer aldehyde or ketone groups
• EC 2.3 includes acyltransferases
• EC 2.4 includes glycosyltransferases
• EC 2.5 includes enzymes that transfer alkyl or aryl groups, other than methyl groups
• EC 2.6 includes enzymes that transfer nitrogenous groups (transaminase)
• EC 2.7 includes enzymes that transfer phosphorus‐containing groups (phosphotransferase,
including polymerase and kinase)
• EC 2.8 includes enzymes that transfer sulfur‐containing groups (sulfurtransferase and
sulfotransferase)
• EC 2.9 includes enzymes that transfer selenium‐containing groups
50
Hydrolase (subclasses)
Hydrolases are classified as EC 3 in the EC number classification of enzymes. Hydrolases can be
further classified into several subclasses, based upon the bonds they act upon:
• EC 3.1: ester bonds (esterases: nucleases, phosphodiesterases, lipase, phosphatase)
• EC 3.2: sugars (DNA glycosylases, glycoside hydrolase)
• EC 3.3: ether bonds
• EC 3.4: pepAde bonds (Proteases/pepAdases)
• EC 3.5: carbon‐nitrogen bonds, other than pepAde bonds
• EC 3.6 acid anhydrides (acid anhydride hydrolases, including helicases and GTPase)
• EC 3.7 carbon‐carbon bonds
• EC 3.8 halide bonds
• EC 3.9: phosphorus‐nitrogen bonds
• EC 3.10: sulfur‐nitrogen bonds
• EC 3.11: carbon‐phosphorus bonds
• EC 3.12: sulfur‐sulfur bonds
• EC 3.13: carbon‐sulfur bonds 51
Lyases (subclasses)
Lyases are classified as EC 4 in the EC number classificaAon of enzymes. Lyases can be
further classified into seven subclasses:
• EC 4.1 includes lyases that cleave carbon‐carbon bonds, such as decarboxylases (EC
4.1.1), aldehyde lyases (EC 4.1.2), oxo acid lyases(EC 4.1.3) and others (EC 4.1.99)
• EC 4.2 includes lyases that cleave carbon‐oxygen bonds, such as dehydratases
• EC 4.3 includes lyases that cleave carbon‐nitrogen bonds
• EC 4.4 includes lyases that cleave carbon‐sulfur bonds
• EC 4.5 includes lyases that cleave carbon‐halide bonds
• EC 4.6 includes lyases that cleave phosphorus‐oxygen bonds, such as adenylate
cyclase and guanylate cyclase
• EC 4.99 includes other lyases, such as ferrochelatase
8
Isomerase (subclasses)
Isomerases have their own EC classification of enzymes: EC 5. Isomerases can be further
classified into six subclasses:
• EC 5.1 includes enzymes that catalyze racemization (racemases) and epimerization
(epimerases)
• EC 5.2 includes enzymes that catalyze the isomerization of geometric isomers (cis‐
trans isomerases)
• EC 5.3 includes intramolecular oxidoreductases
• EC 5.4 includes intramolecular transferases (mutases)
• EC 5.5 includes intramolecular lyases
• EC 5.99 includes other isomerases (including topoisomerases)
9
Ligase (subclasses)
Ligases are classified as EC 6 in the EC number classification of enzymes. Ligases can
be further classified into six subclasses:
• EC 6.1 includes ligases used to form carbon‐oxygen bonds
• EC 6.2 includes ligases used to form carbon‐sulfur bonds
• EC 6.3 includes ligases used to form carbon‐nitrogen bonds (including
argininosuccinate synthetase)
• EC 6.4 includes ligases used to form carbon‐carbon bonds
• EC 6.5 includes ligases used to form phosphoric ester bonds
• EC 6.6 includes ligases used to form nitrogen‐metal bonds
10
Enzyme kinetics
• Enzymes function not only to catalyze reactions but also
to enable control of those reactions via sensitivity to
environmental conditions and to the presence of
cofactors, which can increase or decrease rates
• Benefits of enzyme kinetics; i) lead to clearer
understanding of the action of enzymes, ii) useful in
modeling of biochemical processes
• Mostly can be described as Michaelis‐Menten kinetics
11
Enzyme kinetics
• The simplest enzyme system with a single substrate, S and
single product, P, therefore the reaction is:
S+E→P+E
• Reaction rate, r, is the amount of product produced or
substrate consumed per unit time, it is expressed as:
d S  d P 
r =− =
dt dt
12
Enzyme kinetics
The rate of the reaction at
any particular substrate
concentration is
proportional to the total
concentration of enzyme if
the initial substrate
concentration is held
constant
Typical behavior of single
substrate‐enzyme reaction; (a)
constant S 57
Enzyme kinetics
If the total enzyme
concentration is held constant,
the rate of reaction shows a
saturation effect of substrate
concentration. At low
concentration of substrate, the
rate is proportional to
substrate concentration.
However as substrate
concentration becomes large,
Typical behavior of single the rate levels off, until it is
substrate‐enzyme reaction; (b) independent of concentration
constant E 58
Enzyme kinetics
• Enzymatic reaction mechanism:
⎯⎯ k1
S + E ⎯⎯ ES
→k −1
ES ⎯⎯→P + E k2
59
Enzyme kinetics
• Km is known as Michaelis constant:
Et S
ES = K
m + S
• The overall rate of reaction, r can be defined as r = k2 ES
• Therefore,
kE t S
r= k2 = k
K m + S
16
Enzyme kinetics
• Substrate activation; the effect of substrate concentration on reaction

rate shows a sigmoidal shape
• Substrate inhibition; adding substrate beyond an optimum amount
causes a reduction in the reaction rate 17
Enzyme kinetics
• Competitive inhibition; binding to the same active site as the substrate. It

does not change the maximum reacAon rate, but do change Km
• Noncompetitive inhibition; binding at a different site than the substrate and
changing the shape of the enzyme, which causes the substrate cannot
enter or leave the active site. The maximum rate is changed but not Km 18
Enzyme kinetics (effect of pH)

• Enzymes have a variety of acidic and basic group, each with its own
ionization constant
• Optimum activity of enzyme, it must exist in a particular ionization state
E+ ⎯⎯→
K1
⎯⎯→
K2
−
⎯⎯ E 
 ⎯⎯ E
• Fraction of the total enzyme
E  = 1
Et H 
+
+
K2
 
1+ +
K1 H 19
Enzyme kinetics (effect of pH)

• The optimum pH, can be found as: pK1 + pK2
pH optimum =
2
Effect of pH on enzyme
activity for various
ranges of pK
20
Enzyme kinetics (effect of temperature)

• Arrhenius equation would be all that is needed to describe the
temperature effect
• 2 ways effect; i. direct influence on the reaction rate constant, ii.
Thermal denaturation of the enzyme at elevated temperature
• Arrhenius equaAon;  − EA  R, k0, E A are the gas law constant,
 
k = k0e  RT  frequency factor and
activation energy, respectively
• Too low temperature; can be no noticable reaction rate since the

enzyme is operating at a temperature far below its optimum
• High temperature; thermal deactivation‐ limit their lifetime in
processing environments
21
Enzyme kinetics (effect of temperature)
Temperature influences the rate

of enzyme‐catalyzed reactions
22
CHAPTER 7
MICROBIAL ACTIVITIES
23
Microbial activity
• Microbial composition
• Microbial growth
• Substrate utilization
• Environmental factors
• Inhibition
70
Microbial composition
Elemental composition of E. coli (C5H7O2N)

• 70 – 90 %
water on a
mass basis
• Remaining dry
weight are 15
% ash/mineral
and 85 %
volatile/organic
material
25
Elemental composition (mass %) of some important microbial cell components
26
Roles of various elements within microorganisms
27
Roles of various elements within microorganisms (continue)
28
Microbial growth
• Some microorganisms can

reproduce asexually by budding,
and many eukaryotes have a
method of sexual reproduction
• Most microorganisms commonly
reproduce asexually by binary
fission; a single ‘parent’ cell
physically splits into two
genetically identical ‘daughter’
cells 29
Exponential growth
• The cells are able to grow freely without outside constraints

that growing by binary fission
• Generation time (tg); period between each successive split,
producing a new ‘generation’ of daughter cells
• Doubling time (td); the time takes by the cells that has been
growing exponentially (double to its number)
N = N 0  2 = N0e t /td t /t g
td = tg  ln(2) 30
Exponential growth
Theoretical microbial growth

schematic
t
X = X0e
 = 1/ tg
X is biomass concentration (g/L)
31
Exponential growth
The equation for a

straight line is given as:
ln X = t + ln X 0
The differentiatin
equation is given as:
dX
= X
dt  = ln2 /t d 32
Batch growth
• Batch system; all nutrients are present at the

beginning and are not resupplied
• Example, E. coli; it demonstrated exponential
growth cannot continue for very long, depletion of
substrates and/or buildup of inhibitory product
will soon lead to decreasing specific growth rates
33
Batch growth
Typical batch
bacterial
growth curve
80
Batch growth (lag phase)
• The cells take sometime for them to become

adapted to new environment (new nutrients, pH,
oxygen tension, and/or salt concentraAon)
• It may involve production of enzymes to
metabolize new organic substrates or utilize
nutrients in another form, or simply replacement
of cell constitutions that had been depleted
previously
81
Batch growth (exponential growth phase)
• Once the organism adapt to their new

environment, they will grow exponentially
• Plenty of substrate and nutrients are available, and
no harmful products metabolism have yet
accumulated
• Specific growth rate, μ will approach the maximum
specific growth rate, μmax in the particular
environment
36
Batch growth (declining rate of growth phase)
• One of more factors (substrate depletion, product

accumulation, pH shil, nutrient deficiency and/or
something else) trigger an inevitable decline in cell
growth rate
• μ > 0, but decreasing
37
• Most of μ decreasing is the result of substrate

depletion
• Modeled by Monod kinetics
S
 = max
S + KS
38
S
(S  KS ), S + K  1,   max
S
S S max
Half-saturation coefficient
(S  K S ), S + K  K ,   K S
S S S
1
(S = KS ),  = 2 max
39
Batch growth (stationary phase)
• Once the substrate is sufficiently depleted, growth

nearly stops (μ≈0)
• The cells may still be growing slowly, but this is
counterbalanced by the loss in mass through
decay (next phase)
40
Batch growth (decay phase)
• Cell mass decreases (decay), if substrate is

unavailable
• Cell continues to carry on some metabolic process
without external sources of energy (endogenous
metabolism), it must utilize internal ones.
• Net specific growth rate (μn) actually represent a
higher true growth rate combine with decay rate (b)
n =  − b
41
Batch growth (decay phase)
Examples of
Monod Kinetic
Coefficients
42
Overall Monod equation
dX
= n X
dt
= ( − b)X
 S 
= max − b X
 S + KS 
43
Substrate utilization
• Microorganism may utilize substrates as a source of

cellular constituents, to supply energy, and/or as an
electron acceptor.
• The rate of substrate utilization olen is considered
to be proportional to the rate of growth
dS
 −X
dt
90
Substrate utilization
• Yield coefficient , Y, is ratio between given substrate

utilized and amount of biomass transformed
dS 
• =− X
dt Y
• Using Monod kinetics gives:
dS max S
=− X
dt Y S + KS
45
Multiple substrates
• Microorganism need many substrates: energy

source; electron acceptor; sources of carbon,
nitrogen, phosphorus
• Empirical approach used to describe this problem is
an interactive, multiplicative Monod model
(example substrate A and B)
SA SB
 = max
SA + K A SB + K B
46
Environmental factors
• Microbial growth can

clearly affected by other
environmental factors;
temperature, pH,
pressure, moisture,
oxygen level and salinity
• The most important
factors are temperature Dependence of mesophilic microbial
and pH growth on temperature
47
Inhibitions
• The presence of toxic agent; slow down the

reactions or to cause damage to the cell
• Inhibitor can be i) substrate used for growth, if it
at high concentration, ii) product of cell
metabolism and/or iii) another substrate or
external factor
48

Chapter 6 and 7 EPF3111

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Chapter 6 and 7 EPF3111

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CHAPTER 6

EC 2 Transfer of a functional group from one substance to AB + C ➔ A+ BC Transaminase,

• Enzymatic reaction mechanism:

• Substrate activation; the eﬀect of substrate concentration on reaction

• Competitive inhibition; binding to the same active site as the substrate. It

Enzyme kinetics (effect of pH)

Enzyme kinetics (effect of pH)

Enzyme kinetics (effect of temperature)

• Too low temperature; can be no noticable reaction rate since the

Enzyme kinetics (effect of temperature)

Temperature inﬂuences the rate

Elemental composition of E. coli (C5H7O2N)

Elemental composition (mass %) of some important microbial cell components

• Some microorganisms can

• The cells are able to grow freely without outside constraints

Theoretical microbial growth

The equation for a

• Batch system; all nutrients are present at the

Batch growth (lag phase)

• The cells take sometime for them to become

Batch growth (exponential growth phase)

• Once the organism adapt to their new

Batch growth (declining rate of growth phase)

• One of more factors (substrate depletion, product

Batch growth (declining rate of growth phase)

• Most of μ decreasing is the result of substrate

Batch growth (declining rate of growth phase)

Batch growth (stationary phase)

• Once the substrate is suﬃciently depleted, growth

Batch growth (decay phase)

• Cell mass decreases (decay), if substrate is

Batch growth (decay phase)

Overall Monod equation

• Microorganism may utilize substrates as a source of

• Yield coeﬃcient , Y, is ratio between given substrate

• Microorganism need many substrates: energy

• Microbial growth can

• The presence of toxic agent; slow down the

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