GROUP 7

De Castro, Nica Mariel

Lopez, Aubrenica Rose Pauline

Umali, Jonalyn

Simple Enzyme Kinetics

 Enzyme Inhibition

• Inhibitors
- chemicals that reduce the rate of enzymatic reactions
- block the enzyme but they do not usually destroy it
- usually specific and work at low concentrations

Two Classes of Inhibitors in the Extent of Interaction

1. Irreversible Inhibitors – combine with the functional groups of the amino
acids in the active site, irreversibly. Forms weak, non-covalent bond that
readily dissociate from an enzyme.
Examples: nerve gases, pesticides
2. Reversible Inhibitors - The site of attack is an amino acid group that
participates in the normal enzymatic actions in the extent of interaction. Forms
covalent or very strong non covalent bonds.
Types
(a) Competitive inhibitor - These compete with the substrate
molecules for the active site. The inhibitor’s action is proportional
to its concentration.
(b) Non- competitive inhibitor - Not influenced by the concentration
of the substrate. Inhibits by binding irreversibly to the enzyme but
not at the active site
Examples: Heavy metals, Ag or Hg, combine with –SH
groups.

Competitive Non- competitive

Inhibitor does not
Inhibitor competes
bind at the active
with the substrate at
site
the active site
Active site may
Active site stays the change shape
same (e.g. allosteric
inhibition)

 Models for more Complex Enzyme Kinetics – involve multiple binding sites
 Allosteric enzymes kinetics
- generally don’t obey Michaelis-Menten kinetics.
- the binding of one substrate to the enzyme facilitates binding of other
substrate molecules.
 Allosteric enzymes – a class of enzymes that bind small, physiologically
important molecules and modulate activity in ways
Kinds of Allosteric Enzymes
1. Positive – activates enzymes
2. Negative – deactivates enzymes
 Enzyme activation
Enzyme Activators
- compounds that increase enzymatic activity.
- are usually involved in allosteric enzymes for metabolism
regulation
- Example: fructose 2,6-biphosphate, which activates
phosphofructokinase and increases the metabolism rate in
response to the hormone glucagon.

 Factors affecting Enzyme Activity

 Temperature
As the temperature increases, the rate of reaction of enzyme also
increases. But very high temperatures denature enzymes.

 pH
Different enzymes work best at different pH values. The optimum pH for
an enzyme depends on where it normally works.
  Other factors:
a) Metal/ Salt Concentration – each enzyme has an optimal salt
concentration.
b) Concentration of the Substrate – As the concentration increases, the
enzyme reaction rate increases.
c) Concentration of Enzyme – increasing enzyme concentration will
increase the enzyme reaction rate.
d) Steric Hindrance – because of the “separation” or spacing, the
substrate is very difficult to bond with the enzyme in the active site.

Enzyme Immobilization

- restricts the mobility of an enzyme or protein and fixes the enzyme into a state without disturbing
its functional ability
- can reduce the sensitivity of a native enzyme hence increasing the functional efficiency of the
enzyme
- “Amino Cyclase” from Aspergillus oryzae in Japan is the first immobilized enzyme

 Methods of Immobilization
A. Adsorption
- Enzyme is adsorbed on the physical outer surface of the support. It can affect the
functional ability of enzyme by blocking its active site.
Carriers used in adsorption can be
(a) Mineral-based support - aluminum oxide, alginate beads
(b) Organic Bimolecular based support – starch, cellulose
(c) Modified ion exchange resin – sepharose
The absorptive immobilization of enzymes can be done by
1. Static Method - Enzyme is immobilized by allowing it to be in contact with
the carrier without agitation. This is most efficient technique but requires
maximum time.
2. Dynamic Method - This process typically involves the admixing of enzyme
with the carrier under constant agitation using mechanical shaker.
3. Reactor loading Method - The carrier is placed into the reactor and enzyme
solution is transferred to the reactor with agitation of the whole content in the
reactor. This process is employed for the commercial production of
immobilized enzymes.
4. Electro-deposition Method - In this technique, carrier is placed in the vicinity
of an electrode and the enzymes migrate to carrier in presence of electric
current.

B. Covalent Bonding
- The method utilizes chemical groups present on both enzyme and carrier for
immobilization.
- Example of chemical groups of carriers and enzymes used in bond formation
 Carrier: Carboxyl Group | Enzyme: Phenol ring of tyrosine

Carriers used in covalent bonding can be

(a) Biomolecules - carbohydrates like cellulose
(b) Synthetic molecules – polyacrylamide
(c) Protein carriers – collagen, gelatin
(d) Inorganic molecules – porous glass, silica
 Covalent bonding can be done by
1. Diazoation – the reaction occurs between amino group of the carrier and
Tyrosil and Histidyl group of the enzyme.
2. Peptide Bond – occurs in amino and carboxyl groups of enzyme and carrier.
3. Polyfunctional agent – a multi-functional agent like Gluteraldehyde is used
to form bond between amino group of enzyme and carrier.

C. Entrapment
- the enzymes or cells trapped inside the polymer matrix. Entrapment is carried out
by mixing the biocatalyst into a monomer solution, followed by polymerization
initiated by a chemical reaction.
- Matrices used in this method are polyacrylamide, collagen, agar, gelatin, alginate
and carrageenan.

Methods of entrapment are

1. Inclusion in the Gel – enzyme is trapped inside the gel, which is formed by
the polymer.
2. Inclusions in fibers – enzymes are supported on the fibers (skeleton of the
matrix in which the enzyme is trapped) of the supporting material forming
the matrix.
3. Microcapsules – enzymes are trapped in the microcapsules. Most common
microcapsules are polyamines and sodium alginate.

D. Cross-linking process / Copolymerization

- This method is based on the formation of covalent bonds between the enzyme
molecules, by means of multifunctional reagents, leading to three dimensional
cross linked aggregates.
- The most common reagents used for cross-linking are gluteraldehyde and
diazonium salts.

E. Encapsulation
- An enzyme is encapsulated within a capsule made up of semi-permeable membrane
like nitrocellulose, nylon and hemi-cellulosic structures.
- The effectiveness depends on the stability of the enzyme inside the capsule.

 Effect of Mass Transfer Resistance

- Immobilization of an enzyme transforms a homogeneous (soluble) catalyst into a
heterogeneous (insoluble) system.
- Carrier binding techniques introduce external mass transfer effects between the liquid
phase and the solid surface.
- An enzyme immobilized through binding to a carrier bead and placed in a simple flow
may be represented by the following illustration.

- The change in concentration of a reagent A from [A]bulk to [A]surface takes place in a

narrow fluid layer next to the surface of the sphere.
- In all but the simplest cases, we express the mass transfer rate as:
N A  kc Ap ([ A]s  [ A])
where NA = transfer rate: mole/s
kc = convective mass transfer coefficient: m/s
 AP = surface area of the particle: m2
[A] = concentration of solute at the surface and in the bulk,
respectively: mole/m3
 Electrostatic and Steric Effects
When enzymes are immobilized in a charged matrix as a result of a change in the
microenvironment of the enzyme, the apparent bulk pH optimum of the immobilized
enzyme will shift from that of soluble enzyme. The charged matrix will repel or attract
substrates depending on the type and quantity of surface charge.

For an enzyme immobilized onto a charged support, the shift in the pH-activity profile is
given by

pHi= Internal pH value

pHe= External pH value
Z= charge (valence) on the substrate
Nf= 96 500 coulumb/ eq.g (Faraday Constant )
Ψ = Electrostatic Potential
R= gas constant

The activity of an enzyme toward a high-molecular-weight substrate is usually

reduced upon immobilization to a much greater extent than for a low-molecular-
weight substrate. This is mainly because of steric hindrance by the support.

Immobilization also affects the thermal stability of enzymes. Thermal stability

often increases upon immobilization due to the presence of thermal diffusion
barriers and the constraints on protein unfolding.

 Industrial Application of Enzymes

Enzymes are used in the chemical industry and other industrial applications when extremely
specific catalysts are required. However, enzymes in general are limited in the number of reactions
they have evolved to catalyze and also by theur lack of stability in organic solvents and at high
temperatures. As a consequence, protein engineering is an active area of reasearch and involves
attempts to create new enzymes with novel properties, either through rational design or into vitro
revolution. These efforts have begun to be successful, and a few enzymes have now been designed
“from scratch” to catalyze reactions that do not occur in nature.

1. Industrial Production – commercial production of antibiotics, beverages, amino acids

and secondary metabolites of industrial grade
2. Biomedical Applications – commonly used in the fast diagnostic kits like ELISA and
treatment of many pathogenic diseases
3. Food Industry
o Pectinases and Cellulases – used in the production of jams, jellies and
fruit and vegetable syrups
o Lactase immobilized with cellulose fibers – produces lactose-free milk
o Amylases from fungi and plants - production of sugars from starch. Such
as in making high- fructose corn syrup. In baking, catalyzebreakdown of
starch in the flour to sugar. Yeast fermentation of sugar priduces the
carbon dioxide that raises the dough.
 o Lipases - is implemented during the production of Roquefort cheese to
enhance the ripening of the bl;ue- mold cheese.
o Papain - to soften meat for cooking
4. Biodiesel Production – from vegetable oils
5. Waste water Management – treating sewage and industrial effluents using packed bed
reactors
6. Starch Industry
 Amylases, amyloglucosideases and glucoamylases - converts starch into
glucose and various syrups.
7. Brewing Industry
 Enzymes frm barley are released during the mashing stage of beer production. They
degrade starch and proteins to produce simple sugar, amino acids and pepticides that are
used by yeast for fermetatation.

8. Paper Industry
 Amylases, Xylanases, cellulases and ligninases - degrade starch to lower
viscosity, aiding, sizing and coating paper. Xylanases reduce bleach required for
decolorizing; cellulases smooth fibers, enhance water drainage and promote ink
removal, lipases reduce pitch and lignin- degrading enzymes remove lignin to soften
paper.

9. Biofuel Industry
 Cellulases - used to breakdown cellulose into sugars that can be fermented

10. Biological Detergent

 Primarily proteases - produced in an extracellular from bacteria and used for
presoak conditions and direct liquid applications helping with removal of
protein stains from clothes.
11. Rubber Industry
 Catalase - to generate oxygen from peroxide to convert latex into foam
rubber
12. Photographic Industry
 Dissolve gelatin oil scrap film, allowing recovery of its silver content.