Cell Nutrients

Nutrients
A nutrient is a chemical substance that comes from the food you eat. The energy you need for the
metabolic processes in your body and for maintaining a constant internal environment comes from these
nutrients.

Essential Nutrients
Essential nutrients refer to classes of nutrients found in food. Essential nutrients are simply those that are
vital for the normal growth, maintenance and development of the body.

Types of Nutrient
 Macronutrients
 Macronutrients provide the bulk energy an organism's metabolic system needs to function while
micronutrients provide the necessary cofactors for metabolism to be carried out.
 The chemical elements humans consume in the largest quantities are carbon, hydrogen, nitrogen,
oxygen, phosphorus, and sulfur, or CHONPS.
 The classes of chemical compounds humans consume in the largest quantities and which provide
bulk energy are carbohydrates, proteins, and fats. Water and atmospheric oxygen also must be
consumed in large quantities, but are not always considered "food" or "nutrients".
 Calcium and salt (sodium and chloride), magnesium, and potassium (along with phosphorus and
sulfur) are sometimes added to the list of macronutrients because they are required in large
quantities compared to other vitamins and minerals. They are sometimes referred to as the
macrominerals

Substances that provide Macronutrients: energy for the body

 Carbohydrates are sugar compounds that can be simple or complex. Complex carbohydrates can
be found as starch in cereals, pasta and potatoes, but also in fruit and vegetables. Simpler sugar
compounds reach the body, for instance, with sweet dishes and beverages. Glucose is the
simplest sugar compound. Because it does not need to be decomposed by the digestive system, it
is available to the body immediately via the blood circulation. 55 – 60 % of our energy intake
should originate from complex carbohydrates.
 Protein is a basic module of our cells. Via our nutrition, proteins supply our body with important
amino-acids. As a source of energy they are useful to it in emergencies, for example during a
starvation diet with no physical exercise, by decomposing albuminous muscle tissue.
Albuminous (rich in protein) foods are eggs, meat, fish and dairy products, but also pulses, nuts
and cereals.

 Proteins are organic compounds that consist of amino acids joined by peptide bonds. The body
cannot manufacture some of the amino acids (termed essential amino acids); the diet must supply
them. Proteins, in nutrition, are broken down through digestion by proteases back into free amino
acids.

 Fats has a very high energy content. Fats consist of a glycerin molecule with three fatty acids
attached. Fatty acids are unbranched hydrocarbon chains, connected by single bonds alone
(saturated fatty acids) or by both double and single bonds (unsaturated fatty acids). Fats are
needed to keep cell membranes functioning properly, to insulate body organs against shock, to
keep body temperature stable, and to maintain healthy skin and hair. The body does not
manufacture certain fatty acids (termed essential fatty acids) and the diet must supply them.
*Although alcohol provides energy, and can thus be compared to macronutrients, it is not a substance that is
essential for normal function. The acetic acid in vinegar also provides a similar amount of energy per gram, but
again, it is not a nutrient because it is not essential for normal function.
Substances that support metabolism
 Dietary minerals are generally trace elements, salts, or ions such as copper and iron. Some of these
minerals are essential to human metabolism.
  Vitamins are organic compounds essential to the body. They usually act as co-enzymes or cofactors for
various proteins in the body.
 Water is an essential nutrient and is the solvent in which all the chemical reactions of life take place.
Plants absorb nutrients from the soil or the atmosphere, or from water (mainly aquatic plants). The
chemical elements consumed in the greatest quantities by plants are carbon, hydrogen and oxygen. These
are present in the environment in the form of water and carbon dioxide; energy is provided by sunlight.
Nitrogen, phosphorus, and sulfur are also needed in relatively large quantities. Together, the “Big Six” are
the elemental macronutrients for all organisms, often represented by the acronym CHONPS. Usually they
are sourced from inorganic or organic compounds, although elemental diatomic molecules of nitrogen and
oxygen are often used.

Micronutrients
Micronutrients are essential elements required by organisms in small quantities throughout life to
orchestrate a range of physiological functions to maintain health. These nutrients include minerals and vitamins.
Unlike macronutrients, these are required in very minute amounts. Together, they are extremely important for
the normal functioning of the body. Their main function is to enable the many chemical reactions to occur in the
body. Nevertheless micronutrients do not function for the provision of energy. Deficiencies in micronutrients
such as iron, iodine, vitamin A, folate and zinc can have devastating consequences. At least half of children
worldwide ages 6 months to 5 years suffer from one or more micronutrient deficiency, and globally more than 2
billion people are affected.
 Vitamins
 are essential for normal metabolism, growth and development, and regulation of cell
function
 work together with enzymes and other substances that are necessary for a healthy life
 either fat-soluble or water-soluble
 fat-soluble vitamins can be stored in the fatty tissues in the body when in excess, and so
are not excreted easily
 water-soluble vitamins are excreted in urine when in excess and so need to be taken daily
 fat-soluble vitamins are vitamin A, D, E and K
 water-soluble vitamins are vitamin B and C
 Minerals
 are found in ionized form in the body
 classified into macrominerals and microminerals (or trace minerals)
 macrominerals present in the body include Calcium, Potassium, Iron, Sodium and
Magnesium to name a few
 macrominerals constitute a larger percent of the body and are needed in more amounts
 microminerals include Copper, Zinc, Cobalt, Chromium and Fluoride
 microminerals are mostly co-factors, and are necessary for the function of enzymes in the
body, but are needed only in minor quantities
 approximately 4% of the body’s mass consists of minerals
 Iron
 Iron is an essential mineral critical for motor and cognitive development
 Low hemoglobin concentration (anemia) affects 43% of children 5 years of age and 38%
of pregnant women globally
 Anemia during pregnancy increases the risk of maternal and perinatal mortality and low
birth weight
 Flour fortification with iron and folic acid is globally recognized as one of the most
effective and low-cost micronutrient interventions
 Iodine
 Iodine is one of the most important minerals required by a fetus for brain and cognitive
development
 18 million babies are born mentally impaired because of maternal iodine deficiency and
38 million are born at risk of iodine deficiency
 Fortification of salt with iodine has been one of the most successful nutrition
interventions to date–71% of global households have access to iodized salt
 Vitamin A
  Vitamin A is necessary to support healthy eyesight and immune system functions
 Vitamin A supplementation of children 6-59 months has been shown to be highly
effective in reducing mortality from all causes in countries where vitamin A deficiency is
a public health concern
 found in two forms; retinol in foods from animal sources and carotenoids (the most
abundant of which is the beta-carotene) from plant sources
 retinol is found in liver and whole milk
 carotenoids are found in dark green leafy vegetables, carrots and orange coloured fruits
 Vitamin D(Cholecalciferol)
 needed for the absorption of calcium and phosphorous from foods, to keep bones healthy
 found in the diet, but most of our vitamin D is made in the body the action of ultra violet
rays on the skin
 occurs naturally in some animal products, including fish liver oils, oily fish, egg yolk, and
butter
 Deficiency of vitamin D leads to rickets and the formation of soft bones
 Vitamin E (Tocopherol)
 is a group of similar molecules with common properties and functions
 acts as an antioxidant and protects cells in the body against damage
 is mainly found in vegetable oils, nuts, seeds and wheat germ
 vitamin E has a low toxicity, but in very large doses may interfere with absorption of
vitamin A
 Vitamin K
 needed for normal clotting of blood and is also required for normal bone structure
 also produced by the bacteria in the gut
 found in green leafy vegetables e.g. broccoli, lettuce, cabbage, spinach and meat and
dairy products
 Vitamin B1 (Thiamin)
 needed for the release of energy from carbohydrate
 mainly found in whole grains, nuts, meat (especially pork), fruit and vegetables and
fortified cereals
 thiamin deficiency can lead to the development of the disease beri-beri
 Vitamin B2 (Riboflavin)
 needed for the release of energy from carbohydrate, protein and fat
 involved in the transport and metabolism of iron in the body and is needed for the normal
structure and function of skin and body linings
 found in milk, eggs, rice, fortified
 breakfast cereals, liver, legumes, mushrooms and green vegetables
 Vitamin B3 (Niacin)
 important for releasing energy from food, and is important for the normal structure of the
skin and body linings
 needed for the normal functioning of the nervous system
 meat, wheat and maize flour, eggs, dairy products and yeast
 deficiency of niacin can result in the disease pellagra
 Vitamin B12
 needed for the formation of red blood cells and the normal functioning of the nervous
system
 found exclusively in animal products, plant products do not provide any vitamin B12
 Vitamin C (Ascorbic Acid)
 needed to make collagen which is required for the normal structure and function of body
tissues, such as skin, cartilage and bones
 also acts as an antioxidant that protects the body from damage by free radicals
 sources of ascorbic acid include fresh fruits, especially citrus fruits and berries, green
vegetables, peppers and tomatoes
 Zinc
  Zinc is a mineral that promotes immunity, resistance to infection, and proper growth and
development of the nervous system, and is integral to healthy pregnancy outcomes

Growth Media
Cell culture is one of major techniques in the life sciences. It is the general term used for the removal of
cells, tissues or organs from an animal or plant and their subsequent placement into an artificial environment
conducive to their survival and/or proliferation. Basic environmental requirements for cells to grow optimally
are: controlled temperature, substrate for cell attachment, and appropriate growth medium and incubator that
maintains correct pH and osmolality. The most important and crucial step in cell culture is selecting appropriate
growth medium for the in vitro cultivation. A growth medium or culture medium is a liquid or gel designed to
support the growth of microorganisms, cells, or small plants. Cell culture media generally comprise an
appropriate source of energy and compounds which regulate the cell cycle. A typical culture medium is
composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth
factors, hormones, and attachment factors. In addition to nutrients, the medium also helps maintain pH and
osmolality.

Natural Media
Natural media consist solely of naturally occurring biological fluids. Natural media are very useful and
convenient for a wide range of animal cell culture. The major disadvantage of natural media is its poor
reproducibility due to lack of knowledge of the exact composition of these natural media.
Artificial Media
Artificial or synthetic media are prepared by adding nutrients (both organic and inorganic), vitamins,
salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors. Different artificial media have been
devised to serve one or more of the following purposes:
 immediate survival (a balanced salt solution, with specific pH and osmotic pressure)
 prolonged survival (a balanced salt solution supplemented with various formulation of organic
compounds and/or serum)
 indefinite growth
 specialized functions

Artificial media are grouped into four categories:

 Serum-containing media
 Fetal bovine serum is the most common supplement in animal cell culture media. It is
used as a low-cost supplement to provide an optimal culture medium. Serum provides
carriers or chelators for labile or water-insoluble nutrients, hormones and growth factors,
protease inhibitors, and binds and neutralizes toxic moieties.
 Serum-free media
 Presence of serum in the media has many drawbacks and can lead to serious
misinterpretations in immunological studies. A number of serum-free media have been
developed. These media are generally specifically formulated to support the culture of a
 single cell type. These media are also referred to as ‘defined culture media’ since the
components in these media are known.
 Chemically defined media
 These media contain contamination-free ultra-pure inorganic and organic ingredients, and
may also contain pure protein additives, like growth factors. Their constituents are
produced in bacteria or yeast by genetic engineering with the addition of vitamins,
cholesterol, specific amino acids, and fatty acids.
 Protein-free media
 Protein-free media do not contain any protein and only contain non-protein constituents.
Compared to serum-supplemented media, use of protein-free media promotes superior
cell growth and protein expression and facilitates downstream purification of any
expressed product.

ENZYMES

Enzymes: How They Work and What They Do

Enzymes
- Help speed up chemical reactions in the human body
- Bind to molecules and alter them in specific ways
- Essential for respiration, digesting food, muscle and nerve function, and a thousand of other roles
- Are built of proteins folded into complicated shapes: they are present throughout the body
Substrate
- Binds to the active site of an enzyme and is converted into products
What do enzymes do?
 The Digestive System – enzymes help the body break down larger complex molecules into smaller
molecules, such as glucose, so that the body can use them as fuel
 DNA Replication – each time a cell divides, the DNA needs to be copied. Enzymes help in this process
by unwinding the DNA coils and copying the information
 Liver Enzymes – through the use of a range of enzymes, the liver is able to break down toxins in the
body
Working Mechanism of Enzymes
An Overview: Each enzyme acts upon a specific target called substrate, which is transformed into usable
products through the action of the enzyme. In other words, the enzyme reacts with the substrate forming an
enzyme-substrate complex. Once the reaction is complete, the enzyme remains the same, but the substrate
transforms to products.
Factors that Affect Enzyme’s Action
 Temperature - high temperatures boost the rate of reactions involving enzymes. The optimal
temperature for such reactions are said to be around 37 ºC to 40 ºC. Once the temperature rises above
this level, the enzymes get denatured and they are no longer fit for reaction with substrates.
 pH - Variations from that pH level may slow down the activity of enzymes and very high or low pH
results in denatured enzymes that cannot hold the substrate properly.
 Concentration - The rate of enzymatic activities may increase with the concentration of enzymes and
substrates.
Nomenclature of Enzymes
Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use.
The second is the more complete systematic name, which is used when the enzyme must be identified without
ambiguity.
A.) Recommended Name
 Most commonly used enzyme names have the suffix “ase” attached to the substrate of the
reaction or to a description of the action performed
 (attached to substrate) Ex. Glucosidase, urease, sucrase
 (attached to action) Ex. Lactate dehydrogenase, adenylate cyclase
 Note: Some enzyme retain their original trivial names, which give no hint of the
associated enzyme reaction.
 Ex. Trypsin and pepsin
B.) Systematic Name
 According to the International Union of Biochemistry, an enzyme has two parts: the
name of the substrate and the type of reaction catalyzed by the enzyme.
 Example: Lactate Dehydrogenase
Classification of Enzymes
Enzymes are divided into six major classes with several subclasses.
a.) Oxidoreductases are involved in oxidation and reduction.
b.) Transferases transfer functional groups (e.g., amino or phosphate groups)
c.) Hydrolases transfer water; that is, they catalyze the hydrolysis of a substrate
d.) Lyases add (or remove) the elements of water, ammonia, or carbon dioxide (CO2) to (or
from) double bonds
e.) Isomerases catalyze rearrangements of atoms within a molecule
f.) Ligases join two molecules
Classification Distinguishing Feature
1.) Oxidoreductase Ared + Box  Aox + Bred

Oxidases - Use oxygen as an electron acceptor but

do not incorporate it into the substrate
Dehydrogenases - Use molecules other than oxygen (e.g.,
NAD+) as an electron acceptor
Oxygenases - Directly incorporate oxygen into the
Peroxidases substrate
- Use H2O2 as an electron acceptor
2.) Transferases A–B+CA+B–C

Methyltransferases - Transfer one-carbon units between

substrates
Aminotransferases - Transfer NH2 from amino acids to keto
acids
Kinases - Transfer PO3~ from ATP to a substrate
Phosphorylases - Transfer PO3~ from inorganic phosphate
(P,) to a substrate

3.) Hydrolases A – B + H2O  A - H + B – OH

Phoshatases - Remove PO3~ from a substance

Phosphodiesterases - Cleave phosphodiester bonds such as
those in nucleic acids
Proteases - Cleave amide bonds such as those in
proteins

4.) Lyases A(XH) – B  A – X + B – H

Decarboxylases - Produce CO2 via elimination reactions

Aldolases - Produce aldehydes via elimination
reactions
Synthases - Link two molecules without involvement
of ATP

5.) Isomerases A–BB–A

Racemases - Interconvert L and D stereoisomers

Mutases - Transfer groups between atoms within a
molecule

6.) Ligases A + B + ATP  A – B + ADP + Pi

Carboxylases - Use CO2 as substrate

Synthases - Link two molecules via an ATP-
dependent reaction

Commercial Applications of Enzymes

Due to ongoing research by biotechnologists, enzymes now have a large number of commercial applications:
 Textile Industry – enzymes are used for improving production methods and for fabric finishing.
 Cellulase – for stonewashing denim, polishing of cotton
 Catalase – removing hydrogen peroxide
  Pectinase – for bioscouring (a way to scour fabrics)
(Note: Scouring - the cleaning of fabrics by removing any impurities such as waxes, pectins and any mineral
salts from cellulose fibers)
 Amylase – for removal of starch from the threads of fabric
 Food and Drink Industry
o Baking Industry - enzymes are added to the dough when baking bread to ensure that the bread
is high in quality and has a better volume. Enzymes also have the ability to preserve bread.
 Fungal alpha amylase – for dough improvement in the bread making industry
o Dairy Industry - enzymes are used in cheese making to help bring about the coagulation of
milk.
o Brewing Industry - enzymes are added to control the brewing process in alcohol making. This
also helps to produce consistent and high-quality beer.
 Papain enzymes – for fermentation in the brewing industry
o Other enzymes used in food and drinks industry:
 Glucoamylase – used in fermentation
 Beta glucanase – for filtration
 Protease – used in biscuit production and it is also used in the manufacturing of baby
foods to predigest proteins
 Pulp and Paper Industry - In the manufacturing of coated papers, a starch-based coating formulation is
used in order to coat the surface of the paper. Compared with the uncoated paper, the coating provides a
number of benefits, including; improved gloss, a smoother texture, and printing properties. To achieve
this, a variety of enzymes are used in this industry.
 Cellulase – can be used for pulp deinking and pulp refining
 Xylanase – for pulp bleaching
 Alpha amylase – starch modification
 Detergent, Personal Care and Hygiene Industry - This industry, in addition to the food processing
industry is currently one of the largest application areas for enzymes. They contribute to a: better overall
cleaning performance; they are biodegradable so they do not really effect the environment that much;
they reduce water consumption through more effective release of soil.
Enzyme Specificity
Enzyme Specificity – refers to the tendency for enzymes to catalyze a specific set of chemical reactions.
(Specificity depends on active site orientation based on its atomic configuration.)
Active Site – is the region that binds the substrates (and the cofactors, if any)
- It also contains the residue that directly participate in the making and the breaking of bonds. These
residues are called catalytic groups.
- It is a three-dimensional cleft formed by groups that come from different parts of the amino acid
sequence.
- Has a precise amino acid sequence that is never changed. A change in amino acid sequence in active site
generally renders enzyme into a non-functional form.
- Bond breaking and forming reacting groups are in the active site of the enzyme
Lock-and-Key Model
- The active site has a rigid shape
- Only substrates with the matching shape can fit
- The substrate is a key that fits the lock of the active site
- The amino acid R groups of enzymes help to mediate interaction of active site and substrate
- This is an older model, however, and odes not work for all enzymes
Induced Fit Model
- The active site is flexible, not rigid
- The shapes of the enzyme, active site, and substrate adjust to maximize the fit, which improves catalysis
- There is a greater range of substrate specificity
- This model is more consistent with a wider range of enzymes
Six Distinct Types of Specificity
 Absolute specificity - the enzyme will catalyze only one reaction.
 Group specificity - the enzyme will act only on molecules that have specific functional groups, such as
amino, phosphate and methyl groups.
 Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of
the molecular structure.
 Stereochemical specificity - the enzyme will act on a particular steric or optical isomer. Also called
optical specificity.
 Bond Specificity – the enzyme will act on a substrate that are similar in structure and have same type of
bond
 Dual Specificity – the enzyme will act on two substrates by same type of reaction
Enzyme Kinetics: Basic Enzyme Reactions
Enzyme kinetics studies the reaction rates of enzyme-catalyzed reactions and how the rates are affected by
changes in experimental conditions.
Michaelis-Menten Type Kinetics
The Michaelis-Menten model is one of the simplest and best-known approaches to enzyme kinetics. It takes
the form of an equation relating reaction velocity to substrate concentration for a system where a
substrate S binds reversibly to an enzyme E to form an enzyme-substrate complex ES, which then reacts
irreversibly to generate a product P and to regenerate the free enzyme E. This system can be represented
schematically as follows:

The rate of the forward reaction from E + S to ES may be termed k1, and the reverse reaction as k-1. Likewise,
for the reaction from the ES complex to E and P, the forward reaction rate is k2, and the reverse is k-2.
Therefore, the ES complex may dissolve back into the enzyme and substrate or move forward to form product.
Assuming steady state, the following rate equations may be written as:
Rate of formation of ES = k1[E][S]
Rate of breakdown of ES = (k-1 + k2) [ES]
and set equal to each other (Note that the brackets represent concentrations). Therefore:
k1[E][S] = (k-1 + k2) [ES]
Rearranging terms,
[𝐸][𝑆] 𝑘−1 + 𝑘2
= 𝑘
[𝐸𝑆] 1

[𝐸][𝑆]
The fraction has been coined Km, or the Michaelis constant.
[𝐸𝑆]
According to Michaelis-Menten's kinetics equations, at low concentrations of substrate, [S], the concentration is
almost negligible in the denominator as KM >> [S], so the equation is essentially:
V0 = Vmax [S]/KM
At High substrate concentrations, [S] >> KM, and thus the term [S]/([S] + KM) becomes essentially one and the
initial velocity approached Vmax, which resembles zero order reaction.
The Michaelis-Menten equation is:

In the above equation:

V0 is the initial velocity of the reaction.
Vmax is the maximal rate of the reaction.
[Substrate] is the concentration of the substrate.
Km is the Michaelis-Menten constant which shows the concentration of the substrate when the reaction velocity
is equal to one half of the maximal velocity for the reaction. It can also be thought of as a measure of how well
a substrate complexes with a given enzyme, otherwise known as its binding affinity. (Note: A low Km value
indicates a large binding affinity and a high Km indicates that the enzyme does not bind as efficiently with the
substrate.)
Sample Problem:
1.) For a given enzyme catalyzed reaction, the Michaelis constant is 0.6mM and the substrate concentration
is 1.0mM. What is the fractional saturation of the enzyme under these conditions?
Explanation:
The fractional saturation of an enzyme is defined as the amount of enzyme that is bound to substrate divided by
the total amount of enzyme. To calculate the fractional saturation, we'll need to use the Michaelis-Menten
equation:
𝑉 [𝑆]
V0 = 𝐾𝑚𝑎𝑥
𝑀+[𝑆]
In addition, we'll need to define the rate and maximum rate in terms of enzyme concentrations:
V0=kcat[ES]
Vmax=kcat[E]T
From the above equations, we can calculate the fractional saturation of the enzyme:
[ES] 𝑉 [𝑆]
Fractional Saturation = [E] = 𝑉 𝑜 = 𝐾 +[𝑆]
𝑇 𝑚𝑎𝑥 𝑀

[𝑆] 1.0 𝑚𝑀
= = 0.625 or 62.5%
𝐾𝑀 +[𝑆] 0.6 𝑚𝑀+1.0 𝑚𝑀

2.) Given an enzyme with KM of 0.5mM, at what substrate concentration will the velocity of the enzyme
reach 1/4 of the Vmax? (Vmax=200mmol/s)
Explanation:
To solve this, we need the solve for [S] in the Michaelis-Menten equation:
𝑉 [𝑆]
V0 = 𝐾𝑚𝑎𝑥
+[𝑆]
𝑀
We know the following information:
1
KM =0.5mM; Vmax = 200mmol/s; Vo = 4 Vmax = 50mmol/s
Plug in these numbers and solve for substrate concentration.

200[𝑆]
50 = 0.5+[𝑆]

1 [𝑆]
= 0.5+[𝑆]
4

0.5+[S] = 4([S])

0.5 = 3[S]

[S] ≈ 0.17 mM

SIMPLE ENZYME KINETICS

I. 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

II. Models for Inhibitor does not
Inhibitor competes
more Complex bind at the active
with the substrate
Enzyme site
at the active site
Kinetics –
involve multiple Active site may
binding sites Active site stays change shape
the same (e.g. allosteric
inhibition)

 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.

III. 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

 Immobilized Enzyme Systems

 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

Where:
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 their lack of stability in organic solvents and at high temperatures. As a consequence, protein
engineering is an active area of research 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, catalyze breakdown of starch in the flour to sugar. Yeast fermentation
of sugar produces the carbon dioxide that raises the dough.
o Lipases - is implemented during the production of Roquefort cheese to enhance the ripening of the
blue- 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
o Amylases, amyloglucosideases and glucoamylases - convert starch into glucose and various syrups.

7. Brewing Industry
o Enzymes from 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
fermentation.
8. Paper Industry
o 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
o Cellulases - used to breakdown cellulose into sugars that can be fermented

10. Rubber Industry

o Catalase - to generate oxygen from peroxide to convert latex into foam rubber

11. Photographic Industry

o Dissolve gelatin oil scrap film, allowing recovery of its silver content.