ENZYMES AND THEIR APPLICATION

Enzymes

• Enzymes are proteins that act as catalysts in all living organisms -

microorganisms, plants, animals, and humans.
• Very small quantities of enzymes can increase the rate of reactions up
to ten million times.
• Enzymes operate within a narrow set of conditions, such as
temperature and pH (acidity), and are subject to inhibition by various
means.
• Enzymes which decompose complex molecules into smaller units,
such as carbohydrates into sugars, are natural substances involved in
all biochemical processes. Due to the enzymes specificities, each
substratum has a corresponding enzyme.
Sources of Enzymes
Biologically active enzymes may be extracted from any living organism:
Of the hundred enzymes being used industrially,
- over a half are from fungi
-over a third are from bacteria with the remainder divided between animal
(8%) and plant (4%) sources .

Microbes are preferred to plants and animals as sources of enzymes because:

-They are generally cheaper to produce.

-Their enzyme contents are more predictable and controllable.

- Plant and animal tissues contain more potentially harmful materials than
microbes, including phenolic compounds (from plants).
 Fungal Enzymes
Enzyme EC Sources Application

a-Amylase 3.2.1.1 Aspergillus E Baking

Catalase 1.11.1.6 Aspergillus I Food
Cellulase 3.2.1.4 Trichoderma E Waste
Dextranase 3.2.1.11 Penicillium E Food
Glucose oxidase 1.1.3.4 Aspergillus I Food
Lactase 3.2.1.23 Aspergillus E Dairy
Lipase 3.1.1.3 Rhizopus E Food
Mucor
Rennet 3.4.23.6 E Cheese
miehei
Pectinase 3.2.1.15 Aspergillus E Drinks
Protease 3.4.23.6 Aspergillus E Baking

E: extracellular enzyme; I: intracellular enzyme

 Bacterial Enzymes

Enzyme Sources Application

a-Amylase 3.2.1.1 Bacillus E Starch

b-Amylase 3.2.1.2 Bacillus E Starch
Escherichia
Asparaginase 3.5.1.1 I Health
coli
Glucose Fructose
5.3.1.5 Bacillus I
isomerase syrup
Penicillin Pharmace
3.5.1.11 Bacillus I
amidase utical
Protease 3.4.21.14 Bacillus E Detergent
Enzyme Production
Steps In Enzyme Study
1. Determine the source of the enzyme

2. Extract the enzyme.

If the enzyme is:

• Cytoplasmic, extract the enzyme first from the source

• Organellar, isolate the organelle from the rest of the cell component, then extract
the enzyme from the organelle

• Extracellular, remove the cells from the enzyme solution (eg growth medium)

The first enzyme solution that one gets is known as the crude enzyme extract. It is not
pure and contains impurities such as other enzymes and proteins, sugars, vitamins etc.

• If it is intracellular, then the crude enzyme extract is the one obtained after
extraction from the cell.
• If extracellular, then the enzyme is in the growth medium/serum (usually induced by
addition of substrates or substrate analague)

3. Stabilise the enzyme. Enzyme is usually stabilized by buffering the pH of the extraction
solution, keeping at low T and by addition of protease inhibitors.

4. Purify: Determine the level of purity that is required and devise a set of purification
protocol or regime.

5. Determine the activity and characterize the enzyme Information regarding an enzyme is
useful for industrial/medical/food applications.

Extraction Techniques

• Extraction techniques are devised to release an enzyme from the cell, or if organellar,
from the organelle.
The choice of technique is dependent on the source of cell plant, animal or microorganism.
Harsher techniques are used to break cells with cell wall.

• Extraction must be carried out a low T (4°C) and as soon as possible to minimize losses
due to proteolytic activity and general tissue breakdown. Otherwise, keep sample/source
frozen or cold.
• An enzyme is usually extracted into a solution that can stabilize it eg a buffer.

• Some compounds that are present in a cell can reduce the quantity (therefore,
activity) of the enzyme being extracted eg phenolic compounds that are often
present in plants and some fungi.

• Losses due to phenolic compounds can be reduced by the addition of one of the
following in the extraction medium:

1. polyvinylpyrrolidone (PVP)
2. beta- mercaptoethanol
3. ascorbic acid
4. Thioglycolate

• There are many techniques to break a cell. These techniques can be divided into
3 types depending on the structure of the cell:
1. gentle
2. moderate
3. vigorous (harsh/strong)
A. Gentle Cell Disruption Techniques

1. Cell lysis (osmotic shock)

• Principle: Based on the osmotic disruption of cell membrane.
• Cell lysis method using osmotic pressure depends on the nature of the cells and
whether fibrous protein such as myosin and collagen are present.

2. Enzyme Digestion (cell wall)

• Principle: Cell wall is digested using enzyme, leading to osmotic shock that causes
the cell membrane to disrupt.
• Enzymatic digestion/lysis methods minimize denaturation and allow some
selectivity in the release of cellular products.
Major enzymes that can digest the cell wall is β-1,3-glucanase, zymolyase and
lyticase.

3. Chemical Solubilisation/Autolysis (Cell wall and membrane)

• Principle: Cell wall and membrane are partially solubilised chemically, lytic
enzymes completes the release process.
4. Ultra sonication
• Principle: Micro-scale high pressure sound waves (ultra sonic) that cause disruption of
cells by liquid shear forces and cavitation. Air bubbles in the cavity compresses, and
then collapsed forming a shock wave that breaks the cells.
• This method is suitable to be used for cell (microbial) suspensions.
• Sonication remains popular for lysing small quantities of cells, and cannot be used for
large quantities like 50g to 1 kg range because of difficulty in controlling low
temperatures.

Preparation of Crude Extract For Purification

• Before purification, the crude enzyme solution must be separated from other
materials. There are several techniques such as :
Centrifugation
a) low speed at room temperature (< 3000 rpm)
b) moderate/high speed (< 20000 rpm at low T)
c) very high speed(>20000 rpm at low T)

Methods of enzyme purification

The purification of a particular enzyme involves removal of other substances (proteins
as well as non-proteins) present in the preparation.

For enzyme purification, commonly used chromatography techniques are: (i) Ion
exchange chromatography; (ii) Adsorption chromatography; (iii) Gel filtration
chromatography and (iv) Affinity chromatography.
•Most commonly used electrophoretic technique is Polyacrylamide Gel
Electrophoresis (PAGE) for enzyme purification.

•Isoelectrofocusing, protein sample is electrophoresed through a gel having stable

pH gradient.

•Dialysis is used for removal of low molecular weight contaminants, salt etc.

•Ultrafiltration is used to concentrate the sample, ultrafiltration is also capable of

removing low molecular weight substances.

•Protein fractionation by heat treatment : In many cases, the enzyme protein to

be purified is fairly stable at temperature like 55oC or 60oC. At this temperature,
many unwanted proteins get denatured and precipitated out with little or no loss
of enzyme activity.

•Nonionic polymers can be used for precipitation of enzyme proteins.

Polyethylene glycol (PEG) is commercially available .

•Acetone is used for fractionation of the enzyme protein. While using acetone,
extreme care is to be taken otherwise acetone will denature the enzyme protein.
Immobilized Enzyme Systems

Enzyme Immobilization:
To restrict enzyme mobility in a fixed space.
Immobilized Enzyme Systems

Enzyme Immobilization:

- Easy separation from reaction mixture, providing the ability to control

reaction times and minimize the enzymes lost in the product.

- Re-use of enzymes for many reaction cycles, lowering the total production
cost of enzyme mediated reactions.

- Ability of enzymes to provide pure products.

- Possible provision of a better environment for enzyme activity

- Diffusional limitation
Immobilized Enzyme Systems

• Methods of Enzyme Immobilization:

- Entrapment

- Surface Immobilization

- Cross-linking
Immobilized Enzyme Systems

Entrapment Immobilization is based on the localization of an

enzyme within the lattice of a polymer matrix or membrane.
- retain enzyme
- allow the penetration of substrate.

It can be classified into matrix and micro capsule types.

Surface immobilization
According to the binding mode of the enzyme, this method can be further sub-
classified into:

- Physical Adsorption: Van der Waals

Carriers: silica, carbon nanotube, cellulose, etc.
Easily desorbed, simple and cheap,
enzyme activity unaffected.
- Ionic Binding: ionic bonds
Similar to physical adsorption.
Carriers: polysaccharides and synthetic polymers
having ion-exchange centers.

Cross-linking:
It is to cross link enzyme molecules with each other using agents such as
glutaraldehyde.
Features: similar to covalent binding.
 Making improved products
• Since the early 1980s, companies
which produce enzymes have been
using genetic engineering
techniques to improve production
efficiency and quality and to
develop new products.
• There are clear advantages here for
both industry and consumers, with
major improvements in enzyme
production giving better products
and processes.
• However, progress is being slowed
down because the debate on some
other, more controversial
applications of biotechnology -
such as genetic engineering in
animals - is continuing throughout
Europe.
• At present, modern biotechnology can be used to give a range of advances
in enzymatic production technology:

– Improved productivity and cost-effectiveness in existing processes. By

producing enzymes more efficiently, the amount of raw materials,
energy and water needed to make a product can be reduced by as
much as one-half by changing from a traditional strain of microbe to a
genetically modified one.

– Companies can tailor their enzymes more precisely to customer

demands for products with specific properties.

– Manufacturers can supply enzymes which otherwise could not be

produced in large enough quantities, giving the consumer access to a
wider variety of products. An example is the amylase-based product
which makes bread stay fresh for longer.
Some commercially produced enzymes
• Pectinase enzyme:
The enzyme is employed in the food industries for fruit ripening, viscosity
clarification and reduction of fruit juices, preliminary treatment of grape juice
for wine industries, extraction of tomato pulp tea and chocolate fermentation.,
vegetal wastes treatment, fiber degumming in the textile and paper industries
animal nutrition, protein enrichment of baby food and oil extraction.
• Lipases:
Lipolytic enzymes such as lipases and esterases are an important group of
enzymes associated with the metabolism of lipid degradation.
• Lactase:
Popularly known as lactase, beta-galactosidases are enzymes classified as
hydrolases. They catalyze the terminal residue of b-lactose galactopiranosil and
produce glucose and galactose. Beta-galactosidase is highly important in the
dairy industry, in the hydrolysis of lactose into glucose and galactose with an
improvement in the solubility and digestibility of milk and dairy products.
• Cellulases:
These hydrolytic enzymes are not only used in food, drug, cosmetics, detergent
and textile industries, but also in wood pulp and paper industry, in waste
management and in the medical-pharmaceutical industry. In the food industry,
cellulases are employed in the extraction of components from green tea, soy
protein, essential oils, aromatic products and sweet potato starch. Coupled to
hemicellulases and pectinases they are used in the extraction and clarification
of fruit juices. After fruit crushing, the enzymes are used to increase
liquefaction through the degradation of the solid phase.
• Amylases:
large quantities of microbial amylases are commercially available and are
almost entirely applied in starch hydrolysis in the starch-processing industries.
The species Aspergillus and Rhizopus are highly important among the
filamentous fungus for the production of amylases.
• Proteases
Proteases are enzymes produced by several microorganisms, namely,
Aspergillus niger, A. oryzae, Bacillus amyloliquefaciens, B. stearotermophilus,
Mucor miehei, M. pusillus. Proteases have important roles in baking, brewing
and in the production of various oriental foods such as soy sauce, miso, meat
tenderization and cheese manufacture.
Application of enzymes..
MEAT:
• Proteases are used in both tenderizing and marination of meat
• Papain is the traditional enzyme used
• Others such as
bromelain,
trypsin,
chymotryosin and
microbial proteases from Aspergillus are also used.

DAIRY:
• Renin is used to manufacture cheese and hydrolys proteins.
• Lipases are used in ripening of cheese.
• Lactases are used to break down lactose to glucose and galactose.
 FRUIT AND VEGETABLE

• Enzymes increase processing capacity and improve economy in the fruit juice and wine
industries

• The most commonly used enzymes in these industries are pectinases which increase juice
yields and accelerate juice clarification.

• They produce clear and stable single-strength juices, juice concentrates and wines, from not
only core-fruits such as apples and pears, but also stone fruits, berries, grapes, citrus-fruits,
tropical fruits and vegetables like carrots, beets and green peppers

• Specialty Enzymes are pectinases that contain hemicellulase enzyme activities

• These products not only increase juice yields, but also increase the color and health-
promoting antioxidants in fruit and vegetable juices extracted by pressing or decanter
centrifuge

• By reducing fruit and vegetable mash viscosity and improving solid/liquid separation, they
increase color extraction and juice volume

• Pectinase and Amylase enzyme solutions speed up filtration and prevent storage or post-
packaging haze formation by depectinizing and reducing starch in raw juices. Pectin and
starch must be removed from freshly extracted juices prior to filtration, fining and
concentration
• Pectinase and Amylase can reduce starch and pectin in raw fruits and juices, thus achieving
clear and stable juices and juice concentrates
BREAD MAKING

• Bread is the most common and traditional foods around the world

• But bread actually has close links with enzymes. For years, enzymes such as malt
and fungal alpha-amylase have been used in bread making.

• Due to the changes in the baking industry and the ever-increasing demand for
more natural products, enzymes have gained real importance in bread-making.

• Amylases degrade starch and produce small dextrins for the yeast to act.

• Gluten is a combination of proteins, which form a large network during dough

formation. This network holds the gas in dough proofing and baking.

• The strength of this network is very important for the quality of all bread raised by
yeast.

• Enzymes such as proteases, xylanases and lipases directly or indirectly improve the
strength of the gluten network and so improve the quality the bread.
Conclusions and Future Perspectives
• The integration of enzymes in food and feed processes is a well-established
approach.
• Clearly shows that dedicated research efforts are consistently being made as to
make this application of biological agents more effective and/or diversified.
• These endeavours have been anchoring in innovative approaches for the
design of new/improved biocatalysts, more stable (to temperature and pH),
less dependent on metal ions and less susceptible to inhibitory agents and to
aggressive environmental conditions.
• Enhanced performance under operational conditions that minimize the risk of
microbial contamination.
• It also favours process integration, by allowing the concerted use of enzymes
that naturally have diverse requirements for effective application.