Taiz University

Pharmacy department , Forth level

Enzymes biotechnology

Dr: Eyad Al-sabaei

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 introduction
Enzymes are colloidal, amorphous, soluble, biocatalysts which are
fabricated by living cells and are proficient to act independently on
the cells.

 It m e di a t e s cou n t l e ss bi ol ogi ca l r e a ct i on s a s w e l l a s
transformation of component into intermediate products and so
on to the final product.

If we take a look at their activity, whole enzyme will not be

convoluted in any transformation of reactant to a product.

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Mainly, active site of an enzyme plays a vital role to carry out the
reaction. Usually, they are entitled as holoenzyme composed of
protein fragment called apoenzymes and non-protein part called
co-enzyme and metal. The protein fragment attached to non-
protein part either by non-covalent or covalent bond.

Chief ly, enzymes are biodegradable and non-toxic in nature. They

can be fabricated in hefty extents by utilizing microorganisms for
commercial uses. Slight adaptations on to the active site will
facillates alteration in activity of the specif ic enzyme. Moreover,
an enzyme night to lose its action completely.

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Numerous parameters essential to be adjusted to create an
enzyme to display its supreme effectiveness in translation of
reactant in to preferred product.

The parameters including: pH, temperature, concentration

substrate and enzyme, time of reaction, activation, specif icity and
several modif ications require being complete at the genetic level
of that specif ic enzyme to give determined yield of the quality
product.

Enzymes have wide range of applications in analytical, healthcare

(including cosmetics, diagnostics) industry.

 Enzyme biotechnology generally involves fabrication, separation,

purif ication and utilization of enzymes (in immobilized or soluble
form) for the crucial benef it of mankind. Furthermore, protein
engineering and recombinant DNA technology involved in the
manufacturing of more effective and convenient enzymes.

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 Source of enzymes
A. Plant and animals: In the initial period, plant and animal sources
principally impacted to enzymes.

 Nowadays for few enzymes still plants and animals are the major
sources. High yield of enzymes like proteases esterase and lipases
are obtained from animal organs and tissues.

 The eggs are good source of enzymes like lysozyme. Few plants
are f ir st-rate sources for many enzymes like papain (papaya),
amylase (sweet potatoes).

 Mostly enzymes obtained from plant and animal sources are

nontoxic in nature and usually free from any kind of contamination.

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B. Ma m m a l i a n source The re i s a proba bi l i t y t o produce
commercial enzymes straightly by mammalian cell cultures.
However, the cost involved behind the preparation or utilization of
mammalian culture is relatively high. Though, few therapeutic
enzymes like tissue plasminogen activator are produced by cell
cultures.

C. Microbial source: Microorganisms are the most noteworthy and

suitable sources of marketable enzymes. Many enzymes of
commercial applications have been effectively manufactured by
utilizing micro-organisms like bacteria, yeasts and fungi.
Microorganisms can be nurtured by means of economical media
and fabrication can carry out in a short time.

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Commercial enzymes fabricated from microorganisms their
source and application

Application Source Enzymes

Preparation of Cheese, flavor Candida Lipolytica Lipase
production
Aspergillus Niger

Penicillin removal Bacillus Subtillis Penicillinase

Digestive aid Aspergillus Niger Protease
Antioxidant in nature Aspergillus Niger Glucose oxidase
Beer and alcohol production Aspergillus Oryzae Alpha -amylase

Artificial honey production Saccharomyces cerevisiae Invertase

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 General conditions of enzyme production
technology
Basically, the methods used for microbial fabrication of enzymes are
analogous to the techniques utilized for preparation of other
commercial products. Following steps an involved in the production
of commercial enzymes:
 A. Selection of specific microorganism
 B. Preparation of medium
 C. Fabrication technique
 Collection and purification of prepared enzymes

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A. Selection of specif ic microorganism: The key factor before
selecting the microorganism is that the organism should yield
the supreme extents of preferred enzyme within short duration
while the quantities of other metabolite manufactured are
negligible.

B. Preparation of medium: For supporting the acceptable

development of microorganisms that will eventually result in to
a good amounts of enzyme production, the selected culture
medium should comprise all the essential nutrients. The
medium components should be freely accessible, cost effective
and are nutritionally harmless. Yeast extract, corn steep liquor,
molasses soy bean meal and whey are some of the frequently
utilized substrates for the preparation of medium. The pH of the
medium plays a vital role in enzyme. Thus the pH of the
medium should be kept optimum for good microbial growth and
enzyme production.
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C. Fabrication technique:
Commercial fabrication of enzymes is generally carried out by
submerged liquid environments as well as to a smaller amount
by solid-substrate fermentation technique. Submerged culture
method, enzyme yield is more and infection chances are rare.
Therefore, this is most preferred technique. On the other hand,
solid substrate fermentation technique is a traditional approach
and quiet in use for the manufacturing of fungal enzymes like
proteases, pectinases, celluloses, and amylases.
During production, the growth environments like pH, supply of O2,
temperature, addition of nutrient are retained at optimum
heights. The development of froth formation an be reduced by
accumulating antifoam agents. Batch fermentation technique is
most favorable technique for the production of enzymes.
However, continuous process also used at a smaller extent. The
bioreactor arrangement essentially maintained sterile during the
fermentation method. The period of fermentation is adjustable
around 2-7 days, in most production procedures. In addition to
the desired enzymes, numerous extra metabolites are also
manufactured. Only the enzymes have to be recovered and
purified.
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D. Collection and purification of prepared enzymes:
 The required manufactured enzymes perhaps excreted into the
culture medium (extracellular enzymes) or may be exist within
the cells (intracellular enzymes). The marketable enzymes are
hi ghl y pur i f ie d or cr ude de pe ndi ng on t he re qui re m e nt .
Furthermore, product perhaps in the solid or liquid state.
 At commercial level enzymes are fabricated by solid substrate
technique utilizing fungal sources and traditional submerged
culture techniques for bacterial source. However, industrial
enzymes are mostly fabricated by solid state fermentation
technique.

Solid State Fermentation: Fermentation techniques carry out in

the absence of free water. Solid state fermentation (SSP)
technique is utilized for the manufacturing of enzymes, which
encompasses the cultivation of microbes on a solid substrate, like
rice wheat bran and grains. This technique engages natural raw
constituents as carbon source such as barley, sugarcane, bagasse,
cassava, various oil cakes like, ground nut oil cake soybean cake,
coffee husk, palm kernel cake, coffee pulp, and tea waste.
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Submerged fermentation:

In this technique, the substrate utilized for fermentation is

constantly in liquid state which encloses the nutrients which
are vital for growth. The fermenter which holds the substrate
is act ivat e d const ant ly and t he produce d biom ass is
uninterruptedly harvested from the fermenter through diverse
procedures. Afterwards, the product is filtered and centrifuged
and then dried. This technique is a process of fabricating
biom ole cule s in which e nzym e s and fur t he r re act ive
compounds are plunged in a liquid such as oil, a nutrient broth
, or alcohol. The pract ice is e m ploye d for a range of
resolutions, typically in industrial fabrication.

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LIPASE
Lipases are also well known as Glycerol ester hydrolases. They are
a subclass of esterase which separates fats into mono or di-
glycerides and fatty acids.
Normally, these are extracellular enzymes manufactured by using
Fungi like Aspergillus, Penicillium, Mucor, Rhizopus.
Bacteria generating lipases comprise species of Achromobacter,
Staphylococcus, and Pseudomonas. Yeasts like Candida and
Torulopsis are also commercially used.
The preparation of lipase must be prompted by addition of oils as
well as fats. However, in few circumstances the fat originates
negative effect on the lipase fabrication. Glycerol, a product of
lipases action plays major role in inhibiting lipase formation.
Lipases are usually bound to the cells, therefore inhibit an
overproduction but adding of the cation such as magnesium ion
liberates the lipase and primes to a higher enzyme titer in the
manufacturing medium.

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PENICILLINASE
• Penicillinases are also known as B-Lactamase 1, B-Lactamase II,
Cephalosporinase, Penicillin amido-B-lactam hydrolase. Normally,
this is a group of enzymes with varying specif icity for hydrolysis
of B-lactam complexes. Penicillinase is a fusion of two types of B
-lactamases, lactamase I and II. Mutually, enzymes are defined as
metalloenzymes or serine enzymes. Penicillinase produced from
Bacillus cereus is a constitutive periplasmic enzyme with
observed KM of 60 uM (benzyl penicillin).
PROTEASE
• Protease enzyme is a blend of peptidases and proteinases
enzymes that are perform the hydrolysis of Peptide bonds.
Peptide bonds connect the amino acids to provide the f in ally
protein structure. Peptidases are endocellular and Proteinases
are extracellular in nature. Protease is one of the vital enzymes
manufactured on a large scale after Amylase. Neutral Proteases
are relatively unstable in nature and sodium, calcium and chloride
must be added for maximal strength. Neutral Proteases are
unstable at higher temperatures. Acid proteases are similar to
mammalian pepsin. It includes rennin like proteases from fungi
which are primarily utilized in cheese manufacturing
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Steps in fermentation technique involved:
 1. Cultures are deposited in the lyophilized state or under
liquid nitrogen.

 2. Preliminary cultures are carried out in shaken f lasks and

small fermenters (40-100 m) a 30-370C.

 3. Fed-Batch culture is normally employed to keep down the

concentration of ammonium ions and ammino acids as they
may suppress protease production.

 4. High oxygen partial pressure is generally essential for

optimal protease titers involves time span for 48-72 hours
depending upon the organism.

 5. Proteases must be converted in a particulate form before

t h e y a r e a d d e d t o d e t e r g e n t s To m a k e a s u i t a b l e
encapsulated product, a wet paste of enzyme is melted at 50
-70C with a hydrophobic substance such as polyethylene
glycol and then transformed into tiny particles.
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CATALASE
Catalase is a common enzyme establish in nearly all living
organisms visible to oxygen )such as plants, animals and
bacteria(. It catalyzes the decomposition of hydrogen peroxide
to‫ו‬water and oxygen. It is a vital enzyme in protecting the cell
from oxidative damage by reactive oxygen species )ROS(.
Similarly, catalase has one of the chief business statistics of all
enzymes. Single catalase molecule can transform millions of
hydrogen peroxide molecules to water and oxygen each second.
AMYLASE
Amylase catalyzes the hydrolysis conversion of starch into
sugars. It is normally present in the salvia of humans. Hydrolysis
of Starch with amylase will produce a short polymer dextrin and
then the disaccharide maltose to end with glucose. alpha-
Amylase is also called as 1,4-alpha-D-glucan glucanohydrolase.
Calcium metalloenzymes which cannot function in absence of
calcium ions. Breaks down long carbohydrate chains of Amylose
and Amylopectin. Beta Amylase also known as 1,4-alpha-D-
glucan maltohydrolase, fabricated by bacteria, fungi, and plants.

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PEROXIDASE
 Peroxidase (HRP) is a hemoprotein catalyzing the oxidation by
hydrogen peroxide of a number of substrates such as ferro
cyanide, cytochrome C, ascorbate, and the leuco form of several
dyes. Peroxidase suitable for the fabrication of enzyme
conjugated antibodies, also having ability to yield chromogenic
products. Peroxidase characterized immunoglobulins have been
utilized effectively as immune histological examinations for the
demonstration of tissue antigens, and in enzyme improved
immunoassay schemes for the quantitative purpose of soluble
and insoluble antigens.

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 Enzyme Immobilization
Enzyme immobilization well-def ined as constraining the enzyme
fragments to a compact matrix dissimilar from the one in which
substrate or the products are present.

Enzyme immobilization facillated by attributing the enzymes to

or inside some appropriate support material.

 It is vital to point out that the substrate fragments and the

products formed should move spontaneously in and out of the
phase to which the enzymes are reserved.

The application of immobilized enzymes can be utilized in variety

of processes. Recently, plethoras of novel methods have
developed far the immobilization of enzymes that have superior
productivity and extensive applications.

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PROS AND CONS OF ENZYME IMMOBILIZATION
(A) Pros of enzyme immobilization
 Improved functional proficiency of enzyme
 Greater reproducibility of the active process.
 Better process control.
 Cost effective and fewer labor contribution in the procedures.
 High product stability
 Increased enzyme substrate ratio
(B) Cons of enzyme immobilization
 Collection and purif ication of prepared enzymes (supreme
disadvantage)
 High cost isolation.
 Limited industrial applications
 Loss of Catalytic properties after immobilization
 Inactivation of enzymes by heat generation

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 Application of Immobilization enzyme in
Biotechnology
1. PHARMACEUTICAL APPLICATIONS:
A-Fabrication of antibiotics and medicines
 Immobilized enzyme has transformed the medicinal sector, as
they are enormously utilized for analysis and treatment of
numerous diseases with fewer manpower, a smaller amount of
time and with great reliability and accuracy. The characteristics of
biosensors like accuracy. Sensitivity, reliability beside with the
exclusive possessions of enzymes have led to the improvement
of enzyme-based antibiotics and medicines.

 Immobilized enzymes assist to recognize the disease type and

pathological conditions too.

 Moreover, immobilized enzymes are also utilized to examine

antibodies and antigens, for instance, B-lactamase and alkaline
phosphatase. 21
B- Acids, Amino acids production
 Appl i ca t i ons of a m i no a ci ds hav e wi de spr e a d usa ge i n
pharmaceutical sector. Many large scale techniques expending
immobilized enzymes are in process. Amino acid fabrication by
enzymatic resolution using amino acids acylase and fructose
syrups from glucose isomerase.
 Immobilized aminoacylate of Aspergillus oryzae is utilizéd for the
continuous optical resolution of DL-amino acids.
 For enhancement of the ef ficiency of organic acids Immobilized
enzymes have been used.
 Processes have been developed for production of Acetic acid by
Acetobacter aceti immobilized with porous ceramics.

C- Production of steroids
 Bio catalytic conversions facillates the steroid alterations. Several
enzymes engaged for steroid requires cofactors. Entire microbial
cells comprising structures of co-factors are immobilized and
engaged for bulk production of steroids.
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2. Clinical Investigation Applications
A- Biosensors
In last few decades, expansion of biosensors for the detection and
measurement of metabolites is an area of extreme research as it
has been established that constant metabolic checking offers an
early suggestion of various diseases and body disorders.
The electrical, mechanical, chemical, optical devices which can
easily carryout detection of biological species are termed as
biosensors. Simply, it is an analytical device which can detect and,
quantify specific analytes in composites.
The model biosensor detects low concentrations of the analytes,
interprets the outcomes immediately and also has the capacity to
distinguish between classes according to the identif ic ation
molecules that are entrapped on its surface.
Nowadays, several enzyme electrodes are available, enzyme is
immobilized to inner wall of nylon tubing and enzyme reactor is
located in sequence with suitable detector.

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THANK YOU

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