Microbiology and Immunology Department

Pharmaceutical Biotechnology
(PHM452/PM511n/PM401n)
Spring 2024

Lecture 5:
▪ Microbial Enzymes
▪ Biotransformation
References
1. Hall, Stephen J._ Stanbury, Peter F._ Whitaker, Allan - Principles of Fermentation Technology-Butterworth
Heinemann (2017)
2. Reddy, C. A._ Beveridge, T. J._ Breznak, J. A._ Marzluf, G. A._ Schmidt, T. M._ Snyder, L. R. (Eds.) - Methods
for General and Molecular Microbiology-American Society for M (1)
3. Murray Moo-Young, Michael Butler, Colin Webb, Antonio Moreira, Bernard Grodzinski, Z F Cui, Spiros Agathos -
Comprehensive Biotechnology, Second Edition -Pergamon (2011)

4. Waites M.J., et al. - Industrial microbiology_ An introduction-Blackwell (2001)

5. Nduka Okafor, Benedict C. Okeke - Modern Industrial Microbiology and Biotechnology-CRC Press (2017)

6. Berenjian A - Essentials in Fermentation Technology: Springer International Publishing (2019)

Lecture Learning Outcomes
▪ By the end of the lecture, students should be able to demonstrate knowledge of:
1. Microbial enzymes and methods of stabilization.
2. Biotransformation and applications.
Interactive teaching methods and activities:
1. Youtube Video:
▪ https://www.youtube.com/watch?v=W95w3D2ZtUk
▪ https://www.youtube.com/watch?v=psjAe4aTmKI

2. Previous exams questions discussion

3. Quizziz:
https://quizizz.com/admin/quiz/6238e4641e261d001e78c2db?sourc
e=quiz_share
Microbial Enzyme
Technology
Enzymes
• Enzymes are complex protein molecules synthesized by living cells where they act-
- -
as catalysts that facilitate
-
-

chemical reactions → They act as “biological catalysts”.

- -

• Although enzymes are only formed in living cells, many can continue to function in vitro.
Enzyme Technology:
• Production, isolation, purification and use of enzymes in food, medical and household industries.
Advantages of enzymes over non-biological (chemical) catalysts:
very specific
1. ↳
Specificity for reactants (substrates) and for the susceptible bond involved in the reaction.
2. High catalytic power → enhance the rate of the reaction by a factor in the range 105–1014.
3. An enzyme carries out its function without being consumed in the reaction.
-

&
4. -
A minute amount of enzyme can react with a large amount of substrate. *
G
I
&
S
-it
↑

5. They catalyze reactions over a wide range of temperatures and pHs.

6. protein
Non-toxic and biodegradable is

Is
* ↑
S & 5
5
8
Microbial enzymes

◼ Enzymes could be obtained from different biological sources → animals, plants and microorganisms.
----

Advantages of using microbes for enzyme production

✓ They are grown economically in bulk fermenters.

✓ They have high growth rates.

✓ They contain a larger proportion of enzymes in relation to their body mass than plants or animals.

✓ Their growth is not limited by season or climate.

✓ They occur naturally in a wide range of habitats, and so some contain enzymes that will function under

(extremes of temperature or pH.J

✓ They can be genetically manipulated to produce any desired enzyme.
Applications of microbial enzymes

Medical applications
-Stren
1. Fungal acid amylases:
- - -

▪ Treatment of dyspepsia (indigestion), because they are acid resistant.

2. Fungal dextranase: polysaccoride
-

▪ Incorporated into the toothpastes to remove dextrans deposited on the

prevent Biofilm-L
teeth
--
by Streptococcus mutans → to help fighting dental decay.
3. L-asparaginase:
-

▪ Produced from E. coli and other Gram-negative bacteria → treatment of

leukemia → the inability of leukemic cells to synthesize asparagine results in
cytotoxicity specific for leukemic cells that depend on an exogenous source
of the amino acid asparagine for their protein synthesis.
hornal Cell can produce
concer cell connot produce asparagin
it by it self 5) Ic
Applications of microbial enzymes

Medical applications
allergy
4.
-Penicillinases:
-

▪ Emergency cases of penicillin hypersensitivity.

6h only
5. D
Proteases: &I ↑
in

▪- >
Digestion of blood clots by dissolving
-
fibrin (as streptokinase enzyme from
- -

Streptococcus sp.) → Treatment of thrombosis.

6. -
Lactase (ß-galactosidase)
- enzyme:
▪ Production of Lactose-free milk for patients with lactose intolerance.
▪ Milk is passed through a column containing immobilised lactase enzyme,
which hydrolyses lactose to glucose and galactose.
Applications of microbial enzymes

Industrial applications
< &1) % g4-

1. Biological washing powders (lipase enzyme).

-

2. Pre-digesting baby food so babies can eat it more easily.

3. Fruit juice production (pectinase enzyme: for degradation of pectin and facilitating juice
extraction)
4. Meat tenderization (make it easier to chew and digest).
Match the following enzymes to their possible applications:
Fungal Washing powders
dextranase
Fungal amylase Toothpastes
Asparginase Fruit juice
Lipase Leukemia
Pectinase Dyspepsia
Production of microbial enzymes

Producer Organism

◼ Most common Fungi: Aspergillus species, Trichoderma species.

◼ Most common bacteria: Bacillus species, Streptomyces species, and

Kluyveromyces species.

◼ Recombinant organisms: To produce industrial enzymes of very high quantity and purity,
genes coding candidate enzymes for industrial use can be cloned into a more suitable production
host microorganism. fungi
Fermentation process → Submerged or solid state fermentation.

T
bast
Production of microbial enzymes

Fermentation medium

◼ Raw materials → readily available in large quantities at low cost and nutritionally safe. E.g;
starch hydrolysate, molasses, corn steep liquor, whey and many cereals.

◼ Enzyme synthesis in microorganisms is often repressed, i.e. the enzyme will only be produced in

A
the presence of an inducer molecule, most often the substrate. E.g; starch for amylase
added
production. inducer should be
enzine
metabolic ,

substrate
when it find its
make entine
>
only
-

are Found
Production of microbial enzymes

Downstream processing
◼ Microbial Enzymes are either
extracellular or intracellular.
a. Extracellular enzymes (Exoenzymes):
-

▪ E..g; amylase → obtained from the

culture filtrate. Lybse
·

b. Intracellular enzymes (Endoenzymes)

-

▪ E.g; Taq polymerase → obtained

-

from the cell homogenate after cell

lysis.
Enzyme stabilization against inactivation

▪ Although a good industrial catalyst should be stable under operating conditions for a long period of time, most
enzymes are easily inactivated by heat, chemical agents, proteases, or radiations. that cause
-
- -

denaturation
Methods used for enzyme stabilization against inactivation include:

1. Isolation of very stable enzymes from microorganisms capable of living in I extreme environmentsJ
>
-

> Stable microb

(thermophiles and halophiles).
2. Immobilization
-

3. Chemical modification of the molecular protein surface

4. Genetic and protein engineering techniques
substrate of enzyne if it with
the it will stabilite it
5. Addition of stabilizing compounds:
a. Substrate stabilization: E.g; Amylase → stabilized by adding starch.
>
- -

b. Solvent stabilization: Many solvents stabilize the enzyme when used at low concentration. However,
high concentration cause denaturation of enzymes.
c. Cation stabilization: E.g; Ca++ ions → stabilize the tertiary structure of proteases and amylases.
Enzyme immobilization

• Immobilization:
• A procedure specifically designed to limit freedom of movement of a biocatalyst in solutions.
• It involves attachment of the enzyme to or location within, an insoluble support material.
• The enzymes are physically confined during a continuous catalytic process and may be
recovered from the reaction mixture and reused over and over again, thus improving the
economy of the process.
• Immobilized Enzyme
• An enzyme whose movement in space has been restricted either completely or to a small limited
region or converted from a water-soluble mobile state to a water-insoluble immobile one.
Advantages of immobilized enzymes
Advantages

1. Immobilized enzymes are more stable than soluble enzymes.

2. The ability to stop the reaction rapidly by removing the enzyme from the reaction solution.
3. Multiple and repeated use of a single batch of the enzyme → low cost
4. Simple separation of biocatalyst from product after reaction →
Downstream proces
▪- easy for
>
Less expensive extraction
▪ Less contamination of the product by the enzyme
5. Allow development of a multienzyme reaction system.
6. Reduce effluent disposal problems.
Disadvantages of immobilized enzymes
Disadvantages

1. The cost of enzyme is high.

-

2. The activity is sometimes inferior to free enzymes because of undesirable conformational

invobilitation
entine
can be damage during
changes resulting from immobilization.
3. Not all enzymes can be immobilized by general methods. Immobilization protocols must be
developed in such cases. -Some enzymes
need specific immobilizing
methods
unvirsal
> no
-
way
Enzyme immobilization

enzyme (1815. &

• Whole Cell immobilization:
• Enzymes can be immobilized as either the isolated form or the whole cells.
-

• The cells can be immobilized in a viable or a nonviable form.

• Benefits of whole-cell immobilization: benefit of the multienzine
in
Whale Cells
1. It avoids expensive procedures for enzyme purification.

S
2. It preserves enzymes in its natural environment thus protecting them from inactivation.
&

• Drawbacks: The limitation in the diffusion of substrates and products through the cell wall →
This can be solved by permeabilization of intact cells before immobilization.
- avoid Downstream procedures
denaturation of enzyme
- avoid the
enter the tubde Cell
comet
-substrate
should be add
enhancer
cell permeablity enzymatic Lysozyme
Surfactant
eX8 Chemical pencilline or
-
Enzyme immobilization

• Whole Cell immobilization:

X
• Permeabilization of cells before immobilization:
• It allows for enhanced access of substrates or reagents to the intracellular components of
bacteria while retaining the structural integrity of the cells.
• Methods:
1.Physical Methods: Using sonication or electroporation.
- -

2.Chemical Methods: Using one of the following methods:

a. Surfactants: E.g: Triton X-100 or Tween.
- - -

b. Organic Solvents: E.g: Ethanol, methanol, or acetone.

c. Enzymes such as lysozyme.
3.Biological Methods: Using bacteriophages
---
or antimicrobial peptides.
Enzyme immobilization methods

Covelent bond -
Ionic bond
-
no covalent bond

Cross linking
Enzyme immobilization methods
1. Physical adsorption:
use weak bond bond
▪ The simplest method
Enzyme
▪ The enzyme is reversibly attached to theL
support material (carrier) by weak noncovalent

Support material (carrier)

- -

-
-
electrostatic forces such as Van der waals force, hydrogen bonding or ionic bonding. Enzyme
Advantages:
1. Simple, cheap, quick, no chemicals are required. Enzyme
- -

2. Little or no damage to the enzyme.

-

temp or Enzyme
3. Easily reversed to allow regeneration of the carrier. upon chaging
PH
Disadvantages:
▪ Since the interaction between the enzyme and support material is weak, leakage of the
-

enzyme may occur with environmental changes (pH, temperature) → product

-

contamination.
Enzyme immobilization methods

#
2. Entrapment and encapsulation
- -

Enzym
▪ The enzyme is trapped into a gel matrix, such as polyacrylamide gel or e

encapsulated in liposome. Sodium alginate Enzym

= Enzym e
e
Advantages
mixing
▪ No chemical modification of enzyme.
-
physcal Enzym
e
Enzym
e

Disadvantage:
Encapsulation
1. Difficult preparation
2. Leakage of the enzyme
Enzym Enzym
e e
3. Reduced contact between the substrate and the enzyme.
4. Not effective for macromolecular substrates. Enzym Enzym
e e

Entrapment
Immobilization of yeast cells in Sodium alginate beads
https://www.youtube.com/watch?v=Uq6jZZKocgs&t=302s
Enzyme immobilization methods
Chemical reaction between the
3. Covalent bonding surfale
Congroups) in solid support -

Cot e
Contain NH
▪ The most widely used method.
and between
to make conface of enzyne
Enzyme
▪ A covalent bond is formed between: [ reaction must not done active site

Support material (carrier)

this on

1. Functional groups present on the surface of a support material (E.g; Enzyme

-
-OH groups in polysaccharide polymers such as cellulose, dextran, or
agarose) Enzyme

2. Functional groups on amino acid residues on the surface of an

Enzyme
enzyme (E.g; NH2-, -COOH, -OH, -SH) that are not essential for
-
&
=>

catalytic action.
Advantages:
▪ Not affected by pH, ionic strength of the medium or substrate concentration.
Disadvantages:
▪ Active site may be modified. use Cherial
▪ High cost. how d
Enzyme immobilization methods

4. Cross-linking . -

▪ Enzyme molecules are covalently bonded to each other toL

create a Enzym Enzym Enzym
-
e e e
matrix consisting of almost only enzyme.
Leakage
-
Enzym Enzym Enzym
Advantages: chanical no e e e
any
▪ No enzyme leakage. Enzym
e
Enzym
e
Enzym
e

Disadvanatges:
▪ Loss of enzyme activity during preparation.
▪ Not effective for macromolecular substrates.
- -

>
▪ Regeneration of carrier not possible.
--

COVa
Ich bond
in &
Applications of immobilized enzymes: Biosensors
- ja
S
Biosensors
▪ A biosensor is an analytical device, used for the detection of a chemical
-
-

substance.
-
-

▪ Biosensors combine a biological component with a physicochemical detector.

▪ They convert biological reactions into--
-
electrical signals in order to quantify
chemicals.
-

▪ Biosensor are used for assay of substrates such as → urea, amino acids, glucose,
=
-
-

alcohol and lactic acid.

-
Applications of immobilized enzymes: Biosensors

Advantages of biosensors over most ‘traditional’ analytical methods:

-

▪ They incorporate biologically active molecules → high specificity → high discriminatory power →
quantification of particular molecular species from within complex mixtures of other materials
with similar structure that may be present at comparable or higher concentrations → Samples can
be analyzed with little or no prior clean-up. active
Contain biologically
be added
Whale cell can are high
-

holecule which
detect the
enzyme specific can
-

-
antibody -> antigen Substrat within complex
of other similar
mixtures
materials
 What is a biosensor?
https://www.youtube.com/watch?v=W95w3D2ZtUk
Applications of immobilized enzymes: Biosensors

Principle of enzyme electrodes

▪ The electrode is composed of a given electrochemical sensor in close contact with a thin
permeable membrane with embedded enzymes capable of reacting specifically with the given
substrates.
▪ Enzymatic reaction → a small molecule is produced or consumed (e.g. O2, H+, CO2) → detected by
the specific sensor → The magnitude of the response determines the concentration of the substrate
▪ The biological component in a biosensor may also be an antibody, nucleic acid, whole cell or
enzyme.
Glucose Electrode (Biosensor)
Biosensor be used
Why glucose
can

Use in caluase detection in Blood

▪ Determination of “glucose concentration”. without
preprocessing
Composition:
▪ It consists of platinum metal coated with glucose oxidase, an
&
- -

enzyme extracted from cows, and a semi-permeable membrane.

Principles →
Biosenser Function is

▪ The immobilized D
glucose oxidase catalyzes the oxidation of
Based on
entine
glucose by molecular oxygen producing gluconic acid and
enzyme
are very specific
hydrogen peroxide. substrate
for one

▪ As the peroxide reacts with the platinum metal → it produces a

current that is proportional to the concentration of glucose in the
blood → The current is then converted into a glucose
concentration.
 immobilized glucose oxidge
acid + hydrogen peroxide
-

glucose-gluconic interact
hydrogen peroxide
will
>
-
in Biosensor every
metal
with the
platinum glucose concentration
current that are measure as
>
-

produce

Microbial Biotransformation
Biotransformation
▪ Biotransformation = bioconversion = microbial transformation:
▪ The processes in which microorganisms/enzymes convert organic compounds into structurally
related products.
▪ Uses of biotransformation
▪ To synthesize compounds when chemical methods are difficult or more expensive.
▪ To reduce toxicity of chemical compounds (by conversion into less toxic chemicals).
▪ Advantages of biotransformation over chemical reactions
1. Substrate specificity
mark on L mat D
2. Stereospecificity
3. The ability to operate at near neutral pH, ambient temperatures and atmospheric pressures.
- - - -

Unlike chemical reactions that often requires extremes of these conditions.

4. Less or no environmental pollution
-
Biotransformation
Biocatalysts used in biotransformation
1. Enzymes or immobilized enzymes
2. Growing cells → the strain used is cultivated in a suitable medium and a concentrated
substrate solution is added after suitable growth of the culture (6-24h). high activity
3. Resting cells → cells are grown to a desired concentration, harvested and resuspended in a
buffer supplemented with a carbon source and the substrate but not nitrogen source →
unable to synthesize protein → enter a non-growing state.
4. Immobilized cells → the process can be carried out continuously and the cells can be used
over and over again.
5. Spores
Biotransformation Give #
en
=
Spores or resting cells are preferred over growing cultures in biotransformation
- - - -

1. No growth inhibition by substrate will inhibit

2. More resistant to toxic byproducts if the substrate one
ic
the growth
3. High cell densities that increase productivity may be used
T
Do not wait for it to grow
4. Reduction of cost
I
5. -
Less time and more efficient
6. Easier product recovery → Since the transformation reaction occurs predominantly in the buffer
solution. ↑
extraction no metabolits
easy
buffer
only containing
Applications of Biotransformation
1. Conversion of penicillin G to ampicillin

▪ Semi-synthetic penicillins have various advantages over natural penicillins such as:
1. Resistance to stomach acids → can be taken orally
2. A degree of resistance to penicillinase (or β-lactamase)
3. An extended range of activity against some Gram-negative bacteria (broad spectrum
activity).
▪ Semisynthetic penicillins are prepared by biotransformation as follows:
▪ The acyl side chain of natural penicillins obtained by fermentation using strains of P.
chrysogenum is removed by microbial penicillin G acylase (also called amidase) → 6
aminopenicillanic acid (6 APA) is produced.
▪ Chemical or enzymatic addition of acyl side chain gives semisynthetic penicillins with more
desirable pharmaceutical properties (E.g; ampicillin).
Penicillin
G acylase

Penicillin G
acylase
Applications of Biotransformation
2. Sugar biotransformation x
Secret Lac
>
-

▪ Lactose in skimmed milk is hydrolyzed by Lactobacillus bulgaricus

with high -galactosidase (lactase) activity into glucose and
galactose → production of lactose-free milk products → which can
consumed by people lacking “lactase enzyme” who suffer from
“lactose intolerance”.
Applications of Biotransformation
3. Steroid biotransformation

▪ Steroids are complex organic compounds whose skeleton has 17 carbon

atoms in a tetracyclic ring system.
▪ Natural steroids include → cholesterol, bile acids, sex hormones, vitamin
D, corticosteroids, etc.
▪ Steroid-based drugs are widely used as → anti-inflammatory, anti-
microbial, contraceptives, etc.
▪ Chemical derivatives of some steroids are reported to have better
therapeutic advantages than the starting materials. caba
▪ The steroid molecule has several asymmetric centres and it makes the
so has way
-

chemical synthesis of steroid very difficult.

Applications of Biotransformation
1. Steroid biotransformation
▪ Microbial steroid transformation is a powerful tool for production of steroid active pharmaceutical
ingredients for the following reasons:
1. Bioconversion can be done at positions of steroid molecule hardly available for chemical agents.
2. Reactions are regio- and stereospecific
- -

3. Several reactions can be completed in one biotechnological step.

▪ Example:
▪ Chemical steroid synthesis involves 31 reaction steps to yield 1 g cortisone from deoxycholic acid (purified
from beef bile).
▪ Production of cortisone by biotransformation of the precursor using Rhizopus sp. and Aspergillus niger
-

reduced the cost of 1g from $ 200 to $ 1 and reduced 31 chemical steps to 11 steps.
Examples of steroids microbial transformation
Mention the role of microbial enzymes in the management of the following medical conditions:

1. Lactose intolerance
2. Thrombosis >
-

3. Dyspepsia
4. Diabetes mellitus
monitor