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Lecture 3
Microbial Enzymes

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Commercial microbial
enzymes are increasingly
replacing conventional
chemical catalysts in many
industrial processes

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Enzymes function Enzymes are

under relatively specific, often
mild conditions stereoselective,
catalysts
pH
Refining
Temp Pressure
Advantages Purification

Certain
enzymes are enzyme-based
not restricted water– processes are
to aqueous organic ‘environmentally
environments solvent
friendly’
non-aqueous
organic media Biodegradable
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Industrial Bioconversions

Immobilized
microbial cells

Suspended
microbial cells 6

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There are some

Limitations of Whole-
Cell as Biocatalyst

The cells ‘waste’ energy and resources in

growth and/or maintenance activities

Side reactions may lead to a reduction in

the potential yield of the target product

Conditions for microbial growth,

where required, may be different
from those necessary for optimum
product formation

Difficulties may be
encountered in the isolation
and purification of the
product from the cells or
spent fermentation medium 8

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Partially Purified Bulk

Microbial Enzyme

partially purified
‘bulk’ microbial preferred for
enzyme numerous & varied
preparations industrial processes

whole conventional multienzyme

Replaced
microbial fermentation systems

more efficient higher

substrate yields
utilization greater
product
uniformity
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Several thousand tonnes of commercial enzymes are

currently produced each year, which have a value in excess
of US$1500 million

few animal and most commercial enzymes are

plant enzymes obtained from microbial sources

Bacillus spp. extracellular enzymes

Proteases Amylases

Demand for the thermostable enzymes

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Applications of bulk
microbial enzymes

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DIFFERENT TYPES OF ENZYME:

Proteases:
Proteases are the most
widely used enzymes in
the detergent industry.
Proteases hydrolyze
proteins and break them
down into more soluble
polypeptides or free
amino acids. As a result
of the combined effect of
surfactants and enzymes,
stubborn stains can be
removed from fibres.

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Lipases:
oily and fatty stains have
always been troublesome
to remove. The trend
towards lower washing
temperatures has made the
removal of grease spots an
even bigger problem. The
lipase is capable of
removing fatty stains such
as fats, butter, salad oil,
sauces and the tough
stains on collars and cuffs.

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Amylases:
Amylases are used to remove residues of starch-based foods
like potatoes, spaghetti, custards, gravies and chocolate.
This type of enzyme can be used in laundry detergents as
well as in dishwashing detergents.

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Cellulases:
Cellulase enzyme has properties enabling it to modify the
structure of cellulose fibre on cotton and cotton blends.
When it is added to a detergent, it results into the following
effects:

1. Color brightening
2. Softening
3. Soil removal

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Fine High-Purity Enzyme Preparations

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General microbial enzymes of industrial use

ENZYME REACTION SOURCE APPLICATION

α- Amylase Starch Bacillus species Conversion of starch to glucose

hydrolysis or dextrins in food industry

Amyloglucosidase Dextrin Aspergillus species Monomeric glucose production

hydrolysis

Β- galactosidase Lactose Aspergillus sp Hydrolysis of lactose in milk or

hydrolysis whey

Glucose isomerase Glucose to Streptomyces Production of high fructose

fructose Species syrups for artificial honey
conversion

Aminoacylase Hydrolysis of Aspergillus sp artificial sweetners and

acylated L- different mixtures production
amino acids
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Clinical/pharmaceutical prospective of microbial enzymes

Enzyme Reaction Source Application

L- asparaginase Removal of L- E. coli Cancer

asparagine essential chemotherapy
for tumour growth
Urokinase Plasminogen Different Removal of fibrin
activation clots
Amylase and Hydrolysis Fungal or bacterial Wound debridement
protease and digestive aids
penicillin Relieves pain Penicillium sp

Glucose oxidase Glucose oxidation Aspergillus niger Detection of glucose

in blood
Urease Urea hydrolysis to Bacteria Measurment of urea
CO2 and NH3 in body fluids
DNA Polymerase chain Thermus aquaticus DNA synthesis, e.g.,
polymerase reaction in PCR for forensic
analysis
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An example of a
purification
strategy for DNA
polymerase

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Recap….

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Cont…

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IPTG : Isopropyl β-D-1-thiogalactopyranoside- is used to induce protein expression

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Commercial Microbial Enzyme Production

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Microbial enzymes are predominantly produced by…

Extracellular Fungal Enzymes

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The first commercial microbial enzyme preparation

was produced via solid-substrate fermentation

‘Takadiastase’
a fungal amylase

culturing Aspergillus oryzae

on moist rice or wheat bran

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The process was

initially developed by

patented in the USA

in 1884

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large-scale production was possible after improvements to

submerged fermentation technology that followed the
development of penicillin fermentations in the 1940s

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Most industrial
enzymes

Few are currently

produced

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suitable
producer
organism

Development of any
fermentation process,
enzyme production
processes

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Classification of microorganisms based on temperature

3 types: microorganisms are classified in three groups

according to their temperature preferences:

Psychrophilic organisms (psychrophiles) prefer cold

temperatures of about 0°C to 20°C.

Mesophilic organisms (mesophiles) prefer temperatures

at 20°C to 40°C.

Thermophilic organisms (thermophiles) prefer

temperatures higher than 40°C . (our preferable)
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Bulk industrial
enzyme production

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enhance enzyme
productivity

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Immobilization of enzymes
Why is it important to choose a method of attachment?
-prevent loss of enzyme activity

-To avoid reaction

- The structure is retained in the enzyme through hydrogen
bonding or the formation of electron transition complexes:
prevent vibration of the enzyme and increase thermal
stability
Broadly, There are 3 different methods:
Carrier-Binding: the binding of enzymes to water-insoluble carriers
Cross-linking: intermolecular cross-linking of enzymes by bi-
functional or multi-functional reagents.
Entrapping: incorporating enzymes into the lattices of a semi-
permeable gel or enclosing the enzymes in a semi-permeable polymer
membrane
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 Carrier-Binding
 The oldest immobilization technique for enzymes
 Some of the most commonly used carriers for
enzyme immobilization are polysaccharide derivatives
such as cellulose, dextran, agarose, and
polyacrylamide gel.
 The selection of the carrier depends on the nature
of the enzyme itself, as well as the:
- Particle size
- Surface area
- Molar ratio of hydrophilic to hydrophobic
groups
- Chemical composition
 the carrier-binding method can be further sub-
classified into:
– Physical Adsorption
– Covalent Binding
– Ionic Binding 40

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1-1 : Physical Adsorption

Immobilization of the enzyme based on the
surface of water-insoluble carriers.

Advantages : no reagents and only a minimum of

activation steps are required

Disadvantages : the adsorbed enzyme may leak

from the carrier during use due to a weak
binding force between the enzyme and the
carrier. Moreover, the adsorption is non-
specific, further adsorption of other proteins
or other substances
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1-2 : Covalent Binding

Based on the binding of enzymes and water-insoluble
carriers by covalent bonds
The functional groups that may take part in this binding
are Amino group, Carboxyl group, Sulfhydryl group,
Hydroxyl group, Imidazole group (Histidine), Phenolic
group, Thiol group, Threonine group, Indole group
(tryptophan).
Advantages : the binding force between enzyme and carrier
is so strong that no leakage of the enzymes occurs.
Disadvantages : covalent binding may alter the
conformational structure and active center of the
enzyme, resulting in major loss of activity and/or
changes of the substrate

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1-3 : Ionic Binding

Immobilization of the enzyme based on to
water-insoluble carriers containing ion-
exchange residues
Polysaccharides and synthetic polymers having
ion-exchange centers are usually used as
carriers
Advantages : Mild condition is necessary than
covalent bonding.
Disadvantages : leakage of the enzyme due to
altered pH.
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 Cross-Linking
Either to other protein molecules or to
functional groups on an insoluble support
matrix
Advantages : It is used mostly as a means
of stabilizing adsorbed enzymes and also for
preventing leakage from polyacrylamide gels
The most common reagent used for
cross-linking is glutaraldehyde
Disadvantages : Cross-linking reactions are
carried out under relatively severe
conditions. These harsh conditions can
change the conformation of active center of
the enzyme; and so may lead to significant
loss of activity.
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 Entrapping Enzymes

lattice type micro capsule type

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 Entrapping Enzymes continuation

Based on the localization of an enzyme within the

lattice of a polymer matrix or membrane
It can be classified into 2 types: lattice and micro
capsule types.
This method differs from the covalent binding and
cross linking in that the enzyme itself does not bind
to the gel matrix or membrane. This results in a wide
applicability
Disadvantages : The conditions used in the chemical
polymerization reaction are relatively severe and
result in the loss of enzyme activity.
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Lipase

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Glucose Isomerase starch

processing
industry

‘high fructose corn syrup’

(HFCS) or ‘isosyrup’

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Invertase

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Enzymes in Cheese Production

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chymosin, an
aspartic protease

limited proteolysis of
milk protein (casein)
to form curds

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Enzymes in Plant Juice Production

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Arabanases

Araban: - A polymer of the pentose arabinose

-An important component of fruit cell walls.
-Like pectin, it is often extracted during
pressing of some fruits(eg. Pears).
-This may lead to haze formation
But now it can be eliminated by using
commercial arabanases.

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Commercial Microbial Carbohydrases

cellulases

glycosidases
naringinase

Glucose
converts naringin oxidase
to the less bitter
compound prunin
remove molecular
oxygen
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