The document discusses microbial enzymes. It begins by defining enzymes as biological catalysts produced by living organisms. It then discusses the history of enzymes, noting that Wilhelm Kühne first coined the term in 1877. The document outlines various sources of microbial enzymes from soil, food, and degrading areas. Common microorganisms that produce enzymes include bacteria, fungi, and actinomycetes. Examples are provided of specific enzymes produced by different microorganisms. The document then discusses methods for isolating and purifying enzymes, including precipitation, chromatography techniques, and affinity purification. It provides examples of enzyme purification strategies and industrial applications of enzymes in areas like baking, brewing, dairy, pharmaceuticals, and more.

1. MICROBIAL ENZYMES
By
SHILANJALI. B

2. • Enzymes are the biological substance or biological
macromolecules that are produced by a living organism which
acts as a catalyst to bring about a specific biochemical reaction.
•These are like the chemical catalysts in a chemical reaction
which helps to accelerate the biological/biochemical reactions
inside as well as outside the cell. These are generally known as
“Biocatalyst.”
•In 1877, Wilhelm Friedrich Kühne a professor of physiology at
the University of Heidelberg first used the term enzyme, which
comes from a Greek word meaning “in leaven"
•Even many centuries ago enzyme and its use were well known
to the mankind but Wilhelm Friedrich Kühne was the first
person to give a scientific terminology to this biomolecule.
•Use of enzyme has been seen in ancient Egyptians where they
were used for the preservation of food and beverages.
•Cheese making has always involved the use of enzymes, and it
goes as far as back in about 400 BC, when Homer’s Iliad
mentioned the use of a kid’s stomach for making cheese.
Wilhelm
Friedrich Kühne
1837-1900
INTRODUCTION

3. •The catalysts for biochemical reactions that happen
in living organisms are called enzymes.
•Enzymes are usually proteins, though some
ribonucleic acid (RNA) molecules act as enzymes too.
NATURE

4. NATURE
Source:- Soil, fermented food , degrading area
Samples:- Soil sample, fermented food samples etc.
Isolation of Microorganisms for example Bacteria,
fungi, actinomycetes.
Medium:- NA, SDA, SCA
Serial dilution method
ISOLATION

5. SOURCE ENZYME MICROORGANISM
Fungal Amylases Aspergillus oryzae
Glucosidases Aspergillus flavus
Proteases Aspergillus niger
Pectinases Aspergillus niger
Glucose oxidase Penicillium notatum
Catalase Aspergillus niger
Bacterial Amylases
Proteases Bacillus subtilis
Penicillinase
Yeast Invertase Lactase Saccharomyces
cerevisiae
Saccharomyces fragilis

6. • It is important to study enzymes in a simple
system (only with small ions, buffer molecules,
cofactors, etc.) for understanding its structure,
kinetics, mechanisms, regulations, and role in a
complex system
• Also isolating pure enzyme is important to use it
for medical and industrial purposes.
ISOLATION OF ENZYMES

7. TYPES OF ENZYMES
ENZYMES OF TWO TYPES:
(a) Intracellular enzymes or endoenzymes: They act
within the cell. The great majority of plant enzymes
are endoenzymes.
(b) Extracellular enzymes or exoenzymes: They diffuse
out of the cell and act on some outside medium.
•Exoenzymes are present in fungi and bacteria and in
some of the insectivorous plants.
•Endoenzymes are produced by the living protoplasm.
But they do not require environment of a living cell for
their action

8. METHODS OF SEPARATION

9. SIZE AND MASS
 Ultracentrifugation (300,000g)
 Mr is the major factor for separation
 Not very efficient to separate a enzyme from enzyme
pool : Usually used to remove impurities
 Gel filteration (Mr ~ hundreds of thousands)
 Sephadex, Bio-Gel P, Sephacryl, and Sepharose –
expensive and time-consuming
 Usually in later stage of purification
 Dialysis (Mr ~ tens of thousands)
 Usually used for removing salts, organic solvents, etc..
 Ultrafilteration
 Small molecules are filtered out by pressure
 Used for concentrating proteins
 Alternatively, centrifugation with dialysis membrane

10. POLARITY
Ion-exchange chromatography
• Electrostatic property
• Flow through in low salt and at appropriate
pH
• Desorption by changing salt conc’ and pH
• Enzymes can be separated by gradient
condition
• Large scale is possible
• Usually 10-fold increase in purity

11. POLARITY
 Electrophoresis
 Separation by movement of charged molecules
 Capillary electrophoresis (cross section less than 100 m)
 Isoelectric focusing
POLARITY
 Hydrophobic interaction chromatography
 Depending on the nonpolar amino acid on the surface of enzyme
 Octyl- or phenyl-Sepharose with high ionic strength
 Desorption by lowering ionic strength or adding organic
solvents (or detergents)

12. SOLUBILITY
 Change in pH
 Enzymes are least soluble at pI because there is no repulsive
force between enzymes
 Enzyme must not be inactivated in a range of pH
 Change in ionic strength
 Large charged molecules are only slightly soluble in pure
water; Addition of ion promotes solubility (Salting in)
 Beyond a certain ionic strength, the charged molecules are
quickly precipitated (Salting out)
 Ammonium sulfate is popularly used
 10-fold increase in purity
 Fructose-bisphosphate aldolase from rabbit muscle can be
purified in high purity by ammonium sulfate

13. SOLUBILITY
 Decrease in dielectric constant
 Addition of water-miscible organic solvent
(ethanol or acetone)
 Decrease dielectric constant
 Sometimes deactivate the enzyme
 Work at low temperature
 PEG (poly ethylene glycol) ~ Mr 4000 to 6000
is commonly used

14. SPECIFIC BINDING SITES
 Affinity chromatography
 Substrate or inhibitor is linked to a matrix
 Desorbed by a pulse of substrate or changed pH,
ionic strength

15. AFFINITY CHROMOATOGRAPHY
 Problems
 Attaching a suitable substrate or inhibitor to the
matrix can be difficult
 Linking b/n substrate and matrix itself may inhibit
the binding b/n enzyme and substrate: Spacer arm
(diaminehexane) may be needed
 Binding affinity b/n enzyme and substrate must be in
a proper range
 Special attention is necessary to separate the enzymes
using same substrate or using more than one substrate
 Fusing proteins to solve the problems
 Glutathione-S-transferase : glutathione
 Maltose binding protein : maltose
 Hexahisitidine : Ni2+ (Elution by imidazole or thrombine
cleavage site is added after the tag)

16. OTHER CHROMOATOGRAPHIES
 Affinity elution
 Affinity occur at desorption step
 Can solve some problems of affinity chromatography and easy
to scale up
 Dye-ligand chromatography
 Cibacron Blue F3G-A can bind to a number of
dehydrogenases and kinases
 Procion Red HE-3B binds well with NADP+-dependent
dehydrogenase
 Immunoadsorption chromatography
 Immobilize the antibody to CNBr treated Sepharose
 Achieve much higher purity.
 Covalent chromatography
Separation of cysteine containing
protein using thiol-Sepharose 4B

17. CHOICE OF METHOD
 Time/Large scale -> Precipitation by ethanol or
ammonium sulfate or purification based on
solubility
 Small scale/high purity -> Column
chromatography or electrophoresis
 FPLC or HPLC -> Fast and high purity, expensive

18. PURIFICATION
Objectives : maximum possible yield + maximum catalytic
activity + maximum possible purity
 Enzyme Assay procedure
 History
 Crystallization
 Homogenization + large scale separation
 Attach the affinity tag to enzyme using DNA
recombinant technology (ex. (His)6-tag)
OBJECTIVES OF ENZYME PURIFICATION

19. Strategy
STRATEGY

20. EXAMPLES OF PURIFICATION
 RNA polymerase from E. coli
 Bacterial cell extract;
highly viscous -> Deoxyribonuclease
 Oligonucleotide will be eliminated at
step 4

21. STABILITY AND APPLICATION
Several factors affect the rate at which enzymatic
reactions proceed - temperature, pH, enzyme
concentration, substrate concentration, and the
presence of any inhibitors or activators.

22. INDUSTRY APPLICATION ENZYME SOURCE
Baking and milling Bread baking Amylase
Protease
fungal, malt
fungal
Beer Mashing
Chillproofing
Amylase
Protease
malt, bacterial
papain, bromelain,
pepsin, fungal,
bacterial
Oxygen removal Glucose oxidase fungal
Carbonated beverages Oxygen removal Glucose oxidase fungal
Cereals Precooked baby foods
Breakfast foods
condiments
Amylase
Amylase
Protease
Malt, fungal
Malt, fungal Papain,
bromelain, pepsin,
fungal,
bacterial
Chocolate, cocoa
Coffee
Syrups
Coffee bean
fermentation
Coffee concenterates
amylase
Pectinase
Pectinase,
hemicellulase
Fungal, bacterial
Fungal
Fungal
Confectionery, candy
Soft center candies and
fondants
Sugar recovery from
Invertase
Amylase
Yeast
Bacterial, fungal
scrap candy
Dairy Cheese production Rennin Animal
Milk, sterilization with
peroxide Catalase Liver, bacterial
Milk, prevention of
oxidation flavor
Protease Pancreatin
Milk, protein Protease Papain, bromelain,
hydrolyzates pancreatin, fungal,
bacterial
Evaporated milk, Protease Pancreatin, pepsin,
stabilization fungal, bromelain
Fungal
Distilled beverages Mashing Amylase
Malt, fungal,
bacterial

23. Dry cleaning, laundry Spot removal Protease, lipase,
amylase
Bacterial, pancreatin,
fungal
Pharmaceutical and clinical Digestive aids Amylase Fungal, pancreatin
Protease Papain, pancreatin,
bromelain, pepsin,
fungal
Lipase Pancreatin
Cellulase Fungal
Wound debridement Streptokinase-
streptodornase,
Bacterial, animal,
plant
trypsin, bromelain
Injection for bruises,
Inflammation
Streptokinase, trypsin Bacterial, animal