ppt on Production of enzyme

1. ekelle university
f natural and computational science
MAIREG
KEBEDE
PRODUCTION OF
ENZYMES
COMPAILED
BY:-
UNIVERSITY OF
GONDAR
INSTITUTE OF BIOTECHNOLOGY
Advisor Dr. TAMENE M.

2. © Introduction
© Source of enzymes
© Enzymes from animal sources
© Enzymes from plant sources
© Enzymes from microbial sources
© Production of animal and plant enzymes
© Production of microbial enzymes
© Methods of upstream processing
© Downstream processing
© Large scale production of enzymes
© Application of enzymes in industry
© Conclusion
2

3. Enzymes are biological catalysts initiate and accelerate thousands of biochemical reaction by
decreasing activation energy with out being changing it self in the overall process.
Activation energy is the amount of energy required to activate chemical reactions to occur
With the exception of a few catalytic RNA molecules, or ribozymes, the vast majority of
enzymes are proteins
Enzymes catalyze chemical reactions with great specificity and rate enhancements.
3

4. 4

5. Production of Enzyme has greatly expanded since 1960s due to well designed fermentation
technology and genetic engineering
So far more than 2500 enzymes have been isolated and characterized which is only 1/10 of
the full potential enzymes of the nature
More than 1000 enzymes are recommended for various application
More than 60% of the enzyme production occur in Europe, 15% in USA and 15% in Japan
At present the need of enzymes are highly increasing in various sectors
Recombinant microorganisms are now becomes dominant source of enzymes production
Biologically active enzymes may be extracted from any living organism
5

6.  Animals sources
 Plants sources
 Microbial sources
From enzymes being used industrially:
a) More than 50% are from fungi and yeast
b) Over a third are from bacteria (33%)
c) Animal (8%) and
d) Plant (4%)
Approaches used to increase market need of enzymes
 Less expensive production process and cost reduction
 Identification and development of new enzymes from new source
 Finding new uses for existing enzymes
6

7. Around fifty years ago, enzymes were produced strictly from animals
Animal enzymes are enzymes derived from the pancreas, liver, or stomach of animals such
as pigs, oxen, and cows
Pigs or cows were butchered and enzymes were extracted from their pancreases
Animal tissues and organs provide excellent sources for some lipases, esterases and proteases,
rennets, trypsin
Hen egg is also good source of lysozyme
Problems Associated With Animal Enzymes
 Not very stable at the low pH (acidic) environment of the stomach
 If taken orally much of the enzyme destroyed before doing the job
 Animal enzymes were quite temperature sensitive and unfortunately, we don't have the same
body temperature as many other animals
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8. Plant enzymes were discovered and used next to animal
These are animal-friendly enzymes
Much more stable under low pH conditions, such as inside the stomach
Temperature changes don't seem to affect
Most prevalent plant enzymes are papain, proteases, amylases and soya bean lipoxygenase
which are used in various food industries
Papain extracted from papaya fruit is used as meat tenderizer
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9. Although many useful enzymes have been derived from plant and animals, most future
developments in enzyme technology will rely on enzymes of microbial origin
Microbes provide numerous enzymes that perform multiple body functions
 There are two types of microbial enzymes:-
1. Intracellular Enzymes: enzymes that are produced in the cell and needs certain techniques
for extraction
 Its extraction is bit challenging
 Can be obtained by breaking the cell by homogenizer or bead mill and extracting them via
biochemical processes
E.g:- Invertase, Uric Oxidase, Asparaginase
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10. 2. Extracellular Enzymes: excreted out of the cell
 This group of enzyme is very attractive since it is released to external medium making down
stream processing easier. E.g. enzymes from Bacillus sp.
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Intracellular Enzymes Extracellular Enzymes
More difficult to isolate Easer to isolate
Cells have to broken apart to release them No need to break cells secreted in large
amount in a medium surroundings
Have to be separated out from cell debris and
a mixture of many enzymes and other
chemicals
Often secreted on their own or a few other
enzymes
Often stable in the environment in side intact
cell
More stable
Purification /downstream processing is
difficult/expensive
Purification downstream processing is easier
and cheaper

11.  Microbial enzymes are extracted from fermented bacteria or fermented fungal organisms and tested for
effectiveness and safety in humans
 They are chosen for their stability in different environments and for their ability to perform a valuable
enzymatic function in our body
Microbes are preferred from plants and animals as sources of enzymes because:
1. They are generally cheaper to produce
2. Needs short period of time for production
3. can be genetically manipulated to increase the yield of enzymes
4. Capable of producing a wide variety of enzymes
5. Plant and animal tissues contain more potentially harmful materials than microbes
6. Can grow in a wide range of environmental conditions
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12. Production of animal and plant enzymes starts with preparation of biological starting materials
 For animals, organs must be transported and stored at low temprature.
 Organ should be free of fat and other tissues
 Organ is then minced and enzyme extracted with buffer solution
 For plants, plant material can be ground with various grinders at low temprature and enzyme then
extracted with buffer solutions
 Lytic enzymes can also replace grinding step
 Steps of enzyme extraction and purification from plants and animals tissues
1. Extraction of enzymes
2. Preparation of crude enzymes
3. Purification of enzymes
4. Final processing of enzymes
12

13. 1. Extraction of Enzymes
 Enzymes are very sensitive to physiological change
 Slight change may reduces (inactivate) it’s activities.
 Has to isolated under very controlled condition of pH, temp, ionic strength etc…
 Since majority of enzymes are proteins extraction and purification procedures are the same with proteins
 However activity of enzyme needs to be assayed at every step
 If the extract is not homogenous, the extract may be filtered to remove cell debris, and other
components
 All operation of extraction and purification are generally carried out in cold room (0-4 0C)
 First fresh tissue has to crashed into paste with an extraction medium (buffer) in mortar and pestle
 Molarity and pH of buffer should be appropriate to achieve maximum solubility and activity
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14. EDTA is often included in extraction medium, to remove heavy metal that inhibit activity of an
enzyme
 For disrupting membrane and cell organelles; Detergent like triton-X used for solubilizing
membrane
 Many enzyme has disulfide bond (s-s) due to the presence of cysteine residues which may be
broken during extraction and leads to loss of activity
2. Preparation Of Crude Enzymes
Can be done by centrifugation and precipitation
A. Centrifugation
 Enzyme extract centrifuged to remove cell debris, cell organelles and other aggregates leading to
partial purification of enzymes
B. Precipitation
 Enzymes are charged molecules and can be precipitated with appropriate charge neutralizing chemicals
 Ones there charge is broken, they form an aggregate and settle down as precipitate
14

15.  Adjusting the pH to PI with acid or base causes precipitation
 However acid and bases often inactivates the enzyme
 Hence, there use is not highly recommended
 The other alternative to acid and bases are to use ammonium sulfate and other salts to precipitate
by the process of salting out
 Use of neutral salts are recommended than NH4SO4 since it is corrosive and release
NH3 at high temperature
 Some water soluble organic solvents like acetone, methanol, ethanol can also be used for enzyme
precipitation
15

16. 3. Purification Of Enzymes
Once crude enzyme preparation is made, purification can be achieved by common protein purification
methods
 Purification of plant and animal enzyme is similar with techniques employed for
microbial enzyme
4. Final Processing Of Enzymes
Finally purified enzymes can be concentrated and made available for various purposes
Shelf life of enzymes can also be modified and increased
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17. Production of microbial enzymes involves:-
A) Upstream Processing
B) Downstream Processing
A. Upstream Processing:- Production parts
 Isolation of Microorganisms & Strain development
 Medium formulation and Preparation
 Sterilization and Inoculation of medium
B. Downstream Processing: extraction and purifications steps
 Grinding of cell for intracellular enzymes
 Cell separation from media and enzymes usually by
 Centrifugation
 Precipitation
 Various filtration techniques
 Dialysis and chromatographic techniques
 Concentration and final processing 17

18. 1. Isolation of Microorganisms, Strain Development and Preparation of Inoculum
Microorganisms are isolated on culture media following the microbiological techniques
Aim for isolating a suitable microorganism
a. production of enzyme in high amount and other metabolites in low amount
b. completion of fermentation process in short time
c. utilization of low cost culture medium
Once a suitable microorganism is obtained its enzyme producing ability is optimized by improving
strains and formulating culture medium
 pH
 Temperature
 Carbon source
Strains of microorganisms can be developed by using mutagens
Inoculum of strains developed after treatment of mutagens by multiplying it on liquid broth
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19. 2. Medium Formulation and Preparation
Culture medium is formulated in such a way that
 It should provide all nutrients supporting enzyme production in high amount, but not for good
microbial growth
 Ideal medium must have a cheap source of carbon, nitrogen, growth promoters, trace elements and
little amount of salts
 Appropriate fermentation pH, To should be maintained
pH, temperature and formulation of culture medium is optimized prior to inoculation
Production of enzymes increases with the concentration of culture medium
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20. 3. Sterilization and Inoculation of Medium, Maintenance of Culture and Fluid Filtration
Medium is sterilized batch-wise in a large size fermenter
Continuous sterilization method is also now becoming popular
After medium is sterilized, inoculation with sufficient amount of inoculum is done to start fermentation
process
 Two Types of Inoculation/Culture Methods
1. Surface Culture Technique: inoculums remains on upper surface of broth
 still in use for production of some of the fungal enzymes
2. Submerged Culture Method: is most widely practiced because of less chances for infection and
possibility for more yield of enzymes.
After 30-150 h incubation, extracellular enzymes are produced by the inoculated microbe in culture
medium
Most of enzymes are produced when exponential phase of growth completes but in a few cases, they are
produced during exponential phase
When fermentation is over broth is kept at 5°C to avoid contamination 20

21. Intracellular Microbial Enzyme Extraction
The first step of intracellular enzymes extraction starts with cell breakage
Many enzymes of fungi and Gram -ve bacteria can be extracted by toluene or chloroform that help to
disrupt cell membrane and cause enzyme leakage
Water soluble solvents (ethanol and propanol) can also be used to extract enzymes from periplasmic
space
In some cases alkaline lysis, lysozyme and other techniques may be used to remove the membrane
Ultra sonication may also be employed for some microbes
Extracellular Microbial Enzyme Extraction
This group of enzyme released to outside medium
Easy to recover and purify
Often simple cell removal and concentration of enzyme can suit some application
Purification steps are the same with Intracellular enzymes once the enzymes are released from the cell
21

22. 22
What is mean by enzyme
purification and its aim???

23. What is enzyme purification ?
Extraction of a single enzyme/protein from many different proteins and lots of other biomolecules found
in the same crude solution
23
 The maximum possible yield
 The maximum catalytic activity
 No degraded or inactivated enzymes
 The maximum possible purity
 No other enzymes or large molecules
It is important to study enzymes in a simple system
 Small ions, buffer molecules, cofactors etc…
For understanding its
 Structure, kinetics, mechanisms of regulations, role in a complex system

24. 24
Enzyme purification involves three major steps
1. Dialysis
2. Chromatographic Separation
3. Electrophoresis
1. Dialysis
Enzyme precipitate of crude extract is dissolved in small quantity of buffer solution in which the enzyme
was originally extracted
This process used to remove
 Small molecules from Enzymes through the use of semi-permeable membranes usually dialysis bag
Pores in the membrane allow
 Solvents, Salts , small metabolites to diffuse across but block enzymes

25. 25
Use of Dialysis to Separate Small and Large Molecules
The dialysis may takes place for several hours with regular change of outer solution
For large scale purification diafiltration is used instead of dialysis

26. The most common method of enzyme purification
The mixture of substances (enzymes) to be fractionated is dissolved in a;
 liquid
 gaseous fluid
In chromatography, molecules are separated based on
 Size
 Shape
 Charge
 Hydrophobic Interactions
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2. Chromatography
Mobile Phase

27. 27
Types of Chromatography Techniques
1. Adsorption Chromatography
2. Ion Exchange Chromatography
3. Gel Filtration/Size Exclusion
Chromatography
4. Affinity Chromatography
Chromatographic techniques are available
for enzyme purification
1. Adsorption Chromatography
The mixture of proteins to be fractionated is applied to column
This solution is passed through a column consisting of a porous solid matrix called the stationary
phase

28. Solvent (buffer) applied to top, flowed through column
Different proteins interact with matrix to different extents, flow at different rates
This makes advantage of size, charge and binding affinity to stationary phase
Proteins collected separately in different fractions
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2. Ion Exchange Chromatography
Ion exchangers can be cellulosic ion exchangers and gel-type ion exchangers
Cellulosic ion exchangers are most common
Proteins will bind to an ion exchanger with different affinities
As the column is washed with buffer, those proteins relatively low affinities for the ion exchange resin
will move through the column faster than the proteins that bind to the column.
The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in
eluting off the column.
Cation Exchangers bind to proteins with Positive Charges
Anion Exchangers bind to proteins with Negative Charges
If ion exchangers are acidic in nature they interact with positively charged proteins and are called cation
exchangers
CH2-COO-
Cellulose Cation Exchanger
++
+
+
+
+ Positively charged (basic) protein or
enzyme

30. If these groups are basic in nature, they interact with negatively charged molecules and are called anion
exchangers.
CH2-CH2 -NH+(CH2CH2)
DEAE Cellulose Anion Exchanger
-
-
-
Negatively charged
(acidic) protein or
enzyme
-
-
-
-
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31. 3. Gel Filtration Chromatography
Also called size exclusion chromatography or molecular sieve chromatography
Separates molecules based on size /molecular weight
Stationary phase composed of cross-linked gel particles.
Extent of cross-linking can be controlled to determine pore size
Smaller molecules enter the pores and are delayed in elution time.
Larger molecules elutes first since they do not enter the pores
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32. 4. Affinity Chromatography
Stationary phase has a polymer that can be covalently linked to a compound called a ligand
Ligand will specifically bind to protein
Many proteins can bind specific molecules very tightly
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33. 33
3. Electrophoresis
Charged particles migrate in electric field toward opposite charge
Proteins have different mobility:
 Charge
 Size
 Shape
Agarose used as matrix for nucleic acids
Polyacrylamide used mostly for proteins

34. SDS-PAGE is the most methods of separating proteins
Protein is treated with detergent (SDS) sodium dodecyl sulfate
Smaller proteins move faster (charge is similar)
34

35.  The proteins samples are having uniformed structure and charge  the separation will depend
on their molecular weight only.
 SDS-treated proteins have very similar charge-to-mass ratios, and similar shapes. During
PAGE, the rate of migration of SDS-treated proteins is effectively determined by molecular
weight.
 Small proteins migrate faster through the gel under the influence of the applied electric field,
whereas large proteins are successively retarded, due to the sieving effect of the gels.
35

36.  The large scale production of enzymes involves culturing micro-organisms in chambers called
Fermenters or Bioreactors
 They have rapid growth rates and are able to produce larger numbers of enzyme molecules per body
mass than many other organisms
 Micro-organisms can be genetically engineered to improve the strain and enhance yields
 Micro-organisms are found in a wide variety of different habitats such that their enzymes are able to
function across a range of temperatures and pH
 Micro-organisms have simple growth requirements and these can be precisely controlled within the
fermenter
 Micro-organisms can utilise waste products such as agricultural waste as substrates
36
Micro-organisms are suitable for use in the large scale production of
enzymes in fermenters because:-

37. The Biotechnological Process of Enzyme Production
SCREENING – choosing an
appropriate micro-
organism
for the desired enzyme
MODIFICATION – possible
application of genetic
engineering to improve
the microbial strain
LABORATORY SCALE PILOT
– to determine the optimum
conditions for growth of the
Micro-organism
PILOT PLANT – small scale
fermenter to clarify optimum
operating conditions
INDUSTRIAL SCALE
FERMENTATION
37

38. Commercial Enzyme Production - An Example
 Pectin is an insoluble substance found in the cell walls of plants
 In the drinks industry, juice extracted from fruits appears cloudy due to the presence of pectin
 Pectinase is an enzyme that is used in the industry to break down the pectin
 The effect of pectinase is to clarify the fruit juice and to make it flow more freely
 Pectinase is obtained from the fungus Aspergillus niger
 Aspergillus niger produces pectinase as an extracellular enzyme
PRODUCTION OF PECTINASE
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39. PRODUCTION OF PECTINASE
Aspergillus niger is grown in
a fermenter with a source of
nitrogen, with sucrose as the
carbon source and the
substrate
pectin to stimulate pectinase
production by the fungus
Filtration or Centrifugation to obtain
a cell-free system containing
pectinase in solution
Evaporate to concentrate
the enzyme
Precipitate the pectinase
out of the solution and
filter the solid
Dry and purify the crude
pectinase
Pure,
powdered
pectinase
39

40. 40
Application Enzymes Uses
Food processing Amylase, protease To produce sugars and to digest the
proteins in flour
Baby foods Trypsin To pre-digest baby foods
Brewing industry Amylase, glucanases, proteases,
Acetolactate- decarboxylase
To degrade proteins and
polysaccharides, improve the wort and
fermentation process
Fruit juices Cellulases, pectinases Clarify fruit juices
Dairy industry Rennin, lipase, lactase Production of cheese and other diary
products
Meat tenderizers papain To soften meat for cooking.
Starch industry Amylase, glucoisomerases Convert starch into glucose and simple
sugars
Paper industry Amylase, cellulase, xylanase,
ligninases
Degrade starch, aid in sizing,
decolorizing, soften the paper

41.  Enzymes catalyze chemical reactions with great specificity and rate enhancements.
 Production of Enzyme has greatly expanded since 1960s due to well designed fermentation
technology and genetic engineering
 So far more than 2500 enzymes have been isolated and characterized which is only 1/10 of
the full potential enzymes of the nature
 Intracellular Enzymes are enzymes that are produced in the cell and needs certain techniques
for extraction.While, extracellular Enzymes are excreted out of the cell
 Enzyme Purification is the process of extraction of a single enzyme/protein from many
different proteins and lots of other biomolecules found in the same crude solution
41

42.  The large scale production of enzymes involves culturing micro-organisms in chambers
called Fermenters or Bioreactors
 Pectin is an insoluble substance found in the cell walls of plants
 Pectinase is an enzyme that is used in the industry to break down the pectin
 The effect of pectinase is to clarify the fruit juice and to make it flow more freely
 Pectinase is obtained from the fungus Aspergillus niger
 Aspergillus niger produces pectinase as an extracellular enzyme
42

43. “ Our cells engage a protein
production and many of
those proteins are enzymes
responsible for the
chemistry of life”
Randy Schekman
43

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