Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view
2. What are
enzymes
Enzymes are proteins and are the biocatalysts synthesized by living
cells
They classified into Oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases etc…
Enzymes have been used ever since mankind discovered ways to
process food
3. History
The first enzyme produced industrially was a fungal
amylase in 1896, in United States. It was used as a
pharmaceutical agent to cure digestive disorders.
In 1901 Eduard Bucher won the Nobel prize in
biochemistry for proving the existence of enzymes
A German scientist (Otto Rohm) demonstrated in 1905
that extracts from animal organs (pancreases from pig
and cow) could be used as the source of enzymes-
proteases, for leather softening.
4. BIOTECHNOLOGICAL PROCESS
OF ENZYME PRODUCTION
1) Screening
Choosing an appropriate micro-organism for the desired enzyme
2)Modification
Possible application of genetic engineering to improve the microbial strain
3)Laboratory Scale Pilot
To determine the optimum conditions for growth of
micro-organism
4 )Pilot Plant
Small scale fermenter to clarify optimum conditions
5)Industrial Scale Fermenter
5. Different organisms contribution in
the production of enzymes
a)Fungi – 60%
b)Bacteria – 24%
c)Yeast – 4%
d)Streptomyces – 2%
e)Higher animals – 6%
f)Higher plants – 4%
7. Regulation of enzyme productions
A maximal production of microbial enzymes
can be achieved by optimizing the
fermentation conditions
Fermentation
Surface cultures ( solid-substrate cultures)
Submerged cultures ( liquid cultures)
8. Solid state fermentation
Solid state fermentation has been defined as “the
fermentation process occurring in the absence or near
absence of free water utilizing the solid substrate”.
It is a bio-molecule manufacturing process used in the
food, pharmaceutical, cosmetic, fuel and textile
industries. These bio-molecules are mostly metabolites
generated by microorganisms grown on a solid support
selected for this purpose.
This technology for the culture of microorganisms is an
alternative to liquid or submerged fermentation, used
predominantly for industrial purposes
9. Benefits of SSF
Simple and cost effective
Less effluent release, reduce pollution
Aeration is easy
Resembles the natural habitat of some fungi and bacteria
SSF utilizes solid substrate, thus nutrient rich waste materials can be easily
recycled as substrate
Substrate are used very slowly and steadily so the same substrate can be
used for longer fermentation period
SSF is best suited for fermentation techniques involving fungi and
microorganism that require less moisture content
10. Submerged fermentation / liquid
fermentation
Submerged fermentation is the techniques of cultivation of
microorganism in liquid broth which breaks down the nutrient to
release the desired bio-active compound into solution.
In this method, selected microorganism are grown in closed
vessels containing a broth rich in nutrients and high
concentration of oxygen.
In SMF substrate are utilized quite rapidly hence need to be
constantly replaced or supplemented with nutrients
Bacteria that requires high moisture content or high water
activity are best suited for submerged fermentation.
11. Substrates
Submerged fermentation
(SMF)
Solid state fermentation
(SSF)
Soluble sugar Wheat bran
Molasses Rice and wheat straw
Liquid media Fruit and vegetable waste
Fruit and vegetable juices Paper pulp
Sewage / waste water Bagasses
12. Enzyme Formulation
WHY FORMULATION :
Primary task of formulation is to minimize losses in
enzymatic activity during transport, storage and use.
Secondary purposes include, prevention of microbial
contamination, to avoid the precipitation or haze
formation, minimizing formation of sensitizing dust or
aerosols and improving color and odor
13. Different formulations depending on
applications
T-granulates
physical strength and minimum dust. High shear granulation and coating techniques. Detergent
industry.
BG/SG granulates
Smaller particle size, easy incorporation into flour, safety. Spray drying & Fluidized bed drying.
Bakery industry.
Micro granulates
Fluidized bed Drying for finer particle size distribution and safety (Non-dusting) in food industry
CT-Granulates
(coated-Tough) for heat sensitive enzymes to prevent denaturation. Feed Industry.
Immobilized Enzyme
High productivity at low cost. Enzyme immobilized on a carrier or in a matrix, enhancing stability
and preventing leakage into substrate during application. Starch, Oil & fat industry.
Liquid Formulations
Liquid product formulated and stabilized with polyols like glycerol, sorbitol, MPG, sugar, salts
to decrease water activity
14. PACKAGING
One should use tight bottles PACKAGING and
stoppers to prevent access to moisture and should
not release any traces of heavy metals or other
enzyme-inactivating substances into the enzyme
solution or suspension. In some cases, enzymes
must be protected from light and packaged in brown
glass bottles
17. Microorganism, culture conditions,
and substrates
The fungal strain A. oryzae is obtained from the Microbial Type
Culture Collection
The strain is maintained on yeast extract,malt extract ,agar and
Czapek Dox agar, respectively. Potato–dextrose agar is also
used for the growth and maintenance of the cultures. The
cultures are grown at 30 °C for seven days and then stored at 4
°C.
The powdered edible oil cakes, namely groundnut, coconut,
and sesame oil cakes are selected as substrates
The substrates are powdered and sieved using standard sieves
to eliminate the foreign materials and stored in aseptic
conditions
19. Inoculum preparation and solid-
state fermentation
Spores of 7-day-old fungal cultures are scrapped using an inoculation
loop and aseptically transferred to sterile distilled water containing 0.1%
Tween-80(polysorbate 80).
Exactly 1 ml of spore suspension is used as inoculum for the entire
fermentation.
About 5 g of dry oil cake is taken into a 250-mL flask, containing 2 ml of
mineral salts solution, containing 2 g of potassium di-hydrogen
phosphate, 5 g of ammonium nitrate, 1 g of sodium chloride and 1 g of
magnesium di-hydrogen sulfate in a liter of distilled water to adjust the
required moisture level
All the contents are mixed, autoclaved at 121 °C for 20 min, and cooled.
Spore suspensions are inoculated on the sterile solid substrate and
incubated in a solid-state fermenter maintained at 37 °C.
20. Development of a pilot-scale solid
state fermenter
600L SSF is used for optimization
A fan is fixed at the center of the chamber to maintain the
temperature. A relative humidity (RH) sensor is placed inside the
chamber .A compressor and water supply are connected to spray
water inside the chamber whenever RH decreases
Under optimum conditions, the microorganisms or cells are able to
perform their desired functions. Temperature and RH inside the
chamber is monitored and controlled using sensors and controllers
The bioreactor has four trays, an RH sensor, a temperature
controller, and a control system networked together. The pH
electrode is also set and working in the range of 0 to 13 can be used.
When pH change exceeded the set range, 0.1 N HCl or 0.1 N NaOH
can be added based on the measured value
21. Enzyme extraction
A known quantity of fermented substrate is mixed with double
distilled water along with 0.1% Tween 80. The contents are shaken
in a rotary shaker and then centrifuged at 7000 rpm at 4 °C for 10
min
The reaction mixture consist of 1.25 ml of 1% soluble starch, 0.25
ml of 0.1 M acetate buffer (pH 5.0), 0.25 ml of distilled water, and
0.25 ml of crude enzyme extract.
After 10-min incubation at 50°C, the liberated reducing sugars
(glucose equivalents) is estimated using the dinitrosalicylic acid
(DNS) method. The intensity of color develops and measured at
540 nm
One unit (IU) of α-amylase is defined as the amount of enzyme
releasing one μmol glucose equivalent per minute
22. Enzyme purification
The crude enzyme is saturated up to 50% using ammonium
sulfate and incubated at 4 °C overnight for precipitation of
proteins. The sample is centrifuged for 15 min (7000 rpm at 4
°C) in a refrigerated centrifuge
The supernatant-containing residues can be discarded and the
precipitate is used as an enzyme source
23. Reference
Optimization and scale-up of α-amylase production
by Aspergillus oryzae using solid-state
fermentation of edible oil cakes
M. Balakrishnan1*, G. Jeevarathinam1 , S. Kiran
Santhosh Kumar1 , Iniyakumar Muniraj2 and
Sivakumar Uthandi2*
