Generalized overview of Alpha Amylase Upstream and Downstream process.

1. Alpha Amylase Production
Company in Light: Novozymes
Products : Liquozyme and Termamyl
Submitted By-
Name: Rishav Roy
Roll No. 1860140
IMTH-5
Submitted to-
Dr. Gargi Dey
KIIT School of Biotechnology

2. Founders- Harald and Thorvald Pedersen (1925)
The current Novozymes was founded in 2000
Products- Enzymes, Microorganisms, Biopharma
Ingredients
-
Rethink Tomorrow

3. Liquozyme
 A new cost-effective alpha-amylase
from Novozymes is designed for
ethanol plants. Blend of 6 different
alpha-amylases
 provided up to a 40% improvement in
viscosity in slurry, and up to 17%
improvement in liquefaction
 Robust, trusted performance at low pH
conditions.
 Liquozyme® LpH delivers equal or higher
DE (dextrin equivalence)
Termamyl
• Termamyl® is a liquid enzyme preparation
containing an outstandingly heat-stable α-
amylase.
• used for the continuous liquefaction of starch
at temperatures of up to 105–110 °C, taking
advantage of the extreme heat stability of this
enzyme
• produced by a genetically-modified strain of
Bacillus licheniformis.

4. Overview of
Amylase Production
Upsteaming
Product
Packaging and
Storage
Downstreaming

5. • Generally GMOs(Bacteria) are used to produce the products.
• Metabolic and Genetic Engineering-Dr.Nasir (2019) has cloned the gene of Amylase production into pFLDα
expression vector under the control of formaldehyde dehydrogenase (FLD1) promoter before transforming
into a new yeast expression system, i.e., Meyerozyma guilliermondii strain SO.
• Chemical agents like Nitrous acids, Ethyle methane Sulphonate(EMS)
• Radiation
• Bacterial Sources- For the Production of Thermostable Enzyme
• Bacillus amyloliquefaciens UNG-16 ( Radiation Method and EMS is employed for Mutation)
• B. subtilis (YN9- 3 fold increase) (mutated by- N-methyl-N-nitro-N-nitrosoguanidine)
• B.licheniformis
• B.stearothermophylus
• (Inoculum Prep. Parameter- Temp- 45°C-55°C; pH- 6.0-7.0; Incubation period- 48-72 hours)
• Fungal source can be also considered for the production.
• Aspergillus sp- A.oryzae, A.niger, A.kawachii (Temp.- 30°C-90°C, pH-5.0-6.0, Incubation Period- 96hrs)
• Penicillium sp- P.fellutanum, P.roquefortii, P.chrysogenum (Temp- 30°C-50°C, pH-6.0-7.0, Incubation Period-
72hrs)

6. Carbon Source
• Maltose, Sucrose, Glucose
• Organism Specific Substrate like-->
• A.tamarii-Maltose+Starch+Glycogen(Gives 4 folds of yield
than normal)
• B.subtilis- Banana Waste and Glucose is essential
• A.niger and B.licheniformis-Wheat Bran
Nitrogen Source
• Inorganic Sources- Ammonium sulphate, Ammonium
Chloride, Ammonium Hydrogen Phosphate.
• Organic Sources- Peptone, Yeast Extract, Soyabean Meal,
Tryptone, Meat Extract
• Organism Specific Substrates-->
• A.oryzae- Sodium Nitrate(0.9%)+Malt(1%)
• A.fumigatus+A.niger- Peptone+Sodium nitrate+Casein
hydrolysate
• Thermomyces lanuginosus- Peptone
Metal Mix and Buffer
• Metal Mix (Ca-chloride; Mn chloride;Mg chloride;water),
sterile-filtered or made from pre-sterilized stock
solutions
• Fe-solution-Fe(III)-chloride+HCl+Water
• Phosphate Buffer-KH2PO4,K2HPO4 and Water
Sterilization
(Prev. Slide) and

7.  Specific Organisms- B.licheniformis GCB-U8, P.fellutum, Halobacillus MA-2
 Most Conventional Way of Amylase Production.
 Fermentation Yielded Amylase are secreted into Fermentation Broth
 Substrated utilization is Rapid
 Suitable procedure for the microorganisms that require high moisture content
 Utilization of GMO is much more suitable in this process (than SSF)
 unwanted metabolites are not produced and purification of enzymes takes place
in an easy way.
 Control of the process parameters like- Temperature, pH, Aeration, Oxygen
Transfer and Moisture are very convenient.
Submerged fermentation is a method of
manufacturing biomolecules in which
enzymes and other reactive compounds
are submerged in a liquid

8.  Much More recent Process and an alternative to SmF.
 Specific Microorganisms- B.licheniformis, B.vulgaris, B.megaterium,
B.subtilis - These microbes require less amount of moisture content
for growth.
 Substrates used- Bran, Bagasse, Paper pulp,de-oiled oil seed cakes
etc.
 Substrate Utilization- Very Slow and Steady process
 Bioreactor- GROWTEK Bioreactor (to circumvent many problems
associated with Conventional Tray Reactor)-a side tube with silicon
membrane that permits replenishment of nutrient medium without
disturbing much the fermenting organism.
 Advantages- 1. Nutrient Rich waste materials(Oil Cakes) can be easily
recycles. 2. SImpler Equipments 3. High Volumetric Productivity 4.
Higher Concentrated Products 5. Lesser Effluent Generations
Solid State Fermentation (SSF) is a
fermentation methods to produce
metabolites of microorganisms using
solid support in place of the liquid
medium.

9. • Ultrafiltration- Widely used
technique in concentrating
and purifying proteins by
their molecular weight.
• SDS-PAGE - 55.4 kDa
• The crude alpha amylase
present in the fermentation
broth is ultrafiltrated twice
against 100kDa and 30kDa
Molecular Weight Cut-off
(MWCO)- ultrafiltration
membrane.
• Fold Purification- 3.4
• Yield Recovery- 20.61
• De-Salting Method: After
applying the salting
method when the proteins
are precipited de-salting
method is applied.
• In the presence of 100mM
Phosphate Buffer (pH-6.0)
dialysis is done which
leads to-
• 4.75 fold purification
• 16.66% Yield
• Salt Precipitation- To purify proteins from
the crude enzymes by increasing the salt
concentration gradually and at 60% level
mainly all types of proteins gets precipited.
• Most common salt used- Ammonium
Sulphate(80%)

10. • Based on the Ionic bonds between Cation and
Anion.
• Mobile Phase- Crude Enzyme
• Stationary Phase- Inert Organic Matrix
• Separates Molecules by Their Surface
Charges
• Competitive Ion Binding and Ion Exclusion due
to Similar Charged Ions
• Ions are fixed in a column.
• To provide charge in the proteins- Dissolve
into the buffer with pH lower and higher than
Isoelectric point (the pH at which a molecule
carries no net electrical charge or is
electrically neutral) isoelectric point-9.05
• Desorption or Elution of Enzymes.
• Column- DEAE-Sephadex-A50 and Q-
Sepharose(HiTRAP) (Developed by Cytiva)
• Binding Buffer- Tris-HCl Buffer (pH 8.0)
• Elution Buffer- Tris-HCl Buffer (pH 8.0)
• Result- 9.31 fold Purification and 12.61% yield
`
• Proteins of varying sizes are separated by
Columns consisting of a matrix of beads-
which contains Sieves of Particular sizes.
• SEC-separates molecules according to their
size in solution
• Larger molecules are eluted earlier than
smaller molecules.
• Beads are crosslinked with various
chemicals.
• Most Frequently equipped SEC matrix-
Sephadex G-100
• But for Bacillus sp. special arrangement
required
• Column- Toyopearl Chromatography Resins
by Tosoh Corporation
• Running Buffer- Tris-HCl Buffer (pH 8.0)
• Elution Buffer- Tris-HCl Buffer (pH 8.0

11. DNSA Method
Indian Pharmacopoela
Method

13. References:-
• http://www.ebiosis.co.kr/Novozymes%20Product%20Sheet/Liquozyme%20Supra.pdf
• https://bioresources.cnr.ncsu.edu/resources/purification-and-characterisation-of-thermostable-
%CE%B1-amylases-from-microbial-sources/
• http://pubs.sciepub.com/jaem/2/4/10/
• https://www.hindawi.com/journals/bmri/2017/1272193/
• https://www.sigmaaldrich.com/analytical-chromatography/analytical-
products.html?TablePage=101277530
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768732/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335993/
• https://www.researchgate.net/publication/6262674_Comparative_profiles_of_alpha-
amylase_production_in_conventional_tray_reactor_and_GROWTEK_bioreactor
• nlm.nih.gov/21234823/