The amylase enzyme production by fermentation process

1. AMYLASE PRODUCTION
By Fermentation Technology
By
J. Divya sree
lll-Biotechnology

2. INTRODUCTION - Amylase
Amylases (E.C.3.2.1.1) are starch
degrading enzymes that catalyze
the hydrolysis of glycosidic bonds
in polysaccharides.
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3. 1952 1930
-Amylase
Named - Kuhn
Amylase -Kirchoff -Amylase - Ohlsson
HISTORY
1811

4. -amylase (E.C. 3.2.1.1) 1
2
3
4
-amylase (E.C. 3.2.1.2)
- amylase (E.C. 3.2.1.3)
-Glucosidase (E.C. 3.2.1.20)
Types of Amylases

5. 5
It is a calcium metalloenzyme which depends
on metal cofactor for its activity.
1 Starch is degraded in to glucose by
the amylase enzyme.
2 Present in saliva of humans for
digestion.
3
4
Amylase is broken down into
maltriose and maltose.
Hydrolysis of starch with amylase results
in formation of dextrin and maltose and
finally glucose.

6. Plants
Not considered a
significant source for
the commercial
production of amylase.
Agricultural wastes are
used in the production.
Ptyalin , a salivary
-amylase
(40%total salivary
protein).
Animals
Fungal (Aspergillus niger,
A .oryzae, Pencillium ,
Thermocyces lanuginosus)
Bacterial (Bacillus spp.)
Microbial
Source of production

7. In this, enzymes and other reactive compounds are
submerged in a liquid such as alcohol , oil or a
nutrient broth. The process is used for a variety of
purposes, mostly in industrial manufacturing.
Fermentative production
Used to convert moist agricultural polymeric into fermented
food products. It is simple technique , low capital investment ,
lower levels of catabolic repression and end product
inhibition ,low waste water out put ,better product recovery ,
high quality production.
Solid state fermentation
Submerged fermentation

8. Wheat bran
Saw dust
Oil cakes
Substrates used in Solid state fermentation
Sugar cane
baggasse
Tea powder
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9. Organism :Aspergillus oryzae
Media :Wheat bran is moistured and steamed for 1-2 h to extract the soluble
starch . The substrate contains 8% starch, sodium nitrate, magnesium
sulphate , Kcl, FeSo4, malt extract are added to optimize the media.
Inoculum : Fungus spores are produced in solid ,media to use as inoculum.
Temperature: It is maintained at 28-30C.
pH: It is maintained about 2-3
Cont…..

10. Product recovery:
 The fermentation broth is separated into liquid and solid part by means of
filtration or centrifugation.
 For fungal amylase, filtration alone is sufficient to separate solid from
liquid .
Cont…..
Duration :
 The duration is 3-5 days.
 Fungal molds are grown in specially designed trays equipped with
perforated covers.
 High rates of aeration are provided to encourage growth

11. Steps involved in Amylase production
Selection of Microorganism : Aspergillus niger, A. oryzae as they produce amylase
and it secrets acid so it easily degrades complex raw material .
Selection of Raw materials : Fungi cannot grow on natural media ,so we provide
synthetic or artificial media
CHEMICALS QUANTITY USED
Corn starch 24g/L
KCl 0.2g/L
Na2HPO4 4.7g/L
CaCl2/CaCo3 1g/L
MgCl 2.6H2o 0.2g/L

12. Fermentation process
Fermentor is filled with raw material or fermentation medium
Inoculate mycellium of A.niger
Maintain temperature at 28 C- 30 C
pH is neutralisied by adding basic solutions
After 72 hours recovery process is carried out

13. Recovery process
Filter contains amylase ,synthetic media and mycelia of fungi
By filtration ,mycelia is removed
Amylase is separated from synthetic media by adding ammonium hydroxide which forms
precipitate with amylase
By filtration collect the precipitate
Precipitate is subjected to Crystallization at 4C
Amylase in the form of crystals are separated and collected

14.  Boidin & Effront ,1917 were first to use Bacillus subtilis and Bacillus
mesentricus for the production of -amylase on commercial scale using
large fermentors in submerged fermentation.
 Due to the rapid growth rate of Bacillus species, it is more predominant in
the production of -amylase enzyme industrially.
 The most commonly used strains are Bacillus subtilis, Bacillus licheniformis,
B.amyloliquefaciens, B. mesentricus
Production of amylase by Bacteria

15.  These are improved by mutations to yield enzyme of about 250 times
greater than that of wild times.
 Different techniques such as cell immobilization in the matrix, such as
alginate, polyacrylamide, agar, and k-carrageenan are being used for
continuous and replaced batch fermentation of amylase
Production of amylase by Bacteria

16. Media :
Mostly the liquid media is used . Mostly the strains undergoes catabolite repression with g
lucose.
Yeast extract has been proven to be the most preferred organic nitrogen source for -amyl
ase production as reported by many studies (Alam et al.,1989:Asgher et al.,2007).
Yeast extract not only serves as a source of nitrogen but it is also considered as a rich
source of vitamins and many growth factors.
Thiamine ,Cysteine and pyridoxine are the most suitable for the production(Rajagopalan a
nd Krishnan, 2008 a)
Cont…..

17. Cont…..
Composition of media :
5% Starch
Ammonium nitrate
Sodium citrate
MgSo4 . 7H2So4
CaCo3. 2H2O
Peptone
Yeast extract
Inoculum preparation: Preapared by Shake flask culture method .The
inoculum is grown in small fermenters before final fermentation

18. pH: Optimum pH is maintained for the production of amylase is 6-8.
Temperature: It is maintained at 27-30C, but it also depends on the
bacterial strain used.
Aeration : It is done at a range of 0.8-11 vvm.
Duration :It takes about 48 h
Cont…..

19. Production profile
The enzyme fermentation rate is very low
during the exponential phase of growth
but just before the rate of growth decreases
and spore formation begins, amylase
production increases.

20. Comparision….
Bacillus subtilis Bacillus mesentricus
Mode of fermentation Smf Smf
pH 6-8 7.0
Temperature 27-30 37
Duration 48 h 48-78 h

21. Applications

22. Thank you