Enzyme- Amylases

1. Enzyme-Amylases
Vaishali S.Patil
Professor, Department of Botany
Shri Shivaji College of Arts, Commerce & Science Akola

2. Introduction
Microorganisms in particular have been regarded as treasure
of useful enzymes. There is a great variation between various
genera as to their ability to produce a specific enzyme for the
production of particular enzymes varies with the particular
medium and pH. The first enzyme produced industrially
was an amylase from a fungal source in 1894, which was
used for the treatment of digestive disorder.. Microbial
amylases have successfully replaced chemical hydrolysis of
starch in starch processing industries. Amylases have been
produced by submerged fermentation. The solid state
fermentation (SSF) processes applied for the production of
this enzyme. Amylases are a group of enzymes that have been
found in several microorganisms like bacteria and fungi.

3. Characters
•Fungal Amylase is an alpha amylase enzyme preparation
produced by Aspergillus oryzae.
•It readily degrades a wide variety of starch-containing
substrates. Amylase is an enzyme that breaks down starch
into sugar.
•It is available as a liquid or as a powder formulation. This
rapid acting hydrolase is active throughout the acidic pH
range, through the neutral
pH range & well into the mildly
alkaline range.

4. •This product is not extremely heat-resistant compared with
enzymes from bacterial sources but exhibits adequate heat
resistance for many industrial and most agricultural
applications where its pH range is often complementary to
alpha amylase from bacterial sources.
•Amylase is present in human saliva, where it begins the
chemical process of digestion.

5. •This enzyme is also used for the commercial production of
glucose.
•Besides their use of starch saccharification, they also find
potential application in a number of industrial processes such
as in food, baking, brewing, detergent, textile and paper
industries with the advent of new frontiers in biotechnology,
the spectrum of amylase application has expanded into many
other fields such as clinical, medical and analytical
chemistry.

6. a) Biochemical Properties of Amylases Substrate Specificity: 𝞪 -
amylases display highest specificity towards starch
b) pH Optima and Stability: The pH optima of 𝞪 –amylases vary from
2 to 12. 𝞪 - Amylases from most bacteria and fungi have pH optima in
the acidic to neutral range.
c)Temperature Optima and Stability: The lowest temperature
optimum is reported to be 25 to 30°C for Fusarium oxysporum amylase
and the highest of 100 and 130°C.
d)Molecular Weight: Molecular weights of amylases vary from about
10 to 210 kDa.
e) Inhibitors: Many metal cations, especially heavy metal ions,
sulphydryl group reagents, N-bromosuccinimide, hydroxyl
mercuribenzoic acid, iodoacetate, BSA, EDTA and EGTA inhibit
amylases.
f) Calcium and Stability: Amylase is a metalloenzyme which contains
at least one Ca2+ ion.

7. Classification of Amylase-
1.𝞪 Amylase: The 𝞪 amylases are calcium metalloenzymes, along with
the starch chain 𝞪 -amylase breaks down long-chain carbohydrates,
ultimately yielding maltotriose and maltose from amylose, or maltose,
glucose and "limit dextrin" from amylopectin. In animals, it is a major
digestive enzyme and its optimum pH is 6.7-7.0. In human physiology,
both the saliva and pancreatic amylases are 𝞪 –amylases & also found
in plants (adequately), fungi (ascomycetes and basidiomycetes) and
bacteria (Bacillus).It is used in clinical, medicinal and analytical
chemistry. Besides their use in starch saccaharification they also find
applications in food, baking, brewing, detergent, textile, paper and
distilling industry. Production of fungal alpha amylases is through
submerged (SMF) and solid state fermentation (SSF).

8. 2.𝞫Amylase: Another form of amylase,𝞫 -amylase is also
synthesized by bacteria, fungi and plants. During the
ripening of fruit, 𝞫 -amylase breaks starch into maltose,
resulting in the sweet flavor of ripe fruit. Both 𝞪 -amylase and
, 𝞫 -amylase are present in seeds;
𝞫 –amylaseis present in an inactive
form prior to germination,
whereas 𝞪 -amylase and proteases
appear once germination has begun.
3.ϒ -amylase will cleave 𝞪 (1-6) glycosidic linkages. Unlike
the other forms of amylase, ϒ -amylase is most efficient in
acidic environments and has an optimum pH of 3.

9. Fungal Amylases
Bacteria and fungi secrete amylase to the outside of the cells to carwhich
are large, tightly packed, globular and may be blackry out extra-cellular
digestion. Ellaiah et al. identified amylolytic activity from several fungal
species isolated from soil and Aspergillus sp. was found to possess the
highest amylase activity produced extracellular amylase using bran
(wheat bran, rice bran, black gram bran) as carbon source in shake
flask cultures of a hemophilic strains of Aspergillus niger.
Fungal amylases are used for hydrolyzing carbohydrate,
protein and other constitutes of soybeans, wheat into peptides, amino
acid, sugars and other low molecular weight compounds. The
amylase producing strains of Aspergillus niger have spore bearing heads
or brownish black. They are considered to be mesophilic with optimal
temperature for growth between 25°C and 35°C. They are aerobic in
nature and can grow over a wide range of hydrogen ion
concentration.

10. These organisms can utilize different kinds of agricultural wastes from
simple to complex ones, which make them easy to cultivate and maintain
in the laboratory. Filamentous fungi are microorganisms secrete large
amounts of protein in culture medium. Aspergillus niger
has been described as secreting an 𝞪-amylase and glucoamylase of a
number of different molecular weights in submerged culture.
Filamentous fungi have been used for the industrial production of a wide
variety of native products, such as antibiotic (e.g., penicillin and
cephalosporin), organic acid (Citric and acetic acid) and commercial
enzymes (e.g., protease, catalase, amylase). Detergent industries are the
primary consumers of enzymes.
Aspergillus sp. Penicillium sp. Helminthosporium sp.

11. Fermentation Studies on Amylase Production
In the pH range 4 to 5, both pellets and freely dispersed hyphal
fragments were observed. The optimum growth temperature was found
to be 35°C, using Aspergillus oryzae. Different oil cakes such as
coconut oil cake, sesame oil cake, groundnut oil cake, wheat bran
and olive oil cake, starchy soils, corncob leaf, rye straw, wheat straw
, soya bean flour, (maize), root starches (yam, cassava, sweet
potatoes) paddy to rice flakes, cassava peels, yam peels, banana
peels, plantain peels and brewery spent grains were screened to be
used as substrate for the enzyme production.
Fungal strains of Aspergillus oryzae ,Penicillium chrysogenum,
Penicillium fellutanum, Aspergillus niger, Helminthosporium
oxysporum, Aspergillus fumigatus, Aspergillus flavus, Penicillium
frequestans can be used for the enzyme production.

13. Fungal α -Amylase:
Fungal α-amylase is produced commercially by employing either
Aspergillus oryzae or Aspergillus niger. Stationary culture method is
used when A. oryzae is employed, while submerged culture method is
used when Aspergillus niger is employed.
The process of submerged culture method-
(a) Preparation of Inoculum:
Suitable and pure seed culture is generally selected as inoculum. In
certain cases mutants capable of giving higher yield are selected as
inoculum.
(b) Preparation of Medium:
The following medium is generally employed for submerged
fermentation:
.

14. Amylase biosynthesis is inhibited when there is glucose in the medium.
The medium is steam sterilized. The sterilized medium is passed into a
production fermenter for α-amylase production.
(c) Fermentation Process:
A cylindrical fermenter made up of stainless steel is generally used in the
fermentation process. It is equipped with an agitator, an aerating device,
a cooling system and other ancillary equipment like a device for foam
control, monitoring of pH, temperature and control of oxygen tension
etc. A sufficient quantity of pre-sterilized production medium is taken in
the fermenter and is inoculated with spores of the selected species of the
fungus. The spores are allowed to germinate and produce sufficient
mycelium by controlling the fermentation conditions. Control of
fermentation conditions plays a vital role in the success of the process,
which include pH, temperature, aeration, agitation, oxygen supply etc.
The optimum pH for the fermentation is 7.0. Calcium carbonate is
used as buffer to maintain pH. The fermentation process is generally
operated at a temperature of 30 to 40°C. Aeration and agitation of the
production medium is needed because of high viscosity of the medium.

15. due to the presence of mycelial mat.
(d) Harvest and Recovery:
After the completion of fermentation the recovery of the enzyme was
done . In order to avoid denaturation of the enzyme, the fermentation
broth is subjected to rapid cooling at 5°C temperature immediately and
the enzyme is extracted.
1. Separation of fungal mycelium is accomplished by filtration of the
refrigerated broth.
2. The suspended particles present in the broth are removed with
flocculating agents like calcium phosphate.
3. The enzyme is precipitated, in order to get high degree of purity, by
using acetone or alcohol or even inorganic salts like ammonium sulphate
or sodium sulphate.
4. Sometimes fractional precipitation of the enzyme is done to obtain it
in purest form.

16. Uses of Amylase
1.As a chemical industries & pharmaceutical aid for the treatment of
digestive disorders. This enzyme facilitates the contact between the
digestive enzymes and chyme to improve nutrient absorption and
utilization
2. In food, detergents, textiles and in paper industry, for the hydrolysis of
starch.
3.Amylase enzymes find use in bread making and to break down
complex sugars such as starch (found in flour) into simple sugars.
4. Amylase is also used in clothing ,dishwasher detergents, fermentation
pre-treatment, and animal feed supplements. It can supplement the
deficiency of endogenous amylase of young animals.It can also increase
the utilization of feedstuffs by hydrolyzing the starch contained in feed to
generate dextrins and sugars. In addition fungal amylase lowers feed
conversion ratio (FCR) and improves animal production performance
and utilization of energy.
.
..

17. 5.Blood serum amylase may be measured for purposes of
medical diagnosis,potential diet aid,including acute
inflammation of the pancreas, perforated peptic ulcer, torsion
of an ovarian cyst, strangulation isles, macroamylasemia and
mumps, increased risk of occupational asthma.

18. Maltose manufacture
Bakery
Fermentation in a distillery
Starch
Animal feedstuffs
Paper industry
Textile
Detergent
pharmaceutical aid