Production of Amylase enzyme

Amylase enzyme:

 Starch, a glucose polymer is one of the most widely available plant

polysaccharides which ishydrolysed by an enzyme called amylase.
 One of the main uses of amylases is in the production of sweetener for the
food industry.
 The hydrolysis of starch with amylase result first in the production of short
chain polymers called dextrins then disaccharide maltose and finally glucose.

 The most important enzyme in the starch saccharification process are 

 α-amylases, glucoamylases, glucose isomerases, pullulanases, and

isoamylases. 

 α-amylase: (1-4 α -Dglucan-glucanohydrolases)

 α-amylases are extracellular enzymes which hydrolyse  α-1,4-

glycosidic bond.
 These enzymes are endoenzyme splitting the substrate in the interior of
the molecule.

 Bacteria which produces α -amylases are Bacillus subtilis, B.

cereus, B. amyloliquefaciens, B. coagulens, B. polymyxa, B.
acidocaldarins, B. lichiniformis, Lactobacillus micrococcus,
Pseudomonas, Arthrobacter, Escherichia, Proteus, Thermomonospora,
Serretia
 Fungi- Aspergillus, Penicillium, Cephalosporium,
Neurospora and Rhizopus.
  The most important  -amylase producer are Bacillus
amyloliquefaciens, B. lichiniformis, and Aspergillus oryzae.

 β-amylase: (α -1,4-D glucan-maltohydrolases)

 β -amylases are usually of plant origin but some micro-organisms

also produce.

 This enzyme catalyzes the hydrolysis of the second  α-1,4-

glycosidic bond from non-reducing end.

 E.g. of β -amylase production micro-organisms are B. polymyxa, B.

cereus, B. megaterium,
Streptomyces spp., Pseudomonas spp., Rhizopus spp. etc.

Bacterial α-amylase production:

(a) Preparation of Inoculum:

Pure culture of any of the above-mentioned species of Bacillus is selected as

inoculum. Mutants which produce 250 times greater yields than the wild strain are
preferred as inoculum.

(b) Preparation of Medium:

The formulation of the production medium and control of fermentation conditions

play a major role in the success of enzyme fermentations. The production medium
should basically contain an energy source, a carbon source, a nitrogen source and
growth requirements such as essential amino acids or vitamins. For obtaining high
yields of enzyme, the production medium should also contain certain inducers like
lactose.
Sometimes certain compounds like glucose present in the production medium act
as repressor for certain enzymes like α-amylase. In such conditions either the
concentration of glucose should be kept low or it should be fed intermittently.

(c) Fermentation Process:

The type of fermenter that is used for fungal α-amylase is also used for bacterial α-
amylase production. About 1000 to 30,000 gallons of production medium is taken
in the fermenter and inoculated. The fermentation is continued upto 4-6 days. The
pH of the medium is maintained at 7.0.

Calcium carbonate is used as a buffer for maintaining neutral pH. The temperature
is maintained at 30-40°C. The production of α-amylase starts when the bacterial
density reaches 109-1010 cells ml-1. However, the enzyme production increases just
before the growth rate of the microorganism decreases and spore formation begins.

(d) Harvest and Recovery:

 Centrifugation at 3000 rpm for 30 mins or 5000 rpm for 20mins at 4 oC and
collect the supernatant for crude enzyme.

Fungal α-amylase production:

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 is only described here:

(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:

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 due to the presence of mycelial mat.

(d) Harvest and Recovery:

The following steps are followed during the recovery of the enzyme after the
completion of fermentation. 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.

Application of α -amylase:

 Amylases have potential application in a number of industrial processes such

as in food, textiles, paper industries, bread making, glucose and fructose
syrup, detergents, fuel, ethanol, from starches, fruit juices, alcoholic
beverages, sweatness digestive aid, spot remover in dry cleaning.
 Bacterial  α-amylase are now also used in areas of clinical, medicinal and
analytical chemistry.
 In most wildly used thermostable enzymes are the amylases in starch
industry.

Industry
(field) Applications

Starch industry Liquefaction of starch for production of glucose, fructose, maltose

Milling Modification of  -amylase deficient flour

Liquefaction of starch before the addition of malt for

Alcohol saccharification

Bakery Increase in the production of fermentable carbohydrates

Preparation of barley, liquefaction of additives, enhanced

Brewing fermentability of grains, modification of beer characteristics

For the liquefaction of starch without sugar production for sizing of

Paper industry paper

Textile industry Continuous de-sizing at high temperature

Improvement of utilization of enzymatically barley in poultry and

Feed industry calf raising

Improvement of filter-ability of can sugar juice through breakdown

Sugar industry of starch in juice.

Laundry and
detergent Increase in cleaning power additive in dish washer detergent