Industrial Production of Alpha- amylase

Production of alpha -amylase:

Amylase is the enzyme which hydrolyzed alpha bond of large polysaccharides linked by alpha bond yielding glucose and maltose. The large polysaccharides may be starch and glycogen .Or in another word amylase are the enzyme that hydrolyzed the starch resulting in the production of dextrin,then maltose and finally glucose molecules. They are used for various propose.Amylase is most prominent in the humans and mammals .But it is also found in the seeds of plants .It is present in seeds containing starch as reserve food material. Although it is found in many tissues ,plants, seeds and animals it is abundantly found in the pancreatic juice and saliva, The amylase enzyme found in the saliva is known as 'Ptyalin' that breaks starch into maltose. The optimum pH for salivary amylase is 7.0 and the temperature is normal human body temperature ie; 37°C. The activators like chloride and bromide are the active activators ,iodine is less active and sulphates and phosphate are least effective towards the reaction with Ptyaline. Pancreatic amylase cleaves alpha(1-4) glycosidic linkage of amylose to form dextrin and maltose . It cleaves polysaccharides in a random manner and adopts double displacement method . Amylase is widely used for various propose and they are mostly found in microbes . Alpha -amylase and Beta-amylase are the major type of amylases produced by microorganisms, However ,glucoamylase is also produced by microorganisms.

A) Alpha- Amylase :

These are endo 1,4 alpha -D-glucan glucohydrolases . They are extracellular and randomly cleave alpha 1,4 glycoside bond . They are derived from several bacteria , Yeast, and fungi.

Alpha-amylase producing bacteria :

Bacillus subtilis

B. amyl liquefacient

B. polymyxin

B. coagulans

B. Licheniformis

Micrococcus, Lactobacillus

Alpha- amylase producing fungi:

Aspergillus

Penicillium

Cephalosporium

Mucor

Cladosporium

Candida

Among them commercially used strains are :

Bacillus subtilis

B. licheniformis

B. amyloliquefaciens

A. Oryza

B) Beta- amylase :

These are Beta-1,4 glucanmaltohydrolase. It is usually of plant origin. However, certain microbes such as: B. polymyxa, B, cerevs, B. megaterium. Streptomyces sp, Pseudomonas spps,Rhizopus japonicum produces beta- amylase and they are involved in cleavage of non -reducing chain ends.

C) Glucoamylase :

They are also called as amino glucosidase and it is Exo 1,4 alpha -Dglucanglucohydrolase and hydrolyzed the single glucose unit from the non- reducing ends . Organisms responsible to produce it are: A . niger ,A.oryzae, Rhizopus niveus.

Production method of alpha-amylase;

Commercially ,alpha amylase is produced by improved strains of bacteria and fungi and the mode of operation may be batch or fed-batch type .

Production of bacterial alpha-amylase ;

Bacterial alpha-amylase is produced by either solid state fermentation or by submerged state fermentation. The process of production of enzyme may be discussed under the following headings;

1)Producing organism;

:Amylase is produced by bacteria ,fungi, plants, and animals but due to the advantages of microorganism in the production process microorganisms have substantial potential to contribute different and many industrial applications, Due to the rapid growth rate of Bacillus species it is more predominant in the production of amylase enzyme industrially.

The most commonly used commercial strains are Bacillus substilis, Bacillus licheniformis, B. amyloliquefaciens. These strains are improved by mutation to yield enzyme of about 250 times greater than that of wild strains.

2) Media: Mostly complex wheat bran, a rice bran , gram husk, mustard oilseed cake which are known as agro-industrial wastes are used in the production of amylase in the case of solid state fermentation but production by submerged state method requires media optimization and includes 5% starch, ammonium nitrate,sodium citrate, MgSo_4.7H₂S0₄., Caco₃.2H₂o , peptone, Yeast extract etc are used.

3) Inoculum preparation:

Inoculum is prepared by shake flask culture method . The inoculum is then grown in small fermenters before final fermentation.Thus selected microbial strains are ready for inoculation and inoculated in nutrient broth and then followed by the incubation for 24 hours at temperature 37° C to obtain standard inoculum.

4) Fermenters:

The fermenter used in the submerged state fermentation process may betray or stirred type.

5) Fermentation :

a) MOde of fermentation; It is carried out either in batch or fed-batch manner.

The difference between batch fermentation and fed-batch fermentation.

b) Size of fermenter;

A 100m3 volume of the tank in submerged state fermentation is used.

c) pH :

The optimum pH for the production of amylase is 6-8

d) Temperature:

Maximal enzyme production occurs at a relatively lower temperature of about 27- 30° C. but it is strain specific eg; Thermophilic such as Thermonospora produces the maximum enzyme when the temperature is 53° C.

e) Aeration ;

Aeration is done in range of 0.8-1 vvm

f) Duration:

It should be kept for 1-2 days that means for 48 hours.

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.

Production of fungal amylase;

The production of fungal alpha-amylase is mostly carried out by solid state fermentation using wheat bran as the substrate. However, the substrate may contain 8% starch, sodium nitrate ,magnesium sulphate,KCL, FeSo₄., malt extract are added to optimize the media.

Production method:

a) Inoculum; Fungal spores are produced in solid media to use as inoculum. The major fungus used is Aspergillus oryzae

b) Fermenter; Drum fermenter or tray chamber are used.

c) Fermentation: The inoculum of Aspergillus oryzae is inoculated into the fermenter and the temperature is maintained at 28 -30°C. The duration of fermentation is 3-5 days.

d) Product recovery: The fermentation broth is separated into liquid and solid part by means of filtrattion or centrifugation and is purified in subsequent steps for fungal amylase filtration alone is sufficient to separate solid from liquid while for bacterial amylase filtration and centrifugation are carried out. The recovery and purification are done as below;

Fig: Flow chart of the product recovery of amylase enzyme;

Demonstration of Enzymatic Activity of alpha -amylase:

After the enzyme has to be extracted and purified,it is tested for its enzymatic activity . The simple protocol is given below;

• 1ml of enzyme is precipitated in a test tube
• 1ml of 1% soluble starch in citrate phosphate buffer is taken and the pH of the media is maintained at 6.5 in the test tube containing the enzyme.
• It is incubated in a water bath at 40°C for 30 minutes .
• A blank containing 2ml of enzyme boiled for 20 minutes ( for incubation of enzymes ) is added to the starch solution and treated with the same reagent in an experimental tube.
• The reaction is stopped by adding 2ml of DNS reagent ( dinitro salicylic acid),NaoH and sodium potassium tartrate
• The tubes are boiled for 5 minutes
• It is cooled and 2ml of distilled water is added to each tube.
• The colour intensity is read at 540nm by using calorimeter .

Enzyme activity is defined as the amount of glucose produce per ml in the reaction mixture per unit time ,knowing the standard absorbance of glucose that of the test solution, the concentration of glucose can be estimated.