Readers will come to know the basics of Microbial Alpha-Amylase Production.

1. Microbial α-amylase production
Introduction
Alpha-amylase is a crucial enzyme that plays a pivotal role in various industrial processes,
particularly in the food, pharmaceutical, and detergent industries. It is responsible for the
hydrolysis of starch, a complex polysaccharide, into simpler sugars like maltose and dextrin. This
process is essential for the efficient utilization of starch as a carbon source by microorganisms, as
well as to the production of various products derived from starch.
The production of alpha-amylase has been the subject of extensive research due to its wide-ranging
applications. Traditionally, alpha amylase was obtained from natural sources such as plants and
animals. However, with advancements in biotechnology, recombinant DNA technology has
enabled the production of alpha-amylase through microbial fermentation. This method is not only
more efficient but also allows enzymes with specific properties tailored to the desired application.
In recent years, there has been a growing interest in the use of genetically modified microorganisms
for α-amylase production. Genetic engineering techniques allow for the modification of
microorganisms to enhance their enzyme production capabilities, improve enzyme stability, and
optimize the fermentation process.
The production of microbial α-amylase is a complex process that requires careful optimization of
various factors to achieve high enzyme yields. This enzyme has a wide range of applications in
various industries, making it an important target for research and development efforts.
α-Amylase
α-Amylase is a hydrolase enzyme that catalyzes the hydrolysis of internal α-1, 4-glycosidic
linkages in starch to yield products like glucose and maltose. It is a calcium metalloenzyme i.e., it
depends on the presence of a metal cofactor for its activity.
There are 2 types of hydrolases: endo-hydrolase and exo-hydrolase. Endo-hydrolases act on the
interior of the substrate molecule, whereas exo-hydrolases act on the terminal non-reducing ends.

2. The substrate that α-amylase acts upon are starch. Starch is a polysaccharide composed of two
types of polymers – amylose and amylopectin. Amylose constitutes 20-25% of the starch molecule.
It is a linear chain consisting of repetitive glucose units linked by α-1,4- glycosidic linkage.
Amylopectin constitutes 75-80% of starch and is characterized by branched chains of glucose
units. The optimum pH for activity is found to be 7.0.
Production of α-Amylase
Sources
α-Amylase can be isolated from plants, animals, or microorganisms. The enzyme has been
isolated from barley and rice plants. It has been found that cassava mash wastewater is a source of
α- Amylase which is active in a wide range of pH and temperature. The most widely used source
among the bacterial species is the Bacillus spp. B. amyloliquefaciens and B. licheniformis. Fungal
sources of α-Amylase are mostly Aspergillus species and only a few species of Penicillium, P.
brunneum, penicillium fellutanum
Production Methods
There are mainly two methods that are used for the production of α-Amylase on a commercial
scale. These are
1) Solid State fermentation. 2) Submerged fermentation
1. Solid State Fermentation [SSF]
The method used for microbes that require less moisture content for their growth. The solid
substrates commonly used in this method are bran, bagasse, and paper pulp. The main advantage
is that nutrient-rich waste materials can be easily recycled and used as substrates in this method.
2. Submerged Fermentation [SMF]
It employs free-flowing liquid substrates, such as molasses and broths. The products yielded in
fermentation are secreted into the fermentation broth. The substrates are utilized quite rapidly,
hence the substrates need to be constantly replenished. SmF is primarily used for the extraction of
secondary metabolites that need to be used in liquid form.
Production Media

3. Composition
Na2HPO4, KH2PO4, NH4Cl, NaCl, CaCl2, MgSO4.7H2O, Casein hydrolysate, Yeast extract and
Starch.
Carbon Source:
Common carbon sources used as substrates include maltose, sucrose, and glucose. A. tamarii
actively synthesizes α-Amylase when cultured on maltose, starch, and glycogen under static
conditions. The aim is to use substrates that are waste or by-products of other processes in order
to make the process of enzyme production environment-friendly. One such substrate is oil cake.
Wheat bran has been used as the substrate for α-Amylase production by B.licheniformis and
A.niger.
Nitrogen Source
The nitrogen source used for the production of α-Amylase may be organic or inorganic. A few of
the inorganic nitrogen sources include ammonium sulfate, ammonium chloride, and ammonium
hydrogen phosphate. The most commonly used organic sources of nitrogen include peptone, yeast
extract, and soybean meal.
Process Parameters:
The optimum process control parameters vary depending on the microbial source, desired end
product, method of fermentation employed, and many other such factors.
1. Temperature: There are two temperatures that need to be in the optimum range during
production. They are the temperature for the growth of the microbial source and the
optimum temperature at which maximum production of enzyme takes place. The optimum
temperatures for growth and α-Amylase production were found to be 45°C to 46 °C and
50 °C, respectively.
2. pH: Optimum pH is a critical factor for the stability of the enzyme produced. Enzymes are
pH sensitive and hence care must be taken to control the pH of the production process.
Pyrococcus furiosus produces α-Amylase which shows activity at an optimum pH of 6.5–
7.5

4. 3. Moisture: Optimum levels of initial moisture content may vary depending on the microbial
source used. For fungal sources, the moisture content required is less whereas bacterial
sources need more moisture content for a high yield of the enzyme. production of α-
Amylase by Penicillium janthinellum the moisture content was varied within a range of
20- 80% by varying the amount of salt solution used in moistening the substrate particles.
Duration of fermentation
This is a crucial factor in the fermentation process. The enzyme activity increases with an increase
in incubation time till it reaches the optimum duration. In most cases, the production enzymes
begin to decline if the incubation time is further increased. This could be due to the depletion of
nutrients in the medium or the release of toxic substances. Bacillus subtilis, show a high yield of
alpha-amylase after 48 hours of fermentation. Penicillium fellutanum isolated from mangrove
rhizosphere soil. The culture when incubated at 96 h, showed the maximum activity.
Recovery and purification of enzymes
The desired enzyme produced may be excreted into the culture medium (extracellular enzymes)
or may be present within the cells (intracellular enzymes). Depending on the requirement, the
commercial enzyme may be crude or highly purified. Further, it may be in solid or liquid form.
The steps involved in downstream processing i.e. recovery and purification steps employed will
depend on the nature of the enzyme and the degree of purity desired.
In general, recovery of an extracellular enzyme that is present in the broth is relatively simpler
compared to an intracellular enzyme. For the release of intracellular enzymes, special techniques
are needed for cell disruption. Microbial cells can be broken down by physical means (sonication,
high pressure, glass beads). The recovery and purification steps will be the same for both
intracellular and extracellular enzymes, once the cells are disrupted and intracellular enzymes are
released. The most important consideration is to minimize the loss of desired enzyme activity.
Removal of cell debris: Filtration or centrifugation can be used to remove cell debris.
Removal of nucleic acids: Nucleic acids interfere with the recovery and purification of enzymes.
They can be precipitated and removed by adding poly-cations such as polyamines, streptomycin,
and polyethyleneimine.

5. Enzyme precipitation: Enzymes can be precipitated by using salts (ammonium sulfate) organic
solvents (isopropanol, ethanol, and acetone). Precipitation is advantageous since the precipitated
enzyme can be dissolved in a minimal volume to concentrate the enzyme.
Liquid-liquid partition: Further concentration of desired enzymes can be achieved by liquid-liquid
extraction using polyethylene glycol or polyamines.
Separation by chromatography: there are several chromatographic techniques for separation and
purification of enzymes. These include ion-exchange, size exclusion, affinity, hydrophobic
interaction and dye ligand chromatography. Among these, ion- exchange chromatography is the
most commonly used for enzyme purification.
Drying and packing: The concentrated form of the enzyme can be obtained by drying. This can
be done by film evaporators or freeze dryers (lyophilizes). The dried enzyme can be packed and
marketed.
Conclusion
The increasing importance of sustainable development has inspired man to use enzymes for
various reactions as they are biodegradable and can be produced using biological sources. α-
Amylase can be produced using microbes by SSF which employs by products and waste products
of other processes. The enzyme has crucial applications including the production of fructose syrup,
environmentally safe detergents, and baked products.
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Elyasi Far B, Ahmadi Y, Yari Khosroshahi A, Dilmaghani A. Microbial Alpha-Amylase
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10.34172/apb.2020.043. Epub 2020 May 11. PMID: 32665893; PMCID: PMC7335993.

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