Objectives of this proposed research work are as follow:

1. To standardized protocol for commercial production of alpha amylase.

2. To compare the production and thermal stability of amylase secreted from parent and
mutant strains of B. amyloliquefaciens.
3. To improve strain by physical mutagenesis or chemical mutagenesis.
4. To increase production of enzyme on stress condition by supplementation of minimum &
maximum carbon and nitrogen sources, compare it with control (grown normally).
5. To generate the mathematical kinetic models and simulate the production process to
understand the parameters for production rate improvements and the bottlenecks in
production

processes.

Hypothesis of the study

1. Amylase production would be increase by providing various stress condition with
minimum & maximum supplementation of carbon and nitrogen sources.
2. Mutant strain would show much greater amylase production than parent strain.
3. After UV irradiation, strain would be thermo tolerant and pH tolerant.

Methodology
1. Selection of Bacillus amyloliquefaciens
We will use identified bacterial strain of Bacillus species.
2. Biochemical Test for screening of Microbe
The IMViC tests are combination of biochemical test (MacFaddin, 2000).
Indole test
In this test, the organism under consideration is grown in peptone water broth. It contains
tryptophan, which under the action of enzyme tryptophanase is converted to an Indole molecule,
pyruvate and carbon dioxide. The indole is then extracted from the broth by means of xylene. To
test the broth for indole production, Kovac's reagent is added. A positive result is indicated by a
Pink/Red layer forming on top of the liquid.
Methyl Red test
VogesProskauer test

These tests both use the same broth for bacterial growth. The broth is called MRVP broth. After
growth, the broth is separated into two different tubes, one for the Methyl Red (MR) test and one
for the Voges-Proskauer (VP) test. The pH indicator Methyl Red is added to one tube and a red
color appears at pHs lower than 4.2, and indicated positive test. The VP test uses alpha-naphthol
and potassium hydroxide to indicate a positive or negative test.
Citrate Test
This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate as
its sole carbon source. The citrate agar is green before inoculation, and turns blue as a positive
test indicator.
Sugar Fermentations - Glucose, Lactose, Dextrose
Bacterial cells are able to generate energy from nutrients through respiration or through
fermentation. Respiration uses an external electron acceptor, like oxygen (aerobic respiration) or
some other exogenous source (anaerobic respiration) to generate high yields of ATP through
complete oxidation of an organic compound. Fermentation, on the other hand, only partially
oxidizes the substrate and generates a relatively small amount of ATP. The terminal electron
acceptor is usually produced as an intermediate in the pathway and so is internal instead of
external.
Catalase Activity
One of the by-products of oxidation-reduction in the presence of O2 during aerobic respiration is
hydrogen peroxide (H2O2). This compound is highly reactive and must be degraded in the
cytoplasm of the cell producing it. It can be especially damaging to molecules of DNA. Most
aerobes synthesize the enzyme catalase, which breaks down H2O2 into water and oxygen.
3. Strain improvement by mutation
Culture improvement: The bacterial culture (24 h old) is prepared in nutrient broth medium
and centrifuged aseptically at 8,782g for 15 min. The bacterial cells are resuspended in 50 ml of
saline water and diluted up to 106 times (Haq et al, 2010).
Ultraviolet (UV) irradiation: Ten milliliter of the diluted suspension is transferred in a
sterilized Petri plate (180C for 2 h). The Petri plate is placed under a UV lamp for 15, 20, 25

min at different interval. After time intervals, 0.5 ml of the bacterial suspension is transferred to
the petriplates containing nutrient starch agar medium. The plates are placed in a cooled
incubator at 37C for 24-48 h (Haq et al, 2010).
4. Fermentation technique
Alpha-amylase fermentation is carried out by submerge fermentation. 10% from the bacterial
inoculums was inoculated to 500 ml shake-flasks containing 100 ml of a defined medium
(Horikoshi medium II) (Horikoshi, 1999). Soluble starch 1%(w/v), peptone 0.5%(w/v), yeast
extract 0.5%(w/v), MgSO 4.7H 2O 0.02%(w/v), K 2HPO 40.1%(w/v), and Na 2CO 31%. The
flasks were incubated at 37 C in an orbital shaker at 150 rpm for 24 to 48 hr with an initial pH of
7.0. The culture broth was then centrifuged at 3000 rpm for 30 mins at 4 C; the free-cell
supernatant was used as an extracellular crude enzyme.
5. Saccharification activity: Dinitrosalisylic acid (DNS method)
The DNS method used involved estimating the amount of reducing sugar produced (Miller et al,
1959), using 1% soluble starch as substrate. Maltose was used as standard. One unit of enzyme
activity was defined as the amount of enzyme that formed 1 mg of reducing sugar in 1 min.
Maltose is a reducing sugar (those carbohydrates which have a free aldehyde or keto group can
reduce Fehling's and Benedict's reagents are called reducing sugars). Maltose can be used as a
standard for estimating reducing sugar in unknown samples. Constructing a standard curve /
graph for maltose helps us to estimate concentration of reducing sugars present in an unknown
sample and for determining the activity of amylase enzyme in forthcoming experiments. The
standard curve for maltose is usually constructed using 3, 5-Dinitro salicylic acid (DNS) as the
reagent. Maltose reduces the pale yellow colored alkaline 3, 5-Dinitro salicylic acid (DNS) to the
orange- red colored, 3 amino, 5 nitro salicylic acid.
6. Optimization of -amylase activity assay conditions (DNS)
The influence of pH, time of incubation, temperature and soluble starch concentration is
measured to optimize the enzyme assay conditions (DeMoraes et al, 1999).Optimum pH will
determine at different pH range (5 to 10). Reaction time will determine at different time intervals
ranging from 3 to 20 min with 2% soluble starch in phosphate buffer at pH 6.0 as earlier

obtained at 60 C. The optimum temperature was determined by incubation of 2% (w/v) soluble

starch in phosphate buffer pH 6.0 with the crude enzyme at various temperatures ranging from
40 to 100 C for 10 min. Substrate concentration was determined at various ranges from 1 to 8%
(w/v).

7. Estimation of Protein by Bradford and Lowry assay

The estimation of protein done by Bradford and Lowry assay (Lowry et al, 1951).
Bradford Assay
The dye binding protein assays are based on the binding of protein molecules to Coomassie dye
under acidic conditions. The binding of protein to the dye results in a spectral shift, the color of
Coomassie solution changes from brown (absorbance maximum 465nm) to blue (absorbance
maximum 610nm).
The change in color density is read at 595nm and is proportional to the protein concentration.
Lowry Assay
The determination of protein concentration is an essential technique. Under alkaline conditions
cupric ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to
cuprous ions (Cu+).
The

Cuprous

ions

can

also

be

detected

with

Folin

Ciocalteu

Reagent

(phosphomolybdic/phosphotungstic acid. Cuprous ions (Cu+) reduction of Folin Ciocalteu

Reagent produces a blue color that can be read at 650-750nm. The amount of color produced is
proportional to the amount of peptide bonds, i.e. size as well as the amount of protein/peptide.

8. Partial purification of amylase

Gel Filtration chromatography
Chromatography is used to separate organic compounds on the basis of their charge, size, shape,
affinity, or solubility. A chromatography set up consists of a mobile phase (the solvent and the
molecules to be separated) and a stationary phase either of paper (in paper chromatography), a
porous solid matrix or a resin, (in column chromatography) through which the mobile phase
travels. Because of their chemical properties, molecules travel through the stationary phase at
different rates and are separated from one another (Fischer, 1980).

In gel filtration chromatography microscopic, polyacrylamide beads containing small pores are
packed into a column. A protein sample is applied to the top of the column and passes through
the column with the solvent. As the sample moves through the column, smaller molecules pass
through the pores in the beads talking longer path through the column than larger molecules that
cannot enter the beads and simply travel around them. Therefore proteins are separated based
upon their size with the larger the molecules passing through the column faster than smaller
ones.
Ammonium Sulphate Precipitation
The crude broth obtained after fermentation is centrifuged at 5000gfor 30mins to remove the
cell biomass. Solid ammonium sulphate was added slowly to the culture supernatant to get60%
saturation, stirred for 60min, and left for overnight at 4C. The precipitate is harvested by
centrifugation at10,000g for 10 min, dissolved in 50mM glycine-sodiumhydroxide buffer and
dialyzed against same buffer overnight(4C). The dialyzed sample is then assayed for amylase
activity and glucose content (Yandri et al, 2010).
9. Molecular weight determination by SDS-PAGE
SDS-PAGE is performed using 12% polyacrylamide gel under non-reducing conditions. The
protein bands are visualized by staining coomassie brilliant blue (Laemmli, 1970).The molecular
weight of the purified enzyme is determined by comparing with RF valves of standard molecular
weight markers.
10. Mathematical modeling and process simulation studies.
Modeling and simulating microbial fermentation process has become a popular means of
aiding in the understanding of how the process of production operates, and/or predicting how
it would perform under different sets of conditions.

Number of authors have reported

kinetics of alpha-amylase production (Baig et al, 1984; Pazlarova et al, 1984; Ponzo and
Weigand, 1991; Uchiyama and Shioya, 1999; Vortruba et al, 1984; Yoo et al, 1988). Most
researchers have approached modeling using the Logistic for biomass formation. The most
widely used kinetic model for product formation in biotechnology processes is the Luedeking
Piret model (Zeng, 1995). This model assumes that product formation may be attributed to
growthassociated and/or non growthassociated mechanisms. The

non-growth-associated

mechanism is often considered to be conditional on maintaining the functions of cells.

Substrate consumption for the fermentation processes can be described by applying the

modification of Luedeking and Piret model (Weiss and Ollis, 1980). In present work the
last three mathematical models were applied to experimental results of the batch culture of
alpha-amylase production in shake flasks and in the fermentor. Further the production
process is simulated to find the parameters that maximize the production and bottlenecks that
need to overcome to enhance the production.
Discussion
Discussion will be drowned with the help of results.
Conclusions
Conclusion of research work would be highlighted throughout as per formulated hypotheses and
statistical results.
Scope and limitations:
Amylases have potential application in a number of industrial processes such as in the food,
textiles, paper industries, bread making, , detergents, fuel ethanol from starches, fruit juices,
alcoholic beverages, sweeteners digestive aid and spot remover in dry cleaning. Bacterial amylases are now also used in areas of clinical, medicinal, and analytical chemistry. The limiting
factors are environmental parameters and chemical parameters affecting production as well as
growth of parent and mutant strains. One of the main problems affecting the action of these
enzymes in detergent formulations is the possible influence of surfactants on enzymatic
hydrolysis, due to possible interactions of the surfactant both with the reaction substrate as well
as with the enzyme.
Utility:
This study will be helpful to improve the rate of production of -Amylase and strain
improvement by mutation. Amylases constitute a class of industrial enzymes having
approximately 25% of the enzyme market. New and exciting enzyme applications are likely to
bring benefits in other areas: less harm to the environment; greater efficiency; lower costs; lower
energy consumption; and the enhancement of a products properties.