AMYLASE
PRODUCTION AND ENZYME ASSAY
AIM:
To
isolate amylase producing organism from the soil sample and assay their
production.
PRINCIPLE:
Amylase is the enzyme which breaks down starch into glucose molecules and
commonly called as glycoside hydrolase enzymes. Amylase is an enzyme that is
used in various industries to rapidly degrade complex polysaccharides (e.g.
starches) into smaller oligosaccharides. Starch is an abundant carbon source in
nature, and -amylase (1, 4-a-D- glucanohydrolase), which hydrolyzes a-1,
4-glucosidic linkage in starch-related molecules, is one of several enzymes
involved in starch degradation. Amylases are among the most important
industrial enzymes and also have great significance in Microbiology studies. We
screened soil bacteria to produce an amylase in media. Bacterial and fungi
strains isolated from garden soil were tested for its abilities to hydrolyze
the structural polysaccharides. The strain grows well at 37o C and
the 2% starch concentration, with PH near neutral. The enzyme activities were
observed at 2% starch concentration. Amylase activity was assayed by measuring
the amount of reducing sugars released from starch using dinitro salicylic acid
method.
MATERIALS
REQUIRED: Starch agar, Iodine Solution, Dinitro salicylic
acid, Maltose, Sodium Phosphate Buffer, Starch solution
PROCEDURE:
1.
Isolation and primary screening for
amylase producers was done by using starch agar (containing 1% starch and 2%
agar) plate method.
2.
Sediment samples were serially diluted
up to 10-4 and 0.1 ml the diluted samples were spread over the
surface of starch agar medium.
3.
Plates were incubated at 30oC
for 24 hrs. Morphologically different colonies were selected for the secondary
screening.
4.
In screening, 50 µl of cell free culture was inoculated
in the wells made in starch agar medium. The plates were incubated at 30oC
for 48 hrs.
5.
After incubation, the plates were
flooded with 1% of iodine solution for 5 min and washed with water to remove
the excess color.
6.
Based on the highest size of zone of
clearance around the well the potential strain was selected and maintained on
starch agar slant.
7.
Enzyme assay by DNS method: The crude
enzyme obtained after centrifugation was assayed for amylase activity by
measuring the release of reducing sugar following the DNS method.
8.
Preparation of Maltose standard curve: A
stock solution of 1mg/ml maltose was prepared in 0.1M sodium phosphate buffer
(pH 7.0) and diluted. The graph was plotted between different concentration of
maltose and their respective O.Ds
9. Enzymatic assay
of amylase: One ml of crude enzyme supernatant was taken in test tube and 1.0
ml of substrate (starch solution) was added in test tube. The test tubes were
covered and incubate at 35°C for 15 minutes in water broth. Then 2.0 ml of DNS
reagent was added in each tube and the reaction was stopped by boiling the
reaction mixture in water bath for 10 minutes. After cooling at room
temperature, the absorbance (O.D) was measured at 540 nm by spectrophotometer
and the released sugar was determined from maltose standard curve. One unit of
amylase activity was defined as the amount of enzyme that released 1µmol
reducing sugar equivalent maltose per minute under the assay condition. The
amount of enzyme produced was expressed as μgm / ml.
10. Calculation: Calculate the amount of reducing sugar present in the
sample using standard graph.
11.
Estimation of glucose: The amount of glucose present per ml in amylase assay mixture was
calculated from the standard graph.
RESULTS AND
DISCUSSION:
Morphologically different strains were selected for amylase production
screening. Based on the screening, No. ------------------- Isolates were
selected for the amylase production.
Table 1:-No. of strains and their characteristics
The
screening using well assay to isolate efficient strain and performed enzyme
assay per ml of production by DNS method.
Table
2: The OD values were tabulated based on their respective dilution.
Photos:
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the appropriate title to the figures and tables
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