MATERIALS AND METHODS

The details of materials used and methods to be adopted for conducting the present investigation are
described in this chapter under appropriate heads.
3.1 Experimental Site: The experiment was conducted in Biochemistry and Molecular Biology
laboratory of MGM College of Agricultural Biotechnology, Aurangabad.
Lab Condition:-The experiment was conducted under controlled lab condition.
Treatment details:
Statistical Design : - Completely Randomize Design (CRD)
1. No. of treatments :9
2. Total no. of replication : 3
3. Treatment details : - Different ranges of pH.
No. of Treatments
Treatments
T1 pH4
T2 pH5
T3 pH6
T4 pH6.5
T5 pH7
T6 pH7.5
T7 pH8
T8 pH9
T9 pH10

Sample collection

Soil samples were collected in containers under sterile conditions from garri processing site in
Ogige market, Nsukka, Enugu State, Nigeria.

Inoculation of PDA plates

One milliliter (1 ml) each from the above dilutions was transferred into the PDA plates with the
aid of a pipette. The plates were observed for 72 h at room temperature. Morphologically,
distinct colonies were observed and were purified by repeated sub culturing on separate PDA
plates.

Screening for amylase activity and identification of the isolates

The colonies on PDA plates were transferred onto soluble starch agar medium flooded with
iodine. Isolates with clear zone are selected. The selected amylase producing fungal isolate
were characterized and identified on the basis of morphological feature and microscopy using
lactophenolcotton-blue stain. Among the characteristics used were colonial characteristics
such as surface appearance, texture and colour of the colonies. In addition, microscopy
revealed vegetative mycelium including presence or absence of cross-walls, diameter of
hyphae.
Subculturing of penicillium culture
The colonies on PDA plates were transferred into PDB (potato dextrose broth) and the
culture were kept for 72 hours at 30c in incubator.
Production Medium for Amylase Enzyme

Fermentation medium comprising of starch 20 g/L, yeast extract 0.5 g/L, KH 2PO4 10 g/L,
(NH4)2SO4 10.5 g/L, MgSO4.7H2O 0.3 g/L, CaCl2 0.5 g/L, FeSO4.7H2O trace element,
MnSO4.7H2O 0.004 g/L and ZnSO4.H2O trace element were prepared with incubation
temperature.The production medium were inoculated with loopfull of spores of penicillium sp.
from PDB. The media were inoculated at 30c in orbital shaker at 100 rpm for 72 hours.
Enzyme Extraction
After complete growth of penicillium on Production medium the medium with culture were
filtered out with the help of filter paper. Now the filtrate were Centrifuged at 7000 rpm for 15
min. The supernatant were taken as enzyme.

3.6 Preparation of Standard Maltose Curve:

3.6.1Materials required:-

Phosphate buffer:-

Prepare 500ml of each of 1M Sodium dihydrogen phosphate (Na H2 Po4) and Disodium Hydrogen
phosphate (Na2 H Po4) solutions separately. Adjust the pH 6.9 of the buffer using pH meter by adding
drop wise Disodium Hydrogen Phosphate(Na2 H Po4) to the solution of Sodium Dihydrogen
Phosphate(Na H2 Po4) then dilute the final buffer to required dilutions.

Substrate: -Starch (Dissolve 1gm starch in 100ml of 1 M phosphate buffer)

Dinitrosalicylic acid (DNS):-

Suspend 1 gm of DNS in 20 ml of 2 N NAOH and add the suspension carefully to 50 ml of water

containing 30gm sodium potassium tartarate and made it up to 100 ml using water store the solution in
brown bottles .

Maltose solution: - Dissolve 50mg of Maltose in 50ml of distill water

3.6.2 Procedure:
1} Different aliquot of standard maltose ranging from 0.4-4ml was pipette out in to clean test tubes.
2} final volume was made up to 4ml with distilled water.
3} 2ml of alkaline DNS reagent was added in all the tubes.
4} then the tubes were incubated in boiling water bath for 5 min and cooled.
5} 10 ml of distilled water was and added in all the tubes and O.D was read at 540nm.

3.8 Determination of optimum Temperature of amylase enzyme

3.8.1 Procedure
1}6 clean test tubes were take. Mark them with specific temperature
(Blank,20c,25c,30c,35c,40c).
2} Add o.5To each tube at 0.5 ml Enzyme and 0.5ml of substrate was add and tubes were incubated for
5min at respective temperature.
3} after 5min. the reaction was arrested by pipetting 1ml of DNS solution in all the tubes and mix well
4} The tubes were then incubated in boiling water bath for 10min.
5} 5ml of distilled water was added in all the tubes and O.D was measured at 540nm.