Date: _________ Expt. No: ____ Page No.

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AIM: PRODUCTION OF FUNGAL AMYLASE BY SOLID STATE FERMENTATION

AND ITS PARTIAL PURIFICATION.

Introduction:
Amylase is an extra cellular enzyme, which catalyzes hydrolysis of internal α-1,4-O- glycosidic
bonds in starch and related polysaccharides liberating α-anomeric sugars. Fungal and bacterial amylases
are widely used for the commercial applications in food processing industries. Fungal amylases find
various applications in antistaling (baking industry), haze clarification in fruit juices and alcoholic
beverages, glucose and maltose syrup production and other food products. These amylases have a high
efficiency in saccharification of starch when compared to bacterial amylases.
Solid-state fermentation (SSF) is widely established for the production of enzymes by
filamentous fungi. Morphology and physiology of these molds enable them to penetrate and colonize
various solid substrates. SSF utilizes various agro-industrial wastes as substrate that acts both as physical
support and source of nutrients. Food and agricultural wastes can serve as substrates for the production of
various fermented products and enzymes. SSF offers advantages such as high volumetric productivity,
better product recovery and product characteristics, low capital investment, reduced levels of catabolite
repression, value addition of agricultural industrial wastes reducing pollution problems and less effluent
generation.
Principle:
Solid-state fermentation (SSF) involves the growth of microorganisms on moist solid substrates
in the absence of free water. Different solid substrates viz.; rice bran, wheat bran, coconut oil cake,
gingely oil cake, ground nut oil cake, sesame oil cake, black gram bran, cassava bagasse etc. rich in starch
can be used for amylase production. Organisms inoculated grows on the solid matrix by secreting
extracellular enzymes which acts on the complex solid substrate and releases the simpler molecules which
can be absorbed by the organisms. Secreted enzymes or product of interest remains in the solid matrix
medium which can be recover later.
Requirements:

1) 0.1% Tween20 solution

2) Amylase production culture of Aspergillus spp.
3) Basal Salt Solution
4) 0.1M Sodium Phosphate Buffer (pH=7)
5) 1% Starch solution
6) Standard glucose solution (1 mg/ml)
7) DNSA reagent
8) Standard Bovine Serum Albumin (BSA) solution (200µg/ml)
9) Folin-Lowry Reagents: Alkaline Copper Sulfate
Folin-Ciocalteau Reagent
10) Flasks, glass plate and tray
11) Rotary shaker, centrifuge, spectrophotometer
12) Regular filter paper, whatmann filter paper, charcoal

Composition of Basal Salt Solution.

Magnesium sulphate 0.20

Calcium chloride 0.02
Monopotassium phosphate 1.00
Dipotassium phosphate 1.00
Ammonium nitrate 1.00
Ferric chloride 0.05
Final pH (at 25°C) 7.0
Procedure:
1) Use wheat or rice bran as solid substrates for solid state fermentation (SSF).
2) Weigh 10 g of solid substrate (Rice bran ) and hydrate with 10 ml of basal salt solution and
adjust with moisture content. Sterilize it by autoclaving.
3) After sterilization add 1% inoculum of the Aspergillus niger to each different media of solid
state fermentation. (Prepare the inoculum by suspending fungal spores in 10 ml of 0.1%
Tween20 solution.
4) Incubate at room temperature for six days.
Enzyme extraction: -
1) Add 22 ml of 0.1M sodium phosphate buffer (pH 7) to each of the inoculated substrate beds.
2) Shake vigorously in rotary shaker for 1 hour at 120rpm.
3) Filtere the mixture through muslin cloth/ filter paper and collect the filtrate.
4) Centrifuge the collected filtrate at 10,000 rpm at 40C for 10min.
5) Then, filter the supernatant with whatmann filter paper covered with the charcoal bed for
more transparency.
6) Use supernatant/ filtrate as the crude enzyme preparation.
7) Enzyme amylase was assayed by Dinitrosalicylic acid method and total protein content by folin
lawry’s method.
8) Calculate the specific activity of enzyme amylase.
PARTIAL PURIFICATION OF AMYLASE ENZYME

Introduction and Principle

Ammonium sulfate precipitation is one of the most commonly used methods for protein
purification from a solution. Ammonium sulfate is an inorganic salt with a high solubility
that disassociates into ammonium (NH4+) and sulfate (SO42−) in aqueous solutions.
Ammonium sulfate is especially useful as a precipitant because it is highly soluble,
stabilizes protein structure, has a relatively low density, is readily available, and is
relatively inexpensive.
Proteins are usually stored in ammonium sulfate because it inhibits bacterial growth. With
the addition of ammonium sulfate, proteins unfolded by denaturants can be pushed into
their native conformations. This can be seen with the folding of recombinant proteins. The
solubility of proteins varies according to the ionic strength of the solution, thus according
to the salt concentration. At low ion concentrations (<0.5 M), the solubility of proteins
increases with increasing salt concentration, an effect termed "salting in". As the salt
concentration is further increased, the solubility of the protein begins to decrease. At a
sufficiently high ionic strength, the protein will precipitate out of the solution, an effect
termed "salting out".
When the ammonium (NH4+) and sulfate (SO42−) ions are within the aqueous solution they
are attracted to the opposite charges evident on the compound that is being purified. This
attraction of opposite charges prevents the water molecules from interacting with the
compound being purified, leading to the precipitation or "salting out".
Proteins differ markedly in their solubilities at high ionic strength, therefore, "salting out" is
a very useful procedure to assist in the purification of the desired protein. Ammonium
sulfate is commonly used for precipitation because of its high solubility with the
mechanism of salting-out, there is an omission of the salt from the layer water, which is
closely associated with the surface of the protein, known as the hydration layer. The
hydration layer plays a vital role in sustaining solubility and suitable natural conformation.
There are three main protein-water interaction: ion hydration between charged side chains,
hydrogen bonding between polar groups and water, and hydrophobic hydration. Once salt
is added to the mixture, there is an increase in the surface tension of the water, thus
increasing hydrophobic interactions between water and the protein of interest. The protein
of interest then reduces its surface area, which diminishes its contact with the solvent. This
is shown by the folding and self-association, which ultimately leads to precipitation. The
folding and self-association of the protein pushes out free water, leading to an increase in
entropy and making this process energetically favourable.
Ammonium sulfate precipitation is a useful technique as an initial step in protein
purification because it enables quick, bulk precipitation of cellular proteins.

DIALYSIS
Dialysis Tubing is a type of semi- or partially permeable membrane tubing made from
regenerated cellulose or cellophane. The solution to be dialyzed is placed in a sealed
dialysis membrane and immersed in a selected buffer; small solute molecules then
equilibrate between the sample and the dialysate. Concomitant with the movement of small
solutes across the membrane, however, is the movement of solvent in the opposite
direction. This can result in some sample dilution (usually
<50%). It is used in clinical circumstances to ensure a filtered flow of molecules,
preventing the flow of larger solute molecules.
Dialysis membranes are available in a number of thicknesses and pore sizes. Thicker
membranes are tougher, but restrict solute flow and reach equilibrium more slowly. Pore
size is defined by “molecular weight cut-off” (MWCO) i.e., the size of the smallest particle
that cannot penetrate the membrane. For most protein dialyses, a MWCO of 12,000 to
14,000 is appropriate, whereas a MWCO of 3500, 2000, or even 1000 is appropriate for
peptides. Most dialysis membranes are made of derivatives of cellulose. They come in a
wide variety of MWCO values, ranging from 500 to 500,000.

Requirements
a. Amylase producing culture of Aspergillus spp.
b. Sterile Tendler’s non synthetic medium
c. 500 ml Erlenmeyer’s flask, sterile pipettes and test tube
d. Rotator shaker, high speed centrifuge
e. Alkaline copper reagent, 2N Folin – Ciocalteu reagent
f. DNSA reagent, phosphate buffer
g. 500 maltose solution (For standard)
 h. Magnetic stirrer
i. Ice cubes
j. Dialysis bag and clamps
k. Spectrophotometer
l. Bovine serum albumin solution
m. Centrifuge tube
n. Flask and beaker
o. Anhydrous ammonium sulphate
p. 0.1 M Sodium phosphate buffer
q. 0.2 M Sodium bicarbonate
r. 10 mM EDTA
s. 1 Mm EDTA

Procedure:
1. Prepare the Tendler’s non-synthetic medium containing starch and distribute about 150 ml
amounts in 500ml flask.
2. Inoculate the above medium with 15ml activated culture.
3. Place the inoculated medium on a rotator shaker at 120 r.p.m for 48-72 hr at 30ºC.
4. After 72 hrs aseptically take 10 ml of medium and filter it through Whatman paper.
5. The filtrate is used for checking the amylase activity and measure the total protein by
DNSA and Folin-Lowery’s method respectively. This is crude enzyme activity.
6. Take remaining broth (125 ml) in centrifuge tube. Centrifuge it at 10000 rpm for 15
minutes.
7. Collect supernatant i.e. 100 ml in small beaker add clean magnet and put on magnetic
stirrer.
8. Place this small beaker in large beaker containing 4-5 ice cubes (for maintain low temp).
9. Add grinded fine powder of ammonium sulfate up to 70% concentration and continuously
stirring it. (As per table). Add only pitch of grinded ammonium sufate once it dissolved
again pitch of grinded ammonium sulfate. Maintain cooling condition during precipitation
process.
10. After complete dissolution of ammonium sulfate, precipitation will observe. Put beaker at
4ºC overnight and collect the precipitates by centrifugation at 10000 rpm for 15 minutes at
4ºC temperature (stored the precipitates at 4ºC temperature).
11. If you add 50% ammonium sulfate first time, then rest of 20% you can add similarly
described in above steps
12. Redissolve precipitates in 1 to 2 ml of the 0.1 M sterile sodium phosphate buffer (pH 6.0 to
6.5). Store it to 4 to 8 ºC temperature
 Dialysis for removal of salts
1. Cut the dialysis bag as per the volume of your resolved precipitates. Keep the bags in
distilled water for 1 to 2 minutes
2. Open dialysis bag by keeping into 0.2 M sodium bicarbonate in boiling condition for 10
min.
3. Wash the bag and deep in 10mM EDTA in boiling condition for 10 min followed by same
treatment in 1mM boiled EDTA solution.
4. Wash the bags with distilled water (keep in dH2O for maximum 20 minutes)
5. Fill up the dialysis bag with precipitated protein. Tightly clamp the both ends of the bags.
6. Keep the bag into 1 lit of 100 times diluted 0.2 mM sodium phosphate buffer overnight in 4
to 8 ºC.
7. Next day, remove the excess water filled in bag by placing the bag over table sugar /
sucrose for 1 hr.
8. After removal of water, withdraw the remaining content in the bag.
9. Dissolute the remaining content before checking the enzymes activity and protein (usually
50 times or 100 times dilution is required)
10. Perform DNSA method to check enzyme activity and estimate total protein by Folin-
Lowery method.
11. Calculate the specific activity & fold purification of partially purified enzyme.
 Table 1. Ammonium sulphate saturation table using solid ammonium sulphate
* Initial concentration of ammonium sulphate (% saturation at 0°C)
* Final concentration of ammonium sulphate (% saturation at 0°C)
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Grams solid ammonium sulfate to add to 100 ml of solution/ broth
0 10.7 13.6 16.6 19.7 22.9 26.2 29.5 33.1 36.6 40.4 44.2 48.3 52.3 56.7 61.1 65.9 70.7
5 8 10.9 13.9 16.8 20 23.2 26.6 30 33.6 37.3 41.1 45 49.1 53.3 57.8 62.4 67.1
10 5.4 8.2 11.1 14.1 17.1 20.3 23.6 27 30.5 34.2 37.9 41.8 45.8 50 54.4 58.9 63.6
15 2.6 5.5 8.3 11.3 14.3 17.4 20.7 24 27.5 31 34.8 38.6 42.6 46.6 51 55.5 60
20 0 2.7 5.6 8.4 11.5 14.5 17.7 21 24.4 28 31.6 35.4 39.2 43.3 47.6 51.9 56.5

25 0 2.7 5.7 8.5 11.7 14.8 18.2 21.4 24.8 28.4 32.1 36 40.1 44.2 48.5 52.9
30 0 2.8 5.7 8.7 11.9 15 18.4 21.7 25.3 28.9 32.8 36.7 40.8 45.1 49.5
35 0 2.8 5.8 8.8 12 15.3 18.7 22.1 25.8 29.5 33.4 37.4 41.6 45.9
40 0 2.9 5.9 9 12.2 15.5 19 22.5 26.2 30 34 38.1 42.4
45 0 2.9 6 9.1 12.5 15.8 19.3 22.9 26.7 30.6 34.7 38.8

50 0 3 6.1 9.3 12.7 16.1 19.7 23.3 27.2 31.2 35.3

55 0 3 6.2 9.4 12.9 16.3 20 23.8 27.7 31.7
60 0 3.1 6.3 9.6 13.1 16.6 20.4 24.2 28.3
65 0 3.1 6.4 9.8 13.4 17 20.8 24.7
70 0 3.2 6.6 10 13.6 17.3 21.2

75 0 3.2 6.7 10.2 13.9 17.6

80 0 3.3 6.8 10.4 14.1
85 0 3.4 6.9 10.6
90 0 3.4 7.1
95 0 3.5
100 0

# Adopted from Dawson et al. (1986).

#Values given are the amount of solid (NH4)2SO4 required to bring a solution of known initial
saturation to a desired final saturation. For the data listed, the saturation is relative to full saturation
at 0°C.
Result:

Conclusion:

Reference:

Suganthi, R., Benazir, J. F., Santhi, R., Ramesh Kumar, V., Hari, A., Meenakshi, N., ... & Lakshmi, R.
(2011). Amylase production by Aspergillus niger under solid state fermentation using agroindustrial
wastes. International Journal of Engineering Science and Technology, 3(2), 1756-1763.

Swetha, S., Nampoothiri, K. M., Soccol, C. R., Pandey, A., & Dhanya, G. (2007). Alpha amylase
production by Aspergillus oryzae employing solid–state fermentation.

Flickinger, M. & Drew, S.(1999) Encyclopedia of Bioprocess Technology,(Volumes 1 - 5) : ISBN 0-471-

13822-3.

Padmavathi, T., Bhargavi, R., Priyanka, P. R., Niranjan, N. R., & Pavitra, P. V. (2018). Screening of
potential probiotic lactic acid bacteria and production of amylase and its partial purification. Journal of
Genetic Engineering and Biotechnology, 16(2), 357-362.

Teacher’s Sign__________
Observation: Amylase Production

Standard Estimation of Maltose:

40%
Sr. Standard Concentration D/W Rochelle O.D. at
No Maltose (ml) (μg/ml) (ml) salt (ml) 540 nm

1 Blank 0 1 1.0 0
2 0.1 20 0.9 1.0 0.21
3 0.2 40 0.8 Keep in 1.0 0.418
boiling
4 0.3 60 0.7 2 ml water 1.0 0.546
5 0.4 80 0.6 DNSA bath for 1.0 0.675
6 0.5 100 0.5 10 1.0 0.742
minutes
7 0.6 120 0.4 1.0 0.886
8 0.7 140 0.3 1.0 0.978
9 0.8 160 0.2 1.0 1.012
10 0.9 180 0.1 1.0 1.125
11 1 200 0 1.0 1.202

standard graph of maltose

1.4 y = 0.0057x + 0.1391

R² = 0.966
1.2

1
OD At 540 nm

0.8

0.6

0.4

0.2

0
0 50 100 150 200 250
concentration (μg/ml)
 Total Protein Estimation:

Total Protein Estimation for Partially Purified Enzyme :-

Sr Volume Volume Conc. of Volume of Volume of O.D.

no. of of Standard Alkaline Folin- at
Standard Distilled BSA Copper Ciocalteau 660
BSA Water solution Sulfate Reagent nm
solution (ml) (µg) Reagent(ml) (ml)
(ml) Incubate all
B 0.0 1.0 - 5.0 Incubate all 0.5 tubes in
1 0.2 0.8 40 5.0 tubes at 0.5 dark at 0.20
2 0.4 0.6 80 5.0 room 0.5 room 0.35
3 0.6 0.4 120 5.0 temperature 0.5 temperature 0.46
4 0.8 0.2 160 5.0 for 15 mins. 0.5 for 30 mins. 0.55
5 1.0 0.0 200 5.0 0.5 0.70
Unknown Sample
1
2

Standard curve for Folin-Lowry

0.8
y = 0.6017x + 0.0977
0.7
R² = 0.9928
0.6

0.5
OD At 750 nm

0.4

0.3

0.2

0.1

0
0 0.2 0.4 0.6 0.8 1 1.2
concentration
Amylase enzyme assay

Tube 1% Buffer Enzyme DNSA 40% O.D.

No. Starch (ml) Solution Incubate Reagent Incubate Rochelle Cool at At
(ml) (ml) at 370 C (ml) for 10 Salt room 540nm
for 30 minutes (ml) temp-
Blank - 2.0 - minutes 2.0 in 1.0 erature
Test 1.0 - 1.0 2.0 boiling 1.0
Substrate - 1.0 1.0 2.0 water 1.0
blank bath

Enzyme activity

Days Dilution aliquot O.D. Concentration Dilution Final Enzyme

factor concentration Unit

Protein estimation

Days Dilution Aliquot O.D. at Concentration Final Average

750nm concentration protein

Specific activity

Enzymes Enzyme unit Total protein Specific activity

(µg/ml)
O.D. = O.D. of Test Sample – O.D. of Substrate Blank

= _____ - _____

= _____

Conc. of sugar in terms of glucose(µg/ml) = ______

Enzyme activity per min. = Concentration of sugar

Time of incubation

= ___________ µg/ml min

Specific Activity = Enzyme Activity

Average conc. of Total Protein

= ___________U/min.
 Observation: Amylase Partial Purification

Fraction Volume Total Activity Total Specific Fold %Yield

(ml) protein (U/ml/min) activity activity purification
(µg)