Appendix - Shodhganga

Appendix – I
Appendix – I
List of Media
Actionmycete Isolation Agar:
Sodium caesinate
L-Aspargine
Sodium propionate
Dipotassium phosphate
MgSO4.7H2O
FeSO4.7H2O
Agar
Distilled Water
pH
Bennet’s Agar
Glucose
Tryptone
Meat extract
Yeast extract
Agar
Distilled Water
pH
X+P+ Medium
Xylose
Peptone
KNO3
K2HPO4
NaCl
MgSO4.7H2O
FeSO4
CaCO3
Agar
Distilled Water
pH
X+P- Medium
Xylose
KNO3
K2HPO4
NaCl
MgSO4.7H2O
(gm/100ml)
0.20
0.01
0.40
0.05
0.01
0.0001
3.0
100mL
8
(gm/100ml)
1.0
0.1
0.1
0.1
3.0
100mL
8
(g/100ml)
1.00
0.03
0.20
0.20
0.20
0.05
0.01
0.02
2.00
100mL
8
(g/100ml)
1.00
0.20
0.20
0.20
0.05
231
Appendix – I
FeSO4
CaCO3
Agar
Distilled Water
pH
0.01
0.02
2.00
100mL
8
Wheat Bran Media
(gm/100ml)
Wheat bran
Peptone
KNO3
K2HPO4
NaCl
MgSO4.7H2O
FeSO4
CaCO3
Agar
Distilled Water
pH
1.00
0.03
0.20
0.20
0.20
0.05
0.01
0.02
2.00
100mL
8
Medium used for qualitative GI Production on plate
iiiiii-
X+P- Medium
X+P+ Medium
Wheat Bran Media
Medium used for qualitative Amylase Production on plate
1% Starch incorporated in Bennett’s Agar
Medium used for qualitative Cellulase Production on plate
1% Cellulose incorporated in Bennett’s Agar
Medium used for qualitative Protease Production on plate
iii-
1% Casein incorporated in Bennett’s Agar
1% Gelatin incorporated in Bennett’s Agar
Medium used for qualitative Lipase Production on plate
Tributyrine Agar
(g/100ml)
Peptic digest of animal tissue
Yeast extract
Tributyrin Oil
Agar
Distilled Water
pH
5.00
3.00
1.00ml
3.00
100mL
8
232
Appendix – I
Medium used for qualitative Pectinase Production on plate
1% Pectin incorporated in Bennett’s Agar
Media combinations used for screening of best production medium
Medium No. 1- Bennett’s Broth [Modi, 1993]
(g/100ml)
Glucose
1.0
Tryptone
0.1
Meat extract
0.1
Yeast extract
0.1
Distilled Water
100mL
pH
8
Medium No. 2 - 1% Xylose incorporated in Bennett’s Broth
Medium No. 3 [Manhas et al., 2004]
Xylose
Peptone
KNO3
K2HPO4
NaCl
MgSO4.7H2O
FeSO4
CaCO3
Agar
Distilled Water
pH
Medium No. 4[Pawar et al., 1994]
Glucose
Potassium hydrogen phosphate
MgSO4.7H2O
Yeast extract
CoCl2
Distilled Water
pH
Medium No. 5[Kasumi et al., 1981]
Xylose
Glucose
NaCl
MgSO4.7H2O
Peptone
Meat extract
Yeast extract
CoCl2
(g/100ml)
1.00
0.03
0.20
0.20
0.20
0.05
0.01
0.02
2.00
100mL
8
(g/100ml)
1.00
0.05
0.25
0.50
0.005
100mL
8
(g/100ml)
0.70
0.30
0.50
0.05
1.00
0.50
0.25
1mM
233
Appendix – I
Distilled Water
pH
Medium No. 6[Chen et al., 1979]
Straw hemi cellulose
Corn Steep Liquor
MgSO4.7H2O
Distilled Water
pH
Medium No. 7[Lobanok et al., 1998]
Glucose
Xylose
Peptone
Yeast extract
K2HPO4
MgSO4.7H2O
Distilled Water
pH
Medium No. 8[Strandberg et al., 1971]
Xylose
Dextrose
Beef extract
Yeast extract
Peptone
NaCl
MgSO4.7H2O
CoCl2.6H2O
Distilled Water
pH
Medium No. 9[Srih-Belghith et al., 1998]
Xylose
Peptone
Yeast extract
MgSO4.7H2O
CoCl2.6H2O
Distilled Water
pH
Medium No. 10[Hasal et al., 1992]
Corn steep liquor
Yeast Extract
NH4NO3
100mL
8
(g/100ml)
1.00
2.50
1.00
100mL
8
(g/100ml)
0.30
0.70
1.00
0.50
0.30
0.10
100mL
8
(g/100ml)
0.70
0.30
0.50
0.25
1.00
0.30
0.05
0.024
100mL
8
(g/100ml)
1.00
3.00
0.50
0.10
0.01
100mL
8
(g/100ml)
1.2ml
0.12
0.08
234
Appendix – I
MgSO4.7H2O
CoCl2.6H2O
Distilled Water
pH
Medium No. 11[Dhungel et al., 2007]
Xylose
Tryptone
Yeast extract
MgSO4.7H2O
Distilled Water
pH
Phenol Red Broth Base
Proteose peptone
Beef extract
Sodium chloride
Phenol red
Distilled Water
pH
0.05
0.024
100mL
8
(g/100ml)
1.00
1.00
0.70
0.10
100mL
8
(g/100ml)
1.0
0.1
0.5
0.0018
100mL
7.4
Sugar Discs used were from Hi media
Maltose
Mannitol
Mannose
Melibiose
Raffinose
Rhamnose
Salicin
Sorbitol
Sucrose
Trehalose
Xylose
Adonitol
Arabinose
Cellobiose
Dextrose
Dulcitol
Fructose
Galactose
Inositol
DD005
DD006
DD007
DD030
DD029
DD010
DD001
DD012
DD013
DD031
DD014
DD025
DD001
DD028
DD002
DD003
DD017
DD016
DD027
235
Appendix – I
Inulin
Lactose
DD026
DD004
Nitrate Reduction Broth
Peptic digest of animal tissue
Beef extract
KNO3
Distilled Water
pH
Confirmation of Nitrate reduction test
Sulphanillic acid
α-naphthylamine
0.5
0.3
0.1
100mL
7.0
236
Appendix - II
Appendix - II
List of Reagents
A. Visualisation of Zone for amylase and cellulase production
Lugol’s iodine solution
Iodine
1g
Potassium iodide
2g
Distilled Water
100mL
B. Visualisation of Zone for gelatinase production
Commassie Brilliant Blue
0.25% commassie brilliant blue dye in methanol: acetic acid: water in a
ratio of 50:10:40.
C. Visualisation of Zone for pectinase production
Alcoholic CTAB
1g CTAB was dissolved in 15% Ethyl Alcohol.
D. Glucose Isomerase Assay:
1. 0.2 M Sodium Phosphate Buffer (pH : 7):
a. NaH2PO4.2H2O
0.2 M NaH2PO4.2H2O is prepared by dissolving 3.1202 g in 100 ml of
distilled water.
b. Na2HPO4
0.2 M Na2HPO4 is prepared by dissolving 2.8392 g in 100 ml of distilled
water.
Mix the components in an appropriate ratio to obtain phosphate buffer of pH:
7.
2. 1.0 M Glucose:
Dissolve 18.016 g of D-Glucose in 100 ml of distilled water to prepare a solution
of 1 M.
3. 0.1 M Magnesium Sulphate:
Dissolve 24.072 g of MgSO4 in 100 ml of distilled water to prepare a solution of
0.1 M.
4. 0.01 M Cobalt Chloride:
Prepare a 0.01 M of CoCl2 in distilled water by dissolving 0.2379 g of CoCl2 in
100 ml of distilled water.
237
Appendix - II
5. 0.5 M Perchloric acid:
A 0.5 M solution of perchloric acid can be prepared by taking 4.7 ml of acid and
making up the volume to 100 ml with distilled water.
E. Estimation of the Fructose by Cysteine Carbazole Method:
1. Standard Fructose Solution:
Prepare a 100µg/ml of fructose in distilled water by dissolving .01 g of DFructose in 100 ml of distilled water.
2. 1.5 % Cystein Hydrochloride:
Dissolve 1.5 g of Cystein Hydrochloride in 100 ml of distilled water to prepare a
solution of 1.5%.
3. 70% H2SO4:
Prepare a 70% H2SO4 solution in distilled water by mixing 70 ml of acid with 30
ml of distilled water.
4. 0.12% Carbazole
A 0.12% carbazole solution is prepared in 95% ethanol.
F. Estimation of the Total Protein by Folin Lowry Method:
1. Standard BSA Solution:
Prepare a 30 µg/ml of BSA in distilled water by dissolving 0.03 g of BSA in
distilled water.
2. 2% Na2CO3 in 0.1 N NaOH:
Dissolve 2 g of Na2CO3 solution in 0.1 N NaOH.
3. 1% CuSO4.5H2O:
Dissolve 1 g of CuSO4.5H2O in 100 ml of Distilled water.
4. 2% Sodium Potassium Tartrate:
Dissolve 2 g of Sodium Potassium Tartrate in 100 ml of Distilled water.
5. 1N Folin-Ciocalteu Reagent:
Dilute the commercially available 2N or 3N FC reagent to 1N with distilled
water.
G. Amylase Assay:
1. Starch solution – 1% (w/v) Starch
2. DNS Reagent
Dissolve the following constituents in 100ml of Distilled water:
DNS
0.748 g
NaOH
1.398 g
238
Appendix - II
Sodium Potassium Tartrate
Phenol
Sodium Meta bi Sulphite
2.161 g
0.536 g
0.586 g
3. Standard Sugar Solution
Prepare standard maltose and glucose solution of strength 5mg/ml in distilled
water.
H. Cellulase (Filter Paper) Assay:
1. Cellulose- Whatman Filter papers strip of 50mg.
2. Citrate Buffer (pH4.8):
a. Citric Acid
0.2 M Citric Acid is prepared by dissolving 4.20g in 100 ml of distilled water.
b. Sodium Citrate
0.2 M Sodium Citrate is prepared by dissolving 5.88 g in 100 ml of distilled
water.
Mix the components in an appropriate ratio to obtain citrate buffer of pH: 7.
2. DNS Reagent
As prepared for amylase assay.
I. Estimation of Protease Activity by Nagase’s Method:
1. Acetate Buffer (pH 7.5):
a. Acetic Acid
0.2 M Acetic Acid is prepared by taking 1.15 ml of acetic acid and then the
volume is made up to 100 ml with distilled water.
b. Sodium Acetate
0.2 M Sodium Acetate is prepared by dissolving 2.7216 g in 100 ml of
distilled water.
Mix the components in an appropriate ratio to obtain acetate buffer of pH: 7.5.
2. 0.05 M Na2HPO4:
0.05 M Na2HPO4 is prepared by dissolving 0.7098 g in 100 ml of distilled water.
3. Casein Solution (pH : 7.5)
Dissolve 0.65g casein in 80mL of 0.05M Na2HPO4 adjust the pH to 7.5 with 0.1N
NaOH and makeup the volume to 100mL.
4. 0.11 M Tri-Chloro Acetic Acid
This solution contains 0.11M tri-chloro acetic acid, 0.22M sodium acetate and
0.33M acetic acid in distilled water.
239
Appendix - II
5. 0.55 M Na2CO3.3H2O
The solution can be prepared by dissolving 5.8294 g of Na2CO3.3H2O in distilled
water.
6. 1N Folin-Ciocalteu Reagent:
Dilute the commercially available 2N or 3N FC reagent to 1N with distilled
water.
J. Reagents Used in Thin Layer Chromatography:
1. Solvent System:
The solvent system used for the separation of various components by thin layer
chromatography was the mixture of Chloroform, Glacial acetic acid and water in
the ratio of 6:7:1.
2. Visualizing Agent:
The visualizing agent used was a mixture of methanol and H2SO4 in the ratio of
8:2.
K. Reagents used in SDS- PAGE:
1. 30% Acrylamide Solution:
100 ml of 30% Acrylamide solution can be prepared by dissolving 1g of Bis
Acrylamide and 29 g of Acrylamide in 100 ml of distilled water. The solution has
to be stored at 2-8°C in a dark glass bottle as it is light sensitive.
2. 0.5 M Tris-Cl, pH 6.8:
100 ml of 0.5 M Tris-Cl can be prepared by dissolving 6.0 g of Tris in 60 ml of
distilled water. The pH is then adjusted to 6.8 using 1M HCl or 1M NaOH and
then the volume is made up to 100ml with distilled water.
3. 1.5 M Tris-Cl, pH 8.8:
Dissolve 18.0 g of Tris in 80 ml of distilled water. Adjust the pH to 8.8 using 1M
HCl or 1M NaOH and make up the volume to 100ml with distilled water.
4. 20% Sodium Dodecyl Sulphate:
The solution can be prepared by dissolving 20 g of SDS in distilled water.
5. 10% Ammonium per Sulphate:
Dissolve 10 g of Ammonium per Sulphate in 100 ml of distilled water to prepare
a solution of 10% Ammonium per Sulphate.
6. 2X SDS Gel Loading Buffer:
The SDS gel loading buffer consists of the following components (for 100 ml):
Glycerol
20.0 ml
Tris Base
1.512 g
SDS
4.0 g
BPB
0.04 g
240
Appendix - II
Dissolve Tris Base in 70 ml of distilled water and adjust the pH to 6.8. Mix all
the components and make up the volume to 100 ml with distilled water. Before
sample preparation, dilute sample buffer 1:2 with distilled water each time
fresh.
7. SDS Gel Tris - Glycine Running Buffer (10 X):
For preparing 100 ml of SDS Gel Tris Glycine running buffer dissolve the
following components in 100 ml of distilled water:
Tris Base
Glycine
SDS
3.03 g
14.4 g
1.0 g
8. SDS Gel Tris - Glycine Buffer (1 X) Working Solution:
For preparing 1 Lt of working solution, take 100 ml of the stock SDS Gel Tris Glycine Running Buffer and make up the volume to 1 Lt with distilled water.
9. Silver Staining Solution:
Following solutions are involved in silver staining solution, the compositions of
which are mentioned below:
i.
Solution A:
The solution A consists of 50 % Methanol, 10 % Acetic acid and 40%
Distilled water.
ii.
Solution B:
Solution B is a 5 % Methanol solution in distilled water.
iii.
Solution C:
Solution C is 0.02 % Sodium thiosulphate.
iv.
Solution D:
This solution is a 2 % silver nitrate solution prepared by dissolving 2 g of
silver nitrate in distilled water.
v.
Solution E:
Solution E consists of 3 % Sodium Carbonate. Dissolve 3 g of sodium
carbonate in 50 ml of distilled water. Add 50 µl of 37 % Formaldehyde
solution and 2 ml of 0.02%
Sodium thiosulphate solution to above sodium carbonate solution and make
up the final volume to 100 with sterile distilled water.
vi.
Solution F:
A 1.4 % Na-EDTA solution is prepared by dissolving 1.4 g of EDTA in 100 ml
of distilled water.
10.
The Running Gel:
The Gel in which the separation of various protein samples is brought about is
composed of Separating Gel and the Stacking Gel. The compositions of which
are discussed below:
241
Appendix - II
i.
Separating Gel (13% Gel):
Add the reagents into a clean glass beaker in the same sequence as given
below:
For 20 ml
Water
Acrylamide mix (30%)
1.5 M Tris Cl pH: 8.8
20% SDS
Ammonium Persulfate (10%)
TEMED
5.985 ml
8.6 ml
5.0 ml
100 µl
200 µl
8 µl
Mix the above contents by swirling gently in glass beaker and pour it into
the casting unit using a suitable pipeete till 1-2.5 cm from the top edge of
the plate. After addition of APS and TEMED, the solution should be mixed
and poured quickly or it will begin to polymerize. Overlay 200 µl water
saturated butanol on the top of separation gel gently and allow the gel to
polymerize for 45 mins.
ii.
Stacking Gel (4 %):
Decant the water saturated butanol and rinse the gel with distilled water.
Prepare the stacking gel by mixing the reagents in the same sequence as
mentioned below:
For 5 ml (1 gel)
Water
Acrylamide (30%)
0.5 M Tris Cl pH: 6.8
20 % SDS
Ammonium Persulfate
TEMED
1.8 ml
0.6 ml
2.5 ml
25 µl
50 µl
5 µl
L. Buffers used for determination of GI activity at different pH
1- Acetate Buffer (0.2M) for pH 4 and 5
Prepared as described for protease assay by Nagase’s Method
2- Phosphate Buffer (0.2M) for pH 6 and 7
Prepared as described for glucose isomerase assay
3- Tris HCl Buffer (0.2M) for pH 8 and 9
a. Tris
0.2 M Tris was prepared by dissolving 2.42 g in 100 ml of distilled water.
b. HCl
0.2 M HCl was prepared by taking 1.76mL and making up the volume to
100 ml with distilled water.
Mix the components in an appropriate ratio to obtain the buffer of required
pH.
242
Appendix - II
4- Glycine NaOH Buffer (0.2M) for pH 9 and 10
5- Glycine
0.2 M Glycine was prepared by dissolving 15.014 g in 100 ml of distilled
water.
6- NaOH
0.2 M NaOH is prepared by dissolving 0.8 g in 100 ml of distilled water.
Mix the components in an appropriate ratio to obtain the buffer of required
pH.
M. Divalent Metal salt solutions for checking their effect on GI activity
123456789-
MnSO4
NiCl2
CaCl2
ZnSO4
BaCl2
CuSO4
FeSO4
HgCl2
Ag(NO3)2
1.89g in 100mL
2.37g in 100mL
1.47g in 100mL
2.87 g in 100mL
2.24 g in 100mL
2.49 g in 100mL
2.87 g in 100mL
2.71 g in 100mL
1.69 g in 100mL
243

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