APPENDIX
1.
Horikoshi I medium (g/L)
Glucose
10
Peptone
5
Yeast extract
5
KH2PO4
1
MgSO4.7H2O
0.2
Na2CO3
10
Agar
20
Na2CO3 (10 % w/v) was sterilized separately and added to the autoclaved
medium. pH of the media was 10.
2.
Horikoshi II medium (g/L)
Soluble starch
10
Peptone
5
Yeast extract
5
KH2PO4
1
MgSO4.7H2O
0.2
Na2CO3
10
Agar
20
Na2CO3 (10 % w/v) was sterilized separately and added to the autoclaved
medium. pH of the media was 10.
3.
Nutrient agar (NA) (g/L)
Peptone
5
Yeast extract
1.5
Beef extract
1.5
Sodium chloride
5
Agar
20
Na2CO3 (10 % w/v) was sterilized separately and added to the autoclaved
medium. pH of the media was 10.
Rebecca Thombre, Ph.D. Thesis, 2012.
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4.
Starch agar (g/L)
10 g Soluble starch in 1 L Nutrient agar.
5.
Davis Mingioli’s synthetic medium +0.5% peptone as nitrogen source
K2HPO4
7
KH2PO4
3
MgSO4.7H2O
0.1
Peptone
1.0
Trace element solution
(FeSO4.7H2O 0.5, ZnSO4.7H2O 0.5, MnSO4.3H2O 0.5, H2SO4 0.1N
10ml, pH adjusted to 10.0 with 10 % Na2CO3.
6.
Phenolphthalein methyl orange medium (g/L)
Soluble starch
Peptone
10
5
Yeast extract
5
K2HPO4
1
MgSO4.7H2O
0.2
Na2CO3
10
Phenolphthalein
0.3
Methyl Orange
0.1
pH adjusted to 10.0 with 10 % Na2CO3.
7.
Estimation of β- CGTase (Goel and Nene, 1995)
Procedure
i.
Pipette out different volumes of β- CD (20, 40
,60,100,120,140,160,200 µg/ml) into a series of test tubes and
make up the volume to 1 ml with 50 mM Tris Cl buffer of pH 8.5.
ii.
To each tube add 4 ml of phenolphthalein reagent and mix well.
iii.
Cover the tubes with aluminium foil on top and keep them at room
temperature for 20 min.
iv.
Measure the O.D. at 550.
v.
Plot a standard graph of concentration of β- CD vs. % decrease of
OD
Rebecca Thombre, Ph.D. Thesis, 2012.
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Fig. A1 :
Standard graph for calculation of CGTase activity
% OD decrease X amount of β- CD equivalent to % decrease X 106
CGTase activity = -------------------------------------------------------------------------------Molecular weight of (β- CD, 1135) X time (20 min)
% OD decrease = {OD of blank (control) – OD test / OD of blank }X 100
8.
Estimation of proteins (Bradford, 1976)
i.
Pipette out into a series of tubes (40, 80,120, 160, 200 µg/ml)
standard protein solution (Bovine serum albumin, 200 µg/ml) and
make the total volume upto 1ml with distilled water
ii.
To each tube, add 5 ml of Bradfords reagent (Biorad ready to use
Bradfords reagent) and mix for 5 min.
iii.
Measure the blue colored formed at 595 nm
iv.
Use a reagent blank without protein is used (distilled water +
reagent)
v.
Plot a standard graph of concentrations of standard BSA vs. O.D at
595 nm
vi.
Calculate the protein content from the standard graph
Rebecca Thombre, Ph.D. Thesis, 2012.
Page 183
Fig. A2 :Standard plot of concentration of protein vs OD at 595 nm
9.
Estimation of starch (Smith et al., 1948)
i.
Pipette out into a series of tubes (2, 4, 6, 8, 10 mg/ml) standard
starch solution (soluble potato starch, 10 mg/ml) and make the total
volume upto 1ml with distilled water
ii.
To each tube, add 4 ml of Iodine reagent [0.01 M Iodine (0.3 8 g)
in 0.25 M KI (12.45 g in 300 ml distilled water) ] and mix for 5
min.
iii.
Add 5ml of distilled water to the blue colored formed and measure
at OD at 465 nm.
iv.
Use a reagent blank without starch (distilled water + reagent).
v.
Plot a standard graph of concentrations of standard starch vs. O.D at
465 nm.
vi.
Calculate the starch content from the standard graph.
Rebecca Thombre, Ph.D. Thesis, 2012.
Page 184