Experimental Work: Chapter No.3

Chapter No.
3

EXPERIMENTAL WORK
PDF created with pdfFactory Pro trial version www.pdffactory.com

MATERIALS AND METHODS

The study was divided into two parts.
(A) Production of glucose oxidase from Aspergillus niger.
(B) Commercial application of glucose oxidase.
3.1 (A) Production of glucose oxidase from Aspergillus niger
Glucose oxidase was produced from Aspergillus niger by the submerged fermentation
following the methods of Fidurek and Gromada (1996) with some changes. The study of
this part includes following aspects:
3.1.1 Screening of sources for the microbial strain producing highest GOX activity
along with optimizing pH and carbon source.
3.1.2 Optimization of other fermentation conditions for improved GOX production like
fermentation period, MgSO
4
, KH
2
PO
4
, and urea.
3.1.3 Enzyme kinetics
3.1.4 Enhanced GOX production by UV mutation
3.1.5 Purification of GOX.
3.1.1 Screening for the fungal strain producing the highest GOX activity along
with optimizing carbon source and pH

For commercial production of enzyme first step is the screening of different native strains
for the optimal enzyme production.
So in this section different strains of A. niger were examined along with optimization of
two growth conditions pH and carbon source, (i.e. glucose concentration) for the optimal
production of glucose oxidase. This will help the commercial production of glucose
oxidase in Pakistan.
3.1.1.1 Microorganism
The strains of A. niger were isolated from five different sources (bread, potato, grapes,
pickle, sugar beet) identified from the fungal culture bank (FCB) at Punjab University
Lahore. They were grown on malt extract agar medium at pH 5.5 and with following
composition (gl
-1
).


1. Malt extract 20 g
2. Peptone 2.5 g
3. Agar 10 g.
The medium was prepared by weighing accurately 1-3 and dissolving them in 500 ml of
distilled water. The medium was boiled, with constant stirring for 15 minutes and was
poured into clean test tubes. The tubes were plugged with sterilized cotton and autoclaved
at 20 lbs PSI at 100C for 15 minutes. After autoclaving the tubes were placed in slanting
position for 24 hours. Transfer of strains on fresh medium continued after every two
weeks. These pure and identified colonies were kept in the refrigerator at 4C for storage.
3.1.1.2 Substrate (carbon source)
Glucose obtained from biochemistry Lab. Chemistry Department, GC University Lahore
was used as carbon source for the growth and production of glucose oxidase.
3.1.1.3 Fermentation
The enzyme was produced by submerged fermentation of A. niger in 250 ml shake flask.
The fermentation medium with the following composition was used.
Table 3.1 Composition of fermentation medium.
No. Compounds Amount g/100 ml
1. NaNO
3
12.5
2. Urea 0.2
3. MgSO
4
.7H
2
O 1.25
4. FeSO
4
.7H
2
O 0.025
5. KH
2
PO
4
2.5
6. CaCO
3
1.75/25 ml H
2
O

(Petruccioli and Federici, 1993) with some amendments i.e. urea used instead peptone
and no KCl was added in the medium.

Table 3.2 Composition of components used in the 250 ml flask.
No. Ingredients Amounts
1. CaCO
3
1.75 g/25 ml
2. NaNO
3
2 ml
3. Urea 2 ml
4. MgSO
4
.7H
2
O 2 ml
5. FeSO
4
.7H
2
O 2 ml
6. KH
2
PO
4
2 ml
7. Glucose 10 ml
8. Spore suspension 5 ml

(i) Substrate level (carbon source):
The required glucose amount (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0%(w/v)
was dissolved in 100 ml water. After sterilization, 10 ml of above different
concentrations of glucose solution was added to the flasks containing 35 ml of the sterile
fermentation medium containing the compounds mention in table 3.2 (2ml each) and
were inoculated with 5 ml spore suspension (10
7
10
8
spore/ml) (Collins et al; 1984).
Now the flask contained sterilized growth media of about 50 ml (shown in table 3.2)
which was already autoclaved at 20 lbs. PSI at 100C for 15 minutes.
(ii) pH of media
The required pH of the medium (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) was adjusted
by 1M HCl and 1M NaOH. These flasks were then incubated in shaking incubator at 100
rpm and 30C for 48 hours (Willis, 1966). The fungal spores were also counted by
Thoma Counting Chamber (Collins et al; 1984). All the experiments were carried out in
triplicate.
3.1.1.4 Enzyme extraction and partial purification
After 48 hours of incubation in shaking incubator at 30C, the mycelia were collected by
filtration and washed with distilled water. The mycelia collected were weighed. The
washed mycelia were crushed in sodium citrate buffer (0.1 M pH = 5) In Mortar and
Pestle for 30 minutes and centrifuged at 10,000 rpm for 10 minutes mycelium debris was

separated and kept for dry mass estimation. The filtrate was precipitated with (NH
4
)
2
SO
4

and kept at 4C overnight. The precipitate was collected by centrifugation at 10,000 rpm
for 10 minutes. Each sample were dissolved in 2 ml of distilled water and refrigerated.
3.1.1.5 Assay of GOX
The glucose oxidase activity was determined by the fast spectrophotometric method by
following the enzymatic reduction of benzoquinone to hydroquinone at 290 nm using
glucose as substrate (Ciucu and Patroescu, 1984, Dr. Abdul Hameed and Khawar)
One unit (u) of enzyme activity was defined as the amount of enzyme producing one
micromole of hydrogen peroxide per minute at 30C.
It was also defined as the amount of enzyme catalyzing the decomposition of one
micromole of hydrogen-peroxide per minute at 30C or One unit catalysis the oxidation
of 1 mole glucose to gluconic acid per minute at 25C pH 5 coupled with peroxide and
1-4 benzoquinone. Glucose oxidase oxidize glucose to gluconic acid and H
2
O
2
produced
in above reaction reduces benzoquinone to hydroquinone. Both spectrometer cell (A and
B) were filled up with 2 ml of 1 M glucose solution which was prepared a day before at
room temperature so that and anomers formed at equilibrium.
In cell A, 1.0 ml of 0.1% benzoquinone solution (0.01 g in 10 ml distilled water) and 1.0
ml of buffer solution (sodium citrate buffer of 0.1 M, pH = 5) were added. This cell was
taken as standard. In cell B, 1.0 ml of 0.1% benzoquinone solution and 0.9 ml of sodium
citrate buffer were added. The mixtures of both cell (A and B) were allowed to
equilibrate at 25C for 5 minutes.
In cell B, at time zero 0.05 ml of glucose oxidase solution was added and mixture was
stirred. An increase in absorbance at 290 nm was recorded for 1 2 minutes.

(UV-Cecil CE 7200 Spectrophotometer)
This rate was used to calculate the enzyme activity as follows:
Enzyme activity at 25C =
mM
290
A V
I A u

V
moles HQ/min/ml
Where
V = 4.0 ml, volume of reaction mixture
= 0.05 ml, GOX solution
A
290
mM
= 2.31, molecular absorption co-efficient of HQ
(hydroquinone) at ) = 290 nm
I = 2 cm, length of cell.
*A = the actual absorbance of sample after
sub starting the control.
Above equation become.
Enzyme activity = 17.316 x *A
= moles HQ/min/ml
Specific activity =
17.316 A
mgprotein/ml
V
= mole HQ/min/mg Protein.

3.1.1.6 Protein estimation
Proteins were estimated according to the method described by Moss and Bond (1957).
3.1.1.6.1 Reagents
Solution A = 40 gm sodium carbonate in 500 ml distilled water.
Solution B = 0.38 gm CuSO
4
.5H
2
O and 0.6 gm potassium-sodium tartarate
in 500 ml distilled water.
Solution C = 1 part Folin reagent (Folin ciocaltems phenol reagent BDH) and 2
parts of H
2
O (prepared just before use)
Solution D = 1N NaOH.
3.1.1.6.2 Standard bovine serum albumin solution (500 g ml
-1
)
5.0 mg of Bovine Serum Albumin (BSA) was dissolved in 10 ml of distilled water. The
serum albumin dissolved very slowly. The standard was made two days before use and
kept at 4C.

3.1.1.6.3 Procedure
0.5 ml NaOH 1N (sol. D) was added in each tube containing protein sample (250-750 g)
and were shaken in water bath (37C) for 30 minutes. After cooling 2.6 ml of solution A
and 2.5 ml of solution B was added in each sample as well as in standard tube. The
solution in each tube was thoroughly mixed and incubated at 37C for 30 minutes. Then
0.5 ml of solution C was added, mixed well and kept for 20 minutes at room temperature.
BSA (0.05 1.5 ml) was simultaneously used in experiment. The optical density was
measured at 661nm on spectrophotometer (UV-Cecil CE 7200) and a standard calibration
curve was drawn and with its help the proteins were estimated.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Concentrations (mg/mL)
A
b
s
o
r
b
a
n
c
e
(
O
D
)

Fig-3.1 Standard curve for bovine serum albumin for protein estimation

3.1.2 Optimization of fermentation conditions for max. GOX
production

The conditions for glucose oxidase production by A. niger were also optimized other than
substrate level (carbon source i.e. glucose) and pH optimization was carried out along
with screening of fungal strains in the previous section (3.1).

The medium containing glucose was cultured with A. niger isolated from potato
(screened in previous section) for different incubation periods with varying
concentrations of urea, CaCO
3
, MgSO
4
.7H
2
O and KH
2
PO
4
at 30C in shake flask.
3.1.2.1 Submerged fermentation
Triplicate flasks of 250 ml containing 50 ml of sterilized growth media along with 5 ml
of spore suspension were incubated at 30 on shaking incubator (100 rpm) for each
condition to be optimized.
3.1.2.2 Sample preparation
After 48 hours incubation experimental flasks (in each experimental process) were
harvested. The process of enzyme extraction is described in section 3.1.
3.1.2.3 Glucose oxidase assay
GOX activity was measured by method of which was Ciucu and Patroescu (1984),
described in section 3.1.
3.1.2.4 Fermentation period (incubation time)
The growth media containing 10% (w/v) glucose as substrate at pH = 5.5 (both
conditions optimized in previous section) contained in 10 flasks were sterilized,
inoculated and were incubated at 30C under continuous shaking condition (100 rpm).
Triplicate growth media were incubated for 12, 24, 36, 48 and 60 hours.
3.1.2.5 CaCO
3

The effect of different concentrations of CaCO
3
(i.e. 1.5, 2.5, 3.5 and 4.5%) in the
medium were studied for maximum production of GOX.
3.1.2.6 Nitrogen source (urea)
The concentrations of urea (nitrogen source i.e. 0.1, 0.2, 0.3, 0.4 and 0.5%) have a
considerable influence on GOX production. Different concentrations of urea were added
into the fermentation medium having 10% glucose at pH 5.5.
3.1.2.7 KH
2
PO
4

The effect of different concentrations of KH
2
PO
4
(i.e. 0.2, 0.4, 0.6, 0.8 and 1.0%) were
studied on GOX production in the medium containing optimum concentration of glucose
(10%), Urea (0.2%), and CaCO
3
(3.5%) at pH 5.5.

3.1.2.8 MgSO
4
.7H
2
O
Different concentrations of MgSO
4
.7H
2
O i.e. 0.01, 0.02, 0.03 and 0.04%were examined
for GOX activity in pre-optimized culture medium with 10% glucose, 0.4% KH
2
PO
4
,
0.2% Urea, 3.5% CaCO
3
at pH 5.5 and 30C.
3.1.3 Enzyme kinetics
The effect of temperature on the enzyme activity was also investigated. Effect of aeration
on GOX production in shake flask (oxygen supply) was also investigated.
The effect of aeration rate on GOX production by A. niger (source potato) was studied by
changing the shaking speed as well as the volume of the medium in the flasks. Different
volumes of the medium in 250 ml conical flasks (such as 20, 30, 40, 50, 60 and 80 ml)
and different speeds of the shaker such as (50, 100 and 150 rpm) were tested (Table
4.17).
3.1.4 Enhanced GOX production by UV mutation
A mutation is a permanent change in the DNA sequence of gene. Mutation is a genes
DNA sequence can alter the amino acid sequence of the protein encoded by the gene.
How does this happen? The DNA sequence of each gene determines the amino acid
sequence for the protein it encodes. The DNA sequence is interpreted in groups of three
nucleotide bases, called codons. Each codon specifies a single amino acid in the
polypeptide chain.
3.1.4.1 Microorganism
The fungal strain of A. niger was isolated from potato source and identified from FCB.
Punjab University Lahore and was grown on malt extract agar medium at pH 5.5 as
described earlier in the section (3.1.1.1). The cultures were stored in the refrigerator at
4C for further studies.
3.1.4.2 Preparation for mutagenesis
One ml of prepared conidial inoculums was transferred to 100 ml of sterilized water. This
conidial suspension was further diluted up to 10-30 by serial dilution method.
3.1.4.3 Ultraviolet treatment
Diluted conidial suspension (10 ml) was transferred in the sterilized petri plates. The Petri
plates were placed under the UV lamp (emitting the energy of 2.6 x106 J/m2/S) in the
laminar flow for 5- 60 minutes. After different time intervals 0.5 ml of the irradiated

conidial suspension was transferred to the petri plates containing malt extract agar with 2-
Deoxy D- glucose medium. The Petri plates were then placed in the incubator at 30
o
C for
3-4 days. The young colonies of Aspergillus niger on the petri plates were picked up and
transferred to the malt extract slants
3.1.4.4 Fermentation technique
Submerged fermentation technique was employed process is same as described in
previous section (3.1.1.3).
3.1.4.5 Enzyme extraction:
The enzyme extraction was carried out using the method explained in previous section
(3.1.1.4)
3.1.4.6 Enzyme assay
The process details have been described in previous section (3.1.1.5).
3.1.5 Purification of glucose oxidase
Following steps are involved for the purification of glucose oxidase from A. niger.
Filtration
(NH
4
)
2
SO
4
Precipitation
Dialysis
Gel filtration using Sephadex G-75
3.1.5.1 Filtration
After 48 hours of incubation at 30
o
C and 120 rmp the culture medium was filtered
through nylon gauze to remove the mycelia. The mycelia were washed with distilled
water and the whole solution then filtered through filter paper. The pH of filtrate was
adjusted at 5.0 by using citrate buffer and NaOH and filtrate was placed at 4
o
C for 60
minutes and enzyme activity was calculated.
3.1.5.2 (NH
4
)
2
SO
4
Precipitation
Ammonium sulphate precipitation (salting out) is a technique used to precipitate proteins
from solution by increasing the ionic strength of the solution. The technique is reliant on
the hydrophobic nature of proteins, since they contain hydrophilic and hydrophobic
groups. When the proteins are dissolved, water is forced into contact with the proteins
hydrophobic groups and in the process becomes ordered around the proteins. Increasing

the concentration of salt ions by the addition of ammonium sulphate causes the water to
be removed from around the protein exposing the hydrophobic portions of the proteins.
Precipitation of the proteins will then occur due to the aggregation of proteins via the
exposed hydrophobic portions. This technique is used to fractionate the proteins from
solution since proteins with larger or more hydrophobic portions will aggregate and
precipitate before those with smaller or fewer proteins of hydrophobic groups.

Procedure
Solid ammonium sulphate is added to 100ml of solution of glucose oxidase at the
concentration of 30% (w/v). The suspension was stirred for half an hour at 4
o
C. After
sufficient shaking the precipitates were collected by centrifugation at 10,000 rpm for 30
minutes. Enzyme activity was determined for each concentration and precipitates were
collected for further purification. Enzyme solution was then treated with 40, 50, 60, 70
and finally 80% (w/v) (NH
4
)
2
SO
4
.
3.1.5.3 Dialysis
The precipitates obtained after ammonium sulphate precipitation procedure, were
suspended in small volume of Citrate buffer (pH 5) and dialyzed by using 12,000 d
molecular weight cut off dialysis bag , which was placed in 2 liters of Citrate buffer (pH
5) for 24 hours at 4
o
C against three changes. GOX activity of the dialyzed material was
determined (section 3.1.1.5).
3.1.5.4 Gel filtration using Sephadex G-75
The principle of gel filtration is based on elution of proteins on the basis of size and
shape of the molecules. Molecules of smaller size pass through the beads of gel and the
remaining larger sized pass through spaces between the gel beads. Larger molecules are
eluted earlier and smaller which travel inside the gel beads eluted later.
Reagents
0.1 M Citrate buffer (pH 5.0)
Dextrin blue (molecular weight 2x10
6
) solution (0.5% w/v)
Sephadex-G-75

Procedure
Sephadex-G-75 was soaked in 500 ml of 0.1 M citrate buffer (pH 5.0) containing 0.1 g of
sodium azide and incubated at room temperature for 24 hours. After soaking, the gel was
deaerated by direct drive rotary vacuum pump and then poured in a 0.9 x 60 cm column.
The packed column was washed with 0.1 M citrate buffer (pH5.0). Dextran blue (0.5%
w/v) was used for the determination of its void volume. Dialyzed enzyme extract was
applied on the column and fractions each of 3 ml were collected. Each fraction was then
assayed for enzyme activity and the amount of the protein present. Fractions containing
enzyme activity were pooled and lyophilized (Jakoby 1971).
3.2 Commercial applications of glucose oxidase
In the present research projects two commercial applications of GOX were investigated;
3.2.1 Estimation of glucose by standardization of conditions using GOX.
3.2.2 The production of calcium gluconate, gluconic acid and its derivatives using
glucose oxidase.
3.2.1 Estimation of glucose by standardization of conditions using GOX
The disease involved in the elevation of blood glucose level is known as diabetes
mellitus. It is a very common disease now a days. It is a metabolic problem and is
prevalent in many parts of the world. One fundamental aspect of diabetes is an
abnormality of glucose metabolism due to in sufficient action of insulin, owing either to
its absence or to resist in action (Murray et al 2001). Blood glucose level in diabetes
becomes so elevated that the glucose spills over into urine, providing a convenient
diagnostic test for the disease (Voct et al 1999).
In the process of determination of glucose level there are involved three enzymes i.e.
mutarotase, glucose oxidase and peroxidase. The overall reaction mechanism is as
follows:
-D-glucose
Mutarotase
-D-glucose
-D-glucose
Glucose Oxidase
!-D-gluconolactone + H
2
O
2

2H
2
O
2

Peroxidase
2H
2
O + O
2
.

The enzymes and their kits for glucose estimation are being imported at high cost and a
lot of foreign exchange is required. Economy of our country can not afford such high cost
kits so there is a need to optimize the conditions for such methods. So this part of the
project was designed to develop the technology to optimize the conditions for glucose
estimation using these 3 enzymes and to prepare low priced local kits as compared to
imported kits.
The three enzymes glucose oxidase, mutarotase and peroxidase were produced/extracted
and purified for the preparation and optimization of glucose estimation kit.
3.2.1.1 Glucose oxidase
Glucose Oxidase was produced by submerged fermentation using glucose as substrate
and Aspergillus niger (source potato) as the fermentative organism. GOX extraction,
purification and assays were described earlier in the section (3.1)
3.2.1.2 Mutarotase
Bovine kidney cortex was used to extract the enzyme mutarotase. It was purified by
ammonium sulfate precipitation, dialysis and gel filtration chromatography.
3.1.2.2.1 Enzyme extraction
The method of Bentley (1962) and (Zia M. A.) was applied to prepare extracts. A bovine
kidney cortex of about 100g was mixed with 100 ml of 0.1M phosphate buffer of pH 5.8
and 50 ml of chloroform and was homogenized in a blender for 2 minutes. This extract
was centrifuged at 8,000 rpm for 15 minutes and the supernatants were dialyzed for 2
hours against 0.1 M phosphate buffer (pH 5.8) with continuous stirring.
3.2.1.2.2 Enzyme assay
The activity of mutarotase was determined by following the procedure of Calzyme
Laboratory Manual (1998).
i) Determination of spontaneous mutarotation
At time zero, 100 mg -D-glucose was dissolved in 10 ml of 5mM sodium EDTA and
transferred to polarimeter. Rotation was recorded at 1 minute intervals for the first 10
minutes. After 10 minutes rotation was recorded every 5 minutes intervals. After 30
minutes the rotation was noted at 15 minutes intervals. Until the rotation become
constant.

ii) Graph rotation vs time
The Initial rotation corresponds to an -D-glucose concentration of 555 mole. The data
obtained were extra plotted to zero to obtain the initial rotation.
iii) Blank rotation graph
Graph rotation was determined against time in 1 minute intervals, up to 5 minutes.
iv) Standard curve
Graph rotation Vs mole of -D-glucose using data from spontaneous mutarotation was
plotted. It was between 555 mole (initial and 195 mole (Final rotation).
v) Determination of test rotation
At time zero, 0.1 ml of enzyme sample was added to 9.9 ml of 5 mM sodium EDTA
solution and dissolved in 100 mg -D-glucose. The rotation was determined at 30 second
intervals for 10 minutes.
vi) Calculation
a = Initial rotation from spontaneous rotation graph.
b = Rotation for 5 minutes from graph (ii)
c = Blank rotation for 5 minutes (c = a-b)
d = Conversion of c to moles of -D glucose from standard curve
graph.
e = 555 mole -D glucose at initial rotation.
f = Spontaneous rate in micromoles/minute (f = e-d/5 minutes)
g = Test rotation per 5 minutes from test graph.
h = Test rotation after 5 minutes (h = a-g)
i = Conversion of h to micromole from standard curve.
j = Test rate in mole/minute i.e. [j = (e-i)/5 min f].
Units/mg protein = j/mg protein/10.0 ml reaction mixture.
3.2.1.2.3 Protein estimation
Protein were estimated according to the method of Moss and Bond (1957) as mentioned
earlier in section (3.1). The optical density was measured at 661 nm wavelength and a
standard curve was plotted between concentration (mg/ml) and absorbance. Total protein
concentrations were estimated from that curve.


3.2.1.2.4 Partial purification of mutarotase by (NH
4
)
2
SO
4
precipitation
technique
The enzyme extract was precipitated by ammonium sulfate for partial purification as
described by the method of Bentley (1962). Solid ammonium sulphate was added to 100
ml of solution of glucose oxidase at the concentration of 30% (w/v). The suspension was
stirred for half an hour at 4
o
C. After sufficient shaking the precipitates were collected by
centrifugation at 10,000 rmp for 30 minutes. Enzyme activity was determined for each
concentration and precipitates were collected for further purification. Enzyme solution
was then treated with 40, 50, 60, 70 and finally 80% (w/v) (NH
4
)
2
SO
4
.
3.2.1.2.5 Dialysis
The precipitates obtained by ammonium sulphate precipitation were measured in 0.1 m
phosphate (pH 5.8) buffer and dialyzed in dialysis bag with constant stirring for 2 hours
and then were subjected to enzyme assay (3.2.1.2.2) and protein estimation (3.2.1.2.3).
3.2.1.2.6 Gel filtration using sephadex G-75
Reagents
0.1 M Phosphate (pH 5.8)
Dextran blue (molecular weight 2x10
6
Sephadex-G-75
Procedure
Sephadex-G-75 was soaked in 500 ml of 0.1 M phosphate (pH 5.8) containing 0.1 g of
sodium azide and incubated at room temperature for 24 hours. After soaking the gel was
The packed column was washed with 0.1 M phosphate (pH 5.8). Dextrin blue (0.5% w/v)
was used for the determination of its void volume. Dialyzed enzyme extract was applied
on the column and fractions each of 3 ml were collected. Each fraction was then assayed
for enzyme activity and the amount of the protein present (Jakoby 1971).
3.2.1.3 Peroxidase
Peroxidase isolated from horse Radish was purified by ammonium sulfate precipitation
technique, Dialysis and gel filtration chromatography for utilization in glucose estimation
kit.

3.2.1.3.1 Enzyme extracts preparation
About 100g horse Radish, was thoroughly washed with water, cut into small pieces and
homogenized in a blender with 500 ml distilled water. It was centrifuged at 10,000 rpm
for 15 minutes at 4C and filtered (Civello et al 1995) and (Zia M.A.).
The filtrate was heated in water bath at 65C for 3 minute to inactivate catalase and
cooled quickly in ice cold water. Enzyme assays were performed and protein was
estimated as explained in section (3.2.1.2.3).
3.2.1.3.2 Enzyme assay
The activity of peroxidase enzyme was determined by the method as described by Civello
et al (1995).
Buffered substrate solution was prepared as follow:
Phosphate buffer (pH 6.5) = 46.6 ml
H
2
O
2
(30%) = 0.32 ml
Guaiacol = 1.00 ml
The reagents were mixed in agitator and completely covered till the whole day. In the
blank solution preparation, H
2
O
2
was not added only Guaiacol (1 ml) and phosphate
buffer (46.6 ml) were mixed in agitator and used as blank spectrophotometer was set to
zero at 470 nm wavelengths after in setting blank solution in it. Then in UV cell 1 ml of
buffered substrate solution was taken along with 0.02 ml of enzyme extract and kept in
spectrophotometer and absorbance was recorded after 3 minutes which is directly
proportional to the enzyme activity.

3.2.1.3.3 Partial Purification of Peroxidase by (NH
4
)
2
SO
4
precipitation
techniques

The enzyme extract was precipitated by ammonium sulfate for partial purification as
described by the method of Bentley (1962). Solid ammonium sulphate was added to 100
ml of solution of glucose oxidase at the concentration of 30% (w/v). The suspension was
stirred for half an hour at 4
o
C. After sufficient shaking the precipitates were collected by
centrifugation at 10,000 rpm for 30minutes. Enzyme activity was determined for each
concentration and precipitates were collected for further purification. Enzyme solution
was then treated with 40, 50, 60, 70 and finally 80% (w/v) (NH
4
)
2
SO
4
.

3.2.1.3.4 Dialysis
The precipitate obtained by (NH
4
)
2
SO
4
were dialyzed in the dialysis bag with 0.2 M
phosphate buffer (pH 6.5) with constant stirring for 2 hours and then the sample was
subjected to enzyme assay (3.2.1.3.2) and protein estimation (3.2.1.2.3).
3.2.1.3.5 Gel filtration using Sephadex G-75
Reagents
0.2 M Phosphate (pH 6.5)
Dextran blue (molecular weight 2x10
6
Sephadex-G-75
Procedure
Sephadex-G-75 was soaked in 500 ml of 0.2 M phosphate (pH 6.5) containing 0.1 g of
sodium azide and incubated at room temperature for 24 hours. After soaking the gel was
The packed column was washed with 0.2 M phosphate (pH 6.5). Dextrin blue (0.5% w/v)
was used for the determination of its void volume. Dialyzed enzyme extract was applied
on the column and fractions of three ml were collected. Each fraction was then assayed
for enzyme activity and the amount of the protein present (Jakoby 1971).
3.2.1.4 Optimization of conditions for glucose estimation
Glucose oxidase, mutarotase and peroxidase enzymes were used to estimate glucose
level. The conditions of these partially purified enzymes were optimized. The standard
solution of -D-glucose 100 mg dL
-1
was prepared in distilled water. 2 ml of this solution
was used in all the parameters to be optimized and the three enzymes added at the same
time. Following parameters were optimized.
3.2.1.4.1 Guaiacol time
Timing for the addition of Guaiacol as chromogen was before adding peroxidase.
3.2.1.4.2 Enzyme concentrations
Different concentrations were optimized for three sets of the enzymes as follow:
Set A Mutarotase (5 L) + GOX (15L) + Peroxidase (10L)
Set B Mutarotase (10 L) + GOX (30L) + Peroxidase (20L)
Set C Mutarotase (20 L) + GOX (60L) + Peroxidase (40L)

3.2.1.4.3 Wavelength
Different wavelengths were optimized at spectrophotometer i.e.
i) 470 nm wavelength
ii) 290 nm wavelength
iii) 546 nm wavelength
3.2.1.4.4 Incubation period
Two different incubation periods were investigated at 37C for best results i.e.
i) 10 minutes
ii) 20 minutes
3.2.1.4.5 Sensitivity of glucose estimation kit
Different concentration of glucose was used to determine the sensitivity of glucose
estimation kit. The different concentration were used as: 200, 170, 140, 110, 80 and , 50
mg dL
-1
and their absorbance were calculated with the help of standard curve and the
sensitivity was measured as shown in Table 4.39 and Fig. 4.67.
3.2.1.4.6 Comparison with standard kit
All the optimized conditions (mentioned above) were used for preparation of glucose
estimation kit and then this kit was compared with standard estimation kit. The blood
samples of seven diabetic patients were investigated with both kits and comparison was
made as shown in Table 4.38 and Fig. 4.66

3.2.2 Production of calcium gluconate, gluconic acid and its
derivatives by GOX method

In the present research project gluconic acid, and its derivatives (metal salts) such as
sodium, magnesium, copper, nickel and cobalt gluconates were synthesized from calcium
gluconate which were earlier obtained by A. niger.
3.2.2.1 Production of calcium gluconate
Calcium gluconate was produced by submerged fermentation.

3.2.2.1.1 Microorganism
Aspergillus niger strain (source potato) was obtained and identified from fungal Cultural
Bank Punjab University Lahore, Pakistan and was used in the present study. The fungal
culture was grown on malt extract agar medium at pH 5.5 as described in previous
section in (3.1).
3.2.2.1.2 Preparation of growth media
The growth medium for A. niger was prepared by mixing the following quantities of
ingredients in each 250 ml Erlenmeyer flask.

Table 3.3 Composition of growth medium
INGREDIENTS AMOUNT (gl
-1
)
MgSO
4
.7H
2
O 0.5
FeSO
4
.7H
2
O 0.01
KH
2
PO
4
1.0
Glucose 100.0
Urea 0.2
NaNO
3
5

Table 3.4 Composition of each 250 ml flask
Ingredients Amounts
CaCO
3
1.75 g/25 ml
KH
2
PO
4
2 ml
NaNO
3
2 ml
MgSO
4
.7H
2
O 2 ml
FeSO
4
.7H
2
O 2 ml
Urea 2 ml
Glucose 10 ml
Spore suspension
5 ml
(10
7
10
8
spores/ml)

The above quantities were mixed in the flasks except CaCO
3
and spore suspension,
autoclaved at 121C for 15 minutes and 15 PSI. CaCO
3
was autoclaved separately and
mixed with the media under sterile condition, the flasks were then plugged after the

addition of inoculums (5 ml).The pH of medium was adjusted at 6.0 by using 1M NaOH
or 1M HCl.
3.2.2.1.3 Fermentation technique
The flasks containing 50 ml of fermentation medium were placed on a shaker for
incubation at 30C with speed of 100 rpm for 48 hours. All experiments were carried out
in triplicates. The samples were drawn at the end of fermentation and the contents of
flasks were filtered and filtrate was centrifuged at 5000 rpm for 5 minutes. The
supernatant was used for the estimation of calcium gluconate and glucose. The dry cell
mass was obtained by drying the wet mycelial mass in oven at 105C for 24 hours
according to method of Haq and Daud (1995).
3.2.2.1.4 Assay methods
(i) Estimation of glucose
Glucose was estimated by DNS method. (Tasun et al 1970) DNS solution was prepared
as:
Distilled water = 1500 ml
NaOH = 20 g
3,5 dinitrosalicyclic acid = 10 g
Above ingredients were dissolved in water and heated in water bath at 80C until clear
solution was obtained. The following chemicals were than added.
Rochelle salt = 300 g
Sod. metabisulphate = 10 g
Phenol (melted at 60C) = 5 ml
The solution was filtered and stored at room temperature.
Standard curve of glucose
One gram of glucose was dissolved in small quantity of distilled water and the volume
was raised to 100 ml this stock solution of glucose contained 10 mg/ml glucose. Five
dilutions (0.2 1.2 mg ml
-1
) were made. From the stock solution 2 ml of each dilution
and 2 ml of DNS solution was transferred in each test tube. Blank was also run in parallel
with 2 ml DNS and 2 ml of distilled water. Test tubes were placed in boiling water for 5

minutes and cooled at room temperature and finally diluted with distilled water upto 20
ml. The absorbance was measured at 546 nm by U.V. spectrophotometer. The standard
curve was made by using each absorbance at respective range of glucose concentration
0
0.1
0.2
0.3
0.4
0.5
0.6
1 2 3 4 5
Concentration
A
b
s
o
r
b
a
n
c
e

Fig. 3.2 Standard curve of glucose
To determine glucose concentration (mg ml
-1
) the optical density was measured at 546nm
on spectrophotometer.
(ii) Calcium gluconate estimation
The analysis of calcium gluconate was made by the method of Pharmacopoeia (1990)
UK.
In 1.0 ml of sample, 2.0 ml of 1M HCl was added and water was added upto 200 ml
while stirring. Approximately 20 ml of 0.05M EDTA was added from burette. Then 20
ml of 1M NaOH and 300 mg of Hydroxy naphthol blue indicator was added upto blue
end point. Each ml of 0.05 M EDTA is equivalent to 2.004 mg of calcium gluconate.
1 ml of 0.05 M EDTA = 2.004 mg of calcium gluconate
AND

Percentage yield of calcium gluconate =
Ca. gluconate Produced
x 100
Glucose added


3.2.2.1.5 Optimization of conditions to enhance calcium gluconate production

The following parameters were studied:
(i) Effect of Incubation period on calcium gluconate production
The effect of incubation period on calcium gluconate production was studied at an
interval of every 12 hours upto 96 hours.
(ii) Effect of different pH on calcium gluconate production
The effect of pH on ca. gluconate production was studied within the range of 4.0 7.0
pH. The pH was adjusted by 1M HCl or 1M NaOH.
(iii) Effect of glucose concentration on calcium gluconate production
To examine the effect of glucose concentration, different concentrations of glucose were
added to fermentation medium (6-22%).
(iv) Effect of different types of carbonates and CaCO
3
concentration
Submerged fermentation was carried out using different metal carbonate such as MgCO
3
,
CaCO
3
, ZnCO
3
and FeCO
3
in the medium. The various levels of CaCO
3
(0 7 %) were
further optimized.
(v) Effect of different nitrogen sources and urea concentration
Different nitrogen sources were used at a level of 2-5 gl
-1
in the medium for improving
the expression of calcium gluconate. Different sources are peptone, Urea, NH
4
NO
3
,
NH
4
Cl and NaNO
3
. The various levels of urea (0.1-0.4 gl
-1
) were further optimized.
(vi) Effect of different PO
4
source
To study the effect of different phosphates like K
2
HPO
4
and KH
2
PO
4
on Ca. gluconate
production, different concentration of respective phosphates in fermentation medium i.e.
0.15, 0.20, 0.25, 0.30 were added.
(vii) The effect of temperature
Effect of temperature on calcium gluconate production was also evaluated. The influence
of temperature was studied over the range of 15 to 50C.
3.2.2.2 Preparation of gluconic acid from calcium gluconate
Gluconic acid was prepared from calcium gluconate. Following methods were employed
for its preparation (Prescott and Dunn 1959) and (M. Iqbal and Maqbool).

3.2.2.2.1 Oxalic acid
Exactly 43.0 gm calcium gluconate was dissolved in 180 ml boiling water. 12.6 gm
crystallized oxalic acid was dissolved in minimum quantity of water. Both the solutions
were mixed at 60C with constant stirring. It was filtered to remove calcium oxalate.
Gluconic acid was crystallized under vacuum at 30 - 40C on a rotary evaporator.
Reaction
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ca
+
COOH
COOH
COOH
(CHOH)
4
CH
2
OH
+
COO
COO
Ca
.
2
H
2
O 2
+
2H
2
O
Calcium gluconate Oxalic acid Gluconic acid Calcium oxalate

3.2.2.2.2 Sulphuric acid method
Sulphuric acid was employed in place of oxalic acid to remove calcium as calcium
sulphate and thus releasing the gluconic acid.
215.0 gm calcium gluconate was dissolved in 900 ml boiling water. This solution was
placed in an ice bath and 33.4 ml 30 N sulphuric acid was added to it drop wise. It was
stirred constantly for about five minutes and was filtered through a Buckner funnel to
remove precipitated calcium sulphate. Gluconic acid solution was obtained as filtrate.
This solution was used for subsequent preparations of gluconates.

Reaction:
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ca
+
COOH
(CHOH)
4
CH
2
OH
+
CaSO
4
H
2
SO
4
2

Calcium gluconate Sulphuric acid Gluconic acid Calcium sulphate


3.2.2.2.3 Identification of gluconic acid
About 5 ml of a warm aqueous solution of gluconic acid (9.03%) were added to 1 ml of
freshly distilled phenylhydrazine. The mixture was taken in a test tube and heated on a
water bath for 30 minutes. After cooling, the inner surface of the tube was scratched with
a glass rod. Crystals of gluconic acid phenylhydrazide were formed (Eric and Cook,
1956).
3.2.2.3 Preparation of gluconic acid derivatives
The derivatives of gluconic acid such as magnesium gluconate, sodium gluconate, etc
were prepared either from the calcium gluconate or directly from the gluconic acid.
Calcium gluconate on treatment with the metal sulphate under suitable conditions gave
the desired gluconate. Metal carbonates while treated with gluconic acid gave the metal
gluconate with the evolution of carbon dioxide (Baronnet R. 1948). The appropriate
conditions and detailed procedures are given as follows:
3.2.2.3.1 Sodium gluconate
(i) Double decomposition method
Sodium gluconate was prepared by the double decomposition of sodium sulphate and
calcium gluconate as given below:

CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ca
+
CH
2
OH
(CHOH)
4
COONa
+
Na
2
SO
4
COONa
(CHOH)
4
CH
2
OH
+
CaSO
4

Calcium gluconate Sodium sulphate Sodium gluconate Calcium sulphate

The 68.8 gm of calcium gluconate was added to 250 ml boiling water and stirred to
dissolve it. The solution of calcium gluconate was treated with sodium sulphate (51.52
gm). This solution was heated to boiling. The precipitate of calcium sulphate was
removed by filtration while hot. Sodium gluconate solution was concentrated under
vacuum at 30C on a rotary vacuum evaporator. Ethanol was added to crystallize sodium
gluconate. It was filtered and the crystals were dried in a desicator over anhydrous
calcium chloride.
(ii) Gluconic acid method
Sodium gluconate was prepared by the interaction of sodium carbonate and gluconic
acid. The reaction is given by the following equation:

COOH
2(CHOH)
4
CH
2
OH
+
COONa
(CHOH)
4
CH
2
OH
+
Na
2
CO
3
+
H
2
O CO
2
2

Gluconic acid Sodium Sodium Carbon Water
carbonate gluconate dioxide

Sodium carbonate (21.2 gm) was added to 50% gluconic acid (156.8 ml corresponding to
78.5 gm). The solution was heated to expel carbon dioxide. Sodium gluconate solution
was concentrated under vacuum at 30C. Ethanol was added to precipitate the salt. It was
recrystallized from water solution and the crystals were dried in a desicator over
anhydrous calcium chloride.

3.2.2.3.2 Magnesium gluconate
(i) Procedure
Calcium gluconate (86.0 g) was dissolved in minimum quantity of boiling water.
Magnesium sulphate (49.2 g) was added to the gluconate solution and it was boiled,
filtered, while hot, to remove calcium sulphate. Magnesium gluconate solution was
concentrated at 40 - 50C and the salt was precipitated from the concentrated solution
with alcohol.
Reaction
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ca
+
MgSO
4
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Mg
+
CaSO
4

Calcium gluconate Magnesium Magnesium Calcium
sulphate gluconate sulphate
(ii) Procedure
Magnesium carbonate (84.0 g) was added to 50% gluconic acid solution (784.0 ml
corresponding to 392.0 g). The solution was heated to expel carbon dioxide and
concentrated under vacuum. Magnesium gluconate was precipitated with ethanol.
Reaction
COOH
2(CHOH)
4
CH
2
OH
+
MgCO
3
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Mg
+
CO
2 +
H
2
O

Gluconic Acid Magnesium Magnesium Carbon Water
gluconate gluconate dioxide


3.2.2.3.3 Copper gluconate
(i) Procedure:
Solution of calcium gluconate (86.0 g) was made in minimum quantity of boiling water.
This solution while boiling was treated with copper sulphate (49.8g) and filtered to
separate calcium sulphate. Copper gluconate solution was concentrated to crystallization
and the crystals were dried in a desicator over anhydrous calcium chloride.
Reaction
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Cu
+
CaSO
4
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ca
+
CuSO
4

Calcium gluconate Copper sulphate Copper gluconate Calcium sulphate
(ii) Procedure
A 50% Gluconic acid solution (78.4 ml equivalent to 39.2 g) was treated with cuprous
carbonate (22.1 g). Carbon dioxide was removed by heating and the resulting cuprous
gluconate solution was concentrated under vacuum. Cuprous gluconate was precipitated
with alcohol.
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Cu
+
CO
2
COOH
2(CHOH)
4
CH
2
OH
+
CuCO
3
+
H
2
O

Gluconic acid Copper carbonate Copper gluconate Carbon dioxide Water

3.2.2.3.4 Nickel gluconate
56.2 g Nickel sulphate was treated with 86.0 g calcium gluconate previously dissolved in
minimum quantity of water. The solution was heated, filtered to remove calcium

sulphate. Nickel gluconate solution was concentrated and precipitated with alcohol. The
precipitate of nickel gluconate was dried by placing it in a desicator.
Reaction
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ni
+
CaSO
4
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ca
+
NiSO
4

Calcium gluconate Nickel sulphate Nickel gluconate Calcium sulphate
(ii) Procedure
The 12.6 g of nickel carbonate was reacted with 78.4 ml (equivalent to 39.2 g) 50%
gluconic acid solution. It was heated to release carbon dioxide and concentrated. Nickel
gluconate was crystallized from the concentrated solution with ethanol.
Reaction
CH
2
OH
(CHOH)
4
COO
COO
(CHOH)
4
CH
2
OH
Ni
+
CO
2
COOH
2(CHOH)
4
CH
2
OH
+
NiCO
3
+
H
2
O

Gluconic acid Nickel carbonate Nickel gluconate Carbon Water
dioxide.
Percentage Yield
Formula used
Actualyield x 100
%ageYield =
Theoretical Yield

These salts of gluconic acid have been prepared and used in view of their comparatively
high water solubility.


Experimental Work: Chapter No.3

Загружено:

Сведения о документе

Оригинальное название

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

Experimental Work: Chapter No.3

Загружено:

Авторское право:

Доступные форматы

Chapter No.

Вам также может понравиться