Keywords (Extracted from title, table of contents and
abstract of thesis)
Microbial, Production, Glucose, Oxidase, Commercial,
Applications, enzyme, gluconic, peroxidase, mutarotase |
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Abstract
Glucose oxidase (EC
1.1.3.4) is an important enzyme that oxidizes glucose to gluconic
acid. It is present in all aerobic organisms and has become a very
useful enzyme for its wide applications especially in food industry
and in clinical analysis. The most important application for GOX is
the determination of glucose using biosensor technology. GOX belongs
to a large group of enzymes oxido reductase and is also called as
glucose aerodehydrogenase Glucose oxidase was produced from
different microorganisms. Both fungi and bacteria produce glucose
oxidase during fermentation. The present project was planned for the
optimum production of glucose oxidase by Aspergillus niger and its
utilization for estimation of glucose and for the production of
calcium, gluconate, gluconic acid and its derivatives. The project
was divided into two parts, in the first part production of glucose
oxidase from Aspergillus niger was investigated and the second part
consists of commercial applications of glucose oxidase. Here the aim
was to improve GOX production using mutagenesis of A. niger, to
optimize the conditions of fermentation, screen fungal strains
producing highest GOX activity, and to medium composition.
Mutagenesis was carried out on several strains at different time
intervals. GOX enzyme purified by (NH4)2SO4 precipitation technique
was dialysed and subjected to gel filtration chromatography. The
enzyme was found to be intracellular. Five strains of A. niger
isolated from grapes, bread, potato, pickle and sugar beet sources
were screened for maximum GOX production. It is clear from our
results that the A. niger strain isolated from potato was best for
GOX production. This strain showed the maximum enzyme activity in
medium containing 10% (w/v) glucose and at pH 5.5. Different
conditions like the fermentation period, varying concentrations of
urea, MgSO4.7H2O, CaCO3 and KH2PO4 were optimized by conducting
different experiments. The maximum activity of glucose oxidase was
recorded after 48 hours of continuous shaking fermentation of
optimum growth medium containing 3.5% (w/v) CaCO3, 0.2% (w/v) Urea,
0.4% (w/v) KH2PO4 and 0.01% (w/v) MgSO4.7H2O. It was observed that
addition of Urea, CaCO3, and KH2PO4 in the medium enhanced the GOX
production whereas addition of MgSO4.7H2O decreased the GOX
production. The GOX was found to be quite active upto 60oC with
optimum temperature at 30oC. The batch fermentation volume of 50 ml
at 100 rpm speed shaker was found to be the optimum for GOX
production. Among mutant, it was found that mutant (9) had maximum
activity and growth. The UV induced mutation gave a stable and
viable culture for hyper production of GOX as the production was
enhanced. Then the enzyme was purified by (NH4)2SO4 precipitation
technique, Dialysis and Gel filtration chromatography. It was
observed that enzyme activity was increased by increasing (NH4)2SO4
concentration. Enzyme activity also increased by Dialysis and Gel
filtration chromatography from 11.90 to 37.24 µ/ml. Purification was
11.55 folds than simple precipitation at this final step. In the
second part of project two commercial applications of GOX were
investigated i.e. estimation of glucose by standardization of
conditions using GOX and the production of calcium gluconate,
gluconic acid and its derivatives using GOX. In the first
application the three enzymes GOX, mutarotase (EC # 5.1.3.3) and
peroxidase (EC # 1.11.1.) were produced, extracted and purified for
the preparation and optimization of glucose estimation kit. The
enzyme concentrations of 5 µL mutarotase, 15 µL glucose oxidase and
10 µL of peroxidase with chromagen Guaiacol added before peroxidase,
proved to be best for estimations of glucose in blood samples. The
sensitivity of the best kit was as low as 50 mg/dL glucose. The
wavelength of 470 nm was best for the test. The results were
comparable with standard kit of Medisense Abbott (UK). In the second
application, calcium gluconate and gluconic acid and its derivatives
were produced by glucose oxidase from Aspergillus niger. The time
course during fermentation showed that the calcium gluconate
production was maximum at 48 hours after conidial inoculation. The
cultural conditions optimized for maximum calcium gluconate
production were, glucose concentration 10% (w/v), pH 5.5, 7% (w/v)
CaCO3, 0.2% (w/v) urea 0.15% (w/v) KH2PO4 concentration at 35oC.
Different nitrogen, phosphate and metal carbonate sources were also
optimized. The present study also described the production of
gluconic acid and its derivatives on the laboratory scale. Gluconic
acid and its metal salts such as sodium, magnesium, copper and
nickel gluconates were synthesized from calcium gluconate which was
produced by fermentation. The gluconic acid was released by the
action of oxalic acid and sulphuric acid on calcium gluconate.
Sulphuric acid gave better yields i.e. (90%) as compared to oxalic
acid (80%). So the organic acid was obtained by H2SO4 in the present
work because it was cheap and readily available in local market.
Metal gluconates were also produced by both the double decomposition
and gluconic acid methods respectively. It is clear from the study
that the gluconic acid method gave greater yields compared to the
double decomposition method. This project will help in the
commercial production of products using GOX in Pakistan.
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