Abstract: The enzyme glucose oxidase was produced by fermentation technology, using Aspergillus niger as fermentation organism and rice polishing as substrate. Maximum production of enzyme (3.42 U mL-1) was recovered at substrate level of 2% after 36 h of submerged fermentation. The pH for the optimal production of enzyme was found to be 4. Addition of salts such as urea (0.3%), CaCO3 (0.04%) and KH2PO4 (0.6%) into the fermentation medium enhanced enzyme production while MGSO4.7H2O was found to inhabit microbial growth and glucose oxidase production by A. niger.
Introduction
Enzymes can be defined as soluble colloidal organic catalysts which are produced by living cells but are capable of acting independently of the cells (Rao, 1993). Glucose oxidase (EC. 1.1.3.4) belongs to class oxidoreductase and is also called as glucose aerodehydrogenase (Sidney and Northon, 1955). It is a βavo-enzyme that catalyzes the oxidation of β-D-glucose to O-D- gluconolactone and H2O2 is also formed in this reaction. It is highly specific for β-D-glucose while a-anomer is not acted upon. Glucose oxidase has a molecular weight of 160,000 and consists of two identical polypeptide chain subunits linked by disulfide bonds, optimum pH for the enzyme is 5.5 with broad range of 4-7 (Bentley, 1959).
Glucose oxidase is produced from most of the microorganisms such as Penicilluim notatum, Penicillium chrysosporium, Aspergillus niger and Botrytis cinerea (Liu et al., 1998). The glucose oxidase from Aspergillus niger is an intracellular enzyme present in the mycelium of the organism (Willis, 1966). The present research project was designed for optimization and production of enzyme based glucose oxidase from Aspergillus niger for ultimate use in glucose estimation kit.
Materials and Methods
Microorganism and fermentation
Pure culture of fungus Aspergillus niger procured from NIBGE, Faisalabad
was raised on potato starch-agar slants, sporulation medium. It was incubated
aerobically at pH 4 and 30oC for 72 h.
Conical βasks with 100 mL of rice polishing medium containing different concentrations of micro-nutrients were inoculated with 5 mL of homogenous spore suspension (107-108 spores mL-1). The βasks were incubated at pH 4 and 30°C on a shaker (120 rpm) for optimum fermentation period (Zubair et al., 2002). The fermented biomass in each case was filtered and then blended to extract intra-cellular enzyme finally the filtrates were centrifuged. The supernatant was ultra- filtered through filter paper and the filtrate was assayed for glucose oxidase (Zia, 2002).
Optimization of culture conditions
The growth medium of rice polishing was fermented with Aspergillus niger
for different fermentation periods with varying levels of substrate, pH,
urea (nitrogen source), CaCO3, MGSO4.7H2O and KH2PO4 in shake βask. The experiments
were carried out in such a way that the parameter optimized in one experiment
was maintained in the subsequent investigation.
Enzyme assay
The glucose oxidase activity in crude enzyme extract was determined by a
spectrophotometric method at 460 nm wavelength using glucose as substrate and
o-dianisidine buffer mixture as coupling reagent (Worthington, 1988).
Results and Discussion
Fermentation period
For the optimization of fermentation period duplicate growth media containing
2% (w/v) rice polishing as substrate were autoclaved, inoculated (5% v/v) and
incubated for 24, 36, 48, 60 and 72 h at pH 4 and 30°C. The maximum glucose
oxidase activity (3.42 U mL-1) was noted in enzyme solution harvested after
36 h. It was observed that production of glucose oxidase increased with an increase
in fermentation period from 12-36 h, reached its maximum after 36 h and decreased,
thereafter (Fig. 1).
The results of Willis (1966) are in line of this work, who optimized the fermentation medium for the production of glucose oxidase by Aspergillus niger. He obtained highest glucose oxidase yield after 48 h of fermentation.
Substrate level
The maximum activity of glucose oxidase (3.42 U mL-1) was observed with
2% rice polishing in continuous shaking culture medium. All other substrates
lower are higher than 2% gave lower enzyme productivity (Table
1).
Results are in line with those of Willis (1966) who grew Aspergillus niger in a submerged culture fermentation in mineral medium containing urea as organic nitrogen source and rice polishing (as carbohydrate source) and observed that growth media containing 2.5% rice polishing produced maximum glucose oxidase.
| Table 1: | Production of glucose oxidase with varying substrate level |
| LSD value = 0.01757 | |
| Table 2: | Glucose oxidase production at different pH |
| Table 3: | Production of glucose oxidase with varying urea |
| LSD value = 0.02143 | |
Effect of pH
In this experiment duplicate media of rice polishing (2%) were adjusted
at different pH values i.e 2, 3, 4, 5 and 6. The results showed maximum activity
of glucose oxidase (3.45 U mL-1) at pH 3 (Table 2).
The results of Rando et al. (1997) accord with our results when they produced glucose oxidase by Penicillium pinophilum. They determined that the optimum pH for glucose oxidase production was in the range pH 4-4.6.
| Fig. 1: | Effect of fermentation period on glucose oxides production |
| Fig. 2: | Effect of different concentrations of MGSO4. 7H2O on glucose oxidase production |
Effect of urea
The concentration of nitrogen source in the growth media has a considerable
inβuence on enzyme production. The results showed maximum activity of glucose
oxidase (4.66 U mL-1) with 0.3% urea (Table 3) as additional
nitrogen source.
| Fig. 3: | Effect of different concentrations of CaCO3 on glucose oxidase production |
| Fig. 4: | Effect of varying concentrations of KH2PO4 on glucose oxidase production |
Further increase in nitrogen concentration resulted in a decrease in enzyme yields.
The results of Pazlarova and Votruba (1996) accord with this work. They used synthetic and natural ammonium-sorbing zeolite to control the ammonium (NH4) level in the medium to growth Bacillus amyloliqueficiens.
Effect of MGSO4.7H2O
Four different concentrations of MGSO4.7H2O were tested for glucose oxidase
production in the growth medium containing optimum level of substrate (2%) and
urea (0.3%) at pH 4 and 37oC temperature. It was observed that addition of MGSO4.7H2O
into the medium glucose oxidase production decreased gradually (Fig.
2). So it was recommended that MGSO4.7H2O should not be added in the medium.
The results of present study are in line with the work of Yang et al. (1996) who studied the production of glucose oxidase from waste mycelium of Aspergillus niger and effect of metal ions on the activity of glucose oxidase. The results showed that addition of Mg2+ in the medium strongly inhibited the production of glucose oxidase.
Effect of CaCO3
To enhance the rate of fermentation and glucose oxidase production by Aspergillus
niger CaCO3 was adduced into the optimum rice polishing medium. The results
regarding the effect of CaCO3 on glucose oxidase production in rice polishing
(2%) containing urea (0.3%) showed that the addition of CaCO3 into the growth
medium enhanced enzyme production and showed maximum glucose oxidase activity
(5.57 U mL-1) with 0.04% level of this salt.
These results accord with Hatzinikolaou and Macris (1995) who reported factors regulating production of glucose oxidase by Aspergillus niger. They identified CaCO3 as a particularly strong inducer of glucose oxidase activity.
Effect of KH2PO4
Effect of different levels of KH2PO4 was studied on the production of glucose
oxidase in growth medium containing optimum concentrations of rice polishing,
urea and CaCO3 at pH 4.0.
Results indicated maximum glucose oxidase activity (7.49 U mL-1) with 0.6% KH PO in the medium under pre-optimized culture conditions. Glucose oxidase production was found to be enhanced by the addition of KH2PO4 upto 0.6% and decreased by its further addition (Fig. 4).