Abstract: Production cost of enzyme is largely determined by the type of the strain and raw material used to propagate the strain. Hence, selection of the strain and raw materials is crucial in enzyme production. For Glucose oxidase (GOx), previous studies showed Aspergillus terreus UniMAP AA-1 offers a better alternative to the existing sources. Thus, a lower production cost could be logically anticipated by growing the strain in a cheaper complex media such as molasses. In this work, sugar cane molasses, supplemented with urea and carbonate salt and a locally isolated strain Aspergillus terreus UniMAP AA-1 were used to produce a crude GOx enzyme in a small scale. A statistical optimization approach namely Response Surface Methodology (RSM) was used to optimize the media components for highest GOx activity. It was found that the highest GOx activity was achieved using a combination of molasses, carbonate salt and urea at concentration 32.51, 4.58 and 0.93% (w/v), respectively. This study provides an alternative optimized media conditions for GOx production using locally available raw materials.
INTRODUCTION
Glucose oxidase (Gox) oxidizes glucose to D-glucono-1,5-lactone and hydrogen peroxide. The enzyme has found several commercial application including removal of oxygen for food and beverage industries (Wong et al., 2008), detection of glucose for development of biosensor (Yoo and Lee, 2010) and as an enzymatic electrode for biofuel (Kim et al., 2009). As the need of glucose oxidase increases for various fields, there is need to search for fungus glucose oxidase producer in the presence of economical carbon and nitrogen sources.
Extensive work has been made to improve the production of GOx from fungus (Bankar et al., 2009a). However, minimal effort has been focused on the improvement of GOx in terms of processing cost using locally avaliable materials and statistical optimization approach.
Ahmad and Arbain (2012), reported a locally isolated and promising glucose oxidase-producing strain, Aspergillus terreus UniMAP AA-1. This strain produces GOx extracellularly and exhibits a pelleted form in submerged fermentation, thus allowing easy product recovery and purification. These two typical behaviours of the strain would prove useful in lowering the manufacturing cost of the enzyme. However, having a good strain alone would not guarantee a cheaper production cost because a predominant cost factor of enzyme production and in many bio-based productions lies on the type of raw materials used. Typically, raw material cost in fermentation may reach to 60% of the total manufacturing cost. Thus, it is deemed important to select and to optimize the raw material used for a production scale of fermentation. As far as media is concerned, it is not economical to use a defined media for production scale. Instead, it is preferable to use complex media such as molasses, corn-steep liquor, meat extract and agricultural wastes. In this context, to further justify the usefulness of the locally isolated strain, Aspergillus terreus UniMAP AA-1 for GOx production, here we are reporting the optimization of complex fermentation media for production of GOx by Aspergillus terreus UniMAP AA-1. Thus, in this study, Molasses and urea, locally available materials are used as a relatively inexpensive and economic carbon and nitrogen source respectively alternative to synthetic media for the production of economic glucose oxidase enzymes. Molasses is found to be an attractive economic component as it has been reported to enhance enzyme activity up to 40 folds (El-Sherbeny et al., 2005; Hatzinikolaou and Macris, 1995). Apart from the molasses and urea, CaCO3 is used in the production media to control the pH which is deemed necessary for optimal production of glucose oxidase (Petruccioli and Federici, 1993).
Response Surface Methodology (RSM) is a statistical approach used to optimize the conditions of the fermentation media by using a generated model (Panda et al., 2007). RSM is preferable than one-factor-at-a-time (OFAT) approach to optimize the fermentation media since the method is effective in terms of time, analysis and cost. It is thus expected that this approach will improve the production of GOx in terms of processing cost and productivity.
MATERIAL AND METHODS
Material: Molasses are obtained from Fermpro Sdn. Bhd, bioethanol production factory located at Chuping, Perlis, Malaysia while urea and CaCO3 are procured from grocery at Kangar, Perlis, Malaysia.
Microorganism: Aspergillus terreus UniMAP AA-1 strain (Ahmad and Arbain, 2012) is maintained on Malt Extract Agar (MEA) at 4°C at the culture collection of School of Bioprocess Engineering, University Malaysia Perlis.
Pre-treatment of molasses: The pH of the molasses solutions was maintained at 3.5 by treating with 35 mL of 1N H2SO4 per litre. Then, the treated molasses was heated at 60°C for 2 h. After centrifugation at 8000 g for 15 min, the supernatant was adjusted to pH 6.0 with 2 M NaOH.
Production of crude Gox: All the culture media were carried out in 100 mL Erlenmeyer flasks with 50 mL working volume. The culture media consist of molasses, CaCO3 and urea. The flasks were inoculated with 1 mL (5.17x107 spores mL-1) of inoculums and incubated in a rotary shaker operating at 200 rpm and 30°C for 110 h.
Assay of GOx activity: Glucose oxidase activity in the supernatant was measured spectrophotometrically using the coupled o-anisidine-peroxidase reaction method explained by Bankar et al. (2009b).
Optimization of media components by response surface method (RSM): The Central Composite Design (CCD) under RSM (Box and Wilson, 1951) was employed in order to find the optimum levels of significant media components.
| Table 1: | Values of independent variables at different levels of CCD |
CCD was generated using a statistical analysis package Design-Expert Software (Stat-Ease Inc., Statistic made easy, Minneapolis, MN, USA, version 6.0.8) and the statistical analysis of experimental data was also performed using this software.
Molasses, CaCO3 and urea were optimized and examined at three different levels (low, middle, high) concentration coded (-1, 0, +1) as shown in Table 1. The center point value was set at molasses; 30.0% (v/v), CaCO3; 4.0 % (w/v) and urea; 1.0% (w/v).
According to CCD for the three variables, 19 experimental runs were executed and observations were fitted to the following second order polynomial:
| (1) |
where, Y is the predicted response (enzyme activity U mL-1), A, B and C are independent variable (molasses, CaCO3 and urea); βo is the regression coefficient at center point; β1, β2 and β3 are linear coefficients; β11, β22 and β33 are the quadratic coefficients; and β12, β13 and β23 are the second order interaction coefficients.
Validation of the model: In order to verify the adequacy of the developed CCD model, confirmation runs were performed.
Crude Glucose oxidase analysis by Fourier transforms infrared spectroscopy (FT-IR): The formation of D-glucono-1,5-lactone as the result of GOx enzymatic reaction was analyzed using Spectrum 65 Perkin-Elmer FT-IR spectrophotometer. The formation of D-glucono-1,5-lactone was reflected in the peaks corresponding to the C = O and C-O bonds.
RESULT AND DISCUSSION
Optimization of media components by CCD: Table 2 shows the result of GOx activity for the experimental data (using Eq. 1) for the respective parameters. The highest GOx activity was found from the center point value. This indirectly reflects the closest of the values to optimal media conditions.
Regression analysis: The experimental data through polynomial regression analysis was used to develop the following second-order empirical model:
| (2) |
where, GOx production as yield (Y) is a function of molasses (A), CaCO3 (B) and urea (C).
| Table 2: | Observe value of GOx activity obtained from CCD |
| *Gox activity (U mL-1) was expressed as the mean of 3 replicates | |
| Table 3: | ANOVA of response surface model, RSM |
| p<0.05 indicate the model terms are significant | |
Summary of the analysis of the variance (ANOVA) is presented in Table 3. The fit of the model was checked by the coefficient of determination R2, which was calculated to be 0.9035. This indicates that the model is able to explain 93.50% of total variations and only 6.5% is not explained. Besides, the goodness of the model was also supported by the high adjusted coefficient of determination that is 0.87. This high value indicated the significance of the model.
ANOVA result presented in Table 3 reveals that the linear effect of molasses (A) and CaCO3 (B) and the interactive terms and quadratic terms of AB and molasses (A2), respectively were significant at the level p<0.05. These significant variables indicate that they can act as limiting factors for GOx production and the small changes of the components in the fermentation media could affect the glucose oxidase activity.
| Fig. 1: | 3D plot of interaction between molasses, A (%) and urea, C (%) at constant CaCO3, B (4.00%) on GOx production (U mL-1) |
The result shows the linearly-formed molasses has the highest F-value and lowest p-value which indicates the significance of molasses in maximizing the production of glucose oxidase.
Effect of interaction between media components on GOx activity: A 3D response surface allows the user to study the effects of the selected media components and their mutual interaction on the GOx production. From Fig. 1, the response surface is elliptical in the entire region where this result reflected the perfect interaction between the molasses and urea. The maximum predicted value of glucose oxidase activity is referred by the surface confined in the smallest ellipse in the diagram where the concentration of molasses is near 32.50 % (v/v) and urea near 1.00% (v/v) (Fig. 1).
Similar observation is found in the effect of molasses and CaCO3 on maximum production of glucose oxidase at their center and onward values (Fig. 2). An increment in CaCO3 concentration when molasses concentrations near the value of 32.50% (w/v), the GOx productions increased. Likewise in Fig. 3, the production of GOx is increased when an increment in CaCO3 concentration at urea concentrations near 1.0% (w/v). CaCO3 is in turn, significant in GOx production because the addition of CaCO3 prevents pH reduction during cultivation for optimal GOx production (Petrucoili et al., 1995).
| Table 4: | Validation and optimum media compositions for developed quadratic model with predicted and observed results |
| Fig. 2: | 3D plot of interaction between molasses, A (%) and CaCO3, B (%) at constant urea, C (1.00%) on GOx production, (U mL-1) |
| Fig. 3: | 3D plot of interaction between CaCO3, B (%) and urea, C (%) at constant molasses, A (30.00%) on GOx production, (U mL-1) |
This result also is in agreement with Liu et al. (2001) and Hatzinikolaou and Macris (1995) who found that addition of CaCO3 being a strong inducer in GOx production by A.niger. Thus, CaCO3 strongly induced the enzyme production by Aspergillus sp.
| Fig. 4: | FT-IR spectra of the enzymatic reaction where spectra shows the wavelength within the range of 4000-650 cm-1 |
Validation of the model: The results of optimum media compositions are presented in Table 4. The experiment yields 1.056 U mL-1 as the highest GOx activity obtained from the optimized media conditions. The experimental value is lower than the predicted value by 0.47% only which is a very good agreement. These results confirm the validity of the model. This small error is probably due to the nature of the microorganism and its biochemical reaction with complex media comprising of molasses, CaCO3 and urea.
Analysis of crude glucose oxidase, GOx by Fourier Transform Infrared Spectroscopy (FT-IR): The ability of the crude Gox to catalyze the oxidation of β-D-glucose into D-glucono-1,5-lactone was analyzed by FT-IR spectra. The spectra in Fig. 4 indicate that the product formation resulted from the enzymatic reaction of crude glucose oxidase with β-D glucose. The spectra at 1728 cm-1 and at 1204 cm-1 show the presence of C = O and C-O band respectively as a reflection of the formation of D-glucono-1,5-lactone in the enzymatic reaction.
CONCLUSION
The result is clearly shows that a relatively cheaper complex material such as molasses can be used as a substrate for a new locally isolated strain for production of Gox. The use of statistical approach such as RSM is also proved helpful for obtaining the optimum concentration of the complex media for highest GOx production. Additionally, apart from the assay used to monitor the GOx production, FTIR spectra provides support for the oxidation of glucose by the crude enzyme excreted by A. terreus UniMAP grown in molasses as a complex media supplemented with urea and carbonate salt. The highest GOx activity was achieved using a combination of molasses, carbonate salt and urea at 33.96, 4.56 and 0.83% (w/v), respectively. To this end, although no attempt has been made to actually compare the production cost between the combination strain and media used in this study and the combination used in commercial production of GOx, but nonetheless, this study provides an alternative production of GOx which explore some benefits carried by locally isolated strain and a locally obtained complex substrate.
ACKNOWLEDGMENT
The authors thank to the Ministry of Higher Education (MOHE) for financing this research under Fundamental Research Grant Scheme (FRGS 9003-00268) and to School of Bioprocess Engineering, Universiti Malaysia Perlis for their support.