Enzymatic activity of Glucose Oxidase from Aspergillus niger IPBCC.08.610 On Modified Carbon Paste Electrode as Glucose Biosensor

Glucose oxidase (GOx) has been developed as glucose sensor for measuring blood glucose level because of its specificity to glucose oxidation. This research aimed to determine kinetic parameters of GOx activity voltametrically and further test its potential as a glucose biosensor. GOx, in this research, was produced by local fungi Aspergillus niger IPBCC.08.610 which was isolated from local vine in Tarakan, East Borneo, Indonesia. GOx was immobilized with glutaraldehyde, which cross-linked onto modified carbon paste electrode (MCPE) nanofiber polyaniline. Intracellular GOx activity was higher than extracellular ones. Immobilized GOx used glutaraldehyde 2.5% and dripped on the surface of MCPE nanofiber polyaniline. MCPE have a high conductance in copper with the diameter of 3 mm. The concentration of glucose in the lowest concentration of 0.2 mM generated a current value of 0.413 mA while 2 mM of glucose induced a current of 3,869 mA value. Km and Imax of GOx in MCPE activities polyaniline nanofiber were 2.88 mM and 3.869 mA,respectively, with turnover (Kcat) of 13 s-1. Sensitivity was 1.09 mA/mM and response time to produce a maximum peak current was 25 seconds. Km value was then converted into units of mg/dL and obtained 56.4 mg/dL. GOximmo-IPB|MCPE electrode is potential to be able to detect blood glucose level in a normal condition and hypoglycemia conditions


Introduction
Indonesia is a tropical country, which consists of diverse microorganism. Microorganisms may be valuable and potential sources for various purposes, which can be revealed through comprehensive research. Production of various enzymes using microorganisms is one thing that can be developed. Ironically, to date Indonesia has not been able to produce enzymes industrially yet still fully dependent on imported products [1]. One of widely used enzymes is glucose oxidase (GOx). In this research, GOx was produced directly from local Aspergillus niger which was isolated from local vine Dryobalanops in Tarakan, East Borneo, Indonesia. Previous study showed that this enzyme has activity and yield of 27.77 U/mg and 72.86%, respectively. GOx is an enzyme that catalyzes the oxidation of β-D-glucose into D-gluconolactone and hydrogen peroxide using molecular oxygen as an electron acceptor [2]. Nowadays, GOx is generally applied to biosensor and other commercial applications. In addition, the enzyme is also used in biomedicine for determining blood glucose levels. Determination of blood glucose levels using GOx through either colorimetric or amperometric assay is more effective than that non-enzymatic methods because GOx has a high substrate specificity towards β-D-glucose [3]. Thus, biosensor based on GOx could be used in analyzing glucose content of a blood. GOx can oxidize glucose in blood to gluconic acid and hydrogen peroxide by using oxygen molecule as the electron acceptor which will be taken as the signal, and it is then decoded in computer software [4].
Application of GOx onto electrode in biosensor system needs immobilization process [5]. GOx immobilization into polyaniline-nanofiber at carbon pasta electrode surface is presumably to rise electrode conductance in biosensor. Nanometer-sized polyaniline widens electrode surface so that its application may be more economically efficient. GOx immobilized into carbon graphite can be repeatedly used so that it is cheaper and more efficient.
This research has a novelty as a biosensor because of GOx produced from local fungi A. niger IPBCC.08.610. This research aimed to determine kinetic parameters of GOx activity voltametrically, to further test its potential as glucose biosensor in blood. It can be developed potential diversity of microorganisms to make high value product.
2.1.1. Glucose oxidase production, isolation, and purification. This assay was done based on Rohmayanti 2016 with modifications. Production media of A. niger contained 0.4 g/L (NH 4 ) 2 HPO 4 ; 0.2 g/L KH 2 PO 4 ; 0.2 g/L MgSO 4 .7H 2 O; 40 g/L CaCO 3 ; 3.3 % sucrose; and 0.35 % glucose. Culture was then incubated at 37 0 C on 120 rpm for 40 hours. After being incubated, mycelium was filterred by using gauze. The supernatant contained the crude extract of extracelullar glucose oxidase. Filtered mycelium was homogenized using quartz sand and sodium phosphate buffer 0.1 M pH 6.0, then centrifuged at 12,000 x g temperature of 4 0 C for 20 minutes. The supernatant obtained was crude extract of intracellular. The purification of both extracellular glucose oxidase and intracellular crude extract was done through precipitation with saturated 80% (NH 4 ) 2 SO 4 . Protein content was measured according to Bradford method (1976) and its activity based on Bergmeyer method (1988).

Measurement of Carbon Paste Electrode (CPE) and Modified Carbon Pasta Electrode (MCPE)
Cyclic Voltammogram. This experiment was conducted based on Colak et al. [6] with modifications. CPE was prepared by mixing 100 µL paraffin and 0.15 gram graphite powder into mortar until pasta shaped. Furthermore, the carbon paste electrodes was inserted into the prepared tube of glass capillary size variation copper diameter of 1 mm and 3 mm. The best diameter copper size of EPK was then modified by the addition of polyaniline nanofiber by 2 mg polyaniline. Whereas, MCPE was made of 2 mg polyaniline which mixed into mortar with 100 µL paraffin and 0.15 g graphite powder. Furthermore, each graphite pasta was added into electrode tube which prepared from capillary glass with its diameter and height were respectively 3 mm and 10 mm. CPE and MCPE (as working electrode alternately), Ag|AgCl electrode (reference electrode) and platina electrode (counter The prototype electrode is showed at figure 1.  be large enough round cells and white. Then GOx intracellular crude extract was purified by saturated (NH 4 ) 2 SO 4 80%. Precipitation of intracelullar GOx crude extract was the initial step to purify enzyme that significantly increased protein concentration, reduced the volume, and separate the protein target from partial undesired contaminant. Indeed, the purity level of precipitated GOx was 20 times higher than GOx crude extract, with specific activity and yield of 27.77 U/mg and 72.86%, respectively.

GOx Fraction of Intracellular and Extracellular Crude Extract and
Based on the chart Lineweaver-Burk, the activity of the GOX is glucose concentration dependent. Enzymatic activity at low glucose concentration showed linear pattern in corresponds to the increased glucose concentrations. However, enzymatic activity at more than 0.06 M glucose was reduced V maks and Km value of precipitated GOx was 20.747 U/mg and 56 mM, respectively. Moreover, the GOx k cat value of ammonium sulfate 80% fraction was predicted about 53 s -1 .

Cyclic Voltammogram of CPE and MCPE
Cyclic Voltammogram may visualize the produced current by electrochemical cell as current value. Conductance change of this electrode was measured by using cyclic voltammetry method because it could evaluate electrochemical reaction on the surface of electrolite solution and electrode [7]. Figure  2 shows that cyclic voltammogram of CPE and MCPE has higher current evolving than cyclic voltammogram of MCPE. The increasing current is conceived by the change of the width area of cyclic voltammogram. This is due to an improvement of polyaniline into nanofiber that had role to raise its conduction capacity. Moreover, the variation of the size would affect the mass and thickness of carbon graphite which added to electrode capillary, so that it causes distinct electron transfer that could be analyzed from its current and potential.  According to Michaelis-Menten curve, below 2 mM of glucose concentration the GOx immo-BAU |MCPE electrode performance to its subtrate would evolve by the increasing concentration of glucose, on the other hand, enzymatic activity increased following elevated concentration of glucose.However, when glucose concentration was about 2 mM, GOx activity has reached its maximum activity, thus additional glucose concentration had no effects to the oxidation current value of GOx immo-BAU |MCPE electrode ( figure 3).    Km value of GOx immo-IPB |MCPE was 56.4 mg/dL, thus it is potential for this electrode to be used as glucose biosensor. . According to the Km values, GOx immo-IPB |MCPE electrode is able to detect blood glucose level in a normal condition, hyperglycemia, and hypoglycemia conditions. Human normal blood glucose level is about 70-110 mg/dL, and hypoglycemia blood glucose condition to be in a state if glucose level under 60 mg/dL and in a state of hyperglycemia when glucose level above 270 mg/dL.

Conclusion
Biochemical characteristics of GOx of A. niger (IPBCC.08.610) consists of enzymatic activity, kinetics parameter and its immobilization in carbon pasta electrode. The enzymatic activity of intracellular GOx was higher than extracellular one. MCPE have a high conductance value in copper with the diameter of 3 mm. Linear region of the electrode performance GOx immo-IPB |MCPE, which is half of the maximum speed, is at a concentration of 1.6 mM or 56.4 mg/dL. GOx immo-IPB |MCPE electrode is hihgly potential to be used as glucose biosensor to to detect blood glucose level in a normal condition and hyperglycemia.