P T Kalaichelvan

Vijayakumar Aruldoss

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IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) 
e-ISSN: 2278-3008, p-ISSN:2319-7676.  Volume 9, Issue 1 Ver. IV (Jan. 2014), PP 51-54 
www.iosrjournals.org 
www.iosrjournals.org                                                     51 | Page 
 
Biosensor application of extracellular catalase from Ganoderma lucidum 
AVK-1 and Acinetobacter calcoaceticus AV-6 and Comparative study of 
commercial catalase from Bovine liver catalase 
 
Vijayakumar Aruldoss  ⃰and P.T.Kalaichelvan  
Centre for Advanced Study in Botany,  University of Madras, Guindy, Chennai-600 025 and India.  
 
Abstract: An amperometric biosensor based on catalase enzyme was developed for the investigation of the effect of calcium 
ions on the activity of the enzyme. Calcium plays an activator role for the catalase enzyme that catalysis the degradation of 
hydrogen peroxide to O2 and H2O. Determination method of the effect of calcium ion on the activity of the enzyme was based 
on the assay of the differences on the responses of the biosensor in the absence and the presence of calcium in the reaction 
medium. The biosensor had a linear relation to calcium concentrations and good measurement correlation between 0.05 and 
15 mM with 3 min response time. PPB–HCl buffer (pH 6.8; 20 mM) and 37ºC were obtained as the optimum working 
conditions. In the application studies, the biosensor was used determination of calcium level of real samples such as cow 
milk, Aavin (green) and MD water. In the characterization studies of the biosensor some parameters such as reproducibility, 
substrate specificity, operational and storage stability were carried out. Finally, by using the biosensor developed and 
enzymatic-spectrophotometric method alcohol concentrations of some alcoholic drinks were determined and results were 
compared. 
Keywords: Acinetobacter calcoaceticus, Ganoderma lucidum, Catalase, Biosensor, Milk and calcium  
 
I. Introduction 
Calcium (Ca) is the most abundant mineral in the human body. It is important for intracellular metabolism, bone 
growth, blood clotting, nerve conduction, muscle contraction and cardiac functions [1 & 2]. Calcium and calcium salts are 
also very important minerals used in the food industry. Different calcium salts have been studied for decay prevention, 
sanitation and nutritional enrichment of fresh fruits and vegetables. Calcium carbonate and calcium citrate are the main 
calcium salts added to foods in order to enhance the nutritional value. Calcium lactate, calcium propionate and calcium 
gluconate have shown some of the benefits of the use of calcium chloride, such as product firmness improvement, and avoid 
some of the disadvantages[3]. Also, the use of calcium salts other than calcium chloride could avoid the formation of 
carcinogenic compounds (chloramines and trihalomethanes) linked to the use of chlorine [4]. 
 
II. Experimental 
Preparation of catalase enzyme  
Ganoderma lucidum AV-1 and Acinetobacter calcoaceticus AV-6 those organisms were used for this study. 
Synthesis and extraction of catalase was purified and applied for biosensor technology and commercial Bovine Liver 
Catalase also tested.  
 
Chemicals 
Catalase (hydrogen peroxide: hydrogen peroxide Oxidoreductase). (EC 1.11.1.6.) from bovine liver 1.9 U mg-1, 
CaCl2, NaCl2, KCl2, MgSO4, NiCl2, CuSO4, MnCl4, KH2 PO4, K2HPO4, calf skin gelatin, glutaraldehyde (25%), Na EDTA, 
and all other chemicals were purchased from Sigma Chemical Co. (USA). All solutions were prepared with double distilled 
water just before their use.  
 
Apparatus 
In these experiments, a YSI Model 5300 Biological Oxygen Meter (Yellow spring instrument co. inc., Yellow 
spring Ohima 45387 USA) with 0.01-mg/l dissolved oxygen (DO) concentration sensitivity, YSI 5700 Model DO probes 
(with YSI 5740 cable) as transducers, standard teflon membranes (YSI, Yellow Springs, OH, USA), Accupipet P100 and 
P1000 automatic pipets (USA), Yellow-Line magnetic stirrer (Germany) and Nuve model thermostat (TR) were used. 
 
Preparation of the biosensor 
First of all, a DO probe was covered with a standard teflon membrane using an O-ring and then the membrane 
which is sensitive for oxygen was pretreated with 0.5% SDS (sodiumdodecylsulphate) in phosphate buffer (50 mM, pH 7.0) 
to reduce the tension on the membrane surface of the DO probe. After this step, 250 µl of catalase enzyme solution and 
gelatin were mixed and dissolved at 38ºC for a few minutes. Two-hundred microlitre of the solution was spread over the DO 
probe membrane surface and allowed to dry at 4  ºC for 30 min. At the end of the time, the bioactive layer was treated with 
glutaraldehyde (2.5%, in phosphate buffer; 50 mM, pH 7.0) for 3 min to form chemical covalent bonds (Schiff bases) 
between gelatin, enzyme and glutaraldehyde molecules for the immobilisation of the enzyme on the surface of the DO probe. 
 
Measurements 
In the reaction, catalase converts hydrogen peroxide to hydrogen dioxide and carbon dioxide in the presence of 
oxygen. There is an intermediate surface between the bioactive layer and the teflon membrane of the DO probe and during 
Biosensor application of extracellular catalase from Ganoderma lucidum AVK-1 and Acinetobacter 
www.iosrjournals.org                                                     52 | Page 
0
20
40
60
80
100
120
4 5 6 7 8 9
 Different pH
R
el
at
iv
e 
ac
tiv
ity
 (%
)
the enzymatic reaction dissolved oxygen concentration in the intermediate surface decreased relative to the substrate 
concentration added into the reaction medium. The measurements with the developed biosensor were carried out at steady-
state conditions.  DO is the differences of the dissolved oxygen concentration when the substrate is not in the reaction 
medium and after addition of substrate into the reaction medium to obtain a new steady-state DO concentration. It is well 
known that calcium is a cofactor for catalase and it plays an activator role for the catalase so when the calcium was injected 
into the reaction medium it increased the activity of the enzyme and in this case the dissolved oxygen concentration changed 
relative to the calcium concentration added into the reaction medium. 
The principle of the measurement of the biosensor was based on the determination of these changes in the 
dissolved oxygen concentration related to calcium concentrations used in the enzymatic reaction. As a result, the differences 
between the first and the final dissolved oxygen concentrations related to calcium concentrations were detected by the 
biosensor to obtain a standard curve for the determination of calcium. All the measurements were carried out at 37º C using a 
thermostatic reaction cell and oxygen saturated tris–HCl buffer (50 mM, pH 7.0). 
 
III. Results and discussion 
Detection of calcium effect as an activator on the biosensor responses 
At the beginning of the study, some 
experiments were carried out for the determination of 
the effect of calcium as an activator on the catalase 
enzyme biosensor. For this purpose firstly, the 
developed biosensor was used only for hydrogen 
peroxide detection using standards with 
concentration between 1 and 10 mM in the absence 
of calcium and a linear curve was obtained. After 
that, by using the same hydrogen peroxide standards 
but in the presence of 5 mM calcium a new standard 
curve was obtained. Fig. 1 shows the results obtained 
from the experiments. According to the figure the 
biosensor responses increased very efficiently in the 
presence of calcium.                                               Figure1. Effect of CaCl2 with catalase on break down of H2O2 
Optimisation of the bioactive surface of the biosensor 
Effect of the enzyme activity on the biosensor response Different enzyme amounts were used for determination of 
the effect of the enzyme activity on the biosensor response. For this purpose, three biosensors which contain 0.5, 0.75 and 
1.0 Ucm-2 catalase were prepared by immobilising with gelatin (5.31 mg cm2) and glutaraldehyde (2.5%).  
Estimation of calcium using enzyme electrode biosensor 
  Catalase, the most useful calibration curve was obtained. Calcium was detected with a linear range between 1 and 
10 mM concentrations by this biosensor. Increase in the catalase activity from 0.75 to 1.0 cm2 contained 0.75 U cm-2 resulted 
in higher biosensor responses. When the bioactive layer of the biosensor contained 0.5 and 1.0 U cm-2 activity of catalase we 
didn’t obtain any suitable standard curve for calcium. In this case, if we consider the results obtained from the experiments it 
can be said that the most suitable biosensor responses were obtained by the biosensor which contained 0.75 U cm-2 activity 
of catalase. 
 
Detection of the effect of gelatin amounts on the biosensor response 
To determine the effect of the amount of gelatin on the biosensor response different gelatin amounts were used in 
the construction of the biosensor. Biosensors contained 0.25, 0.50, 0.75 and 10 mg cm-2 gelatin and 0.75 U cm-2 catalase. All 
the biosensors were immobilised by 2.5% of glutaraldehyde. The measurements were made in order to obtain standard 
curves for calcium. The most suitable curve was obtained with the biosensor prepared using 10.0 mg cm -2 gelatin amounts.  
From the experiments when the gelatin amount increased from 2.5. to 7.5 mg cm-2, we obtained higher biosensor 
responses but there was deviation from linearity. Decreases of the gelatin amounts from 0.50 to 0.75 mg cm-2 results in a 
lower biosensor response. The reason of this effect was the reducing in the forming of cross-linked bonds between enzyme–
gelatin–glutaraldehyde. Therefore the enzyme escaped from the bioactive layer. From the results it can be uttered that the 
most suitable biosensor responses were obtained by the biosensor which contained 10.0 mg cm -2 gelatin. 
 
Effect of the percentage of glutaraldehyde on the biosensor response 
For the determination of the effect of 
the percentage of glutaraldehyde on the 
biosensor response different glutaraldehyde 
amounts were used in the construction of the 
biosensor. For this purpose we prepared 
biosensors which contain 0.75 U cm
2
 catalase 
and 0.75 U cm
-2 
gelatin. The biosensors were 
treated with 1.25%, 2.5% and 3.75% 
glutaraldehyde solution prepared in phosphate 
buffer (50 mM, pH 7.0) for the immobilisation. 
After the immobilisation procedure, experiments were carried out to obtain standard curves for hydrogen 
Biosensor application of extracellular catalase from Ganoderma lucidum AVK-1 and Acinetobacter 
www.iosrjournals.org                                                     53 | Page 
peroxide using the prepared biosensors. 
Experiments showed that the higher biosensor responses were observed at 2.5% glutaraldehyde 
percentage. The biosensors those were prepared with 1.25% and 3.75% glutaraldehyde, showed lower biosensor 
responses. As a result of the good linear range and high biosensor responses the biosensors were prepared with 
2.5% glutaraldehyde. 
  
Optimisation of working conditions 
Effect of pH on the biosensor response  
To determine the effect of the pH value on the biosensor response different buffer systems were investigated. 
For this aim 50 mM con-    Figure 2.Effect of different pH on the activity of catalase for break down of  H2O2 
-centration of citrate (pH 5.0–6.0), tris–
HCl (pH 7.0– 8.0) and glycine (pH 9.0–
10.0) buffers were used in the 
experiments.The optimum pH value was 
determined to be 7.0 Fig. 2. From the 
experiments below and above this pH 
value decreases in the biosensor 
responses were observed. If we consider 
the optimum pH value (pH 7–8) of the 
free catalase it can be uttered that the 
immobilisation procedure did not affect 
the optimum pH value of the enzyme.                                                
                                     Figure 3.  Standard curve for calcium determination 
Effect of temperature on the biosensor response  
The enzyme activity depends on the temperature and the medium conditions. For determination of the effect of 
temperature on the biosensor response, experiments were carried out between 15 and 40 ºC. The highest biosensor responses 
were observed at          
 37 ºC. Below and above this degree, decreases  in the biosensor responses that probably resulted from the changes in the 
enzyme structure, were recorded. 
 
Analytical characteristics of the biosensor 
Linear range of the biosensor 
The results obtained for the determination of detection limits for calcium are given in Fig. 3. When we consider the 
figure, it can be said that the biosensor responses depended linearly on the calcium concentration between 1 and 10 mM with 
(y = 0.02x + 7E-17), and R2 = 1 the detection limit of the biosensor was determined to be 1 mM. 
Reproducibility 
 The reproducibility of the biosensor was also investigated for 5 mM calcium concentration (n=7). The average 
value (x), the standard deviation (SD) and variation coefficient (CV %) were calculated to be 5.1 mM, ±0.106 mM and 
2.07%, respectively. 
Effect of some metal ions on the activity of catalase 
 In order to determine the effect of different compounds on the catalase activity some experiments were carried out 
using 5 mM calcium and various substances such as CaCl2, NaCl2, KCl2, MgSO4, NiCl2, CuSO4   and MnCl2  at the same 
concentration with calcium in the presence of 5 mM hydrogen peroxide. The increase in the biosensor response obtained 
with calcium was compared to other biosensor responses obtained in the presence of the other substances.  
In the other words, for the investigation of these metal ions on the catalase activity in the presence of calcium ion, 
some experiments were made. Table 1 also shows these results obtained from the experiments. 
According to the results, although MnCl2, KCl2, MgSO4 and NiCl2 played activator role on the catalase activity in 
the absence of calcium ion, they showed negative effects on the catalase activity in the presence of calcium. 
Table 1 Detection of the effects of some metal ions on the activity of catalase in the presence and absence of calcium ion. 
 
Metal ions            Response% 
Cacl2  100 
MgSO4   80 
NiCl2  70 
 Nacl  30  
KCl  10 
CuCl2  05 
 Values are triplicates 
 
Application  
 In this section of the study the calcium content of some drinks were detected by using the biosensor. The results 
obtained from the biosensor were compared to results obtained using a reference procedure [5]. For the same samples in 
order to complete the validation of the new method. For this goal, some drinks such as milk, water and mineral water which 
Biosensor application of extracellular catalase from Ganoderma lucidum AVK-1 and Acinetobacter 
www.iosrjournals.org                                                     54 | Page 
contains different quantity of calcium, were used. Results obtained from the experiments were given in Table 2. From the 
experiments when we compare the results of two methods it can be said that calcium in the drinks can be determined 
sensitively by using the biosensor. 
 
Table 2.  Determination of Calcium level of some commercial, raw milk and liquid by using the amperometric 
biosensor method. (E. Akyilmaz et al., 2009) 
Sample            Reported (mg/l)     Found (mg/ml by the biosensor) Recovery (%  SDS 
Milk-1   120  110    99 ±700 
Milk-2   127  126    99 ±1.410 
Milk-3   170  167    98 ±0.866 
GD- water   335  133    98 ±1.000  
Hot water   80  290    101 ±0.100 
Values are triplicates 
 
                              Overal experiment figure Comparison of biosensor application of commercial catalase and purified                                                          
                                          Acinetobacter  calcoaceticus AV-6 catalase                    
                               a. Biosensor unit of YSI model 5300 
                                  b. Oxygen electrode with thermo static cell   
                                  c. Enzyme coating on oxygen electrode and treated with SDS (reduce the tension) 
                                  d. Thermostatic cell of oxygen electrode during reaction time tested for activity  (2µL) 
                                  e. Acinetobacter calcoaceticus AV-6 catalase on activity staining 
                                  f. Bovine liver catalase activity expression on native gel 
I. Conclusion 
In this study, an amperometric biosensor was developed in order to investigate the effect of calcium on the activity 
of catalase enzyme. From the experimental studies we detected a positive effect of calcium on the enzyme activity and this 
effect increased in higher calcium concentrations. By using the biosensor we detected a linear concentration range for 
calcium in the presence of constant concentration of hydrogen peroxide. The biosensor is really original for Ca 
determination especially liquid samples and there is no any study like this in the literature. It does not need any expensive 
equipments, materials or laboratory conditions. The biosensor is portable so it can be used in everywhere for Ca analysis. 
For all liquid samples it is not necessary any pre-treatment for the samples except dilution (if it is necessary) in the Ca 
determination. The biosensor developed can be used not only food samples also clinical purpose. In dealing with a large 
number of samples, the biosensor is rapid, accurate, and precise and with low operation cost is required. By using the 
biosensor because of the specificity of the enzyme we can determine calcium concentration in the presence of the other metal 
ions such as Cu+and K2+, Mg
2+, Ni2+, Na2+, Mn2. Reproducibility of the biosensor is very well and from the application 
studies of real samples it can be said that the biosensor can be used as a sensitive alternative method for analysis of calcium. 
 
Acknowledgment 
The authors express thanks to .The authors acknowledge the financial assistance provided by Council of Scientific 
and Industrial Research (CSIR), New Delhi. (Meritorious Fellowship). The authors thank Dr. Ravichandran, Nuclear physics 
department for the technical support on the X-ray Diffraction.  The authors thanks to Centralised Instrumentation 
Laboratory, Tamil Nadu and Animal Science University for the technical support on the transmission electron microscope.  
 
References 
[1].  Caceres, E., Garcia, M. L., & Selgas, M. D. (2006). Design of a new cooked meat sausage enriched with calcium. Meat Science, 73, 368–377. 
[2].  Akyilmaz E, Kozgus O, Turkmen H, Cetinkaya B. (2010) Bioelectrochemistry 78: 135-140. 
[3].  Alzamora, S. M., Salvatori, D., Tapia, M. S., Lopez-Malo, A., Welti-Chanes, J., & Fito, P. (2005). Novel functional foods from vegetable 
matrices impregnated with biologically active compounds. Journal of Food Engineering, 67, 205–214. 
[4].  Manganaris, G. A., Vasilakakis, M., Diamantidis, G., & Mignani, I. (2007). The effect of postharvest calcium application on tissue calcium 
concentration, quality attributes incidence of flesh browning and cell wall physicochemical aspects of peach fruits. Food Chemistry, 4, 1385-
1392 
[5].  Kamal, T. H. (1960). Complexometric titration of calcium and magnesium in the presence of phosphate in milk and blood plasma. Journal of 
Agricultural and Food Chemistry, 8, 156–158. 
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Biosensor application of extracellular catalase from Ganoderma lucidum AVK-1 and Acinetobacter calcoaceticus AV-6 and Comparative study of commercial catalase from Bovine liver catalase

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Biosensor application of extracellular catalase from Ganoderma lucidum AVK-1 and Acinetobacter calcoaceticus AV-6 and Comparative study of commercial catalase from Bovine liver catalase

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