Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

A Potential Peroxidase Obtained from the Juice of Beta vulgaris (Beet)

Pankaj Kumar Chaurasia, Shashi Lata Bharati*, Sunil Kumar Singh, Rama S.S. Yadav

Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh, India

Abstract: In this communication, authors have reported the peroxidase enzyme in the juice of Beta vulgaris (beet) and studied its different enzymatic properties. The steady state enzyme kinetics of this peroxidase was studied using two substrates i.e. guaiacol and hydrogen peroxide. The Km values of this peroxidase for the substrates guaiacol and hydrogen peroxide were 900 and 30 μM, respectively. The pH and temperature optima were 6.0 and 60°C, respectively. The enzyme has maximum stability at pH 6.0. The enzyme was most stable at 45 °C and the activation energy for thermal denaturation of the enzyme was 38.6 kJ/mole/K. Activity of this peroxidase is inhibited completely by sodium azide at the concentration of 16 mM.

Keywords: Beta vulgaris, peroxidase, H₂O₂, guaiacol, sodium azide

Received August 15, 2013; Revised September 17, 2013; Accepted September 21, 2013

Citation: Pankaj Kumar Chaurasia, Shashi Lata Bharati, Sunil Kumar Singh, Rama S.S. Yadav, A Potential Peroxidase Obtained from the Juice of Beta vulgaris (Beet). American Journal of Food Science and Technology, 1 (3). 30-35. January 2013. doi:10.12691/ajfst-1-3-4

1. Introduction

Peroxidase [E.C. 1.11.1.7] is a heme-containing enzyme, which catalyses the oxidation of different organic and inorganic substrates using hydrogen peroxide as the electron acceptor [1,2]. Peroxidases are widely distributed in living organisms such as microorganisms, plants and animals [3]. It is mainly located in the cell wall [4]. It is one of the key enzymes controlling plant growth and development. Peroxidases also involved in various cellular processes including construction, rigidification and eventual lignifications of cell walls [5], suberization [6], organogenesis [7], phenol oxidation [8], crosslinking of cell wall proteins [9] and protection of tissue from damage and infection by pathogenic microorganisms [10,11,12]. It is also used in clinical diagnosis and microanalytical immunoassays because of its high sensitivity. Apart from these applications, peroxidases have been used for biotransformations in organic synthesis [13]. In enantioselective reduction of hydroperoxides [14], hydroxylation of arenes [15], halogenation [16], N-oxidation [17], and sulfoxidation [18] and epoxidation of olefins [19], peroxidases play promising roles.

In this manuscript, authors have identified the juice of B. vulgaris (beet) as a good source of peroxidase enzyme and studied the different enzymatic properties as steady state enzyme kinetics, temperature optimum, thermal stability, pH optimum, pH stability, activation energy for thermal denaturation of enzyme and inhibition of peroxidase. Since peroxidase enzyme is very active biocatalyst in several types of organic syntheses and this source of peroxidase is very easily available, thus, authors have decided to do the several possible organic biotransformations by this enzyme obtained from this source in future. In order to know the efficiency of the enzyme Km has been determined for two different substrates along with its different properties.

2. Materials and Methods

2.1. Chemicals

Guaiacol was from Sigma Chemical Company, St. Louis USA. All other chemicals used in these investigations were either from Himedia Laboratory Ltd, Mumbai or from E. Merck (India) Ltd., Mumbai and were used without further purifications.

2.2. Isolation of the peroxidase

The enzyme was isolated by cutting the Beta vulgaris (beet) into the small pieces, crushing the pieces in mortar with pestle and extracting the juice by keeping the pieces in four layers of cheese cloth and squeezing it. The juice was centrifuged using Sigma (Germany) model 3K-30 refrigerated centrifuge at 14000 rpm for 30 min at 4°C to remove the cloudiness of the juice. The clear juice was stored at 4°C. The enzyme stored in this way has reasonable activity even after 2-3 months.

2.3. Peroxidase Activity Assay

Peroxidase activity of the enzyme was measured in 1 mL reaction solution containing 50 mM sodium phosphate buffer pH 7.0 at 30°C using 5 mM guaiacol, 0.6 mM hydrogen peroxide as the substrates and by monitoring the absorbance changes at λ = 470 nm using molar extinction coefficient value of 2.66 × 10⁴ M⁻¹ cm⁻¹ for the product tetraguaiacol formed during the enzymatic reaction [20,21]. The reaction solution was allowed for thermal equilibration for 10 minutes, 20 μL of the enzyme was added and activity measurement was started immediately and was continued for 180 seconds. All spectrophotometric measurements were done with UV/Vis spectrophotometer Hitachi (Japan) model U-2000 which was fitted with electronic temperature control unit for variation of temperature in the cuvettes. The least count of the absorbance measurement was 0.001.

2.4. Steady State Enzyme Kinetics

The steady state kinetics of the enzyme was studied for the peroxidase obtained from the juice of B. vulgaris using guaiacol and H₂O2 as the variable substrates and monitoring the steady state formation of tetraguaiacol as mentioned in the assay section. We have studied the kinetic properties by varying the concentration of guaiacol (keeping the concentration of H₂O2 constant at enzyme saturating value 0.6 mM) and again by varying the concentration of H₂O2 (keeping the concentration of guaiacol constant at the enzyme saturating value 5 mM.). The Km values were calculated in both cases (guaiacol and H₂O₂) using linear regression analysis of the data points of double-reciprocal plots. Each point of steady state velocity was an average of triplicate measurements and the percentage standard deviation was less than 5%.

2.5. pH Optimum and pH Stability

The pH optimum was determined by measuring the relative activity of the enzyme in the pH range 3.0–9.0 using buffer prepared with H₃PO₄/NaH₂PO₄/Na₂HPO₄.The composition of the reaction solution was the same as mentioned in the peroxidase activity assay. pH optimum for the peroxidase present in the juice of B. vulgaris was 6.0 that means it is most active at pH 6.0.

pH stability of the enzyme was tested by incubating an enzyme aliquot at a particular pH for every 10 minutes time intervals upto one hour, assaying its activity and plotting the activity against time intervals for that particular pH.

2.6. Temperature Optimum and Temperature Stability

The temperature optimum was determined by measuring the relative activity of the peroxidase in the temperature range 10-80°C at fixed pH 7.0 using the reaction solution of the composition mentioned in peroxidase activity assay. Temperature optimum was 60°C.

For thermal denaturation of the enzyme, rate constant at different temperatures were determined by keeping the enzyme aliquots at different fixed temperatures and assaying the enzyme activity at regular time intervals and plotting activity against time intervals. Rate constants were calculated from t1/2 values using equation k = 0.693/t1/2. Energy of activation for thermal denaturation was calculated by Arrhenius plot.

2.7. Inhibition Study

The effect of sodium azide on the activity of the peroxidase was also studied for the different concentrations of sodium azide ranges from 0-16 mM and activity of peroxidase obtained from juice of B. vulgaris was completely diminished at 16 mM.

3. Results and Discussion

The rapid increase of absorbance at λ = 470 nm, ΔA₄₇₀ is due to the conversion of guaiacol to tetraguaiacol with time in a peroxidase assay solution extracted from B. vulgaris. There is no increase in absorbance at λ = 470 nm in assay solutions containing no enzyme or the denatured enzyme which was obtained by one hour boiling in water. These results indicated that B. vulgaris juice contained a good peroxidase activity.

Figure 1. (a) Michaelis-Menten and (b) double-reciprocal plots for the peroxidase obtained from the B. vulgaris (beet) juice using guaiacol as the variable substrate

Figure 2. (a) Michaelis-Menten and (b) double-reciprocal plots for the peroxidase obtained from the juice of B. vulgaris (beet) using hydrogen peroxide as the variable substrate

Figure 3. Dependence of the peroxidase activity on pH of the assay solution at fixed temperature 30°C

The Michaelis-Menten and double reciprocal plots for the peroxidase using guaiacol as the variable substrate at the saturating concentration of the other substrate, H₂O2 (0.6 mM) are shown in Figure 1(a) and Figure 1(b), respectively. The Michaelis-Menten and double reciprocal plots using hydrogen peroxide as the variable substrate at the fixed enzyme saturating concentration of guaiacol (5 mM) are shown in Figure 2(a) and Figure 2(b), respectively. The calculated Km values for guaiacol and hydrogen peroxide of peroxidase were 900 μM and 30 μM, respectively. The corresponding values of Km for horseradish peroxidase [22], Turkish black radish [23] and Solanum melongena fruit juice [21] were 800 μM and 100 μM, 36.0 μM and 8.4 μM, and 6500 μM and 330 μM, respectively. The reported enzyme has lower affinity for both the substrates than the peroxidases of Turkish black radish (Raphanus sativus) while has lower affinity for guaiacol and higher affinity for hydrogen peroxide than horseradish peroxidise [22]. Peroxidase obtained from the fruit juice of B. vulgaris has higher affinity for both the substrates as compared to the peroxidases of S. melongena fruit juice. Since peroxidases are known to follow double displacement type kinetics [24], B. vulgaris juice peroxidase was also analysed for this type of kinetics by measuring the steady state velocity of the enzyme-catalyzed reaction at three different fixed concentrations of the hydrogen peroxide and varying the concentration of guaiacol at each hydrogen peroxide concentration and also at three different fixed concentrations of guaiacol and varying the concentration of hydrogen peroxide. Double reciprocal plots (not shown here) in both cases have been found to be parallel straight lines confirming that the reported peroxidase also follows double displacement type mechanism observed in case of other peroxidases [24].

The results of the variation of the activity of the this peroxidase enzyme with the variation of pH of the reaction solution are shown in Figure 3. The determined pH optimum of the enzyme was 6.0. Plant peroxidases of S. melongena fruit juice [21], Musa paradisiaca stem juice [25] and peroxidase of fruit juice of L. aegyptiaca [26] have been studied in our laboratory. The pH optima of the peroxidases from these sources have been found to be 5.5, 4.5 and 6.5 pHs, respectively. Thus, the reported peroxidase can be used effectively near neutral pH. The first two peroxidases of three, have pH optima in more acidic regions while third one has pH optimum near neutral pH as peroxidase obtained from the fruit juice of B. vulgaris i.e. pH 6.0. pH optimum for the peroxidase present in the juice of B. vulgaris was 6.0 that means it is most active at pH 6.0. When this enzyme was exposed for different time intervals (10 min.) upto one hour at different pH then it was found to be most stable at pH 6.0 (Figure 4).

Figure 4. Stabilities of peroxidise at different pH when exposed for one hour at 10 minutes time intervals. (pH 4.0 (o),, 5.0 (◊), 6.0 (∆), 7.0 (□), 8.0 (♦))

Figure 5. Dependence of the peroxidase activity on temperature of the assay solution at fixed pH 7.0

The results of variation of the activity of peroxidase enzyme obtained from the juice of B. vulgaris, as a result of variation of the temperature of the reaction solution of the enzyme catalyzed reaction are shown in Figure 5. The calculated temperature optimum was 60°C. The temperature optima of the peroxidases from S. melongena fruit juice [21], M. paradisiacal stem juice [25] and fruit juice of L. aegyptiaca [26], peroxidases reported from our laboratory, were 84°C, 62.5°C and 60°C respectively. In this way, we see that the peroxidase from B. vulgaris juice has the temperature optimum toward the higher temperature side as like S. melongena fruit juice, M. paradisiacal stem juice and L. aegyptiaca fruit juice. When this enzyme was exposed for different time intervals (10 min.) for one hour at different temperatures then it was found to be most stable at 45°C. The result of the studies on the thermal stability of the peroxidase is shown in Figure 6. Activation energy of the thermal denaturation of the peroxidase calculated from Arrhenius plot was found to be 38.6 kJ/mole/K.

Figure 6. Thermal denaturation of the enzyme at different temperatures. (45 ⁰C (□), 50 ⁰C (∆) and 55 ⁰C (o))

Figure 7. Inhibition of the activity of the peroxidase obtained from the fruit juice of B. vulgaris (beet) by sodium azide

Studies on inhibition of the peroxidase has also been performed for various concentrations of sodium azide started from 0.0 mM to 16 mM. Activity of this peroxidase has been found to be completely diminished at 16 mM. It is shown in Figure 7.

Peroxidases are the active biocatalysts which catalyses the several types of organic conversions like epoxidation, halogenation of oleifins, oxygenation of heteroatoms, several enantioselective organic reactions and oxygenation of racemic hydroperoxide, etc. Due to the significant scope of peroxidases, the peroxidase enzyme obtained from the juice of B. vulgaris may be effective because peroxidase obtained from this source is very active for hydrogen peroxide, especially, and does not lose activity at room temperature for several hours.

4. Conclusions

This communication demonstrates mainly the kinetic, thermal and pH properties. Activation energy for this peroxidase has also been calculated. It is clear from the Km values determined for different substrates as guaiacol and hydrogen peroxide, clearly explain the fact that this peroxidase can be used as potential biocatalyst for various biotransformations and organic synthetic reactions using hydrogen peroxide as substrate in comparison to guaiacol.

Acknowledgement

Pankaj Kumar Chaurasia acknowledges the financial support to CSIR-HRDG, New Delhi, in the form of CSIR-SRF (NET), award no. 09/057(0201)/2010/EMR-I, under which this research work has been done. S.K. Singh is also thankful to CSIR New Delhi for the award of RA, award no. 09/057(0206)-2012/EMR-I. Authors are also thankful to Prof. K.D.S. Yadav, Emeritus Scientist, UGC, New Delhi for his valuable suggestions.

References

[1]   | Banci, L., Structural properties of peroxidises, J. Biotechnol. 53(2-3), 253-263 (1997).
[2]   | Yemenicioglu, A, O¨zkan M, Cemeroglu B. Partial purification and thermal characterization of peroxidase from okra (Hibiscus esculentum). J. Agric. Food Chem. 46(10). 4158-4163. 1998.
[3]   | Rodriguez-Lopez, J.N., Smith, A.T., Thorneley, R.N.F., Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding. J. Biolog. Inorg. Chem. 1(2). 136-142. 1996.
[4]   | Chen, E.L., Chen, Y.A., Chen, L.M., Liu, Z.H., Effect of copper on peroxidase activity and lignin content in Raphanus sativus. Plant Physiol. Biochem., 40(5), 439-444, (2002).
[5]   | Quiroga, M., Guerrero, C., Botella, M.A., et al. A tomato peroxidase involved in the synthesis of lignin and suberin. Plant Physiol. 122(4). 1119-1127. 2000.
[6]   | Bernards, M.A., Fleming,, W.D., Llewellyn, D.B., et al. Biochemical characterization of the suberization-associated anionic peroxidase of potato. Plant Physiol. 121(1). 135-145. 1999.
[7]   | Lee, D.J., Kim, S.S., Kim, S.S., The regulation of Korean radish cationic peroxidase promoter by a low ratio of cytokinin to auxin. Plant Sci., 162(3). 345-353. 2002.
[8]   | Lagrimini, L.M., Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidise. Plant Physiol., 96(2). 577-583. 1991.
[9]   | Schnabelrauch, L.S., Kieliszewski, M., Upham, B.L., Alizedeh, H., Lamport, D.T.A., Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular cross-link site. Plant Journal, 9(4). 477-489. 1996.
[10]   | Sakharov, I.Y., Castillo, J.L., Areza, J.C., Galaev, I.Y., Purification and stability of peroxidase of African oil palm Elaies guineensis, Bioseparation, 9(3). 125-132. 2000.
[11]   | G¨ulcin, I., Yildirim, A., Purification and characterization of peroxidase from Brassica oleracea var. Acephala, Asian J. Chem., 17(4). 2175-2183. 2005.
[12]   | S¸at I.G., The effect of heavy metals on peroxidase from Jerusalem artichoke (Helianthus tuberosus L.) tubers, African J. Biotechnol., 7(13). 2248-2253. (2008).
[13]   | Adam W., Lazarus M., Saha-M¨oller C.R., et al. Biotransformations with peroxidases, Adv. Biochem. Eng. Biotechnol., 63. 73-108. 1999.
[14]   | Akasaka, R., Mashino, T., Hirobe, M., Hydroxylation of benzene by horseradish peroxidase and immobilized horseradish peroxide in an organic solvent. Bioorg. Med. Chem. Lett. 5(16). 1861-1864. 1995.
[15]   | Jacobsen, E.N., Asymmetric catalytic epoxidation of unfunctionalized olefins, in (Ed. Ojima I) Catalytic Asymmetric Synthesis, p.159, VCH, Weinheim, Germany, 1993.
[16]   | Hofrichter, M., Ullrich, R., Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance. Appl. Microbiol. Biotechnol. 71(3). 276-288. 2006.
[17]   | Yadav, P., Sharma, J.K., Singh, V.K., Yadav, K.D.S. Noxidation of arylamines to nitrosobenzenes using chloroperoxidase purified from Musa paradisiaca stem juice. Biocat. Biotrans., 28(3). 222-226. 2010.
[18]   | Colonna, S., Gaggereo, N., Carrea, G., Pasta, P., The microperoxidase-11 catalysed oxidation of sulfides is enantioselective, Tetrahedron Lett., 3548. 9103-9104. 1994.
[19]   | Sundaramoorthy, M., Kishi, K., Gold, M.H., Poulos, T.L., The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06 °A resolution, J. Biol. Chem., 269(52). 32759-32767. 1994.
[20]   | Whitaker, J.R., Principles of Enzymology for Food Sciences, Marcel Dekker, New York, NY, USA, 1972.
[21]   | Vernwal, S.K., Yadav, R.S.S., Yadav, K.D.S., Musa paradisiaca stem juice as a source of peroxidase and lignin peroxidase Indian J. Exp. Biol. 38(10). 1036-1040. 2000.
[22]   | Rodriguez-Lopez, J.N., Smith, A.T., Thorneley, R.N.F., Role of arginine 38 in horseradish peroxidase: a critical residue for substrate binding and catalysis, J. Biol. Chem. 271(8). 4023-4030. 1996.
[23]   | Sisecioglu, M, G¨ulcin I, Cankaya, M, et al. Purification and characterization of peroxidase from turkish black radish (Raphanus sativus L.), J. Med. Plants Res., 4(12). 1187-1196. 2010.
[24]   | Tien, M., Kirk, T.K., Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization and catalytic properties of a unique H₂O₂-requiring oxygenase, Proc. Nat. Acad. Sci. of the United States of America, 81(8). 2280-2284. 1984.
[25]   | Vernwal, S.K., Yadav, R.S.S., Yadav, K.D.S., Musa paradisiacal stem juice as a source of peroxidase and ligninperoxidase, Indian J. Exp. Biol., 38(10). 1036-1040. 2000.
[26]   | Yadav, R.S.S., Yadav, K.S., Yadav, H.S., Luffa aegyptiaca (Gourd) fruit juice as a source of peroxidise, Enz. Res. 2011.