Introduction

Peroxidase is an oxidative enzyme which is involved in defence reactions in plants [1]. Peroxidases reduce hydrogen peroxide and oxidize a wide number of compounds including phenols, aromatic amines, thioanisoles, halide ions, thiocyanate ions, fatty acids and also degrade hydrogen peroxide [2]. Plant peroxidases are induced by stress for example, pathogen attack, wounding, heat, cold or draught and UV light [3]. Products from these enzymes are also commonly used in pharmaceutical industry, waste water treatment, bio-sensor construction and food industry [4–7]. They also serve many purposes, including lignifications, suberization, wound healing, protection against pathogen attack and the scavenging of damaging hydrogen peroxide from the cell [8]. The characterisation of these enzymes may be of interest, not only for their negative effects on colour, flavour and nutritional values, but also for their positive effects on food [9, 10]. Peroxidase is widely used in health sciences, food industry and for diagnostic purposes [11]. Peroxidase isolated from soyabean was involved in the defence reaction of plant against pathogen attack [12]. Being the most heat resistant enzyme, it is used in food industry as an index in blanching procedures [13].

Peroxidases have received extensive attention in the recent years as biocatalysts for synthetic applications in biotransformation [14–17]. In plant kingdom, they are omnipresent, catalyzing a reaction in which the acceptor is hydrogen peroxide and the hydrogen donor is another compound [18]. Their industrial application has been demonstrated by several authors. Some of them perform peroxide removal from industrial wastes [19]. They are used in ELISA system [20, 21] and waste decolourization [22] and waste water treatment containing phenolic compounds [23, 24]. India is currently the foremost producer, consumer and exporter of commercial tea. The state of Assam is the world’s largest tea growing region.

According to the pre-existing literature, the characterisation of peroxidase enzyme of tea was very limited. Considering this fact in mind, the present research is carried out to characterize the use of the enzyme for industrial purposes. This study optimizes peroxidase enzyme from tea leaves as this will explore a new idea about its use for industrial and agricultural purposes.

Material and Methods

Sample Collection

Tea leaves (Fig. 1) were collected from 5 leading tea gardens, Ethelwood, Moran, Lepetkotta, Borborooah and Anandabag tea estates located in Dibrugarh district, Assam, India (Fig. 2).

Fig. 1
figure 1

Leaves of Camellia sinensis for experimental analysis

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Fig. 2
figure 2

Location of Dibrugarh district in India as well as the location of the five tea estates from where the samples were collected

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Extraction of Peroxidase

1 g of fresh green tea leaves were washed thoroughly with deionised water at room temperature and were homogenised in 4.5 mL of 0.1 M phosphate buffer of pH 6.5 for 15 min [25]. The homogenate was filtered using cheese cloth to remove suspended particles. The clear filtrate was centrifuged at 10,000 rpm for 20 min at 4 °C (Sigma 3–30 K, Germany). The collected supernatant was stored at 4 °C.

Enzyme Assay

The enzyme assay was carried out by taking 0.1 mL of above extract in 0.1 mL o-dianisidine (Conc. 1 mg/mL) maintained at 28 °C for 2 min (Sartorius Stedim biotech, Certomat BS-1, Germany) to carry out the reaction [25]. The reaction was stopped by adding 0.2 mL of H2O2 (30 %). The oxygen liberated reacts with o-dianisidine to give a red chromophore, which can be measured at 430 nm. The reaction is shown as follows:

$$ {\text{H}}_{ 2} {\text{O}}_{ 2} \to {\text{H}}_{ 2} {\text{O }} + [{\text{O}}] $$
$$ o{\text{-dianisidine}} + [{\text{O}}] \to {\text{ oxidised }}o{\text{-dianisidine }}\left( {\text{coloured}} \right) $$

The change in absorbance at 430 nm was recorded for every 30 s for 5 min during the reaction by UV–Vis Spectrophotometer (TCC-240A, Shimadzu corporation, Kyoto, Japan) (Fig. 3).

Fig. 3
figure 3

Peroxidase activity of leaves of Camellia sinensis collected from different tea estates (1 Ethelwood, 2 Borborooah, 3 Lepetkotta, 4 Moran, 5 Anandabag). The data is presented as mean ± SD

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The specific activity of the enzyme was expressed in terms of units/mg of protein/mL of reaction mixture according to the following equation:

$$ {\text{Specific activity }} = \frac{\text{enzyme\,activity}}{{{\text{protein content}} \left( {\frac{\text{mg}}{\text{mL}}} \right)}} $$

Characterisation of Peroxidase Enzyme

The thermal stability of the enzyme was assayed spectrophotometrically (at 430 nm) by incubating the reaction mixture for 25–30 min at the temperature ranging from 10, 20,…,80 °C against suitable blank. The effect of pH was monitored spectrophotometrically by carrying out the reaction at pH 5, 5.5, 6,…,9. The effect of substrate concentration was determined by carrying out the reaction at a substrate (o-dianisidine) concentration ranging from 0.002, 0.006, 0.010,…,0.050 mg/mL at an interval of 30 s for 5 min.

The effect of inhibitor was studied by assaying the reaction by adding 100 μL of 0.1, 0.4, 0.8,…,13 mM EDTA (ethylenediaminetetraacetic acid) solution for the above mentioned duration.

Effect of salt concentration on the isolated enzyme was determined by carrying out the reaction by adding 100 μL of 0.5, 1.0,…,8.0 % (w/v) of NaCl.

Kinetic Constants

Apparent km and Vmax was determined by Lineweaver–Burk equation taking o-dianisidine as substrate. The lower or higher km value indicates higher or lower affinity of tea (Camellia sinensis) leaf peroxidase for substrate o-dianisidine.

All the chemicals used in the study were procured from Merck India Pvt. Ltd. All the tests were performed in triplicate.

Statistical Analysis

The values reported are the mean of at least three independent determinants. The significance of the study was checked by performing t test and f test.

Results and Discussion

Peroxidase was extracted and characterized from tea leaves collected from 5 leading tea estates located in Dibrugarh district of Assam, India. The optimum temperature for the enzyme activity was 30 °C, but the activity decreased significantly after 60 °C (Fig. 4). The optimum pH was 6.5 for the above reaction but the reaction rate significantly decreased after pH 7.5 (Fig. 5). Optimum substrate concentration for the reaction mixture was 0.044 mg/mL. The enzyme activity decreased after adding 4 % NaCl (Fig. 6). The reaction rate decreased gradually after adding 10–13 mM EDTA as inhibitor (Fig. 7).

Fig. 4
figure 4

Change in the activity of enzyme peroxidase in the leaves of Camellia sinensis pooled from above mentioned five different tea estates of Dibrugarh district at different temperature. The data is presented as mean ± SD

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Fig. 5
figure 5

Change in the activity of enzyme peroxidase in the leaves of Camellia sinensis at different pH. The data is presented as mean ± SD

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Fig. 6
figure 6

Change in the activity of enzyme peroxidase in the leaves of Camellia sinensis at different NaCl concentration. The data is presented as mean ± SD

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Fig. 7
figure 7

Change in the activity of enzyme peroxidase in the leaves of Camellia sinensis at different EDTA concentration. The data is presented as mean ± SD

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The enzyme kinetics was determined with the help of Lineweaver–Burk equation (Fig. 8). Km value was 0.01 mg/mL (0.04 mM) and Vmax value was 0.03. The enzyme specific activity was 6.197 U/mg of protein.

Fig. 8
figure 8

Lineweaver–Burk plot for the determination of km and Vmax (where, 1/Vmax = 28.5 or Vmax = 0.03, 1/km = 55 or km = 0.01, Vmax/km = 0.03/0.01 = 3)

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The temperature effect on enzyme activity was studied at 10–90 °C. The optimum activity in terms of optical density was at 30 °C but its decreasing trend was not so pronounced up to 50 °C. The fall was much sharp from 60 °C onwards. The result was similar to that of Burnette [26] who reported peroxidases to be heat stable.

pH is the key factor for enzyme activity as it changes ionisation state of protein and substrate [27–29]. In the present study, the optimum pH was 6.5. The result was in accordance with the findings of Halpin et al. [30], Civello et al. [31] and Rehman et al. [32] who found optimum pH for different vegetables to be between 6 and 6.5.

The optimum activity was found at 0.6 M salt concentration. This activity was almost at par (80 % activity) with the activity in the absence of salt indicating that this amount does not hamper the enzyme activity. This finding was in accordance with the peroxidase activity of radish [33].

The Km value of H2O2 using guaiacol as reducing substrate was 0.9 mM for peroxidase II [34]. The significance of low Km for H2O2 reflects higher hydrophobic interactions between substrate and the heme group at the enzyme active site [35]. In the current study, the Km of o-dianisidine was 0.04 mM for peroxidase enzyme, which also indicated higher interaction between substrate and enzyme active site as reported with turnip peroxidase isoenzyme Cl [36].

EDTA has moderate inhibitory effect on peroxidase [37]. EDTA inhibits the enzyme activity by reacting with the peroxidase active center [38]. In the present study, the peroxidase activity was inhibited by EDTA from 10 mM and was quite prominently decreased at 13 mM.

Conclusion

Plant enzymes such as peroxidases are widely used in medicine as diagnostic tools and in the bioremediation and biobleaching industries among others [39]. Now a days, hazardous chemicals viz; formaldehyde sulphoxylate has been replaced with eco-friendly horseradish peroxidase enzyme in textile discharge printing. Advantages such as elimination of formaldehyde, energy saving, environmental friendly and reduction of strength loss have been observed by using the peroxidase enzyme discharge printing method [40]. Horseradish peroxidase immobilised on colloidal gold modified screen-printed carbon electrode displayed fast amperometric response and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the help of an electron mediator. The biosensor exhibited high sensitivity, good reproducibility and long term stability for the determination of H2O2 [41]. Recently, peroxidases from bitter gourd (M. charantia) immobilised on some cheaper supports have been found highly effective in decolourising reactive textile dyes as compared to its soluble counterpart as the immobilised enzyme looses only 50 % activity even after 10 cycles of usage [42].

Peroxidase enzyme has attained a crucial position in biotechnology as well as in industrial applications. Knowledge on the characterisation of enzyme is therefore very essential to know its stability in temperature, pH, substrate concentration, salt concentration, inhibitors, enzyme kinetics and enzyme specific activity so that such enzyme can be used for the industrial purposes from readily available sources present in particular area such as the case of tea plants in Assam. This will help reduce people dependency on a particular plant for industrial purposes and help in future to explore more from it.