INTRODUCTION:

Fruit browning, as a consequence of bruising, is due to phenolic oxidation. Reaction usually degrades the sensory properties of products because of the associated changes in color, flavour and hardness. These changes shorten the products’ shelf lives and decrease their nutritional and market values of fruits and vegetables. Due to the importance of visual appearance as a produce cosmetic quality parameter, tissue browning has long gained attention from horticultural researchers and in the fields of plant physiology and food science.[1] Enzymatic browning is caused mainly by the activity of Tyrosinase (EC 1.14.18.1; PPO) in the presence of oxygen. It is copper containing, mixed function oxidase, ubiquitous in nature; catalyzes the production of melanin via separate active sites: (i) o- hydroxylation of monophenols and (ii) oxidation of o-diphenols to o-quinones. Tyrosinase acts as a precursor of melanin/browning reactions and possesses unusual kinetic behavior. This behavior of tyrosinase activity have been carried out by several researchers [2].The potential of enzyme is used for the detection of phenols in aqueous and organic media in removal of toxic chemicals including phenols[3,4] and aromatic amines from industrial waste water and drinking water [5] ,in organic synthesis because of its specific regioselectivity [6].

Versatility of it has fascinated to encapsulate activity to minimize the purification cost and time. Therefore attempts were made to immobilize onion leaves tyrosinase on agar: Abelmoschus esculentus composite matrix (3:1). Entrapment and covalent attachment slightly affect the catalytic ability of the enzyme based on the comparisons of Michaelis constant (Km) but provides higher thermal stability and reusability to enzymes. [7, 8, 9]. Catalytic efficacy enables to have broad idea about the performance of the enzyme. Hence, the catalytic properties such as Km, Vmax, pH optima and optimum temperature, activation energy and storage stability of soluble and immobilized onion leaves tyrosinase were studied.

MATERIALS AND METHODS:

Materials:

Fresh onion leaves (Allium cepa) for tyrosinase and Abelmoschus esculentus for isolation of gum for immobilization as source material was purchased from local market, Thane. Agar was obtained from Hi-media, India; L-tyrosine was obtained from S.D. Fine, India. Other reagents used were of analytical grade.

A Shimadzu UV-1601 model spectrophotometer was used for the activity determination of both soluble and immobilized enzyme.

Methods:

1. Enzyme extraction by solvent and salt method:

Fresh onion leaves (300 g) were homogenized with 150 ml of ice-cold acetone, water with Citron X-100 (80:19:1, V/V) for about 24 hrs (-18°C).After filtration through a muslin cloth the residue was washed 3x with 150 ml of acetone (-18°C). The resulting acetone powder was dried for about 30 min. at room temperature and was suspended in 200 ml of 0.05 M phosphate buffer (pH 7.0), containing 0.01mM EDTA, and 0.1 mM ascorbic acid.Sonicated for 1 hr. and then centrifuged at 5600 g for 15 mins. Solid ammonium sulfate was added to 60 % molar saturation, the solution was stirred for about 3 hrs and kept it for about 24 hr. at 4°C, centrifuged at 5600 g for 30 mins. The resulting pellet was homogenized in 50 mM sodium phosphate buffer (pH 7.0) and dialyzed for about 20 hrs against the same buffer at 4°C. The dialysate was used as active enzyme. Tyrosinase activity and protein were measured at each stage.

2. Immobilization of onion leaves tyrosinase in Abelmoschus esculentus gum and agar

Dialysate tyrosinase was entrapped in agar- Abelmoschus esculentus gum composite by method described by Tembe [10] with slight modification. Total volume of 10 ml was prepared containing 1% agar, 3% Abelmoschus esculentus gum and 1 ml of enzyme solution (4000 IU). The mixture was pour into Petri dish allowed to solidify and incubated overnight at 4⁰C. The immobilized enzyme was tested in repeated batches in order to simulate recovery and reuse.

3. IR analysis of agar: Abelmoschus esculentus gum and tyrosinase coupled agar: Abelmoschus esculentus gum:

IR analysis of agar: Abelmoschus esculentus gum and tyrosinase coupled agar: Abelmoschus esculentus gum were carried out to retain its activity.

4. Assay for tyrosinase activity and protein estimation:

The activity of the soluble and immobilized enzyme was assayed by using method of Kahn et.al. 1985 [11]. Immobilized enzyme bead was added to the assay solution. The mixture was briefly but rapidly shaken and monitored to observe increase in the absorbance. Protein in the filtrates and dialysate were estimated by employing Lowry [12] method.

5. Determination of Km and Vmax:

The reaction was carried out by the assay as noted above containing various concentration of substrate L-tyrosine. The K_m and V_max values of for soluble and immobilized tyrosinase were obtained from Line weaver-Burk plot.

6. Optimum pH and temperature:

pH( 5.7 to 8.0) and temperature optima(10^oC to 90^oC) were determined by measuring immobilized tyrosinase activity at particular range using 50 mM of phosphate buffer and compared with soluble enzyme.

Activation energy of PPO: At a more advanced level, the Arrhenius Activation energy term from the Arrhenius equation is best regarded as an experimentally determined parameter which indicates the sensitivity of the reaction rate to temperature. Activation energy may also be defined as the minimum energy required for initiating a chemical reaction. The activation energy of a reaction is usually denoted by E_a, and given in units of kilojoules per mole. The Arrhenius equation relates the activation energy Ea to the rate constant K of a process.

K = A e ^–E_a^{/ RT---------------------- [1]}

e – Log to the base 10 (2.3)

Ea – activation energy of the reaction (J mol)

R – molar gas constant (8.3143 JK^-1mol^-1)

T – Absolute temperature (K)

The values Ea and A for the reaction was calculated from the equation:

Ea = 2.3 R X T₁ X T₂ log K₁/K_{2------------ [2]}

T₁- T₂

7. Storage Stability:

Preparations of soluble and immobilized tyrosinase were stored at 4^oC and assayed for the activity periodically.

RESULTS AND DISCUSSIONS:

Behaviors of immobilized tyrosinase activity in agar- Abelmoschus esculentus gum composite were determined using partially purified enzyme and compared with soluble enzyme (table1). IR of soluble tyrosinase and tyrosinase coupled agar: Abelmoschus esculentus gum spectral studies confirm the coupling and retaining activity of the onion leaves PPO.

Table 1: Comparison of catalytic properties of soluble and immobilized tyrosinase.

Parameters	Soluble tyrosinase	Immobilized tyrosinase
V max (µmol/lit/min)	167	90
Km (µM)	5 X 10 ^-3	8.33 X 10 ^-3
Activation Energy (Kcal/mole)	108.35	78.80
Optimum temperature(^oC)	50	60
Optimum pH	7.2	7.0
Storage stability	65%	75- 80%

IR of free agar: Abelmoschus esculentus gum: 3426.534 cm^-1(Primary amine stretch), 2926.509 cm^-1 (aldehyde stretch), 1637.842 cm^-1(-NH stretch), 1456.23 cm^-1, 1087.407 cm^-1.

IR of tyrosinase coupled agar: Abelmoschus esculentus gum: 3414.224 cm^-1(Primary amine stretch), 2945.535 cm^-1(-OH stretch), 1712.175 cm^-1 (-OH stretch), 1651.986 cm^-1(C=C stretch), 1443.599 cm^-1, 1377.989cm^-1,1257.251cm^-1,1114.936cm^-1(-NH stretch),1024.946cm^-1(-NH stretch), 952.955 cm^-1(C=C out of plane bend) ,895.055 cm^-1(Aromatic out of plane bend),857.874 cm^-1(Aromatic out of plane bend),636.899 cm^-1(Aromatic out of plane bend).

Absence of 1712.175cm^-1 band in free agar: Abelmoschus esculentus gum shows that enzyme amino group forms Schiff’s base. In addition, a new band at 1651.986 cm^-1 can be assigned to amide I band of the enzyme.

Determination of K_m and V_max: According to figure 1, Linearity curve of L-tyrosine oxidation reaction catalyzed by soluble and immobilized tyrosinase. It holds a linear relationship at least up to 187µg of protein which remains steady up to 218µg of protein. Respective optimum concentration for soluble and immobilized tyrosinase was 700 µL and 800 µL. The initial rate of enzyme catalyzed reactions responded proportionally to the enzyme concentration suggesting that the reactions were not interfered with the endogenous phenolic substances present in the partially purified enzyme equally in soluble and immobilized tyrosinase. [13]

Substrate concentration is produced depth knowledge and characteristic of velocity of the enzyme reaction. Line weaver and Burk plot, (1/ ν Vs 1/s) suggest that initial velocity related to the rate of breakdown of enzyme substrate complex indicating a straight line that was corresponded to the equation (Figure 2A. and B.) and this straight line cuts the base line at a point giving -1/K_m.

1 = K_m . 1 + 1

ν V S V [3]

This is easily shown by putting 1/ ν = 0 in the equation 3 which gives 1/s = 1/-K_m.The graph cuts the vertical axis at a point which gives 1/V and has a slope = K_m/V.

Based on the figure 2, a saturation curve illustrates the relation between the substrate concentration [S] and velocity [V]. K_m and V_max for soluble (Km 5X10^-3VmaxµM167µm/lit/min) and immobilized tyrosinase (8.33X10^-3 V_max µM 90µm/lit/min) were calculated at lower substrate concentrations. The correlation coefficient value ( r2) for soluble and immobilized tyrosinase was 0.8921 and 0.9891; indicates r2 is venerable fit between the data points and the regression line further states that increases 1/S increases 1/V_o. Immobilized enzyme gives lesser V_max value than soluble enzyme. Immobilization of enzyme, procured decline in the rate of reaction was not only affects the rate of product formation, but difficulty of enzyme- substrate interaction and also in diffusion of substrate to the matrix as compared to diffusion in solution. Km is inversely proportional to the affinity of the enzyme to substrate. Probably it may be due to structural deformation of the enzyme upon entrapment in the matrix. Elevations of Km values observed upon the immobilization of tyrosinase indicate that the components are loosely bound and form the ES complex more gradually as compared to soluble enzyme.

Optimum pH:

Respective optimum pH (7.2) and (7.0) for soluble and immobilized tyrosinase was observed according to figure 3A, that are in neutral range. At pH < 6 the activity of the immobilized tyrosinase was less sensitive than soluble form and also changes in pH. Signifying a better resistance of the immobilized protein molecules to the ionization, since the ionic state of the functional groups in or close to the active center has a great effect on its activity. It might be due to the partitioning of protons which are entrapped in matrix protecting the enzyme against the high concentration of OH^-ions. This protection makes the pH value around the enzyme is lower than that of the bulk. This negative charge shell attracts H⁺ ions around itself. Those H⁺ ions protect the enzyme immobilized in the matrix from the high concentration of OH⁻ions and give an extra stability to the enzyme in neutral to alkaline range. This alkaline range stability of the immobilized enzyme, which is observed for tyrosinase can be explained by the buffering effect of the dopant ion. [14] The dopant ion used in the electro polymerization penetrates into the matrix as the polymer is oxidized. Resulting polymer has positive charges on its own and can be assumed that it has a negative charge shell around it. This negative charge shell attracts H+ ions around itself.

Figure 1 Linearity curve of L-tyrosine oxidation reaction catalyzed by soluble and immobilized tyrosinase

Optimum temperature:

The temperature optima were 50^oC for soluble tyrosinase and which shifted to 60^oC after immobilization. Soluble tyrosinase lost its activity at 70°C completely however, immobilized tyrosinase lost only 40% of its activity at that temperature. These results indicate that immobilization preserved the enzyme structure from thermal inactivation. Tyrosinase from banana and tomato had a temperature optima 30^oC [15] and 40^oC [16]. Consequently, concluded that onion leaves tyrosinase is more thermophilic, elevated after immobilization.

According to the Arrhenius equation noted earlier equation1, activation energy (Ea) for soluble and immobilized tyrosinase is 108.35 Kcal/mole and 78.80 Kcal/mole. However the plot of natural logarithm of the initial reaction rates against 1/T, where T is the absolute reaction temperature shows biphasic thermal behavior evaluating that the presence of denatured protein forms and groups of tyrosinase molecule that remain active during heating (Figure 3 B). [17]

Figure 2 A. Effect of substrate (tyrosine) on soluble and immobilized tyrosinase B. Lineweaver-Burk plot for soluble and immobilized tyrosinase

Figure 3 Optimum A. pH and B. temperature for soluble and tyrosinase

Figure 4 Storage stability of tyrosinase at 4^oC for 120 days

Storage stability: Immobilized tyrosinase was more stable than soluble enzyme and its storage stability for 120 days when it was stored in phosphate buffer (50 mM, pH 7.0) at 4^oC Figure-4. About 75- 80% in case of immobilized and 65% in case soluble tyrosinase activity was retained during the storage up to 4 months. Since immobilized tyrosinase maintained 90% of its seed activity after 30th days and constant up to 40th day, it can also be used up to 2 months with a lower activity.

CONCLUSION:

Enzyme reusability provides integer of cost advantages, which are often an essential prerequisite for establishing an economically viable enzyme-catalyzed process. Therefore partially purified onion leaves tyrosinase; most potential enzyme/ a precursor of browning reaction were immobilized on agar: Abelmoschus esculentus composite matrix. Behavioral of it was investigated and compared with soluble tyrosinase. The immobilized onion leaves tyrosinase exhibited a broadening in temperature- optimum from 50^oC to 60^oC along with long storage stability. Thus, the use of cheaper source of enzyme and support can be beneficial for the reuse of tyrosinase activity. Matrix is easy to use and convenient for any kinds of enzymes because support is non-toxic, biocompatible, good film forming and adhesion ability and it provides natural ambient to enzyme. It may be utilized alternate source for entrapment of any kinds of enzymes

ACKNOWLEDGMENT:

We greatly acknowledge University Grants commission, New Delhi India for financial assistance through Major research project No.34-284(2008).

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Received on 26.03.2012 Modified on 16.04.2012

Asian J. Research Chem. 5(5): May 2012; Page 663-667