Application of Papain from Paw Paw (Carica papaya) - Unn

MITTEILUNGEN KLOSTERNEUBURG 64(2014) XX-XX www.mitt-klosterneuburg.com

Application of Papain from Paw Paw (Carica papaya) latex in the Hydrolysis of Tiger Nut (C.esculentus) Proteins By Kingsley Ozioma OMEJE1, *Sabinus Oscar O. EZE1., Vincent E.O. OZOUGWU1., Chike S. UBANI1., Emeka OSAYI2, Christopher C. ONYEKE2. and Ferdinand C. CHILAKA1 1 2

1

Department of Biochemistry, University of Nigeria Nsukka

Department of Plant Sciences and Biotechnology, University of Nigeria Nsukka

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Abstract Enzymatic hydrolysis of tiger nut protein homogenate offers many advantages as compared to the use of strong chemical reagents such as O-phthaldialdehyde (OPA) which destroys some amino acids present. Papain was purified to 6.82 purification folds with an activity yield of 531U/min by three steps purification processes of ammonium sulphate precipitation, followed by gel filtration on sephadex G50 and G200. The pH and optimum temperature of the enzyme was determined and found to be pH 7.5 and 90ºC respectively. The enzyme activity of crude enzyme and purified enzyme was 179U/min and 531U/min with Vmax and Km values of 1.133μmole/min and 0.487µg/ml respectively. Tiger nut protein homogenate was hydrolysed using the papain obtained from C. papaya latex and compared with that hydrolysed by O-phthaldialdehyde (OPA) after 10mins incubation. Equally, it was observed from the result of this study that papain hydrolysed the protein more than O-phthaldialdehyde (OPA). Hence, the optimum incubation time of 10mins was obtained at 37 oC and pH 7.5 for all concentrations analysed. This protein hydrolysate could be used in preparing feeds for both livestock and human consumption. Keyword;

2

Papain,

C.

papaya,

Hydrolysis,

Tigernut,

protein

homogenate

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Introduction Milk is an excellent source of all nutrients and has been recognized as an important food for infants and growing children (Obizoba and Anyika, 1995; Ukwuru et al, 2011), yet the cost of dairy milk and their products are prohibitive in Nigeria and other developing countries which decreases their consumption (Kerven, 1986). Similarly, some individuals suffer intolerance of lactose and other dairy milk components such as casein and gluten. Development of milk substitutes extracted from legumes, grains and nuts serve as an alternative source of producing acceptable nutritious milk (Harkins, and Sarret, 1967). Tiger nut milk, a phyto milk is less expensive and possesses no intolerance, hence, serves as a substitute for dairy milk (Iwuoha and Umunnakwe, 1997). In view of the scarcity of dairy milk supply in Nigeria and the ever increasing gap between its requirement and population, efforts are being made to develop alternative milk-like beverage with enriched amino acids from vegetable sources using a plant protease (Papain) extracted from C. Papaya latex. Papain (EC 3.4. 22.2) is an endolytic plant cysteine protease which is isolated from papaya latex (Amri and Mamboya, 2012). The latex of C. papaya is also a source of other cysteine endopeptidases, such as glycyl endopeptidase, chymopapain and carican (Azarkan et al, 2003).Papain has been applied in different biotechnology processes such as surfactants of toiletry, personal cares, soaps, shower gel, food industries (Uhlig, 1998), in the manufacture of protein hydrolysate, confectionary industry, brewing industry, dairy industry, textile and tanning industries (Uhlig, 1998; Chaplins, 2002). Researchers have hydrolysed protein with acid but this destroyed certain amino acids such as tryptophan, asparagine and glutamine (Nielsen et al., 2001); In recent times, enzymatic hydrolysis is being applied in the modification of protein structure in order to enhance the functional properties of protein (Corredig, 1997). Enzymatically hydrolysed proteins possess functional properties such as low viscosity, increased whipping ability and high solubility which make them advantageous for use in many food products (Alder – Nissen, 1979). It shows extensive proteolytic activity towards proteins, shortchain peptides, amino acids, esters and amide linkages (Arnon 1970; Ding et al, 2003). Tiger nut considered as weed in some areas (De Vries, 1991), is an under-utilized crop that contains varieties of nutrients and has been reported to be a health food since its consumption could help prevent heart diseases, thrombosis and activate blood circulation (Chukwuma et al, 2009), and also reduces the risk of colon cancer (Adejuyitan et al, 2009), obesity and gastrointestinal diseases (Anderson et al, 1994). It is used as a composite material for yoghurt production and in the preparation of kunnu (Belewu and Abodurin, 2006; Sanful, 2009).

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The protein content of tiger nut is low (Venkatachalam and Sathe, 2006), and effort is been made to increase the amino acid in the milk- like beverage prepared from tiger nut through its treatment with papain, and hence, the need for this research. This fortifies the milk- like beverage and increase the value- added to tiger nut. It was observed that treatment of plant material with papain produces significant increase in the yield of protein extracts such as in soya beans milk (Pinitglang et al, 2011).. This study therefore focuses on partial purification of papain from the latex of unripe Carica papaya and its application in the hydrolysis of protein in the tiger nut protein homogenate with a view to fortify the amino acid composition of it.

Materials and methods Materials The papaya fruit was freshly collected from Edem-Ani, Nsukka, Enugu state, Nigeria. Tiger nut (C. esculentus) was purchased from Ogige, a local market in Nsukka LGA of Enugu state, Nigeria. Tris salt was purchased from Sigma-Aldrich, USA. All other chemicals used in this work were of analytical grade and were obtained from reputable sources.

Methods Extraction of Latex from C. papaya Latex from C. papaya was extracted according to the method of Chu chi Ming et al., (2002).

Preparation of O- phthaldialdehyde (OPA) O- Phthaldialdehyde (OPA) was prepared as described by (Nielsen et al 2001). A quantity, 200mg of sodium dodecyl sulfate, 7.62g disodium tetraborate decahydrate was completely dissolved in 150ml of distilled water. The reagents were completely dissolved before adding 176mg of Dithiothreitol (DTT) to the mixture, and then the solution was made up to 200ml with distilled water.

Enzyme Extraction and Purification The method used was adapted from that of Chu chi Ming et al., (2002). The latex was obtained by making incisions on the surface of green fruits early in the morning (9:00am), on the same tree for two weeks. A plastic bowl was held under the fruit to collect the latex. The collected latex (373.08g) was dissolved in 1120ml of 20mM EDTA (3:1) ratio. The solution was stirred for 15minutes using a magnetic stirrer to produce a homogenous solution. Then, the latex solution was centrifuged at 4000rpm for 30minutes and filtered with whatman’s No.1 filter paper, hence crude papain. Total volume of crude papain produced was 1,021ml.The crude enzyme extract was made to

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80% ammonium sulphate saturation with solid ammonium sulphate. The solid ammonium sulphate was added gradually at 4oC and stirred using a magnetic stirrer. The solution was kept at 4oC for 12h. This centrifuged at 4000 × g for 30min at 37oC using Wischerfuge Model centrifuge, the precipitate was dissolved in 0.05M Tris HCl buffer pH 7.2 and introduced onto sephadex G-50 gel chromatography column (3.2 ×70cm) pre equilibrated with 0.05M Tris HCl buffer pH 7.2. Fractions were eluted with the same buffer at a flow rate of 1.0ml/min. Active fractions were pooled together and loaded onto sephadex G-200 chromatographic column (3.2 ×70cm) pre equilibrated with the same buffer. Fractions were elution with the same buffer at a flow rate of 1.0ml/min. Active fractions were pooled and stored at 4oC for further studies.

Enzyme Assay Papain activity was assayed by the method of Nitsawang et al., (2006). The change in absorbance at 275nm due to hydrolysis of casein in the presence of Tris buffer pH 7.2 and the enzyme at 37oC was monitored using a Jenway 6405 Spectrophotometer (Jenway Scientific, England). The standard assay solution contains 1.0ml 1% casein, 0.2ml 20mM EDTA, 0.3ml 0.05M Tris HCl buffer pH7.2 and 0.5ml enzyme extract, in a total volume of 2.0ml. After mixing and incubation at 37 oC for 10minutes the reaction was stopped by adding 3ml of trichloro-acetic acid which precipitates the residual large molecular weight material. The precipitate formed was filtered after standing for 60minutes at 25oC. Absorbance was read at 275nm against blank. One unit of enzyme activity was defined as the activity which gives rise to an increase in absorbance of 0.1/min at 37oC.

Protein Estimation Protein content of papain was determined by the method of Bradford (1976), using ovalbumin as the standard protein except where otherwise stated. Protein concentration during chromatography was followed by reading the absorbance at 275nm on Jenway 6405 spectrophotometer.

Preparation of Tiger Nut Protein Homogenate Fractionation of ground tiger nut sample was carried out as described by Bautista et al., (1990).

Kinetics Studies of Papain Km and Vmax of papain were determined from Lineweaver -Burk plots of initial velocity data at different concentration of casein (0.1-1.0gml).

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Optimum temperature The optimum temperature was determined by incubating reaction mixture at different temperature, ranging from 35oC - 110oC for 30min using SSY-H stainless steel Thermostatic water Bath by method of Chu chi Ming et al.,(2002).

Optimum pH Papain activity of partially purified enzyme was measured in the range of pH3.5 to 9.0 using the following buffer, 0.05M Tris HCl buffer 7.5 9.0, Sodium acetate buffer pH 3.5 to 5.5, and Phosphate buffer pH 6.0 to 7.5 by method of Chu chi Ming et al.,2002

Effect of substrate concentration The effect of substrate concentration on papain activity was determined.

Hydrolysis of Tiger nut protein homogenate The hydrolysis was carried out by a modification of methods of Calderon de la Barca et al., (2000); and that of Nelson et al, (2001). This was carried out as in the assay section but casein was replaced with different concentrations of Tiger nut protein homogenate (0.1 – 1.0g\ml). The reaction mixture was also incubated at different period of time (10 – 120mins). This experiment was also carried out in the presence of different concentrations of O- phthaldialdehyde (OPA). Proteins hydrolysis was monitored at different incubation time of 0, 10, 30, 60 and 120mins at 37°C and pH 7.5 at 340nm using Jenway 6405 UV/Visible spectrophotometer model.

Results and Discussion Crude papain extract from an unripe Carica papaya when saturated to 80 with ammonium sulphate resulted in the precipitate of most of the enzyme. Papain precipitated by ammonium sulphate at 40 has been reported by Arnon 1970. Similarly, Nitsawang et al., 2006 reported 45 ammonium sulphate saturation. The specific activity of papain after ammonium sulphate precipitation was found to be 1.31U/mg (Table 1) and , this could be attributed to the ability of ammonium sulphate to aggregate the proteins present from the crude extract. This was subsequently desalted on sephadex G50 chromatographic column (Figure 1).

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Figure 1. Chromatogram of sephadex G-50

Table 1: The purification table for purified papain Purification steps

Volume (mL)

Protein .(µg/mL)

Activity (U/min)

Total activity (U) 179000 72000

% Yield

Purification Fold

179 240

Specific Activity (U/mg) 1.32 1.28

Crude enzyme Ammonium sulphate precipitation Gel filtration on sephadex G50 Gel filtration on sephadex G200

1000 300

136 188

100 94.70

1 0.97

65

109

361

3.31

23465

13.59

2.51

30

59

531

9.0

15930

6.36

6.82

The elution pattern of column chromatography on sephadex G200 clearly indicated that the enzyme was eluted in mid fractions Fig 2. The specific activity of papain increased from 1.31U/mg to 1.48U/mg after G200 gel filtration. This suggests that the gel was able to remove some impurities that could interfere with the activity of papain.

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Fig 2. Chromatogram on sephadex G200 gel filtration column

Analysis of the initial velocity data using Lineweaver -Burk plot Fig.3 showed that papain had a Vmax of 1.133µmol/min and Km of 0.217mg/ml respectively for papain activity at optimal conditions.

Fig 3. Lineweaver-Burk plot of papain from C.papayalatex

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Effect of pH and temperature on partially purified papain

Activity(U/min)

The optimum pH for the activity of papain isolated from C. papaya latex was pH 7.5 (Fig.4). The enzyme showed a gradual increase in its activity with increasing pH up to optimum, followed by a sharp fall in its activity. From the pH studied, as the pH was increased from pH 3.5 to pH 7.5, the papain activity was found to increase successively but further increase to pH 9.0 resulted in the decrease of papain activity (Figure 4). Similar results were obtained for papain using casein as substrate at pH 7.0, by Chu chi Ming et al., 2002; Ding et al.,(2003).

140 120 100 80 60 40 20 0 0

2

4

6

8

10

pH

Fig 4 Effect of pH on papain activity

The effect of temperature on papain activity was studied ranging from 40 °C- 110°C and optimum temperature was obtained at 90°C as observed in Fig 5. Further increase in temperature above 90 °C lead to a decrease in papain activity. The decrease in the enzyme activity at higher temperature may be attributed to protein denaturation of the enzyme. The optimum temperature within the range of 65-80°C was reported by Harton et al (2002). Papain was reported by Schechter and Berger (1968) to be thermophilic.

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Fig 5 Effect of temperature changes on papain activity

The optimum incubation time for the hydrolysis of tiger nut protein concentration of 1.0g/ml with papain was obtained at 10mins (Fig 6). It was clear that among the analysed time of incubation, the highest degree of hydrolysis was obtained at 10mins incubation time at all sample concentrations examined.

Fig 6 Degree of hydrolysis against time at 0.1g/ml tiger nut protein concentration

The improvement in protein extraction is revealed by high degree of hydrolysis as shown in fig 7 which goes up to 24% at 10mins incubation time and a protein concentration of 0.2g/ml. The degree of hydrolysis values obtained after papain treatment were higher than those obtained from OPA at the same reaction conditions of 37°C and pH 7.5 as observed in figures 7 and 8

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. This reveals that the enzyme has a positive proteolytic effect on the protein of tiger nut. Subsequently, the decrease in the degree of hydrolysis at incubation time of 30 and 60min as obtained in this study could be attributed to the denaturation of amino acids present in the solution (Hill and Schmidt, 1962; Nielsen et al., 2001). The degree of hydrolysis by papain was observed to be higher than that of OPA. This could be attributed to the presence of sodium dodecyl sulphate (SDS) in OPA reagent. SDS according to Eze et al., (2012) can affect the force of interaction within a protein and unfold the protein slightly until the protein is denatured. It could be that OPA denatured the protein present in tiger nut thereby reducing the protein that would have been hydrolysed. Figure 9 shows the degree of protein hydrolysis by papain and OPA at 0. 10, 30, 60 and 120 min intervals. It shows that the degree of hydrolysis by papain is higher when compared to OPA.

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Fig 9. Degree of Hydrolysis against time at 0.4g/ml tiger nut protein concentration

The degree of protein hydrolysis by papain is represented in figure 10. At 0.5g/ml protein concentration, the degree of hydrolysis was highest at 10min hydrolysis time by papain.

Fig 10.. Degree of Hydrolysis against time at 0.5g/ml tiger nut protein concentration

The degree of hydrolysis of tiger nut protein at 0.6d/ml protein concentration is shown in figure 11. The degree of hydrolysis was highest at 10min incubation time with papain. As the protein concentration increased to 0.7g/ml, the degree of hydrolysis increased as shown in figure 12. Degree of hydrolysis at 10min incubation time is highest as shown in figure 12.

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Fig. 11 Degree of Hydrolysis against time at 0.6g/ml tiger nut protein concentration

Figure 13 shows the degree of hydrolysis of tiger nut protein at 0.8g/ml papain and OPA. Incubation with papa in for 10min incubation time gives the highest yield.


Figure 13 shows the degree of hydrolysis of tiger nut protein at 0.8g/ml by papain snd OPA. Incubation with papain for 10min incubation time gave the highest yield.


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Figure 14 shows the degree of hydrolysisof tiger nut protein 0.9g/ml concentration at incubation time of10min with papain gave the highest yield.


Figure 15 represents the degree of tiger nut protein (1.0g/ml) hydrolysis at varying incubation time of 0, 10, 30, 60 and 120mins.


Conclusion The results obtained in this research show that papain can be effectively used to hydrolyse tiger nut protein at mild laboratory conditions. The protease, papain, isolated from C. papaya has optimum temperature of 90°C, hence it is thermophilic and hydrolyses proteins with high activity. Papain hydrolytic process is more efficient for protein extraction from tiger nut than the conventional O-phthaldialdehyde (OPA) reagent. The increased degree of hydrolysis achieved by papain may be useful in food industries to which hydrolysates can be added to improve and fortify the nutritional value of foods. Considering broad substrate specificity of the enzyme, it could be used in

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preparing feeds for livestock as well as preparation of food stuffs for human consumption. These are important features relevant in using the enzyme in some food and biotechnology industries that would require higher working temperature and near neutral pH.

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