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Research Article

Studies on the Effect of The Different Modes of Extraction on Enzyme Activity of Proteolytic Enzymes from the Peels of Three Fruit Samples Carica Papaya (Papaya), Ananas Comosus (Pine Apple) and Actinidia Deliciosa (Kiwi) Thanika Vel P* and Shaleesha A. Stanley Department of Biotechnology, Jeppiaar Engineering College, Chennai. Abstract The aim of the present study was to extract proteolytic enzymes from the waste peels of three fruit samples Carica papaya (papaya), Ananas comosus (Pine apple) and Actinidia deliciosa (Kiwi). The primary extraction was carried out with water and the crude enzyme extracts were further subjected to aqueous two phase partitioning with the system containing polyethylene glycol PEG 6000 – K2HPO4 to compare extraction method with that of traditional aqueous extraction method. The crude extract of the three fruit peel samples were subjected for phytochemical analysis and the results were positive. The proteolytic activity of the enzyme extracts was studied using casein as substrate. The results confirmed the presence of proteolytic enzyme in all the three fruit peel samples. The study concludes that the proteolytic enzyme activity of Ananas comosus has the highest range (3.331) and subsequently Actinidia deliciosa (3.131) and Carica papaya (2.125).

Key words: Protease, Proteolytic enzyme, Carica papaya (papaya), Ananas comosus (Pina apple) and Actinidia deliciosa (Kiwi,) Peels, Casein assay. Abbreviations:PEG – Poly Ethylene Glycol, ATPS – Aqueous Two Phase System, K2HPO4 – Dipotassium Hypo Phosphate, USD – United States Dollars, Bovine Serum Albumin (BSA) Introduction

waste poses an environmental issue.

Peptide bond cleaving enzymes are widely referred to as proteolytic enzymes. Protease has a wide range of industrial applications of which some include pharmaceutical applications, leather, textiles, and animal feed (Caygill J.C. 1979 and Feijoo-Siota 2010). Protease represents approximately 60% of all enzymes that are commercially used earns a yearly sum of USD 3 billion (Leary et al., 2009). The fruits of the following plants, Carica papaya, Ananas comosus and Actinidia deliciosa; are rich in protease. Researches concluded that the fruits and leaves of Carica papaya, Ananas comosus and Actinidia deliciosa are rich producing protease (Saranya Chitturi et al., 2013).

In Asian countries, like Thailand, China and India the average vegetable waste from restaurants and food processing industries sums up several thousand metric tons. The African country Nigeria is ranked as the third largest producer of Carica papaya. In a year the total harvest of Carica papaya fruit is 765000 metric tonnes while India is ranked fifth (FAO 2007). Ananas comosus production is 18 million tons (FAO 2005) and Actinidia deliciosa, several thousand metric tons are harvested in Asian and African countries.

Proteolytic enzymes are a mixture of enzymes found naturally in the extracts of leaves, stem and fruits of the plants Carica papaya, Ananas comosus and Actinidia deliciosa. Papain from Carica papaya, bromelain from Ananas comosus and actinidin of Actinidia deliciosa are collectively known as proteolytic enzymes as the enzymes cleaves peptide bonds (Dayanand C D 2013). Generally, peels of Carica papaya, Ananas comosus and Actinidia deliciosa are discarded from fresh juice shops, home, restaurants, and food industries. Annual waste accumulation from peels of these fruits accounts to 2000-3000 tons. In Thailand, largest producer of papaya pickles states that peels of papaya alone add up to more than a 1000 tons every year (Phanuphong Chaiwut et al 2007). This

The peels of the fruits comprises of varying concentration of proteins, carbohydrates and other phytochemicals. Carica papaya, consists of four cystine protease namely: protease, chymopapain, glycyl endopeptidase and caricain (Barrett et al 1998). The peels of Carica papaya and Actinidia deliciosa contain proteolytic enzyme in the vegetative state whilst in Ananas comosus, the enzyme content rapidly increases on maturity and fades over a period of time (Maurer 2001). This study is aimed to find effective measures to extract enzymes from the peels which would reduce the organic wastes.

Received:December 2014 Accepted: January 2015 *Corresponding Author Email: [email protected]

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Research Article Materials and Methods Chemicals (Analytical Grade) Poly Ethylene Glycol (PEG) (MW 6,000), Dipotassium Hydrogen Phosphate (K2HPO4), Casein, Tri Chloro Acetic acid (TCA), Sodium Hydroxide (NaOH), Sodium Carbonate(Na2CO3), Copper Sulphate (CuSO4), Coomassie Brilliant Blue G 250, Bovine Serum Albumin (BSA), Bradfords's Reagent and Folin phenol reagent. Equipments Hot air oven, commercial blender, orbital shaker, heating mantel, spectrophotometer and centrifuge. Biologicals Fruit peels of Carica papaya, Ananas comosus and Actinidia deliciosa. Enzyme Extraction The peels of Carica papaya were dried at 55 C ̊ in a hot air oven for the duration of 4 – 5 hours to reduce the moisture. The dried peels were powdered using a commercial blender. The extraction was carried out with 1:9 (W/V) water. The mixture was left undisturbed for a period of 30 minutes and centrifuged at 4000 RPM at 4 ̊ C for 10 minutes. The supernatant was filtered using a Whatman No.1 filter paper and the resultant is the crude enzyme extract. The extraction procedure for Carica papaya is followed for the peels of Actinidia deliciosa. The Ananas comosus peel was ground using a domestic juicer without water and the contents were centrifuged at 8000 RPM at 4ºC for 30 min. The supernatant was cooled to 4°C. The supernatant was discarded and the precipitate was dissolved in potassium phosphate buffer at pH 7.0 (Iara Rocha 2013)

Protein concentration was estimated using Bradford method (Bradford 1976). Bradford's reagent was prepared by dissolving 100 mg of Coomassie Brilliant Blue G 250 in 50 ml of 95% ethanol and 100 ml of 85% phosphoric acid. It is made upto 1.0 liter by adding sterile distilled water. Protein content was measured by adding 100μl of leaf extract to 2.0 ml of Bradford reagent and 900μl of pH 7 buffer. Absorbance was measured at 595 nm using UV visible spectrophotometer. Protein content was measured using standard graph of Bovine Serum Albumin (BSA). Casein assay (Subathra et al 2012) In a sterile test tube, 5 ml of 1% casein was prepared by dissolving 0.06g of casein in 5ml distilled water taken and 250μl of peel extract (protease) was added. The reaction mixture was incubated for 10 minutes at room temperature (37 ̊ C). After incubation for 10 minutes, 5 ml of 10% TCA was added to stop the reaction and absorbance was measured at 280 nm (Subathra et al 2012).

Results Extraction Methods In Carica papaya, the extraction with water accumulated large protein contamination apart from the papain. Aqueous two phase system was previously studied by Sarote et al 2006. The effect of PEG system was insignificant in partitioning of papain from other protein. Similarly ammonium sulphate precipitation that was compared with the PEG system did not prove efficient. Thus, the crude enzyme activity is comparatively higher compared to the

Phytochemical analysis Plants constitute a wide array of phyto chemical compounds that have potential health beneficial properties (Mohamed Sham Shihabudeen 2010). Similarly, the fruits peels are suspected to be rich in phyto chemicals. To confirm, peel extracts of Carica papaya, Ananas comosus and Actinidia deliciosa were subjected to a detailed qualitative phytochemical analysis (Jigna P 2007). Test for carbohydrates, tannins, saponins, flavonoids, alkaloids, quinonoes, glycosides, cardiac glycosides, terpenoids, phenols and steroids were performed according to standard procedure. Aqueous two phase extraction PEG (MW 6,000) of 50% concentration and 40% K2HPO4 was prepared using pH 7 buffer. In a sterile test tube, 5.0 ml of PEG (MW 6,000), 5.0 ml of K2HPO4 and 2.0 ml of extract was taken and left undisturbed for 10 minutes at room temperature (37 ̊ C). This mixture was centrifuged at 4,000 RPM for 10 minutes. The contents form two aqueous phases with varying protein content. Aqueous two phase system is used to attain high enzyme content. The PEG 6000 – K2HPO4 system binds protease and separates it based on molecular weight. protein assay is performed for estimation of total protein content. The proteolytic activity of the enzyme extracts were assayed with casein as substrate. Protein assay (Bradford 1976)

Figure 1. Result of the Aqueous two phase extract of Ananas comosus (pine apple) peel extract

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Research Article

Figure 2. Results of the Phytochemical analysis of Carica papaya (papaya) peel extract Phytochemical analysis: (Left to right)Read as Tannins, saponins, alkaloids, flavanoids, quinines, glycosides, cardiac glycosides, terpenoids, phenols and steroids

Figure 3. Results of the Phytochemical analysis of Ananas comosus (pine apple) peel extract Phytochemical analysis: (Left to right)Read as Tannins, saponins, alkaloids, flavanoids, quinines, glycosides, cardiac glycosides, terpenoids, phenols and steroids. 29 THE SCITECH JOURNAL VOLUME 02 ISSUE 02 FEBRUARY 2015

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Research Article

Figure 4. Results of the Phytochemical analysis of Actinidia deliciosa (kiwi) peel extract Phytochemical analysis: (Left to right) Read as Tannins, saponins, alkaloids, flavanoids, quinines, glycosides, cardiac glycosides, terpenoids, phenols and steroids. Table 1. Results of the comparative phytochemical analysis performed on the three fruit samples Carica papaya (papaya), Ananas comosus (pine apple) and Actinidia deliciosa (kiwi)

Phytochemicals Tannins Saponins Alkaloids Flavanoids Quinones Glycosides Cardiac Glycosides Terpenoids Phenols Steroids

Carica papaya

Ananas comosus + + + + + + + +

other extraction methods. In the case of Ananas comosus and Actinidia deliciosa the water extraction method was sufficient to obtain the proteolytic constituents from the peels. Iara Rocha 2013, concluded that ammonium sulphate precipitation of bromelain was able to purify the enzyme only partially. Phytochemical analysis The phytochemical analysis of the crude extract revealed the

+ + + + + + +

Actinidia deliciosa + + + + + + +

presence of proteins. The extracts were also rich in other phytochemicals namely saponins, alkaloids, flavanoids, quinines, glycosides and terpenoids. The results of the same are given in table 1 and the figures 2, 3 and 4. Protein assay (Bradford 1976) The assay was performed to quantify the protein present in the crude enzyme extracts of the three fruit peel samples Carica papaya (papaya), Ananas comosus (pine apple) and Actinidia

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Research Article Table 2. Estimation of protein and enzyme activity of Carica papaya (papaya), Ananas comosus (pine apple) and Actinidia deliciosa (kiwi) using Protein assay and Casein Assay Protein Assay

Wavelength

Absorbance

Carica papaya

595

1.2053

Ananas comosus

595

1.3321

Actinidia deliciosa

595

1.0025

Casein Assay

Wavelength

Enzyme activity

Carica papaya

280

2.1251

Ananas comosus

280

3.3310

Actinidia deliciosa

280

3.1913

deliciosa (kiwi). The spectrophotometer absorbance at 595nm for the Carica papaya extract is given in table 2. Casein assay (Subathra et al 2012) The enzyme activity of the crude enzyme extracts was determined using the substrate conversion with casein as the substrate. The varying rate of conversion was observed with different fruit peel extracts. The absorbance analysis with spectrophotometer at 280nm is given in table 2.

Discussion The extraction method comparison between crude extraction and aqueous two phase extraction has struck that the latter does not significantly affect extraction process and purifies the enzymes partially. thus, it can be stated that crude extraction is by far the efficient method to extract high proteolytic enzymes. Purification can be performed by ion exchange chromatography on DEAE cellulose (Gautam S. S 2010). The phytochemical analysis revealed the presence of alkaloids, flavonoids, saponins, steroids, glycosides and terpenoids in all three samples while tannins and phenols were uniformly absent in the samples. Phyochemicals are significant in demonstrating defense mechanism against microorganisms and insects (Mohamed Sham Shihabudeen 2010). Protein content in each sample are closer in terms of value. The Bradford's assay for total protein content revealed that the crude enzyme extracts had comparatively higher protein concentration over aqueous phase extracts. The principle part of the study, enzyme activity of the samples that was studied with substrate conversion process with casein as substrate has undeniably confirmed the presence of proteolytic enzymes. The activity was determined as 3.3310 for Ananas comosus, 3.1913 for Actinidia delicosa and 2.1251 with extracts of Carica papaya. Thus, Ananas comosus has the chief enzyme activity of the three followed by Actinidia delicosa and Carica papaya.

Conclusion The study concluded that Carica papaya (papaya), Ananas comosus (Pina apple) and Actinidia deliciosa (Kiwi,) are rich in proteolytic enzymes. Harnessing this source for enzyme production would at large bring down organic waste accumulation, prevent environmental issues, reduce cost in enzyme production with recombinant microorganisms and also boost the economy. Further, this can be implemented in waste degradation process owing to the crude nature of the enzymes.

Acknowledgements The authors gratefully acknowledge Jeppiaar Engineering College, Chennai for providing with the laboratory infrastucure to carry out the study. We are also immensely thankful to our friends and family for their limitless support.

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To cite this article: Thanika Vel P and Shaleesha A. Stanley, 2015. Studies on the Effect of The Different Modes of Extraction on Enzyme Activity of Proteolytic Enzymes From the Peels Of Three Fruit Samples Carica Papaya (Papaya), Ananas Comosus (Pine Apple) and Actinidia Deliciosa (Kiwi), The Scitech Journal 2(02): 27-32

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