Isolation of Protease Enzyme From Chayote Fruit (Sechium edule (Jacq.) Sw.) With Ammonium Sulfate Fractination Method 1

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Isolation of Protease Enzyme From Chayote Fruit (Sechium edule (Jacq.) Sw.) With Ammonium Sulfate Fractination Method 1 1)

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Ketut Ratnayani , Ni Wayan Lia Kusumaningrum1 Jurusan Kimia, FMIPA, Universitas Udayana, Bukit Jimbaran, Badung 82121 (0361)701954 Ext 255, [email protected]

ABSTRACT Protease is an enzyme that is capable to hydrolyze (breakdown) protein molecules into simpler compounds such as small peptides and amino acids. The aim of the research was to isolate protease enzyme from chayote (Sechium edule (Jacq.) Sw.) using fractination ammonium sulfate method, so that it could be obtained the saturation level of ammonium sulfate which is the optimum condition for protease in chayote fruit precipitation. Protease activity examination of each fraction of ammonium sulfate was performed using Anson method and protein content assay was determined using Biuret method. The results showed that crude extract protease of chayote has specific activity of 3.7338 x 10-3 U/mg. The optimal saturation levels of ammonium sulfate for protease chayote precipitate was found at 40-50% saturation level, because at this saturation obtained the highest enzyme spesific activity were 16.00 x 10-3 U/mg with purity of protease enzyme reached four times more as compared to crude extract protease. Keyword : protease, isolation, chayote, ammonium sulfate

INTRODUCTION

Protease is an enzyme that is capable to hydrolyze (breakdown) protein molecules into simpler compounds such as small peptides and amino acids. The source of protease enzymes can come from microorganism, animal and plant. Plant is the biggest resourches (43.85%) followed by bacteria (18.09%), fungi (15.08%), animal (11.15%), algae (7.42%) and viruses (4.41%) (Mahajan and Shamkant, 2010). Plant protease have some advantages namely, they have higher activity and stability on various temperature, pH, inhibitor and metal ion (Mehrnoush et al., 2011). One of the subtropic plant in Indonesia is labu siam or chayote (Sechium edule (Jacq.) Sw) which its fruits can be used as vegetable food. Plant latex which can cause itching, probably caused by its protease component(Lavinka and Dong, 2013). It is predicted that chayote fruit has protese activity because its latex can cause itching too like papaya latex(Joseph, 2010). This hypothesis can be proved by Juwarni et al(2014) on previous research which found that the chayote latex contain protease with protease activity as much as 0.0264 U/mL. However there is problem in latex collection from chayote fruit cause the latex is rapid clot, few in number and also the latex trickle out (sticking to the blade) when tapped from the fruit. So it is necessary to do another

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alternative method for obtaining protease of chayote by using chayote fruit directly (no need to collect the latex). For characterization and next application purposes, protease enzyme need to be isolated from chayote fruit with fractionation using ammonium sulfate salt. Ammonium sulfate salt often used to fractionate enzyme protein cause by its high solubility, give stabilizing effect to the enzyme and can not disturb protein structure (GE Healthcare Life Sciences, 2011). Protease enzyme isolation using ammonium sulfate have been reported by some reasearcher namely Noda, Koyanagi and Kamiya in 1994 using ammonium sulfate 50% saturation level to isolate protease from melon fruit. Asakura et al. in 1997 extracted and purified oryzasin from rice seed using ammonium sulfate 30-60% saturation level. The aim of the research was to isolate protease enzyme from chayote (Sechium edule (Jacq.) Sw.) using fractination ammonium sulfate method, so that it could be obtained the saturation level of ammonium sulfate which is the optimum condition for protease in chayote fruit precipitation. Specific activity of the enzyme is unit amount of the enzyme every milligram protein, and it can be used in determine the purity level of enzyme, the more specific activity value the more purity of enzyme exctract. MATERIAL AND METHODS Materials Chayote fruit, aquades, ammonium sulfate ((NH4)2SO4), BSA, tyrosine, natrium metabisulfit 1%, casein 0.65% (b/v), K2HPO4 0.05 M, KH2PO4 0.05 M, Na2CO3 0.5 M, TCA (Trichloroacetic Acid) 0.11 M, Biuret reagent and Folin-Ciocalteau reagent.

Methods Crude Extract Preparation (Witono, Y., 2008) The crude protease extract was obtained as the result of enzyme separation from chayote fruit. 200 g chayote fruit was added with 90 mL cold buffer fosfat 0,05 M pH 7 which contain natrium metabisulfit 1% then it homogenized and filtered using a filter paper. The filtrate of filtering results then centrifuged at a speed of 5000 rpm for 15 minutes at a temperature of 4oC. The supernatant separated from the sediment (residue). Supernatant obtained is referred to as supernatant I, which will be used for the fractionation process Protease Isolation Method Using Ammonium Sulfate Fractionation (Noda et al., 1994) The supernatant I (crude extract protease) as much as 100 mL fractionated with ammonium sulfate. Determination of the amount of ammonium sulfate that is used to achieve the expected saturation level refers to a table compiled by the Scopes in 1982. The percentage of saturation of ammonium sulfate used is 0-20%, 20-50% and 50-70%. The addition of ammonium sulfate salts done slowly until a certain saturation percentage with stirring using a magnetic stirrer. The precipitate obtained by centrifugation at 5000 rpm at a temperature of 4 ° C for 15 minutes, then dissolved in 12 mL of cold 0.05 M phosphate buffer pH 7 containing 1% sodium metabisulfite. Ammonium sulfate fraction with the highest specific activity will be carried out prior to fractionation protease such as ammonium sulfate salt saturation narrower ie 20-30%, 30-40% and 40-50%.

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Assay of Protease Activity Determination of enzyme activity using the method of Anson (Anson, 1938) with a colorimetric technique that utilizes absorption of the blue complex formed by reaction of tyrosine with Folin Ciocalteu reagent (Folin and Ciocalteu, 1927). Casein solution 0.65% (w / v) of 2.5 mL in the pre-incubation at 37 ° C for 4 minutes added 1.0 mL sample of this protease results fraksinasi.Campuran incubated at 37 ° C for 30 minutes. Hydrolysis reaction was stopped by addition of 2.5 mL of 0.11 M TCA and allowed to stand for 5 minutes. This mixture was centrifuged for 15 minutes at a speed of 5000 rpm. Protease activity was determined by measuring the levels of tyrosine generated colorimetric reagent Folin-Ciocalteau. As many as 2.0 mL of the supernatant was reacted with 5.0 mL of 0.5 M Na2CO3 and 1.0 mL reagent Folin-Ciocalteau then the mixture was left for 30 minutes. Read the absorbance of this solution using a UV-Vis spectrophotometer at 755.4 nm. As a blank, the addition of 0.11 M TCA conducted before the addition of a protease sample. Protease activity (U / mL) is expressed in units of activity ie one unit (U) is expressed as the amount of protease that can hydrolyze a substrate (casein) and produce a color equivalent to 1 mol tyrosine product (181 mg) every minute incubation period in experimental conditions. Protease enzyme activity can be determined by the formula (Sigma, 1999): Protease Activity (U/mL) =

μmol tirosin × V V ×t ×V

Keterangan : = total volum used on uji aktivitas protease sampel (meliputi volume kasein 0,65%, ekstrak protease kasar dan TCA) (6 mL) = volume sampel ekstrak protease kasaryang digunakan ( 1 mL) = volume supernatan yang digunakan dalam penentuan kadar tirosin secara kolorimetrik ( 2 mL) t = waktu inkubasi (30 menit)

Determination of Total Protein Levels (AOAC, 1995) Determination of protease enzyme protein fractionation results from using fruit squash Biuret method. Proteases as much as 1.0 mL sample was reacted with 5.0 mL of Biuret reagent and then allowed to stand for 20 minutes at room temperature. Mix absorbance was measured using a UV-Vis spectrophotometer at 547 nm. Total protein content is calculated by converting the absorbance to the standard linear regression equation BSA.) Penentuan Aktivitas Spesifik (Sigma,1999) Aktivitas spesifik (U/mg) protease buah labu siam ditentukan dengan membagi aktivitas protease (U/mL) dengan kadar protein total (mg/mL) protease dari buah labu siam.( Determination of Specific Activity (Sigma, 1999) The specific activity (U / mg) protease fruit squash is determined by dividing the protease activity (U / mL) with a total protein content (mg / mL) protease of chayote fruit. Discussion Dalam tahap preparasi ekstrak kasar dari daging buah labu siam diperoleh filtrat (hasil penyaringan jus buah) berwarna hijau pekat. Filtrat disentrifugasi sehingga menghasilkan ekstrak protease kasar (supernatan I) berwarna hijau muda, dari 200 gram daging buah labu siam diperoleh ekstrak kasar protease sebanyak 200 mL.( In the preparation phase of the crude extract of the fruit flesh of squash obtained filtrate (filtering results juice) is solid green. The filtrate was centrifuged resulting in rough protease extract (supernatant I) light green, 200 grams of meat fruit squash protease obtained crude extract as much as 200 mL.) Penentuan aktivitas spesifik dilakukan untuk mengetahui tingkat kemurnian enzim, semakin tinggi nilai aktivitas spesifik maka semakin tinggi kemurnian dari enzim yang telah diisolasi(Lehninger, 1990). Aktivitas spesifik enzim protease merupakan aktivitas enzim untuk setiap miligram protein total ekstrak buah labu siam. Penentuan aktivitas spesifik protease dilakukan dengan membagi antara aktivitas protease enzim

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(U/mL) dengan kadar protein total (mg/mL) (Seidman and Mowery, 2006). Aktivitas spesifik protease buah labu siam pada ekstrak kasar, fraksi 0-20%, fraksi 20-50% dan fraksi 50-70 dapat dilihat pada Tabel 1 dan Gambar 1.( The determination of the specific activities carried out to determine the level of purity of the enzyme, the higher the value of specific activity, the higher the purity of enzymes that have been isolated (Lehninger, 1990). The specific activity of the protease enzyme is an enzyme activity for each milligram of total protein extracts of fruit squash. Determination of the specific activity of proteases is done by dividing the protease enzyme activity (U / mL) with a total protein content (mg / mL) (Seidman and Mowery, 2006). The specific activity of protease fruit squash in the crude extract, fractions 0-20%, 20-50% fractions and fractions 50-70 can be seen in Table 1 and Figure 1.) Tabel 1. Aktivitas spesifik protease labu siam dari ekstrak kasar, fraksi 0-20%, fraksi 20-50% dan fraksi 5070%

Keterangan Ekstrak kasar Fraksi 0-20% Fraksi 20-50% Fraksi 50-70%

Aktivitas Protease Rata-rata (U/mL) 5,13 x 10-3 ± 0,0775 1,09 x 10-3± 0,0000 25,30 x 10-3 ± 0,0002 4,05 x 10-3 ± 0,0000

Kadar Protein Rata-rata (mg/mL) 1,3782 ± 0,0822 0,8431 ± 0,0125 1,4938 ± 0,0125 0,6551 ± 0,0000

Aktivitas Spesifik Rata-rata (U/mg) 3,7338 x 10 ± 0,0018 1,2931 x 10 ± 0,00002 16,9000 x 10-3 ± 0,0003 6,1823 x 10 ± 0,0000

Berdasarkan Tabel 1, aktivitas spesifik tertinggi terdapat pada fraksi 20-50%, sehingga dilakukan fraksinasimenggunakan kejenuhan garam ammonium sulfat dengan kisaran yang lebih sempit yaitu 20-30%, 30-40% dan 40-50%.Data hasil aktivitas spesifik protease dapat dilihat pada Tabel 2. (Based on Table 1, the highest specific activity found in fractions 20-50%, so do fraksinasimenggunakan saturation of ammonium sulfate salt with a more narrow range that is 20-30%, 30-40% and 40-50% .Data results of the specific activity of the protease can be seen in Table 2. Tabel 2. Aktivitas spesifik protease labu siam dari ekstrak kasar, fraksi 20-30%, fraksi30-40% dan fraksi 4050%

Keterangan Fraksi 20-30% Fraksi 30-40% Fraksi 40-50%

Aktivitas Protease Rata-rata (U/mL) 9,6933 x 10-3 ± 0,0074 13,7700 x 10-3 ± 0,0124 5,4933 x 10-3 ± 0,0039

Kadar Protein Rata-rata (mg/mL) 0,7563 ± 0,0821 0,4671 ± 0,0102 0,3588 ± 0,2622

Aktivitas Spesifik Rata-rata (U/mg) 12,30 x 10-3 ± 0,0119 11,05 x 10-3± 0,0303 16,00 x 10-3 ± 0,0049

Tabel 1 menunjukkan bahwa nilai aktivitas protease setelah dilakukan penambahan garam ammonium sulfat ((NH4)2SO4) kejenuhan 0-20% dan 50-70% mengalami penurunan dari ekstrak kasar (5,13 x 10-3 U/mL). Peningkatan aktivitas protease terjadi pada fraksi garam (NH4)2SO420-50% yaitu 25,30 x 10-3 U/mL, sedangkan pada Tabel 2 tampak bahwa aktivitas protease secara keseluruhan mengalami peningkatan dari ekstrak kasarnya(5,13 x 10-3 U/mL). Dari ekstrak kasar sampai penambahan garam (NH4)2SO4 30-40% meningkat lalu menurun pada penambahan garam (NH4)2SO4 40-50%. Terjadinya penurunan dan peningkatan aktivitas protease ini dapat disebabkan oleh adanya variasi jumlah protein enzim protease dan protein non enzim yang mengendap di masing-masing fraksi karena protease hasil fraksinasimasih terdiri dari protein enzim dan protein non enzim (Wang, 2004), serta disebabkan oleh masing-masing jenis protein memiliki kelarutan yang berbeda (Scopes, 1982).( Table 1 shows that the protease activity after the addition of salts of ammonium sulphate ((NH4) 2SO4) saturation of 0-20% and 50-70% decline of the crude extract (5.13 x 10-3 U / mL). Increased protease activity occurs in fractions of salt (NH4) 2SO420-50% ie 25.30 x 10-3 U / mL, whereas in Table 2 it appears that protease activity as a whole has increased from crude extract (5.13 x 10-3 U / mL). From the crude extract to the addition of salt (NH4) 2SO4 30-40% rise and drop upon addition of salt (NH4) 2SO4 40-50%. The decrease and increase in the activity of this protease can be caused by variations in the amount of protease enzyme protein and non-protein enzyme that settles in each fraction as protease results fraksinasimasih consists of protein enzymes and non-enzyme proteins (Wang, 2004), as well as due to the respective each type of protein has a different solubility (Scopes, 1982). Kadar protein pada Tabel 1 menunjukkan terjadinya penurunan pada fraksi 0-20%, 50-70% dan peningkatanpada fraksi 20-50% jika dibandingkan dengan kadar protein ekstrak kasar. Kadar protein pada Tabel 2, mengalami penurunan di setiap penambahan tingkat kejenuhan garam (NH4)2SO4. Hal ini diakibatkan karena setiap protein memiliki kelarutan yang berbeda-beda (Scopes, 1982) dan dalam hal ini diduga protein telah banyak mengendap di fraksi 20-30%, sehingga protein tidak terlalu banyak mengendap di fraksi 30-40% dan 40-50%. Penambahan garam ammonium sulfat mengakibatkan molekul air berikatan dengan ion garam, sehingga jumlah air yang berikatan dengan protein berkurang dan mengakibatkan protein

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mengendap (Seidman and Mowery, 2006).( The protein content in Table 1 shows a decrease in the fraction 020%, 50-70% and 20-50% peningkatanpada fraction when compared to the protein content of the crude extract. The protein content in Table 2, has decreased in each additional level of salt saturation (NH4) 2SO4. This is caused because each protein has a different solubility (Scopes, 1982) and in this case a protein thought to have much sediment in fractions 20-30%, so that the protein is not too much sediment in fractions 30-40% and 40-50% , The addition of ammonium sulfate salt resulting water molecules bind to the salt ions, so that the amount of water that bind to protein is reduced and the resulting precipitate proteins (Seidman and Mowery, 2006). Aktivitas spesifik pada Tabel 1 terlihat sangat jelas bahwa pada fraksi 20-50% memiliki aktivitas spesifik paling tinggi diantara fraksi lainnya, sedangkan pada kisaran yang lebih sempit (Tabel2) terlihat aktivitas spesifik fraksi 40-50% paling tinggi dibandingkan fraksi 20-30% dan fraksi 30-40%. Namun, nilai aktivitas spesifik antara fraksi 20-30%, fraksi 30-40%, dan fraksi 40-50% saling berdekatan satu sama lain. Maka dapat disimpulkan bahwa fraksi 40-50% paling optimal untuk mengendapkan enzim protease buah labu siam. Meskipun nilai aktivitas protease tertinggi ditemukan pada fraksi 20-30% dan kadar protein tertinggi ditemukan pada fraksi 30-40%, namun fraksi-fraksi tersebut memiliki aktivitas spesifik yang lebih rendah. Aktivitas spesifik menunjukkan tingkat kemurnian enzim protease (Lehninger, 1990). Semakin murni enzim protease yang diperoleh, akan memiliki nilai aktivitas spesifik yang makin meningkat (Lehninger, 1990). Selama proses pemurnian enzim, konsentrasi enzim akan meningkat relatif terhadap kadar protein total sampai batas tertentu (Lehninger, 1990). Kemurnian protease yang diperoleh pada fraksi 40-50% (16,00 x 10-3U/mg) mencapai 4 kali aktivitas spesifik ekstrak kasar (3,7338 x 10-3 U/mg). Berdasarkan Tabel 1 dan Tabel 2 diduga protein enzim protease dari buah labu siam yang diisolasi pada penelitian ini bersifat hidrofilik sedang karena protein enzim protease mengendap pada fraksi 40-50%. Hal ini diakibatkan karena setiap protein memiliki kelarutan dan sifat spesifik yang berbeda-beda, protein ada yang bersifat hidrofobik dan hidrofilik. Menurut Scopes (1982), protein hidrofobik akan mengendap pada konsentrasi garam yang rendah dan protein hidrofilik mengendap pada konsentrasi garam yang tinggi. Specific activity in Table 1 looks very clear that the 20-50% fraction having the highest specific activity among other factions, while the narrower range (Tabel2) shown specific activity of the highest fraction of 40-50% compared to 20-30% fraction and fraction of 30-40%. However, the specific activity values between 20-30% fraction, fraction of 30-40%, and 40-50% fraction adjacent to each other. It can be concluded that the most optimal fraction of 40-50% to precipitate the enzyme protease fruit squash. Although the value of the highest protease activity was found in fractions 20-30% and the highest protein content was found in fractions 30-40%, but the fractions has a lower specific activity. The specific activity showed a purity level of protease enzyme (Lehninger, 1990). The more pure protease enzyme obtained, would have a value of specific activity increasing (Lehninger, 1990). During the process of purification of enzymes, enzyme concentration will be increased relative to total protein content to a certain extent (Lehninger, 1990). Purity protease obtained in fractions 40-50% (16.00 x 10-3U / mg) to reach 4 times the specific activity of the crude extract (3.7338 x 10-3 U / mg). Based on Table 1 and Table 2 protease enzyme protein suspected of fruit squash isolated in this study was due to hydrophilic protease enzyme protein precipitated in fraction 4050%. This is caused because each protein solubility and specific nature of different, existing proteins hydrophobic and hydrophilic. According to the Scopes (1982), hydrophobic proteins will settle on a low salt concentration and hydrophilic proteins precipitate at high salt concentrations.

SIMPULAN DAN SARAN Simpulan Berdasarkan hasil penelitian , maka dapat disimpulkan bahwa nilai aktivitas spesifik ekstrak kasar protease buah labu siam (sebelum mengalami tahap salting out) sebesar3,7338 x 10 U/mg dan tingkat kejenuhan garam ammonium sulfat yang paling optimal untuk mengendapkan protease buah labu siam sebesar 40-50%.( Based on the research results, it can be concluded that the value of the specific activity of the crude extract protease fruit squash (before experiencing the stage of salting out) sebesar3,7338 x 〖10〗 ^ (- 3) U / mg and ammonium sulfate salt saturation levels are the most optimal to precipitate protease fruit squash by 40-50%. Saran Perlu dilakukan pemeriksaan kemurnian dari masing-masing fraksi ammonium sulfat dengan metode SDS PAGE, sehingga dapat diketahui ada berapa jenis protein yang mengendap pada tiap fraksi. Suggestion Necessary to check the purity of each fraction of ammonium sulphate using SDS PAGE, so it can be known how many types of proteins are precipited on each fraction.

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Acknoledgement Penulis mengucapkan terima kasih kepada Staf Laboratorium Analisis Hasil Pertanian Fakultas Teknologi Pertanian Universitas Udayana dan Laboratorium Bersama FMIPA Universitas Udayana yang telah memberikan bantuan dan dukungan fasilitas hingga penelitian ini selesai. The author would like to thank the staff Laboratory Analysis of Agricultural Technology Faculty of Agriculture, University of Udayana and Joint Laboratory, State University of Udayana who have provided help and support facilities until these studies are completed. REFFERENCE Anson, M.L., 1938, The Estimation of Pepsin, Trypsin, Papain and Cathepsin with Hemoglobin. Journal of General Physiologi, 22 : 79-89 Asakura, T., Watanabe, H. Abe, H., and Arai, S., 1997, Oryzasin as an Aspartic Proteinase Occuring in Rice Seeds: Purification, Characterization and Application to Milk Clotting, J. Agric. Food Chem., 45 (4) : 1070-1075 AOAC, 1995, Official Methods of Analysis of The Association of Official Analytical Chemist, AOAC, Washinton DC Folin, O., and Ciocalteu, W., 1927, On Tyrosine and Tryptophane Determinations in Proteins, J. Biol. Chem., 73 : 627-650 GE Healthcare Life Sciences, 2011, Instructions 28-9955-33 AB Hydrophobic interaction media CaptoTM Phenyl (high sub), General Electric Company Joseph, J.A., 2010, Menghilangkan Warna Hitam di Tumit,, diakses tanggal 1 Desember 2014 Juwarni, Septri Ayu A.A.,Ratnayani, K., Mayun, Laksmiwati A.A.I.A., 2014, Uji Aktivitas Protease Getah Labu Siam dan Talas Serta Perbandingannya Terhadap Getah Pepaya, Jurnal Kimia, 9 (2): 160-165 Lehninger, A.L., 1990, Dasar-dasar Biokimia Jilid I, a.b. Thenawidjaja, M., Erlangga, Jakarta Mahajan, R.T., and Shamnkant, B.B., 2010, Biological Aspects of Proteolytic Enzyme : Review, India J. Pharm., Research, 3 (9) : 2048-2068 Mehrnoush, A., Mustafa, S., Sarker, M.Z.I, and Yazid, A.M.M.Y., 2011, Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper) Leaves Using Response Surface Methodology, Molecules, 16 : 9245-9260 Noda, K., Koyanagi, M. and Kamiya, C., 1994, Purification and Characterization of an Endoprotease from Melon Fruit, J. Food Sci., 59 (3) : 585-587 Poedjiadi, A., 1994, Dasar-Dasar Biokimia, UI Press, Jakarta Scopes, R.K., 1982, Protein Purification Principle and Practice, Springer-Verlag, New York Seidman, L and Mowery, J., 2006, Salting Out: Ammonium Sulfate Precipitation, The Biotechnology Project, Illinois State University Sigma, 1999, Enzymatic Assay of Protease : Casein as A Substrat, Sigma Quality Control Test Procedure Wang, N.S., 2006, Enzyme Purification by Salt (Ammonium Sulfate) Precipitation, Department of Chemical Engineering, University of Maryland Witono, Y., 2008, Deklorofilasi Ekstrak Protease dari Tanaman Biduri (Calotropis gigantea) dengan Absorben Celite, Berk. Penel Hayati, 13 : 115-121