BAB III. METODE PENELITIAN
4.5. Kadar Protein
Kadar protein yang terlarut dalam ekstrak kasar enzim protease ditentukan dengan analisis secara kuantitatif menggunakan metode Bradford. Persamaan regresi linier kurva standar Bovine Serum Albumin yang diperoleh adalah y = 0,009x + 0,0042 dengan koefisien regresi linier (R2) = 0,995 (Lampiran 3). Nilai absorbansi dari pengukuran kadar protein pada λ595 nm dimasukkan ke dalam persamaan tersebut untuk mendapatkan nilai kadar protein.
Analisis kadar protein dengan metode Bradford melibatkan pewarna Coomassie Brilliant Blue (CBB) G-250 yang membentuk warna kebiruan pada larutan sampel. Hal ini karena CBB G-250 berikatan dengan protein yang terdapat pada larutan enzim dalam suasana asam dan membentuk warna larutan kebiruan (Masri, 2013). Kadar protein diukur pada λ595 karena absorbansi yang dihasilkan sebanding dengan pewarna yang terikat pada protein (Malle et al., 2015).
Gambar 10. Nilai kadar protein enzim protease isolat JE-DP4 menggunakan substrat; (a) kasein, (b) susu skim
Kadar protein yang diukur dari ekstrak kasar enzim protease menunjukkan jumlah substrat yang telah didegradasi oleh mikroorganisme proteolitik. Kadar protein mengggunakan substrat kasein tertinggi sebesar 0,107 mg/mL pada hari ke-1 inkubasi karena jumlah substrat protein masih tinggi. Pada waktu inkubasi berikutnya jumlah substrat kasein terus mengalami penurunan hingga akhir waktu inkubasi hari ke-7 (Gambar 10a). Kadar protein menggunakan substrat susu skim tertinggi sebesar 0,068 mg/mL pada hari ke-1 inkubasi karena jumlah substrat protein masih tinggi. Pada waktu inkubasi berikutnya substrat protein susu skim terus mengalami penurunan hingga akhir waktu inkubasi (Gambar 10b). Kadar protein mengalami penurunan seiring dengan pertambahan umur isolat kapang.
Hal ini karena enzim protease yang dihasilkan oleh kapang endofit proteolitik b
a
mampu mendegradasi substrat protein kasein dan susu skim. Kespesifikan suatu enzim terhadap substrat dapat diketahui dari kemampuan enzim dalam mendegradasi substrat tersebut untuk menyintesis enzim (Sajuthi et al., 2010).
Jain et al. (2012), melaporkan dalam penelitiannya kadar protein yang diuji dari enzim protease yang dihasilkan oleh kapang endofit Acremonium sp. sebesar 0,02 mg/mL. Ramadhani et al. (2015), juga melaporkan dalam penelitiannya kadar protein enzim protease tertinggi yang dihasilkan dalam pengujian aktivitas enzim protease oleh Aspergillus niger PAM18A sebesar 0,460 mg/mL. Hal ini menunjukkan bahwa penggunaan substrat protein untuk menyintesis enzim protease setiap jenis kapang endofit berbeda dipengaruhi oleh variasi gen penyandi protease dan kemampuan kapang dalam mendegradasi substrat protein.
Rata-rata kadar protein total enzim protease isolat kapang endofit JE-DP4 menggunakan substrat kasein sebesar 0,063 mg/mL sedangkan menggunakan substrat susu skim sebesar 0,047 mg/mL. Kadar protein yang diukur pada substrat kasein sebesar 0,063 mg/mL lebih tinggi dari 0,047 mg/mL pada substrat susu skim. Hal ini menunjukkan bahwa aktivitas enzim protease yang dihasilkan menggunakan substrat kasein lebih tinggi dibandingkan susu skim. Kandungan protein dalam enzim berpengaruh terhadap daya katalitik enzim (Ramadhani et al., 2015). Kadar protein yang tinggi tidak berarti menunjukkan kandungan enzim protease yang tinggi. Hal ini karena protein yang mengendap bukan hanya dari enzim protease tetapi dari protein non enzim yang terukur ketika analisis kadar protein (Pasaribu et al., 2018).
41 BAB V
KESIMPULAN DAN SARAN 5.1. Kesimpulan
Berdasarkan hasil penelitian yang telah dilakukan maka dapat disimpulkan:
1. Isolat kapang endofit JE-DP4 memiliki kemampuan terbaik menghasilkan enzim protease dengan indeks hidrolisis protein sebesar 1,45 dibandingkan kedua isolat lain yang tidak menghasilkan zona bening.
2. Substrat media produksi berupa kasein menghasilkan aktivitas enzim protease lebih tinggi dibandingkan susu skim dengan aktivitas enzim tertinggi sebesar 46,72 U/mL.
3. Rata-rata aktivitas enzim protease tertinggi isolat kapang endofit JE-DP4 sebesar 22,64 U/mL pada suhu 37°C, waktu inkubasi pada hari ke-7, dan pH pertumbuhan sebesar 6 menggunakan substrat kasein.
5.2. Saran
Saran dari penelitian ini adalah:
1. Perlu adanya analisis molekuler isolat kapang endofit JE-DP4 yang diisolasi dari daun tanaman pepaya untuk diidentifikasi.
2. Perlu dilakukan pengujian variasi pH aktivitas enzim protease isolat kapang endofit JE-DP4 untuk mengetahui faktor lain yang memengaruhi aktivitas enzim.
3. Kapang endofit daun pepaya yang kemampuan aktivitas enzimnya menurun dapat diinduksi dengan penambahan ekstrak daun pepaya pada media pertumbuhan kapang agar kondisinya sesuai lingkungan tempat tumbuh kapang endofit.
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51 LAMPIRAN
Lampiran 1. Komposisi reagen 1. Kasein 1%
a. Kasein 0,5 g b. Akuades 50 mL 2. Asam trikloroasetat 5%
a. Asam trikloroasetat 2,5 g b. Akuades 50 mL
3. Bufer Tris-HCl 0,2 M pH 7,5
a. Tris Base (hydroxymethyl)-aminomethan 1,211 g b. Akuades 50 mL
c. HCl pekat 4. Reagen Bradford
a. Coomassie Brilliant Blue G-250 25 mg b. Metanol 50 mL
c. Asam fosfat (H3PO4) 85% 50 mL d. Akuades 250 mL
e. Disaring, ditambahkan akuades 175 mL 5. Larutan standar tirosin stok 3 mg/mL
a. L-tyrosine 120 mg b. Akuades 20 mL
6. Larutan standar Bovine Serum Albumin (BSA) stok 50 µg/mL a. BSA 0,25 mg
b. Akuabides 5 mL
Lampiran 2. Hasil pengukuran larutan dan kurva standar tirosin a. Pengukuran larutan standar tirosin
No. L-tyrosine (mg/mL)
Stok (mL)
HCl 0,1 M (mL)
Nilai Absorbansi (λ280 nm)
1. 0,01 0,01 0,99 0,119
2. 0,02 0,02 0,98 0,250
3. 0,03 0,03 0,97 0,364
4. 0,04 0,04 0,96 0,457
5. 0,05 0,05 0,95 0,548
6. 0,06 0,06 0,94 0,637
7. 0,07 0,07 0,93 0,774
8. 0,08 0,08 0,92 0,848
9. 0,09 0,09 0,91 0,939
10. 0,10 0,10 0,90 0,984
b. Kurva standar tirosin
Lampiran 3. Hasil pengukuran larutan dan kurva standar bovine serum albumin (BSA)
a. Pengukuran larutan standar BSA
No. BSA
(µg/mL)
Stok (mL)
ddH2O (mL)
Nilai Absrobansi
(λ595 nm)
1. 2 0,08 1,92 0,0217
2. 4 0,16 1,84 0,0387
3. 6 0,24 1,76 0,0531
4. 8 0,32 1,68 0,0786
5. 10 0,40 1,60 0,1000
6. 12 0,48 1,52 0,1177
7. 14 0,56 1,44 0,1321
8. 16 0,64 1,36 0,1428
9. 18 0,72 1,28 0,1680
10. 20 0,80 1,20 0,1825
b. Kurva standar BSA
Lampiran 4. Nilai pH pertumbuhan kapang endofit JE-DP4 menggunakan substrat kasein dan susu skim
No. Hari Ulangan
Substrat kasein Substrat susu skim
pH ƩpH pH ƩpH
Lampiran 5. Konsentrasi tirosin yang dilepaskan hasil hidrolisis protein
Lampiran 5. Konsentrasi tirosin yang dilepaskan hasil hidrolisis protein
