Hidrolisis Protein dari Biji Jarak Kepyar (Ricinus communis) dengan Kimotripsin dan Uji Aktivitas Antibakteri Peptida yang Dihasilkan

ZEHAN ANDRIANA, Prof. Tri Joko Raharjo, S.Si., M.Si., Ph.D.; Prof. Dra. Tutik Dwi Wahyuningsih, M.Si., Ph.D.

2023 | Tesis | MAGISTER KIMIA

Telah dilakukan penelitian identifikasi peptida antibakteri dari hidrolisat kimotripsin protein biji jarak kepyar Ricinus communis terfraksinasi dengan menggunakan reversed-phase high performance liquid chromatography (RP-HPLC). Protein biji jarak kepyar diekstraksi dengan SDS, TFA, dan akuades. Ekstrak protein dihidrolisis secara enzimatis menggunakan kimotripsin. Hidrolisat protein difraksinasi menggunakan RP-HPLC yang dielusi menggunakan gradien komposisi campuran 6 dan 60% asetonitril dalam 0,1% TFA yang ditingkatkan secara bertahap. Fraksi peptida diuji aktivitas antibakteri terhadap E. coli dan S. aureus menggunakan metode mikrodilusi. Fraksi peptida yang memiliki aktivitas antibakteri diidentifikasi menggunakan high resolution mass spectrometry (HRMS). Ekstraksi protein biji jarak kepyar menghasilkan persen rendemen sebesar 18,55; 59,28; 68,35 dan 69,8% berturut-turut untuk ekstrak SDS dialisis, SDS amicon, TFA dan akuades. Hidrolisis protein biji jarak kepyar dengan kimotripsin menghasilkan nilai derajat hidrolisis berturut-turut untuk SDS, TFA dan akuades sebesar 32; 55,13 dan 83,92%. Fraksi peptida ekstrak akuades memiliki 5 fraksi antibakteri dengan spektrum luas yaitu fraksi 2, 3, 5, 6 dan 10 dengan nilai IC50 masing-masing yaitu 7,48; 12,54; 10,81; 14,12 dan 14,57 µg/mL terhadap E. coli serta 10,07; 9,68; 15,06; 13,37 dan 14,47 µg/mL terhadap S. aureus. Urutan asam amino peptida yang diduga berpotensi sebagai senyawa antibakteri yaitu NVLRGKGMASL.

Studies have been conducted to identify antibacterial peptidas from chymotrypsin protein hydrolysate castor bean seeds (Ricinus communis) fractionated with reversed-phase high performance liquid chromatography (RP-HPLC). Castor bean protein was extracted with sodium dodesyl sulfate (SDS), TFA and water. Protein extracts were hydrolyzed enzymatically using chymotrypsin. Protein hydrolysate was fractionated using RP-HPLC eluted with gradient composition of acetonitrile 6 and 60% in 0.1% TFA which increased gradually. Peptida fractions were tested for antibacterial activity against E. coli and S. aureus using the microdilution method. Peptida fractions with antibacterial activity were identified using high resolution mass spectrometry (HRMS). Castor bean protein extraction yielded 18.55; 59.28; 68.35 and 69.8% yields respectively for dialisis SDS, amicon SDS, TFA and aquadest extracts. Hydrolysis of the protein with chymotrypsin resulted in successive hydrolysis degree values of 32; 55.13 and 83.92% for SDS, TFA and water extract. The peptide fraction of water extract has 5 antibacterial fractions with broad spectrum, namely fractions 2, 3, 5, 6 and 10 with an IC50 value of each fractions are 7.48; 12.54; 10.81; 14.12 and 14.57 µg/mL against E. coli and 10.07; 9.68; 15.06; 13.37 and 14.47 µg/mL against S. aureus. The amino acid sequence of the peptide that is predicted to have the potential antibacterial activity is NVLRGKGMASL.

Kata Kunci : ekstraksi protein, biji jarak, kimotripsin, RP-HPLC, peptida antibakteri/protein extraction, castor bean, chymotrypsin, RP-HPLC, antibacterial peptide.