Characterization of Ammonium Sulphate Fraction Tripsin Isolated from Intestine of Little Tuna: Trypsin from little tuna

Intestine is part of the digestive tract of fish having neutral pH. Thus, fish intestine is a potential source of typsin and can be an alternative source to the commercial trypsin enzyme derived from pork and cattle. This study was aimed to analyze the characteristics of the ammonium sulphates fraction of the trypsin enzyme from tuna intestine. The research began with the extraction of the trypsin enzyme from the little tuna intestine and followed up by fractionation using ammonium sulphates (0-80%). Optimum temperature and pH as well as effects of metal ions, stability to NaCl, optimum substrate and reaction kinetics were determined. Enzyme activity was measured using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The results showed that the trypsin crude extract had an activity of 0.205 U / mL. The highest enzyme activity was found in the ammonium sulphates fraction of 40-50% . The enzyme worked optimally at 60°C and pH 9. Metal ions including ZnCl2 and CaCl2 inhibited trypsin activity, while MnCl2, CuCl2, and NaCl increased trypsin activity. Trypsin activity was stable at 5-30% NaCl. The enzyme had a Vmax value of 0.42 mmol/s and a Km value of 1.12 mM.Usus ikan tongkol merupakan bagian saluran pencernaan ikan yang memiliki pH neteral sehingga berpotensi sebagai sumber enzim, yaitu enzim tripsin. Enzim tripsin dari jeroan ikan bisa menjadi alternatif dari enzim tripsin komersil yang berasal dari babi dan sapi. Penelitian ini bertujuan menganalisis karakteristik fraksi amonium sulfat enzim tripsin dari usus ikan tongkol. Tahapan penelitian diawali dengan ekstraksi enzim tripsin dari usus ikan tongkol lalu difraksinasi menggunakan amonium sulfat (0-80%). Fraksi terbaik dikarakterisasi suhu, pH, pengaruh ion logam, stabilitas terhadap NaCl, dan substrat optimum serta dilakukan analisis kinetika reaksi. Pengukuran aktivitas enzim menggunakan substrat N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA). Hasil penelitian menunjukkan bahwa ekstrak kasar tripsin memiliki aktivitas sebesar 0,205 U/mL. Aktivitas enzim tripsin tertinggi terdapat pada fraksi amonium sulfat fraksi 40-50% sebesar 0,248 U/mL. Enzim bekerja optimum bekerja pada suhu 60°C dan pH 9. Ion logam ZnCl2 dan CaCl2 menghambat aktivitas tripsin, sedangkan MnCl2, CuCl2, dan NaCl dapat meningkatkan aktivitas tripsin. Aktivitas tripsin stabil pada NaCl 5-30%. Enzim tersebut memiliki nilai Vmaks sebesar 0,42 mmol/s dan nilai Nilai Km sebesar 1,12 m