Ekstraksi dan karakterisasi enzim tripsin dari jeroan ikan bandeng (Chanos chanos) hasil purifikasi parsial Extraction and characterization of trippsin enzyme from milkfish (Chanos chanos) partial purification result
Abstract
Fish offal is known for its high content of digestive enzymes, especially proteinases such as trypsin. The characteristics of fish trypsin are influenced by the species and habitat. Milkfish offal is part of the fish digestive tract and has a neutral pH, so it is a potential source of enzymes, especially trypsin. Fish offal can be an alternative to commercial trypsin enzymes derived from pork and beef. The purpose of this study was to determine the characteristics of trypsin enzyme from milkfish offal with ammonium sulfate fraction. The research process began with the extraction of trypsin enzyme from milkfish offal, followed by fractionation with ammonium sulfate (0-80%). Temperature, pH, the effect of metal ions, the effect of NaCl on enzyme activity, and reaction kinetics were all used to determine the best fraction. Enzyme activity was measured using the substrate N-α-benzoyl DL-arginine-p-nitroanilide (BAPNA). The results showed that the crude trypsin extract had an activity of 0.117 U/mL. The highest trypsin enzyme activity was in the 20-30% ammonium sulfate fraction, which was 0.295 U/mL. The enzyme worked best at a temperature of 50°C and pH 6. The metal ions ZnCl₂, MnCl₂, CuCl₂, NaCl, and CaCl₂ inhibited the trypsin enzyme activity. Trypsin activity in 5–30% NaCl increased to a ratio of 10%, which was 0.249 U/mL. The Vmax value of this enzyme was 0.285 mmol/s, and the Km value was 0.374 mM.
References
Arndt, C., Koristka, S., Bartsch, H., & Bachmann M. (2012). Native polyacrylamide gels. Methods in Molecular Biology, 869, 49-53. https://doi.org/10.1007/978-1-61779-821-4_5
Asgeirsson, B., Fox, J. W., & Bjarnason, J. B. (1989). Purificatrion and characterization of trypsin from the polikilotherm Gadus morhua. European Journal of Biochemistry, 180, 85-94.
Atma, Y., & Ramdhani, H. (2017). Identifikasi gelatin dari tulang ikan patin hasil ekstraksi menggunakan kulit nanas dengan elektroforesis vertical. Prosiding Seminar Nasional Sains dan Teknologi, 1-7.
Barkia, A., Bougatef, A., Nasri, R., Fetoui, E., Balti, R., & Nasri, M. (2010). Trypsin from the viscera of Bogue (Boop boops): isolation and characterization. Fish Physiology Biochemistry, 36, 893-902. https://doi.org/10.1007/s10695-009- 9365-z
Burgess, R. R. (2009). Protein precipitation techniques. Di dalam: Burgess dan Murray, editor. Methods in Enzymology volume 463. Wisconsin (US): Elsevier Inc. hlm 331-342.
BPPT [Badan Pengkajian dan Penerapan Teknologi]. 2014. Menuju kemandirian enzim nasional. https://inspektorat.bppt.go.id/.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-254. https://doi.org/10.1016/0003- 2697(76)90527-3
[CCI] Citra Cendekia Indonesia. 2017. Perkembangan Impor Enzim Indonesia. Diperoleh dari http://cci-indonesia.com/perkembangan-impor-enzim- indonesia/
Churry. 2017 Apr 29. BPPT-Petrosida Gresik Resmikan Pabrik Enzim Pertama di Indonesia. It Works.
Falch, E. A. (1991). Industrial enzymes developments in production and application. Biotechnology advances, 9(4), 643-658.
Fitriani, E. (2003). Aktivitas enzim karboksimetil selulase Bacillus pumilus galur 55 pada berbagai suhu inkubasi. Bogor: Kimia FMIPA IPB.
[GVR] Grand View Research. (2020). Enzymes Market Size, Share & Trends Analysis Report by Product (Lipases, Polymerases & Nucleases, Carbohydrase), by Type (Industrial, Specialty), by Source (Plants, Animals), by Region, and Segment Forecasts, 2022 - 2030.
Ketnawa, S., Benjakul, S., Martinez-Alvarez, O., & Rawdkuen, S. (2017). Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: bioactivity and stability. Food Chemistry, 215, 383-390.
Khaled, H. B., Jellouni, K., Souissi, N., Ghorbel, S., Barkia, A., & Nasri, M. (2011). Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita). Food Chemistry, 37, 123–133. https://doi.org/10.1016/ j.foodchem. 2011.05.014
Khandagale, A. S., Mundodi, L., & Sarojini, B. K. (2017). Isolation and characterization of trypsin from fish viscera of oil sardine (Sardinella longiceps). International Journal of Fisheries and Aquatic Studies, 5(2), 33-37.
Khangembam, B. K., Yadavilli, K. S., & Chakrabarti, R. (2012). Purification and characterization of trypsin from the digestive system of carp Catla catla (Hamilton). International Aquatic Research, 4(1), 1 12. https://doi.org/10.1186/2008-6970-4-9
Khantaphan, S., & Benjakul, S. (2010). Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta). Food Chemistry, 120, 658–664. https://doi.org/10.1016/j.foodchem.2009.09.098
Kim, M., & Jeong, Y. (2015). Purification and characterization of trypsin-like protease from faltfish (paralichthy olivaceus) intestine. Journal of Food Biochemistry, 37, 732-741.
Kita, Y., Arakawa, T., Lin, T. Y., & Timasheff, S. (1994). Contribution of the surface free
energy perturbations to protein solvent interactions. Biochemistry, 33,
-15189.
[KKP] Kementerian Kelautan dan Perikanan. (2023). Statistik Kelautan dan Perikanan 2023. Jakarta: Kementerian Kelautan dan Perikanan.
Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B. K., & Saeki, H. (2007). Trypsins from yellowfin tuna (Thunnus albacores) spleen: Purification and characterization. Comparative Biochemistry and Physiology, B (144), 47–56.
Klomklao, S., Benjakul, S., & Visessanguan, W. (2004). Comparative studies on proteolytic activity of splenic extract from three tuna species commonly used in Thailand. Journal of Food Biochemistry, 28(5): 355–372. https://doi.org/10.1111/j.1745-4514.2004.05203.x
Ktari, N., Khaled, H. B., Nasri, R., Jellouli, K., Ghorbel, S., & Nasri, M. (2012). Trypsin from zebra blenny (Salaria basilisca) viscera: purification, characterization and potential application as a detergent additive. Food Chemistry, 130, 467–474. https://doi.org/10.1016/j.foodchem.2011.07.015
Lehninger, A. L. (1997). Dasar-Dasar Biokimia. Thenawidjaja M, penerjemah; Suwarman, editor. Jakarta (ID): Penerbit Erlangga. Terjemahan dari: Principle of Biochemistry.
Liu, H., Zhou, X., Tian, S., Hao, X., You, J., & Zhang, Y. (2014). Two-step transpeptidation of the insulin precursor expressed in Pichia pastoris to insulin ester via trypsin-catalyzed cleavage and coupling. Biotechnol Appl Biochem, 61(4), 408-417.
Liu, H., Zhou, X., Xie, F., You, J., & Zhang, Y. (2013). An efficient trypsin digestion strategy for improving desB30 productivity from recombinant human insulin precursor fusion protein. Process Biochem, 48(5-6), 965-971.
Mao, Y. H., Krischke, M., Hengst, C., & Kulozik, U. (2018). Comparison of the influence of pH on the selectivity of free and immobilized trypsin for beta-lactoglobulin hydrolysis. Food Chemistry, 253, 194–202.
Muallifa, A. Y. (2017). Mengurai hadis tahnik dan gerakan anti vaksin. Jurnal Living Hadis, 2(2), 253-269.
Namjou, F., Yeganeh, S., Madani, R., & Ouraji, H. (2019). Extraction, purification, and characterization of trypsin obtained from the digestive system of yellowfin seabream (Acanthopagrus latus). Archives of Razi Institute, 74(4), 405-411. https://doi.org/10.22092/ari.2018.122854.1229
Nurhayati, T., Nugraha, R., & Lihuana, D. N. (2020). Karakterisasi fraksi ammonium sulfat tripsin yang diisolasi dari usus ikan tongkol (Euthynnus affinis). Jurnal Pengolahan Hasil perikanan Indonesia, 23(2), 372-382.
Nurhayati, T., Arbajayanti, R. D, & Nurilmala, M. (2021). Komponen asam amino dan aktivitas enzim tripsin dari usus tuna sirip kuning (Thunnus albacares) dan kakap merah (Lutjanus campechanus). Jurnal Pengolahan Hasil Perikanan Indonesia, 24(1), 97-106.
Nurhayati, T., Ramadhan, W., & Raharja, T. F. K. (2022, June). Microencapsulation of trypsin from the intestine of yellowfin tuna (Thunnus Albacares). In IOP Conference Series: Earth and Environmental Science (1033(1) p. 012058). IOP Publishing.
Nurhayati, T., Abdullah, A., Rahmawati, S., Kurniawan, R. (2024). Characteristics of trypsin isolated from intestines different of fish and correlation toward trypsin activity. Squalen Bulletin Marine and Fisheries Postharvest and Biotechnology, 19(2), 131-143. https://doi.org/10.15578/squalen.904
Parfitt, K. (1999). Martindale and Complete Drug Reference. Ed ke-13. (USA): Pharmaceutical Press.
Ranasasmita, R. (2015). Kehalalan produk obat-obatan, terutama obat herbal. Di dalam: Ranasasmita R, Roswiem AP, editor. Prosiding Simposium Penelitian Bahan Obat Alami XIV [Internet] terutama obat herbal. Prosiding Simposium Penelitian Bahan Obat Alami. 2014; 14, 552–9.
RISTEKDIKTI [Kementerian Riset, Teknologi, dan Pendidikan Tinggi Republik Indonesia]. 2017. Kemandirian Produk Enzim Indonesia. [Internet]. Tersedia pada: https://www.ristekdikti.go.id/-kabar/kemandirian-produk-enzim indonesia/#cjeI58Ws3gvTW5vt.99.
Silva, J. F., Esposito, T. S., Marchuschi, M., Ribeiro, K., Cavalli, R. O., Oliveira, V., & Bezerra, R. S. (2011). Purification and partial characterization of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus). Food Chemistry, 129(3), 777-782.
Sinaga, M., Nugroho, T. T., & Dahliaty, A. (2014). Pemekatan enzim selulase Penicillium sp. LBKURCC20 dengan pengendapan amonium sulfat 80%. Jurnal Online Mahasiswa Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Riau, 1(2), 283-288.
Trismillah, Sumaryono, W., Malik, A., & Sadikin, M. (2014). Isolasi dan karakterisasi protease serupa (PST) dari Lactobacillus plantarum FNCC 0270. Jurnal Ilmu Kefarmasian Indonesia, 12(1), 57-66.
Zhang, Y., Liang, Q., Zhang, C., Zhang, J., Du, G., & Kang, Z. (2020). Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor. Microbial Cell Factories, 19(1), 1-11.
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