Pemurnian dan Karakterisasi Kitosanase Bacillus coagulans LH 28.38
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Balai Besar Penelitian dan Pengembangan Pascapanen Pertanian
Abstract
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Enzim ekstrasellular kitosanase telah berhasil dimurnikan dan dikarakterisasi dari bakteri termofilik Bacillus coagulans LH 28.38 asal tanah geotermal Lahendong - Sulawesi Utara. Media pertumbuhan mengandung 0,4% koloidal kitosan sebagai sumber karbon dan diproduksi optimum pada suhu 55°C selama tujuh hari. Pemurnian enzim dilakukan dengan teknik filtrasi gel menggunakan matriks Sephadex G-IOO menghasilkan tiga puncak protein sebagai kitosanase fraksi A. B, C dan masing masing memiliki aktivitas sebesar 13.33 U/mg, 38.66 U/mg dan 88 U/111g. Kitosanase fraksi A memiliki suhu optimum pad a 60°C dan bekerja pad a kisaran pH luas 2-12. Kitosanase fraksi B memiliki suhu optimum 70°C dan bekerja pada kisaran pH 2-12. Kitosanase f'raksi C memiliki suhu optimum 60°C dan kisaran pH pH 2-9. Penentuan berat molekul enzim dilakukan dengan cara SDS-PAGE dan anal isis zimogram. Berm molekul enzim yang diperoleh 93, 91, 87, 77.6 dan 74 kDa. Enzim kitosanase ini dapat diaplikasikan untuk memperoleh oligomer kitosan. Oligomer kitosan sangat diminati oleh kalangan medis, karena mamiliki aktivitas anti tumor, anti kolesterol dan anti mikroba. Purification lind characterization of chitosanase from Bacillus coagulans LH 28.38Characterization and purification of the extracellular chitosanase produced by Bacillus coagulans LH 28.38 from geothermal soil origin from Lahendong - North Sulawesi has been successfully done. The enzyme was produced in medium containing 0.4% colloidal chitosan as sole carbon source and reached the highest level after 7"' day incubation at 55°C. The purification of enzyme was done by column chromatography using Sephadex G-IOO as matrix produced three peaks of protein chitosanase named as Chitosanase fraction A, Band C while has 13.3 U/mg; 38.66 U/mg and 88 U/mg activity, respectively. Chitosanase fraction A has optimal temperature at 60°C and work at range pH 2-12. Chitosanase fraction B has optimal temperature at 70°C and work at range pH 2-12. Chitosanase fraction C has optimal temperature 70°C and work at range pH 2-9. Molecular weight of chitosanase was determined by SDS-PAGE and zymograrn analysis. The molecular weight of enzyme was estimated to be 93, 91. 87. 77.6 and 74 kDa. This chitosanase could be applied to produce oligochitooligosaccaride. Oligochitooligosaccaride was used for medical popuse by medical, due to have the hipokolesterolemik, antimicrobial and antitumor activities.
Keywords
kitosanase; Bacillus coagulans; chitosanase.