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J. Math. Fund. Sci., Vol. 48, No. 2, 2016, 149-163 149 Production and Characterization of Chitinases from Thermophilic Bacteria Isolated from Prataan Hot Spring, East Java Ruth Chrisnasari, Steven Yasaputera, Pauline Christianto, Veronika Ivone Santoso Tjandra Pantjajani Department of Biology, Faculty of Biotechnology, University of Surabaya, Jalan Raya Kalirungkut, Surabaya 60292, Indonesia Email: ruth_c@staff.ubaya.ac.id Abstract. Thermophilic bacteria producing chitinase were collected from Prataan hot spring, East Java, Indonesia and screened. The isolated bacterium was analyzed using 16S rRNA gene sequencing analysis and identified as Paenibacillus sp. The molecular identification was confirmed through morphological and physiological analyses. The production of chitinase was conducted at various incubation times, temperatures, pH and concentrations of colloidal chitin. The optimum condition of the isolate to produce the highest chitinase was 0.9% (w/v) of colloidal chitin (pH 7.0) at 48 °C for 24 hours. The obtained chitinases were optimally active at 55 °C and pH 6.0-7.0. The chitinases were gradually purified by ammonium sulfate precipitation, Sephadex G-100 gel filtration, followed by DEAE–cellulose ion exchange chromatography (IEC). The purification method gave a purification factor of 9.43 and a yield of 2.68%. Two protein fractions were obtained from sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS) with molecular weights of 68 and 82 kDa. Keywords: chitin; chitinase; Paenibacillus sp.; Prataan hot spring; thermophilic bacteria. 1 Introduction Chitinases (EC 4) are a