Isolasi Bakteri Keratinolitik Dari Limbah Bulu Ayam Dan Karakterisasi Enzim Keratinasenya

Abstract

Penelitian mengenai isolasi bakteri keratinolitik dari limbah bulu ayam dan karakterisasi enzim keratinasenya telah dilaksanakan. Isolasi dilakukan menggunakan medium Skim Milk Agar dan Feather Meal Agar yang diinkubasi pada suhu 31 oC dan pH 7. Uji indeks keratinolitik dilakukan di medium Feather Meal Agar. Presipitasi enzim kasar dilakukan dengan penambahan amonium sulfat secara bertingkat dan dialisis. Pengaruh pH dan suhu terhadap aktivitas keratinase dilakukan untuk mengetahui karakteristik masing-masing enzim. Sembilan belas isolat dengan karakteristik morfologi yang berbeda diperoleh dari hasil isolasi. Tiga isolat yaitu A4, B4, dan B6 dipilih untuk pengujian lanjut berdasarkan indeks keratinolitik. Aktivitas tertinggi diperoleh keratinase isolat A4 pada konsentrasi amonium sulfat 20% yaitu sebesar 95,9 U/ml, dan keratinase isolat B4 dan B6 pada konsentrasi amonium sulfat 70%, berturut-turut sebesar 57,5 U/ml dan 71,2 U/ml. Aktivitas spesifik keratinase setelah dialisis isolat A4 yaitu sebesar 4,61 U/mg, isolat B4 sebesar 3,13 U/mg, dan isolat B6 sebesar 4,08 U/mg. Aktivitas keratinase isolat A4 optimum pada pH 8 dan suhu 45 oC, keratinase isolat B4 optimum pada pH 7 dan suhu 35 oC, dan keratinase isolat B6 optimum pada pH 7 dan suhu 40 oC. Identifikasi berdasarkan gen 16S rRNA menunjukkan bahwa isolat A4, B4 dan B6 berturut-turut mendekati Leclercia adecarboxylata strain M-X17B dengan homologi 97%, Azotobacter chroococcum strain ABA-1 dengan homologi 85%, dan Stenotrophomonas maltophilia strain BIW dengan homologi 96%.

A study on isolation of keratinolytic bacteria from feather waste and characterization of its keratinase has been done. Isolation was conducted on Skim Milk Agar and Feather Meal Agar incubated at 31 oC and pH 7. Index keratinolytic was measured on Feather Meal Agar. Crude enzyme presipitation was performed by addition the concentration series of ammonium sulfate followed by dialysis. The influences of pH and temperature to keratinase activity were studied to examine the characteristics of each enzyme. Nineteen bacteria with different morphological characteristics were isolated from feather waste. Three isolates, A4, B4, and B6, were chosen for futher studied based on their keratinolitic index. The highest production of keratinase of A4 isolate was shown on 6 th day, B4 isolate was on 4 th day, and B6 isolate was on 5 th day. The maximum keratinase activity was produced by A4 isolate at 20% ammonium sulfate of 95.9 U/ml, B4 and B6 isolates produced it at 70% ammonium sulfate of 57.5 U/ml and 71.2 U/ml respectively. The specific activity after dialysis were 4.61 U/mg for A4 isolate, 3.13 U/mg for B4 isolate, and 4.08 U/mg for B6 isolate. The optimum of keratinase A4 isolate was at pH 8 and 45 oC, B4 isolate was at pH 7 and 35 °C, and B6 isolate was at pH 7 and 40 °C. Identification based on 16S rRNA gene showed that A4, B4 and B6 isolates closed to Leclercia adecarboxylata strain M-X17B with similarity of 97%, Azotobacter chroococcum strain ABA-1 with similarity of 85%, and Stenotrophomonas maltophilia strain BIW with similarity of 96%, respectively