Karakterisasi dan Semipemurnian Enzim Selulase dan Ligninase dari Bakteri Asal Area Tambang Batuan Kapur.

Abstract

Area pertambangan batu kapur di Citeureup memiliki banyak lahan yang menyisakan sisa tanaman dan pepohonan, yang mendukung keanekaragaman hayati, khususnya sebagai habitat berbagai mikroorganisme, termasuk bakteri pengurai lignin dan selulosa untuk proses dekomposisi. Daun, ranting, dan batang tanaman terdiri atas 35% hingga 50% selulosa, 20% hingga 35% hemiselulosa, 10% hingga 25% lignin, serta komponen kecil lainnya. Keberadaan mikroorganisme dapat membantu proses dekomposisi, yaitu perubahan fisik maupun kimiawi yang terjadi secara sederhana. Proses dekomposisi yang dilakukan oleh bakteri dibantu oleh enzim yang secara tidak langsung menguraikan bahan organik seperti protein dan karbohidrat. Penelitian ini bertujuan mengisolasi, mengidentifikasi, melakukan semipemurnian, dan mengarakterisasi enzim selulase dan lakase dari isolat bakteri yang diperoleh dari area revegetasi tambang batu kapur Citeureup. Hasil isolasi dari dua titik sampel tanah di area revegetasi tambang batuan Citeureup didapatkan 9 isolat, dengan nilai indeks selulolitik berkisar antara 1,1 hingga 4,8. Sementara itu, 1 isolat menunjukkan aktivitas lignolitik pada medium guaiacol. Isolat III.1 menunjukkan aktivitas selulase tertinggi pada substrat karboksimetil selulosa (CMC), sedangkan isolat C.1.2 menunjukkan aktivitas lakase tertinggi pada substrat guaiacol. Aktivitas selulase maksimum isolat III.1 diamati pada jam ke-21, sedangkan aktivitas lakase maksimum isolat C.1.2 diamati pada jam ke-48. Dua isolat tersebut juga dipilih untuk dilakukan pemekatan dan karakterisasi enzim. Semipemurnian dilakukan menggunakan amonium sulfat. Aktivitas selulase dan lakase hasil pemekatan menunjukkan aktivitas yang lebih tinggi dibandingkan dengan enzim ekstrak kasar. Hasil pemekatan menunjukkan bahwa selulase dari isolat III.1 mampu mengendap pada konsentrasi 60-70%, menghasilkan peningkatan aktivitas enzim spesifik sebesar 9,25 kali lipat, sedangkan isolat C.1.2 mengendap pada konsentrasi 40-50% dengan peningkatan aktivitas enzim spesifik sebesar 6,99 kali lipat. Hasil dialisis isolat III.1 menunjukkan aktivitas optimum pada pH 6 dan 60 °C. Sebaliknya, isolat C.1.2 menunjukkan aktivitas optimum pada pH 4 dan 40 °C. Enzim hasil pengendapan lebih stabil dibandingkan dengan enzim ekstrak kasar. Analisis bioinformatika mengungkapkan bahwa isolat III.1 berkerabat dekat dengan Bacillus stercoris (kemiripan 99,77%) dan isolat C.1.2 berkerabat dekat dengan Metapseudomonas boanensis (kemiripan 99,88%).

Kata kunci: biodegradasi, lakase, selulase, tambang batuan kapur

The limestone mining area in Citeureup contains extensive areas of remaining plant and tree debris, which support biodiversity, particularly as habitats for various microorganisms, including lignin- and cellulose-degrading bacteria, which are essential for decomposition. Leaves, bark, and plant stems contain 35% to 50% cellulose, 20% to 35% hemicellulose, 10% to 25% lignin, and other minor components. The presence of microorganisms can aid the decomposition process, which involves simple physical and chemical changes. The decomposition process carried out by bacteria is aided by enzymes that indirectly break down organic materials such as proteins and carbohydrates. This study aims to isolate, identify, semi-purify, and characterize cellulase and laccase enzymes from bacterial isolates obtained from revegetation in the Citeureup limestone mining area. The results of isolation from two soil points in the revegetation area of the Citeureup rock mine yielded 9 isolates, with cellulolytic index values ranging from 1.1 to 4.8, while only 1 isolate showed lignolytic activity on guaiacol medium. Isolate III.1 showed the highest cellulase activity on carboxymethyl cellulose (CMC) substrate, while isolate C.1.2 showed the highest laccase activity on guaiacol substrate. The maximum cellulase activity of isolate III.1 was observed at 21 hours, while the maximum laccase activity of isolate C.1.2 was observed at 48 hours. The two isolates were also selected for enzyme concentration and characterization. Semi-purification was carried out using ammonium sulfate. The concentrated cellulase and laccase activity showed higher activity than the crude extract enzymes. The concentration results showed that cellulase from isolate III.1 was able to precipitate at a concentration of 60-70%, resulting in a 9.25-fold increase in specific enzyme activity, while isolate C.1.2 precipitated at a concentration of 40-50% with a 6.99-fold increase in specific enzyme activity. The dialysis results of isolate III.1 showed optimum activity at pH 6 and 60 °C. In contrast, isolate C.1.2 showed optimum activity at pH 4 and 40 °C. The precipitated enzyme was more stable than the crude extract enzyme. Bioinformatics analysis revealed that isolate III.1 was closely related to Bacillus stercoris (99.77% similarity), and isolate C.1.2 was closely related to Metapseudomonas boanensis (99.88% similarity). Keywords: biodegradation, cellulase, laccase, limestone mining area