Isolasi, Karakterisasi, dan Identifikasi Cendawan Penghasil Enzim Lakase dan Selulase dari Area Revegetasi Tambang Batuan Kapur.

Abstract

Penambangan batu kapur dengan sistem terbuka menyebabkan perubahan kondisi fisikokimia tanah melalui hilangnya lapisan tanah atas, rendahnya kandungan bahan organik dan unsur hara, serta terbatasnya kelembapan tanah. Revegetasi menjadi salah satu upaya pemulihan lahan pascatambang yang meningkatkan masukan serasah dan residu akar sebagai sumber biomassa lignoselulosa. Dekomposisi biomassa tersebut memerlukan aktivitas mikroorganisme penghasil enzim ekstraseluler, terutama lakase dan selulase, yang berperan dalam penguraian lignin dan selulosa. Cendawan indigenus dari tanah area revegetasi berpotensi menghasilkan kedua enzim tersebut, tetapi informasi mengenai aktivitas enzim, karakteristik biokimia, dan identitas taksonominya pada tingkat isolat masih terbatas. Penelitian ini bertujuan mengisolasi dan menyeleksi cendawan indigenus yang memiliki aktivitas ligninolitik dan selulolitik, mengarakterisasi lakase dan selulase dari isolat terpilih, serta mengidentifikasi isolat tersebut berdasarkan karakter morfologi dan analisis molekuler daerah internal transcribed spacer (ITS). Sampel tanah diambil dari area revegetasi tambang batuan kapur yang didominasi tanaman jati dan sengon. Cendawan diisolasi melalui pengenceran bertingkat dan ditapis berdasarkan aktivitas ligninolitik dan selulolitik. Aktivitas lakase dan selulase isolat positif diukur selama kultivasi. Enzim dari isolat terpilih dimurnikan secara parsial melalui fraksinasi amonium sulfat dan dialisis, kemudian dianalisis menggunakan SDS-PAGE serta dikarakterisasi berdasarkan pengaruh pH dan suhu. Identifikasi isolat dilakukan melalui pengamatan morfologi, analisis sekuen ITS, dan rekonstruksi pohon filogenetik. Sebanyak 12 isolat cendawan berhasil diperoleh, dengan dua isolat menunjukkan aktivitas ligninolitik dan tiga isolat menunjukkan aktivitas selulolitik. Isolat FT.1.4 menghasilkan aktivitas lakase tertinggi sebesar 0,496 U/mL pada hari ke-9, sedangkan FT.1.1 menghasilkan aktivitas selulase tertinggi sebesar 0,488 U/mL pada hari ke-12. Kedua isolat menunjukkan respons ?-hemolisis. Fraksi amonium sulfat 40–60% menghasilkan aktivitas spesifik tertinggi pada kedua enzim. Analisis SDS-PAGE menunjukkan pita protein berukuran sekitar 56,2 kDa pada fraksi lakase FT.1.4 serta pita berukuran sekitar 39,0 dan 15,1 kDa pada fraksi selulase FT.1.1. Aktivitas lakase FT.1.4 tertinggi diperoleh pada pH 5 dan suhu 50 °C, sedangkan aktivitas selulase FT.1.1 tertinggi diperoleh pada pH 6 dan suhu 50 °C. Analisis morfologi dan filogenetik menempatkan FT.1.4 dalam genus Perenniporia dan menunjukkan kekerabatan dekat dengan P. subtephropora, sedangkan FT.1.1 ditempatkan dalam Fusarium solani species complex dan berkerabat dekat dengan F. falciforme. Hasil penelitian menunjukkan bahwa cendawan indigenus dari area revegetasi tambang batuan kapur berpotensi menjadi sumber lakase dan selulase ekstraseluler untuk kajian dekomposisi biomassa lignoselulosa.

Opencast limestone mining alters soil physicochemical conditions through topsoil removal, low organic matter content and nutrient availability, and limited soil moisture. Revegetation is a post-mining land restoration approach that increases litter and root residue inputs as sources of lignocellulosic biomass. The decomposition of this biomass requires the activity of microorganisms that produce extracellular enzymes, particularly laccase and cellulase, which contribute to lignin and cellulose degradation. Indigenous fungi from revegetated soils have the potential to produce both enzymes; however, information on their enzyme activities, biochemical characteristics, and taxonomic identities at the isolate level remains limited. This study aimed to isolate and select indigenous fungi exhibiting ligninolytic and cellulolytic activities, characterize laccase and cellulase from the selected isolates, and identify the isolates based on morphological characteristics and molecular analysis of the internal transcribed spacer (ITS) region. Soil samples were collected from revegetated limestone quarry areas dominated by teak and sengon trees. Fungi were isolated by serial dilution and screened for ligninolytic and cellulolytic activities. Laccase and cellulase activities of the screening-positive isolates were measured during cultivation. Enzymes from the selected isolates were partially purified by ammonium sulfate fractionation and dialysis, analyzed by SDS-PAGE, and characterized for the effects of pH and temperature on enzyme activity. The isolates were identified through morphological observations, ITS sequence analysis, and phylogenetic tree reconstruction. A total of 12 fungal isolates were obtained; two exhibited ligninolytic activity and three exhibited cellulolytic activity. Isolate FT.1.4 produced the highest laccase activity of 0.496 U/mL on day 9, whereas FT.1.1 produced the highest cellulase activity of 0.488 U/mL on day 12. Both isolates showed ?-hemolysis and were selected for further characterization. For both enzymes, the 40–60% ammonium sulfate fraction yielded the highest specific activity. SDS-PAGE revealed a protein band of approximately 56.2 kDa in the FT.1.4 laccase fraction and two protein bands of approximately 39.0 and 15.1 kDa in the FT.1.1 cellulase fraction. Laccase activity from FT.1.4 was highest at pH 5 and 50 °C, whereas cellulase activity from FT.1.1 was highest at pH 6 and 50 °C. Morphological and phylogenetic analyses placed FT.1.4 within the genus Perenniporia and indicated a close relationship with P. subtephropora, whereas FT.1.1 was placed within the Fusarium solani species complex and was closely related to F. falciforme. These findings indicate that indigenous fungi from revegetated limestone quarry soils have potential as sources of extracellular laccase and cellulase for studies on lignocellulosic biomass decomposition.