Fraksinasi dan Karakterisasi Fraksi Kitinolitik Non-Konvensional Berbasis Chitin-Binding Protein dari Bacillus amyloliquefaciens SLBD yang Diduga sebagai Lytic Polysaccharide Monooxygenase

Abstract

Bacillus amyloliquefaciens SLBD (Ba-SLBD) merupakan isolat lokal yang diperoleh dari feses kuda dan diketahui memiliki kemampuan mendegradasi biomassa lignoselulosa. Karakteristik tersebut menunjukkan bahwa Ba-SLBD berpotensi memanfaatkan berbagai polisakarida struktural, termasuk kitin. Pada bakteri, degradasi kitin umumnya dikatalisis oleh kitinase dari famili glycoside hydrolase 18 (GH18). Namun, analisis whole genome sequencing (WGS) pada Ba-SLBD tidak mendeteksi keberadaan gen penyandi GH18 maupun kitinase hidrolitik konvensional lainnya. Sebaliknya, analisis genom mengidentifikasi satu gen yang awalnya dianotasi sebagai chitin binding protein (CBP) dan kemudian diklasifikasikan sebagai anggota keluarga lytic polysaccharide monooxygenase auxiliary activity family 10 (LMPO AA10). Temuan tersebut mengindikasikan adanya kemungkinan mekanisme degradasi kitin non-konvensional berbasis oksidatif pada Ba-SLBD yang belum banyak dilaporkan. Oleh karena itu, penelitian ini bertujuan memperoleh fraksi murni CBP¬ Ba-SLBD, mengevaluasi karakteristik aktivitas kitinolitik, dan menganalisis struktur serta potensi fungsi CBP Ba-SLBD melalui pendekatan bioinformatika. Protein ekstraseluler dipisahkan menggunakan anion-exchange chromatography untuk memperoleh fraksi protein target, kemudian dikarakterisasi melalui analisis konsentrasi dan kadar protein, profil protein SDS-PAGE, uji aktivitas kitinolitik menggunakan substrat alami dan sintetik, uji aktivitas antifungi, serta analisis bioinformatika yang mencakup pemodelan struktur tiga dimensi, analisis sekuens, dan superimposisi struktur. Hasil analisis menunjukkan bahwa fraksi FP3 memiliki aktivitas kitinolitik tertinggi dan mengandung pita protein dominan berukuran ~21 kDa. Protein tersebut menunjukkan aktivitas optimum pada pH 7 dan suhu 50 °C serta tetap mempertahankan aktivitas relatif yang tinggi pada rentang pH 6–8 dan suhu 37–70 °C. Selain itu, ekstrak protein kasar Ba-SLBD juga menunjukkan aktivitas antifungi terhadap Rhizoctonia solani dan Alternaria alternata. Analisis bioinformatika menunjukkan bahwa protein target memiliki karakteristik struktural yang konsisten dengan anggota keluarga LPMO AA10, yang didukung oleh keberadaan motif histidine brace, konservasi residu pengikat tembaga, serta nilai root mean square deviation (RMSD) yang rendah terhadap struktur LPMO AA10 yang telah dikarakterisasi. Temuan ini menunjukkan bahwa aktivitas kitinolitik pada Ba-SLBD berkaitan dengan keberadaan LPMO AA10 meskipun gen penyandi kitinase GH18 tidak terdeteksi dalam analisis genom, sehingga mengindikasikan adanya sistem degradasi kitin non-konvensional berbasis oksidatif pada B. amyloliquefaciens SLBD. Namun, interpretasi tersebut masih terbatas karena pengujian aktivitas kitinolitik belum menggunakan substrat kitin kristalin yang lebih merepresentasikan substrat alami LPMO, sehingga mekanisme oksidatif yang diusulkan masih memerlukan verifikasi lebih lanjut.

Bacillus amyloliquefaciens SLBD (Ba-SLBD) is a local bacterial isolate obtained from horse feces and has been reported to possess lignocellulolytic activity. This characteristic suggests that Ba-SLBD has the potential to utilize various structural polysaccharides, including chitin. In bacteria, chitin degradation is generally catalyzed by chitinases belonging to the glycoside hydrolase family 18 (GH18). However, preliminary whole-genome sequencing (WGS) analysis of Ba-SLBD did not detect genes encoding GH18 or other conventional hydrolytic chitinases. Instead, genome analysis identified a single gene initially annotated as a chitin-binding protein (CBP), which was subsequently classified as a member of the AA10 lytic polysaccharide monooxygenase family (AA10 LPMO). These findings suggest the presence of a non-conventional oxidative chitin degradation mechanism in Ba-SLBD that has rarely been reported. Therefore, this study aimed to obtain a purified CBP fraction from Ba-SLBD, characterize its chitinolytic activity, and investigate its structural features and potential function using bioinformatics approaches. Extracellular proteins were fractionated by anion-exchange chromatography to obtain the target protein fraction and subsequently characterized through protein concentration and protein content analyses, SDS-PAGE protein profiling, chitinolytic activity assays using both natural and synthetic substrates, antifungal activity assays, and bioinformatics analyses, including three-dimensional structural modeling, sequence analysis, and structural superimposition. The results showed that fraction FP3 exhibited the highest chitinolytic activity and contained a predominant protein band with an approximate molecular weight of 21 kDa. This protein exhibited optimum activity at pH 7 and 50 °C while maintaining relatively high activity over a pH range of 6–8 and a temperature range of 37–70 °C. In addition, the crude extracellular protein extract exhibited antifungal activity against Rhizoctonia solani and Alternaria alternata. Bioinformatics analysis demonstrated that the target protein possessed structural characteristics consistent with those of AA10 LPMOs, as supported by the presence of the conserved histidine-brace motif, conservation of copper-binding residues, and a low root mean square deviation (RMSD) relative to structurally characterized AA10 LPMOs. These findings suggest that the chitinolytic activity observed in Ba-SLBD is likely associated with the presence of an AA10 LPMO despite the absence of detectable GH18 chitinase genes in the genome, thereby indicating the existence of a non-conventional oxidative chitin degradation system in B. amyloliquefaciens SLBD. Nevertheless, this interpretation remains limited because the chitinolytic activity assays were not performed using crystalline chitin, which more accurately represents the natural substrate of LPMOs. Consequently, the proposed oxidative mechanism requires further experimental verification.