PENINGKATAN PRODUKSI XILANASE MELALUI MUTAGENESIS BAKTERI XILANOLITIK SERTA APLIKASINYA PADA AYAM PEDAGING

Chusnul Hanim, Ir.,M.Si., Prof. Dr. Ir. Lies Mira Yusiati, SU.; Dr. Ir. Ali Wibowo, MSc.; Dr. Ir. Muhammad Nur Cahyanto, MSc.

2014 | Disertasi | S3 Bioteknologi

Penelitian ini bertujuan untuk memperoleh mutan bakteri xilanolitik yang memproduksi xilanase lebih tinggi dari tetuanya, mengetahui filogeni mutan berdasarkan analisis sekuen 16S rRNA, serta mengetahui kondisi pertumbuhan optimum untuk menghasilkan aktivitas xilanase tertinggi. Penelitian purifikasi bertujuan mengetahui kadar amonium sulfat yang optimum untuk purifikasi parsial xilanase, mengetahui jenis carrier enzim yang sesuai dengan sifat xilanase, serta menganalisis kestabilam xilanase terhadap pH dan suhu. Pada tahap aplikasi bertujuan mengetahui pengaruh penambahan xilanase terhadap kinerja produksi dan saluran pencernaan ayam pedaging yang diberi pakan basal wheat pollard. Bakteri xilanolitik alkalofilik ditumbuhkan dalam medium yang mengandung substrat xilan. Mutasi terhadap bakteri dilakukan menggunakan etidium bromida (EtBr) dan etil metansulfonat (EMS) konsentrasi 50, 100 dan 150 mg/ml selama 30, 60, 90 dan 120 menit untuk setiap perlakuan. Mutan-mutan tersebut dianalisis kemampuannya memproduksi xilanase di dalam medium xilan yang mengandung xilosa, glukosa atau gliserol. Optimasi pertumbuhan mutan hasil screening dilakukan pada medium pH 6 sampai 11 dan suhu 30 sampai 60�°C. DNA mutan diekstraksi dan diamplifikasi dengan PCR menggunakan primer F8 dan universal reverse R1492, kemudian fragmen hasil amplifikasi dilakukan sekuensing. Xilanase yang dihasilkan oleh mutan bakteri xilanolitik alkalofilik dipurifikasi menggunakan amonium sulfat level 30, 40, 50, 60, 70, 80 dan 90%. Enzim hasil purifikasi selain dianalisis menggunakan sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), juga dianalisis stabilitas dan dipreservasi menggunakan beberapa macam carrier. Tahap ketiga adalah aplikasi enzim secara in vivo dan in vitro. Rancangan penelitian yang digunakan dalam aplikasi xilanase yaitu Rancangan Acak Lengkap Pola Searah dengan satu faktor perlakuan yaitu level penambahan xilanase dalam pakan 0; 0,75; 1,5 dan 2,25 g/kg pakan. Pada aplikasi enzim secara in vitro, dilakukan uji kecernaan pakan secara in vitro dengan mengukur gula reduksi yang dibebaskan dalam supernatan digesta. Residu hasil kecernaan in vitro dianalisis bahan kering, bahan organik dan protein kasar untuk menghitung kecernaan bahan kering (KcBK), bahan organik (KcBO) dan protein kasar (KcPK). Setiap perlakuan level enzim terdiri dari tiga ulangan. Pada penelitian ini digunakan 60 ekor ayam pedaging jantan strain Lohmann. Pada periode starter ayam diberi pakan komersial selama 14 hari, kemudian pada periode finisher (15 sampai 42 hari) ayam diberi pakan hasil formulasi dan dibagi dalam empat level suplementasi xilanase, setiap perlakuan terdiri dari tiga ulangan yang masing-masing terdiri dari lima ayam pedaging jantan strain Lohmann. Enzim yang digunakan merupakan hasil purifikasi dan preservasi menggunakan tepung tapioka serta mempunyai aktivitas xilanase 62,03 U/g. Data yang diperoleh meliputi kinerja produksi, kinerja abdominal dan kecernaan in vitro dianalisis variansi pola searah, jika 18 terdapat perbedaan nyata karena perlakuan dilanjutkan dengan Duncanâ��s new Multiple Range test (DMRT). Berdasarkan pohon filogeni diketahui bahwa spesies terdekat dengan isolat xilanolitik adalah C. butyricum W 4 dengan similaritas 99,86%. Hasil mutasi diperoleh 22 mutan dari penggunaan EtBr dan 24 dari penggunaan EMS. Mutan nomer 19 hasil mutasi menggunakan 50 mg/ml EMS yang dipapar selama 120 menit diketahui mempunyai aktivitas xilanase tertinggi yaitu 0,0598 U/ml, serta mempunyai kondisi optimum pertumbuhan yang menghasilkan xilanase tertinggi pada pH 9,5 dan suhu 55ï�°C. Hasil purifikasi menggunakan 60% amonium sulfat menghasilkan aktivitas tertinggi (62,03 U/g), serta recovery enzim 89,40%, dan tapioka merupakan carrier terbaik yang memberikan aktivitas xilanase tertinggi. Xilanase hasil purifikasi mempunyai berat molekul 137,61 dan 165,34 kDa. Suplementasi xilanase tidak berpengaruh (P>0,05) terhadap kecernaan in vitro pakan serta kinerja, persentase organ pencernaan, karakteristiak karkas dan komposisi kimia daging ayam pedaging umur 42 hari. Berdasarkan hasil penelitian dapat diambil kesimpulan bahwa isolat xilanolitik berkerabat dekat dengan C. butyricum W 4, mutasi menggunakan 50 mg/ml EMS selama 120 menit mampu meningkatkan aktivitas xilanase yang dihasilkan mutan bakteri xilanolitik alkalofilik no 19. Tapioka merupakan carrier terbaik untuk preservasi xilanase, meskipun suplementasi xilanase sampai level 2,25 g/kg belum mampu memperbaiki kecernaan in vitro pakan serta kinerja ayam pedaging.

This study was conducted to obtain xylanolytic mutants that had higher xylanase activity than their wild-type counterparts. Mutant with the best xylanolytic activity was selected and identified based on its 16S rRNA sequence, its optimum growth condition was also characterized and its phylogenetic relations to other xylanolytic bacteria were analysed. Second phase, this study was to find ammonium sulphate saturation that had the highest activity was used as standard for saturation to enzyme production and preservation, using several carriers, as well as to analyze the enzyme stability on pH and temperature. Third phase, the aim of the present study was to examine the influence of xylanase supplementation on the performance and the gastrointestinal morphology of broilers fed wheat-based diets. Wild type xylanolytic alkalophlic bacteria were grown in medium containing xylan as a substrate. Mutation was performed using EtBr or EMS at concentrations 50, 100 and 150 mg/ml and times of exposure 30, 60, 90 and 120 minutes for each treatment. The mutants were analysed for their capability to secrete xylanase into xylan medium containing xylose or glucose or glycerol. Growth optimizations of the screened mutant were done in media with pH range 6 to11 and temperature range 30 to 60�°C. DNA of this mutant was extracted and amplified by PCR using F8 and universal reverse R1492primers. The amplified fragments were sequenced by dideoxynucleotide chain terminator method. A xylanase, which produces xylose from oat spelt xylans, was isolated from the culture medium of xylanolytic alkalophylic bacteria mutan. The enzyme was purified by ammonium sulphate with level of 30, 40, 50, 60, 70, 80, and 90%. The purified enzyme of the final preparation was demonstrated by SDS-PAGE. The third phase was the application of enzymes in vitro and in vivo by using Completely Randomized One-way Design with one treatment factor of the addition of xylanase in feed levels 0, 0.75, 1.5 and 2.25 g/kg of feed. On the application of enzymes through in vitro, the feed digestibility in vitro was tested by using the two-stage method of Bedford and Classen (1993) by measuring the reducing sugars released in digesta supernatants. Residues of in vitro digestibility were measured dry matter, organic matter and crude protein, then dry matter (DMD), organic matter (OMD) and crude protein (CPD) digestibility was calculated. Each of the enzyme levels treatment consisted of three replication. This study used 60 male broiler chickens Lohmann strain that was fed with a commercial feed in the starter period for 14 days and with several levels of xylanase supplementation formulation feed in the finisher period or 15-42 days of age. Enzyme used in the study was semi-purified having xylanase activity 62.03 U/g. The data of in vitro digestibility, production performance, as well as the performance of abdominal of broiler was analyzed by using variance of one-way 20 design, and continued by DMRT if there are differences between the means (Rosner, 1990). The closest related species with xylanolytic isolate were C. butyricum W 4, with 99.86% similarity. Twenty two mutants were obtained from the use of EtBr and 24 mutants from EMS mutageneses. Mutant number 19, which was obtained by treatment using 50 mg/ml EMS for 120 min, had the highest xylanase activity (0.0598 U/ml). This activity was obtained at optimum growth conditions, pH 9.5 and temperature 55ï�°C. In purification phase, addition of 60% ammonium sulphate showed the highest xylanase activity (62.03 U/g), and gave 89.40% enzyme recovery. Tapioca, as a carrier, gave the highest xylanase activity. The molecular masses of the purified xylanase were 137.61 and 165.34 kDa. Xylanase supplementation had no significant effect (P>0.05) on in vitro feed digestibility as well as performance, percentage of visceral organs, characteristic of carcass, and meat composition from broiler age 42 days. In conclusion, xylanolytic bacteria was closest related with C. butyricum W 4, mutagenic improvement of xylanase production from xylanolytic bacteria was obtained using 50 mg/ml EMS for 120 min. Tapioca was the best enzyme carrier for xylanase, eventhough, enzyme supplementation as much as 2.75 g/kg still did not lead to better in vitro digestibility and performance of the experimental birds.

Kata Kunci : produksi xilanase, mutagenesis, bakteri xilanolitik, kinerja ayam pedaging