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"ABSTRAKPululanase (Pululan 6-glukanohidrolase, EC 3.2.1.41) adalah debranching enzyme yang spesifik memotong ikatan α-1,6 dalam pululan, pati, amilopektin, dan glikogen. Enzim ini sangat penting di industri pati yang memproduksi sirup glukosa atau maltosa. Kombinasi pululanase dengan glukoamilase atau β-amilase dalam sakarifikasi pati akan meningkatkan konsentrasi glukosa atau maltosa, mempercepat sakarifikasi dan mengurangi penggunaan glukoamilase atau β -amilase.Mikroorganisme penghasil pululanase masih relatif sedikit, sehingga memungkinkan untuk mengeksplorasi sumber mikroorganisme lainnya, di antaranya adalah mikroba yang terdapat atau hidup dalam jaringan vascular tanaman. Mikroorganisme endofit ini diduga sebagai sumber potensial penghasil enzim. Eksplorasi mikroorganisme endofit dilakukan terhadap 16 tanaman penghasil karbohidrat non biji koleksi Kebun Raya Bogor dan Kebun Plasma Nutfah Puslitbang Bioteknologi, Cibinong. Tujuan yang ingin dicapai dari penelitian ini adalah mendapatkan mikroorganisme endofit lokal (indigenous) penghasil pululanase dan mengetahui sifat-sifat enzim yang dihasilkannya. Penelitian diawali dengan melakukan isolasi mikroorganisme yang dilanjutkan dengan seleksi mikroorgansime penghasil pululanase , produksi dan pemekatan enzim serta karakterisasi pululanase yang dihasilkan. Seleksi mikroorganisme penghasil pululanase dilakukan secara bertahap, diawali pada medium padat menggunakan dekstran T-10, tepung beras ketan atau pululan sebagai sumber karbon dan energi. Seleksi selanjutnya dilakukan dalam medium cair.Dan kegiatan di atas, berhasil diisolasi sebanyak 76 mikroba endofit yang terdiri dari 39 bakteri dan 37 kapang. Di antara 39 bakteri, sebanyak 15 isolat menghasilkan enzim pada dekstran T-10, dan hanya 9 di antaranya menghasilkan enzim pada tepung beras ketan. Di antara 37 kapang, sebanyak 21 isolat dapat menghasilkan enzim pada dekstran T-10 dan 23 isolat menghasilkan enzim pada tepung beras ketan. Namun hanya 16 di antara isolat yang menghasilkan enzim pada dekstran T-10, juga menghasilkan enzim pada tepung beras ketan. Sebanyak 7 isolat kapang hanya menghasilkan enzim perombak tepung beras ketan raja tetapi tidak menghidrolisis dekstran T-10. Adanya aktivitas enzim pada dekstran T-10 dan tepung beras ketan menunjukkan kemungkinan adanya enzim amilase termasuk debranching enzyme. Dan keseluruhan isolat bakteri dan kapang penghasil enzim perombak dekstran T-10, telah terjaring 9 isolat penghasil enzim pada pululan. Hasil seleksi kultur cair terhadap 9 isolat tersebut diperoleh isolat ICSo.4 penghasil pululanase yang spesifik memutus ikatan α -1,6 dalam pululan. Maltotriosa terdeteksi sebagai produk akhir hidrolisis pululan. Enzim ini memiliki aktivitas sebesar 42,34 x10-3 unit /mL. dan stabil selama penyimpanan 21 hari pada 4 °C.Penambahan amonium sulfat 0 - 70% mampu memekatkan enzim sebesar 1,05 kali. Pululanase dari ICSo.4 yang telah dipekatkan aktif pada pH dan suhu optimum 4,0 dan 50 °C , serta stabil pada pH 4,5 - 5,5 dan suhu 30 - 40 °C. Kation Mn2+ mampu meningkatkan aktivitas enzim. Namun demikian elektroforesis filtrat enzim menunjukkan adanya 6 pita protein. Nilai Im terhadap pululan dan V°,o. pululanase berturut-turut adalah 27,8 mg/mL dan 0,81 mg/mL/menit. Pululanase dari isolat ICSo.4 ini ternyata juga dapat menghidrolisis amilopektin, glikogen, amilosa, dan soluble starch
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"Sugar is a very important carbon and energy source for human. Thelocal production of sugar in indonesia is not adequate and alternativesources should be found. Microorganisms (Bacillus amyfoiiquefaciens, B. Iicheniformis, B. cereus, B. circulans, B. megaterium, B. polymyxa, B. stearothermophilus, Pyrococcus woeseg P. furiosus, Clostndium thermosulfurogenes, C. thermohydrosulfuricum, Aspergillus awamorL A. nigen A. oryzae, A. saitoil Mucor rouxianus, Penicillium oxalicum, Rhizopus deleman Aerobacter aerogenes, and Streptomyces) are known as producer ot on-amylase, glucoamylase, and pullulanase enzymes through of starch fermentation which may be converted into a sugar compound. A preliminary study on endophytic bacteria proved their ability to grow on soluble starch, glutinous rice, and pullulan. Pullulanase convert pullulan to maltotriosa. This enzyme may work synergistically with on-amylase and with glucoamylase for a better conversion of starch to glucose. An endophytic bacteria lCMe3 obtained from the Research and Development Centre for Biotechnology LIP! at Cibinong, Bogor was examined on its ability to produce pullulanse _ For this purpose, soluble starch 1%, cassava starch 1%, and pullulan 1% (all wlv), were used as carbon and energy source in Bakshi medium (Bakshi etal., 1993). The concentration of the inoculum_was 1.25 x 10° cells/ml. Incubation was carried out at : 30°C (room temperature) and 37°C (Mapiliandari, 1999), at pH 7.0 (Bakshi et al., 1993) and pH 5.0 (Mapiliandari, 1999). The fermentation process was terminated after 24 - 26 hours. The growth of lCNle3 varied depending on carbon source, temperature, and pH. The best growth was found on pullulan at pH 7.0 and incubation temperature of of 30°C . However, when the pH of the medium was lowered to 5.0 (Mapiliandari, 1999) and the incubation temperature 30°C a higher cell number (79.5) x 108 cells/ml was obtained on pullclan as carbon source. The bacteri was grown on cassava starch medium and the pullulanase activity studied. The synergism of pullulanase with amylase and with glucoamylase to degrade cassava starch was also studied. To obtain the crude enzyme extract, the cell mass was centrifuged with a Sorval RC - 26 Plus centrifuge. The Hltrate was then concentrated with UHF, sedimented with (NH4)2SO4, and dialized with buffer Na-acetat (pH 4.8). Activity of the crude enzyme was examined on cassava starch and onpullulan. The unit activity of enzyme was 1.374 U/ml on cassava starch,1.290 U/ml on pullulan, and the protein content was 0.039 mglml. The activity of the crude enzyme, after treatment with UHF, was 2.225 U/ml for pullulan, 2.527 U/mt for cassava starch, and the protein content was 0.014 mg/ml. The activity of the crude enzyme obtained after sedimentation with 60% saturation of (NH4)2SO4, was 1.156 U/ml for pullulan, 1.162 U/mi for cassava starch, the protein content 0.579 mg/ml. After dialysed with buffer Na-acetate (pH 4.8) the activity was 6.25 U/ml for pullulan, 6.45 U/ml for cassava starch with the protein content of 2.997 mg/ml. To study the optimum pH and temperaturefor the enzyme production, the isolate iCMe3 was grown on Bakshi medium with various pHs, : 4.0, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0 and incubated at various temperatures 30°C, 40°C 50°C, 60°C, 70°C, 80°C, 90°C. The optimum pH for enzyme sinthesis on puliulan was 5.0 (4.81 U/ml) and on cassava starch 4.8 (13.27 U/ml). The optimum temperature for enzyme synthesis on pullulan was 40°C (26416 U/ml) and on cassava starch 50°C (22.34 U/ml). The best synergism of pullulanase with on-amylase for both C sources was 25% (dilution of enzyme), while the synergism with glucoamylase was 100% for pulluian and 50% for cassava starch to convere the starch (pullulanand cassava starch) glucose."