Karakterisasi dan analisis stabilitas xilanase dari isolat bakteri Xilanolitik

HANIM, Chusnul, Prof.Ir. Zaenal Abidin, MSc.,PhD

2002 | Tesis | S2 Bioteknologi

Enzim xilanase merupakan enzim kompleks yang tersusun dari endo-p-D- 1,4-xilanase (EC 3.2.1.8) dan p-D-xilosidase (EC 3.2.1.3 7), menghidrolisis dan menjadi xilo-oligosakarida dan xilosa. Enzim pemecah xilan tersebut dapat dihasilkan oleh mikrobia aerob dan anaerob baik yang mesof3i.k maupun tennofilik. Penelitian ini bertujuan untuk melalcukan karakterisasi dan analisis stabilitas xilanase dari isolat xilanolitik. Selain itu juga untuk mengetahui kondisi optimum bagi pertumbuhan isolat xilanolitik. Isolat xilanolitik di isolasi dari ketam menggunakan metode Hungate dan salah satu isolat yang mempunyai aktivitas tertinggi digunakan untuk penelitian berikutnya. Untuk memproduksi xilanase maka isolat tersebut ditumbuhkan dalam medium pertumbuhan anaerob dengan xilan sebagai substrat. Optimasi pertumbuhan isolat bakteri xilanolitik meliputi pH (6 - 11,5), suhu inkubasi (30 - 6OOc) dan macam substrat (xilan, xilosa, glukosa dan selulosa). Kultur had pertumbuhan tersebut disentrifbgasi dan supernatan yang diperoleh digunakan sebagai sumber xilanase. Proses pemunyian enzim didahului dengan pengendapan enzim menggunakan ammonium sulfat (20, 30, 40, 50, 60, 70, 80 dan 90%) kemudian dilakukan dialisis menggunakan buffer asetat 10 mM dan diuji aktivitas enzim endo-P-D- 1,4-xilanase untuk menentukan persentase penambahan ammonium d a t yang menghasilkan aktivitas enzim tertinggi. Selanjutnya enzim dipurifikasi mennggunakan kromatografi pertukaran ion. Hasil purifikasi enzim diuji untuk karakterisasi enzim dengan pengamatan optimasi kerja enzim yang meliputi pH buffer (4 - 8), suhu (30 - 6OOC) dan waktu inkubasi (10 - 60 menit). Dari hasil pengamatan diketahui bahwa penambahan ammonium sulfat 70% memberikan aktivitas endo-p-D-l,4-xilanase tertinggi. Had pemurnian mennggunakan kromatografi pertukaran iqn memberikan satu puncak &&i protein yang mempunyai aktivitas endo-f3-D-1,4-xilanase dan meningkatkan aktivitas enzim 4,43 kali, memberikan recovery 16,23% serta total protein 3,67%. Enzim tersebut mempunyai pH optimum 4,s (0,962 U/mg), suhu 5OoC (0,846 Ulmg) dan waktu inkubasi 20 menit (2,280 U/ mg). Selain itu diketahui nilai K, dan V, xilanase sebesar 83,33 mg/ml dan 69,93 U/mg. Hasil elektroforesis menunjukkan masih ada empat pita protein dengan BM 106.414 ; 81.846 ; 64.565 dan 57.544 Da. Stabilitas xilanase dari isolat bakteri xilanolitik ini mempunyai kisaran pH cukup luas (4-7).

Xylanases, including endo-p-D- 1,4-xylanase (EC 3.2.1.8) and p-Dxylosidase (EC 3.2.1.37), are complex enzyme that hydrolyse xylan to xylooligosacharide and xylose. The xylan degrading enzyme can be produced by aerobic or anaerobic mesophilic or thennophilic microbe. The objectives of this study were to characterise and analyse the stability of xylanase obtained from xylanolytic bacterial isolate, and also to evaluate its optimum growth conditions. Xylanolytic isolates were isolated from Crabs by using Hungate method and screened for the highest enzymatic activity. The xylanolytic bacterial isolate was then cultivated for xylanase production. To produce xylanases, the isolate was grown in a medium using oat spelt xylan as substrate under anaerobic condition. Growth opthisation was conducted at different pH (6 - 11,5), temperature (30 - 60 "C) and type of substrates (xylan, xylose, glucose and cellulose). The culture supmatant was separated by centrifbgation and used as the source of xylanase. The supernatant obtained was concentrated by the addition of ammonium sulphate at varied levels of saturation (20, 30, 40, 50, 60, 70, 80 and 90%). Concentrated supernatant was then dialysed against 10 mM acetate buffer. The endo-P-D-1,4- xylanase activity was analysed to determine the level of ammonium sulphate concentration resulting in the highest enzyme activity. The highest enzyme activity obtained was purified using ion-exchange (DEAE-cellulose) chromatography. purified enzyme was characterised its optimum activity under different pH buffer condition (4 - 8), temperature (30 - 6OOC) and time of incubation (10 - 60 minutes). The results showed that the hifiest endo-P-D-l,4-xylanase activity was obtained under 70% ammoNum sulphatesaturation. The ion-exchange chromatography resulted in one peak protein fraction having endo-P-D-1,4- xilanase activity, a 16.23% recovery of activity and 3.67 % yield of protein. It was found that the purified enzyme had the highest activity at pH 4.5 (0.962 U/mg), temperature 50°C (0.846 U/mg) and 20 minutes of incubation with the substrate (2.280 U/ mg). The value of K, and V, were 83.33 mg/d and 69.93 Ulmg respectively. Based on the electrophoreses analysis, four protein bands were observable molecular weight of 106.414, 81.846, 64.565 and 57.544 Da. It was also found that xylanase isolated fiom xylanolytic bacteria having stability at pH 4 - 7.

Kata Kunci : Bioteknologi,Isolat Bakteri Xilanolitik,Stabilitas Xilanase,characterisation, stability, bacterial xylanase