PRODUCTION, CHARACTERIZATION AND PURIFICATION OF XYLANASE FROM FROM Staphylococcus aureus MBXi-K4 Indah Wijayanti Bogor Agricultural University 1
INTRODUCTION ABSORBTION OF NUTRIENT PERFORMANCE OF POULTRY FIBER DIGESTIBILITY ON POULTRY DIETS 2
Isolation and characterization of microbes (Inda,S 26) Xylanase production Characterizati on and purification of xylanase 3
Aims The objectives of this research are to produce xylanase in batch system bioreactor to characterize and purify xylanase from Staphylococcus aureus MBXi-K4 in order to explore its possibility as feed additive in pelleting poultry feed. 4
HEMICELLULOSE The second most abundance component of plant cell wall after cellulose. Two main composition of hemicellulose are hetero 1,4-D- xylan and hetero-1,4-dmannan Perez et al. 22 5
XYLANASE (EC 3.2.1.8) Xylanase hydrolyze hemicellulose to its simple sugars (xyl ylos ose and xyl ylooligosa ooligosaccharides ccharides) 6
Xylanase aplication on Industry Production and recycling of paper Delignification of pulp Feed Industry Food and beverage Industry Textile Industriy Biopharmaceutical Production 7
METHODS Screening of bacteria that produce xylanase thermostable Production of Xylanase Xylanase production on 2 L bioreactor (STR) with process condition: aeration1 vvm, agitation 16 rpm, Growth temperature: 37oC, ph: 7 Analysis of Process Kinetics : (µ, Yx/s, Yp/s) Analysis of Enzymatic Kinetic : (Km dan Vmaks) Enzyme Characterization : ph and thermal stability Profil of SDS PAGE and zymogram Purification of Enzyme Ammonium sulfat Precipitation Dialysis Gel Filtration Chromatography 8
XYLANASE PRODUCTION 1 propagation Peremajaan 1 propagation isolat 2 inokulation 3 4 Fermentation Substrate Komposisi (% b/v) Yeast ekstract.2 K 2 HPO 4 1.5 Mg.SO 4. 7H 2 O.25 Oat Spelt xylan.7 NaCl.25 NH 4 Cl.5 Na 2 HPO 4.5 ph 7. Temperature: 37oC ph : 7 Aeration: 1 vvm Agitation: 16 rpm Substrate: pollard xylan 9
ENZYME PURIFICATION Ammonium sulphate concentration 4% - 6% Membran Dialysis, MWCO 12kDa Ammonium Sulphate precipitation Dialysis Gel filtration chromatography Matrix: Sephadex G-1 1
SDS-PAGE dan ZYMOGRAM Substrat Oatspelt Xilan,7 % Staining : Congo Red Zymogram SDS-PAGE 1 volt, 5 ma for 2 hours Silver staining 11
RESULTS 1. Kinetika Pertumbuhan Mikroba dan Produksi Enzim 12
Biomass Production xylanase activity (U/ml) biomass (g/l) 5 4.5 4 3.5 3 2.5 2 1.5 1.5 6 12 18 24 3 36 42 48 72 96 Time (hour).9.8.7.6.5.4.3.2.1 reducing sugar (mg/ml) biomass xylanase activity reducing sugar Maximum biomass produstion of 4,6 (g/l) at 72 hours Maximum xylanase activity of 2,26 (U/ml) at 72 h of fermentation Growth associated xylanase production 13
ph ph 7.1 7 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6 12 18 24 3 36 42 48 72 96 5 4.5 4 3.5 3 2.5 2 1.5 1.5 biomass (g/l) Time (h) ph biomassa Decrease of ph value in related to biomass production 14
Enzyme Production activity of enzyme (U/mg) 12 1 8 6 4 2.25.2.15.1.5 protein (mg/ml) specific 6 12 18 24 3 36 42 48 72 96 hours Aktifitas Spesifik Kadar protein Xylanase production had a same trend with consentration of protein. Xylanase is a primer metabolite product for S.aureus Maximum production of xylanase was at 72 of fermentation = 1,5 (U/mg) 15
Kinetics of Cultivation S.aureus MBXi- K4 ln biomassa (g/l) 1.4 1.2 1.8.6.4.2 y =.17x +.134 R² =.781 6 12 18 24 3 36 42 48 72 96 jam ke ln (X) ln (Xt/Xo) = µδt µ (Spesific Growth of Biomass ) =,17 /jam 16
Maximum of Spesific Growth Rate of Biomassa ( µ max) Ln((X/X Xmaks)/(1-X/Xmaks)) 3 2.5 2 1.5 1.5 y =.38x +.152 R² =.91 12 18 24 3 36 42 48 Time (h) µ max =,38 /hour µ max is costant along the fermentation 17
Yield of Biomass (Yx/s) and Product (Yp/s) Yield of Product (Yp/s) Yield of Biomass (Yx/s) 2.5 y = 2.255x -.337 R² =.639 4. y =.4x -.429 R² =.865 2 3. P-Po 1.5 1.5 X-Xo 2. 1. -.5.2.4.6.8 1 S-So P-Po. -1. 2 4 6 8 1, So-S X-Xo Yp/s = 2,25 (U enzyme /mg substrate) Yx/s =,4 g biomass/g substrat 18
2. CHARACTERIZATION OF XYLANASE Enzyme stability on temperature Relative activity on temperature Enzyme activity (U/ml) 2 1.8 1.6 1.4 1.2 1.8.6.4.2 ' 15' 3' 45' 6' 9' 12' Time (minute) suhu 7 suhu 8 suhu 9 % relative activ vity of xylanase 12 1 8 6 4 2 ' 15' 3' 45' 6' 9' 12' Time (minute) suhu 7 suhu 8 suhu 9 Xylanase activity decrease in related to increase of temperature 19
7 Stability of Enzyme on ph Ketahanan Enzim terhadap Enzyme stability on ph Relative activity of enzyme on ph ph 12 Xylanase activity (U/ml) 6 5 4 3 2 1 ' 15' 3' 45' 6' 9' 12' Time (minute) ph 4 ph 6 ph 8 ph 7.5 Relative act tivity (%) 1 8 6 4 2 ' 15' 3' 45' 6' 9' 12' time (minute) ph 4 ph 6 ph 8 ph 7.5 Maximum activity of xylanase of ph 6 (phosphate buffer) Xylanase more stable on ph 7,5 2
KINETICS OF ENZYMATIC REACTION 12 1 y =.34x +.313 R² =.973 8 1/V 6 4 2-5 -2 5 1 15 2 25 3 1/S Vmaks = 3,195 (µmol xilosa/min.ml) Km = 1,86 (mg/ml). Oatspelt Xylan concentration :,5% - 2% 21
PURIFICATION OF XYLANASE 1.2 Enzyme activity (U/ml) 1.8.6.4.2 4% 5% 6% Aktifitas enzim (U/ml) 1.11.161.58 Purification of xylanase use Ammonium Sulphate precipitation (4% - 6%) Best concentration of ammonium sulphate : 4% 22
GEL FILTRATION CHROMATOGRAPHY.4 1.8 Absorbance 28 nm.35.3.25.2.15.1.5 1 2 3 1.6 1.4 1.2 1.8.6.4.2 Enzyme activity (U/ml) 1 4 7 1 13 16 19 22 25 28 31 34 37 4 43 46 49 52 55 58 61 64 67 7 73 76 79 82 85 88 91 94 fraction number absorbansi aktifitas enzim (U/ml) Terdapat 3 puncak protein (fraksi nomer 4-6, 54-55 dan 68-69) Aktifitas spesifik xilanase tertinggi pada fraksi no 6= 383,9 U/mg Sebagian besar protein yang diperoleh merupakan enzim target (xilanase) 23
Purification Steps Crude enzyme Amm.sulfat precipitation 81 3.32 19.1 32.82 1 1 1.48 18.6 37.39 16.57 1.14 Sephadex G-1 3.12 4.69 383.9 4.31 11.69 24
SDS - PAGE 66 kda 45 kda 1 2 3 4 5 6 7 8 47,9 kda well number 1. markerlmw Pharmacia 2. Gel Filtration. fraksino 6 3. fraksino 5 4. fraksino 4 5. diálysis 6. Ammonium sulphate precipitation 7. Ammonium sulphate precipitation no 2 8. Crude ekstract 25
ZYMOGRAM PROFILE M 1 2 3 4 5 6 7 45 kda 35 kda 25 kda 45,7 kda 28,1 kda 21,6 kda Figure 6. Zimogram profile of purified xylanase from S.aureus MBXiK4, stained with Congo Red (1) LMW standards; (2) crude enzyme,(3) ammonium sulfat precipitation,(4) freeze dried crude enzyme,(5)freeze dried ammonium sulfat precipitation,(6) xylanase after dialysis, (7)fraksi no 4 6 of purified xylanase on Sephadex G-1, (8) fraksi no 6 of purified xylanase on Sephadex G-1. 26
CONCLUSIONS Gel Filtration Chromatography technique can purify xylanase 11.69 times of crude extract enzyme. Kinetic of enzymatic reaction of Km : 1.86 (mg/ml) and Vmax : 3.195 (µmol xilosa/min/ml) S.aureus MBXiK4 had 3 kinds of xylanase with MW of 45.6 kda, 28.1 kda and 21.63 kda Xylanases from S.aureus MBXiK4 are moderate thermostable enzyme which had relative activity more than 7% of its activity at 7oC for 3 minutes and active on ph range 4 8 with max activity at ph 6. Xylanase from S.aureus MBXiK4 may has an application on feed industry 27
Due-Like batch III 28