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EXPRESSION OF XYLANASE GENE FROM Penicillium citrinum IN Pichia pastoris AND Yarrowia lipolytica |
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| รหัสดีโอไอ | |
| Title | EXPRESSION OF XYLANASE GENE FROM Penicillium citrinum IN Pichia pastoris AND Yarrowia lipolytica |
| Creator | Chanika Ouephanit |
| Contributor | Warawut Chulalaksananukul, Sophie Bozonnet |
| Publisher | Chulalongkorn University |
| Publication Year | 2558 |
| Keyword | Gene expression, Xylanases, การแสดงออกของยีน, ไซแลนเนส |
| Abstract | Xylan is the major component of hemicelluloses, the second most abundant polysaccharide on earth, and contains xylose as the major component sugar. Xylanases are the main xylan degrading enzymes which hydrolyze the xylan to produce xylose and short-chain xylooligosaccharides. Xylanases have potential applications in a wide range of industries. The most of common industrial xylanases are produced from filamentous fungi which have ability to produce many xylanolytic enzymes that are difficult to purify. The purpose of this study was to investigate expression of xylanase gene from Penicillium citrinum in yeast expression platform for increasing xylanase expression, purification and determine the properties of xylanase. A synthetic xylanase gene from P. citrinum was successfully cloned and expressed in Pichia pastoris using either constitutive expression system under the control of glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) or inducible expression system under the control of alcohol oxidase 1 promoter (pAOX1). Expression of xylanase under control of the pGAP showed the activity 119.5 U/ml after 48 h which was higher than the native xylanase activity for 34 folds. The recombinant xylanase under the control of the pAOX1 showed the maximum activity of 676 U/ml when induced with 1% v/v methanol every 24 h for 5 days which was higher than the native xylanase activity for 193 folds. Optimal xylanase activity was observed at 55°C and pH 5, respectively. The recombinant xylanase was stable at a pH of 3 to 10. After pre-incubation at 40°C for 1 hour, the enzyme retained around 80% of the original activity. The Km and kcat were 2.8 mg/ml and 243 per s, respectively. Moreover, a synthetic xylanase gene from P. citrinum was successfully cloned and expressed in Yarrowia lipolytica using constitutive expression system under the control of translation elongation factor-1α (pTEF) using either native or preproLIP2 secretion signals. The recombinant xylanase was showed the activity higher than native xylanase for 11 and 52 folds, respectively. The recombinant xylanase using the preproLIP2 secretion signal showed the activity 180 U/ml after 48 h. Glycosylation affected the molecular mass of recombinant xylanase, which increased to larger than 60 kDa. After deglycosylation, the recombinant xylanase displayed molecular mass of 20 kDa. Optimal xylanase activity was observed at 55°C and pH 5, respectively. The recombinant xylanase was stable at a pH of 3 to 9. After pre-incubation at 40°C for 6 hour, the enzyme retained around 80% of the original activity. The Km and kcat were 5.2 mg/ml and 245 per s, respectively. |
| URL Website | cuir.car.chula.ac.th |
