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Journal : ANNALES BOGORIENSES

Construction of pY-Af Vector for Expression of Thermostable α-L-Arabinofuranosidase in Saccharomyces cerevisiae Wirajana, I Nengah; Puspaningsih, Ni Nyoman Tri; Wasito, Eddy Bagus; Kusuma, Soekry Erfan; Kimura, Tetsuya; Sakka, Kazuo
ANNALES BOGORIENSES Vol 14, No 2 (2010): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/51

Abstract

In this research,  construction  of expression  vector  for thermostable α -L-arabinofuranosidase  in Saccharomyces cerevisiae was conducted. BJ1824 was conducted The  Escherichia coli/S. cerevisiae  shuttle vector, pYES2 was  used  as  parental  vector  in  construction.  The  abfA  gene  encoding  α-L- arabinofuranosidase  from Geobacillus  thermoleovorans  IT-08  was  amplified  by  PCR,  in  which  the  plasmid  pTP510 was  used  as  a template. The amplification product was treated with  SacI and XhoI and then subcloned to the pYES2 vector, which was previously digested with  SacI and  XhoI. The recombinant plasmid was designated as pY-Af and propagated  first  in  E.  coli  Top 10,  and  then  transformed  into  S.  cerevisiae  BJ1824.  For  α- Larabinofuranosidase (AbfA) production, the yeast transformants were grown in YNBG selective medium and YPG rich medium, using galactose as an inducer. The AbfA activity was assayed by measuring the amount of p-nitrophenol (pNP) released  from  p-nitrophenyl-α-L-arabinofuranoside  (pNPA) substrate at pH 6.0 and 70 C  for  30  min.  The  recombinant  AbfA  activity  was  detected  in  either  of  culture  medium  (0.98%),  cellassociated (14.17%) and intracellular (84.85%) when recombinant yeast was grown in YPG rich medium.Key words: α-L-arabinofuranosidase; Saccharomyces cerevisiae; expression vector
Construction of pY-Af Vector for Expression of Thermostable α-L-Arabinofuranosidase in Saccharomyces cerevisiae I Nengah Wirajana; Ni Nyoman Tri Puspaningsih; Eddy Bagus Wasito; Soekry Erfan Kusuma; Tetsuya Kimura; Kazuo Sakka
ANNALES BOGORIENSES Vol 14, No 2 (2010): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (822.516 KB) | DOI: 10.14203/ann.bogor.2010.v14.n2.15-20

Abstract

In this research,  construction  of expression  vector  for thermostable α -L-arabinofuranosidase  in Saccharomyces cerevisiae was conducted. BJ1824 was conducted The  Escherichia coli/S. cerevisiae  shuttle vector, pYES2 was  used  as  parental  vector  in  construction.  The  abfA  gene  encoding  α-L- arabinofuranosidase  from Geobacillus  thermoleovorans  IT-08  was  amplified  by  PCR,  in  which  the  plasmid  pTP510 was  used  as  a template. The amplification product was treated with  SacI and XhoI and then subcloned to the pYES2 vector, which was previously digested with  SacI and  XhoI. The recombinant plasmid was designated as pY-Af and propagated  first  in  E.  coli  Top 10,  and  then  transformed  into  S.  cerevisiae  BJ1824.  For  α- Larabinofuranosidase (AbfA) production, the yeast transformants were grown in YNBG selective medium and YPG rich medium, using galactose as an inducer. The AbfA activity was assayed by measuring the amount of p-nitrophenol (pNP) released  from  p-nitrophenyl-α-L-arabinofuranoside  (pNPA) substrate at pH 6.0 and 70 C  for  30  min.  The  recombinant  AbfA  activity  was  detected  in  either  of  culture  medium  (0.98%),  cellassociated (14.17%) and intracellular (84.85%) when recombinant yeast was grown in YPG rich medium.Key words: α-L-arabinofuranosidase; Saccharomyces cerevisiae; expression vector
Co-Authors A. A. B. Putra Anak Agung Istri Agung Mayun Laksmiwati D. Rizkiyanti Darma Asih Yuliana Dewi Andayani Farmawati Dewi, I Gusti Ayu Agung Gangga Samala Diah Suci Eddy Bagus Wasito Gusti A Malelak H.M. Soekry Erfan Kusuma I Gede Mahardika I Gusti Ngurah Bagus Andre Hartawan I Ketut Gede Dharma Dewantara I Made Agus Gelgel Wirasuta I P. J. D. A. Suartama I Putu Mahendra I W BUDIARSA SUYASA I Wayan Gede Gunawan I Wayan Suarsa I. A. Gede Widihati Ida Ayu Ratih Dwi Nugraha Putri Ida Bagus Amertha Putra Manuaba Ida Bagus Putra Manuaba Ida Bagus Putu Eristya Putra Ika Kurniawati Indra Juana Adikara Inten Hardianti Nizar Iryanti Eka Suprihatin James Sibarani Kazuo Sakka Ketut Ratnayani Khisan Qamariya Kimura, Tetsuya Kimura, Tetsuya Kusuma, Soekry Erfan Kusuma, Soekry Erfan Luh De Dwi Jayanthi Luk Ketut Budi Maitriani M. Manurung Made Arsa Made Dharmesti Wijaya Made Rai Dwitya Wiradiputra Mirawati N.K.W. N. M. T. Juliasari N. W. Bogoriani Ni Komang Ariati Ni Komang Lia Wahyuni Ni Luh Md. Widayantini Ni Luh Putu Mustika Praptiwi Ni Made Suaniti Ni Made Yustikarini Ni Nengah Kartini Asih Ni Nyoman Tri Puspaningsih Ni Putu Ariantari Ni Putu Citra Anggryni Sugitha Ni Putu Frida Oktaningtias Widiarthi Ni Putu Rahayu Artini O. Ratnayani Oka Ratnayani P. Suarya Pradnyaniti, D.G Putra, Pramana Kumala Putri, Ni Wayan Prasanthi Swarna Putu Elvira Yulianthi Putu Suarya R. R. Sirait Ro’yal Aini Sagun Chandra Yowani Sakka, Kazuo Sakka, Kazuo Satriya Putra Prakoso Soekry Erfan Kusuma Tetsuya Kimura Wahyu Dwijani Sulihingtyas Y. H. Pratiwi