ANNALES BOGORIENSES
Vol 14, No 2 (2010): Annales Bogorienses

Construction of pY-Af Vector for Expression of Thermostable α-L-Arabinofuranosidase in Saccharomyces cerevisiae

I Nengah Wirajana (Faculty of Mathematics and Natural Sciences, Udayana University, Bali, Indonesia)
Ni Nyoman Tri Puspaningsih (Faculty of Sciences and Technology, Airlangga University, Surabaya, Indonesia)
Eddy Bagus Wasito (Faculty of Medicine, Airlangga University, Surabaya, Indonesia 4 Faculty of Bioresources, Mie University, Tsu Mie, Japan)
Soekry Erfan Kusuma (Faculty of Medicine, Airlangga University, Surabaya, Indonesia)
Tetsuya Kimura (Faculty of Bioresources, Mie University, Tsu Mie, Japan)
Kazuo Sakka (Faculty of Bioresources, Mie University, Tsu Mie, Japan.)



Article Info

Publish Date
09 Dec 2010

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

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Journal Info

Abbrev

annales

Publisher

Subject

Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

Description

The Annales Bogorienses (ISSN: 0517-8452, E-ISSN: 2407-7518) is a peer-reviewed Journal that is published biannually. First published in 1955, it is now one of the oldest scientific journal in the nation. The Annales Bogorienses publishes original articles in basic and applied research as well as ...