한국생물공학회:학술대회논문집

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Cloning of the dextranase gene(lsd11) from Lipomyces starkeyi and its expression in Pichia pastoris.

  • Park, Ji-Young (Department of Material Chemical and Biochemical Engineering, Chonnam National University) ;
  • Kang, Hee-Kyoung (Engineering Research Institute) ;
  • Jin, Xing-Ji (Department of Fine Chemical Engineering, Chonnam National University) ;
  • Ahn, Joon-Seob (Department of Molecular Biotechnology, Chonnam National University) ;
  • Kim, Seung-Heuk (Lifenza Co. Ltd.) ;
  • Kim, Do-Won (Kangnung National University) ;
  • Kim, Do-Man (School of Biological Sciences and Technology, Chonnam National University, Biology Research Center for Industrial Accelerator, Dongshin University, Institute of Bioindustrial Technology, Chonnam National University)
  • Published : 2005.10.27
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Abstract

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

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