Cloning of a gene encoding dextranase from Lipomyces starkeyi and its expression in Pichia pastoris.
A gene (lsd1) encoding dextranase from Lipomyces starkeyi KSM22 has been previously cloned, sequenced, and expressed in Saccharomyces cerevisiae. The gene consisting of 1,824 base pairs and encoding a protein of 608 amino acids was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The dextranase productivity of the P. pastoris transformant (pPIC9K-LSD1, 134,000 U/l) was approximately 4.2-fold higher than that of the S. cerevisiae transformant (pYLSD1, 32,000 U/l) cultured in an 8-l fermentor. Over 0.63 g/l of active dextranase was secreted into the medium after methanol induction. The dextranase of the P. pastoris transformant, as analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. This dextranase of the P. pastoris transformant showed a broad band near 73 kDa. Rabbit monoclonal antibodies against a synthetic LSD1 peptide mix also recognized approximately 73 kDa.
Kang
Hee-Kyoung
HK
School of Biological Sciences and Technology, Chonnam National University, Gwang-ju 500-757, Korea.
Park
Ji-Young
JY
Ahn
Joon-Seob
JS
Kim
Seung-Heuk
SH
Kim
Doman
D
D003001:Cloning, Molecular; D003910:Dextranase / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D055160:Lipomyces / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D011994:Recombinant Proteins; D003910:Dextranase