Cloning of a Gene Encoding Dextranase from Lipomyces starkeyi and its Expression in Pichia pastoris

• Published in: Journal of microbiology and biotechnology Vol. 19; no. 2; pp. 172 - 177
• Main Authors: Kang, H.K., Chonnam National University, Gwangju, Republic of Korea, Park, J.Y., Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Republic of Korea, Ahn, J.S., Gang-Jin City Agricultural Technology Extension Center, Gangjin, Republic of Korea, Kim, S.H., Lifenza Co. Ltd., Anyang, Republic of Korea, Kim, D.M., Chonnam National University, Gwangju, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.02.2009
• Subjects: Biological and medical sciences; Biotechnology; Cloning, Molecular; DEXTRANAS; DEXTRANASA; DEXTRANASE; Dextranase - genetics; Dextranase - metabolism; DEXTRANE; DEXTRANS; expression; Fermentation; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression Regulation, Fungal; Lipomyces - genetics; Lipomyces - metabolism; Lipomyces starkeyi; Pichia - genetics; PICHIA PASTORIS; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ascomycota; Yeast; expression; Enzyme; Dextranase; Pichia pastoris; Lipomyces starkeyi; Fungi; Glycosylases; Dextran; Gene; Glycosidases; Hydrolases
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.0802.100

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 R pastoris transformant showed a broad band near 73 kDa. Rabbit monoclonal antibodies against a synthetic LSD1 peptide mix also recognized approximately 73 kDa.