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

摘要

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.