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Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris
Author(s) -
Roca Hernan,
Garcia Bianca,
Rodriguez Efrain,
Mateu Dania,
Coroas Laura,
Cremata Jose,
Garcia Rossana,
Pons Tirso,
Delgado Julio
Publication year - 1996
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(19960930)12:12<1187::aid-yea986>3.0.co;2-u
Subject(s) - dextranase , biology , complementary dna , pichia pastoris , biochemistry , microbiology and biotechnology , gene , cdna library , genomic library , molecular cloning , gene expression , peptide sequence , enzyme , recombinant dna
The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB‐8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase‐1 ( AOX1 ) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N‐terminus of both proteins were identical.