Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris.
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 3.2 g/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.
Roca
H
H
Bioindustry Division, Centre for Genetic Engineering and Biotechnology, Havana, Cuba.
Garcia
B
B
Rodriguez
E
E
Mateu
D
D
Coroas
L
L
Cremata
J
J
Garcia
R
R
Pons
T
T
Delgado
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003910:Dextranase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D015698:Genomic Library; D007438:Introns; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003910:Dextranase