Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract

A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-a-glucan hydrolase, EC 3.2.1 .l 1) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.

Dextranase (α-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation

“…In fraction rK 15 of the recombinant protein digest ( Fig. 1B), an unexpected peptide with m/z 3231.7 was observed.…”

“…Natural K-1,6 glucan-6-glucanohydrolase was puri¢ed from the culture ¢ltrate of a P. minioluteum Hl-4 strain supplied by the Center for Genetic Engineering and Biotechnology (La Havana, Cuba). The recombinant enzyme was obtained by cloning of the DEX gene from P. minioluteum in the methylotrophic yeast P. pastoris under the control of the AOX promoter using the SUC2 gene signal sequence from S. cerevisiae [15].…”

“…In this report we compare glycosylation pro¢les of a natural K-1,6 glucan-6-glucanohydrolase from the ¢lamentous fungus P. minioluteum and the recombinant protein expressed in P. pastoris [14,15] by means of amine absorption high-performance liquid chromatography (NH 2 -HPLC) and £uorophore-assisted carbohydrate electrophoresis (FACE) methodologies. Occupancy and heterogeneity of glycosylation sites as well as the primary structure of these enzymes were also exhaustively studied using lysyl endopeptidase (LEP) digest and matrix-assisted laser desorption-time of £ight-mass spectrometry (MALDI-TOF-MS).…”

Insights into the reaction mechanism of glycosyl hydrolase family 49

“…Asp353, Glu356, Asp372, Asp373, and Trp402, whose substitutions resulted in the reduction of the activity for both pullulan and panose, were predicted to be located in potential subsites )1 and )2 (a detailed description is given in the next section). Trp31 and Glu273, whose [12]; DEX49A, Penicillium minioluteum dextranase isoform [13]; PFDEXA, Penicillium funiculosum dextranase (DDBJ/EMBL/GenBank No. AJ272066); AGTDEX1 and 2, Arthrobacter globiformis T-3044 endodextranase 1 and 2 [14]; AGCDEX, Arthrobacter sp.…”

“…Interestingly, IPU does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes, such as endo-dextranase (EC 3.2.1.11) [11][12][13][14] and isomaltotrio-dextranase (EC 3.2.1.95) [15]. We have reported the molecular cloning of IPU, and indicated that seven highly conserved regions are found among the primary structures of these dextran-hydrolases and IPU [2].…”