Cloning of thePenicillium minioluteum gene encoding dextranase and its expression inPichia pastoris

Roca, Hernán; Garcı́a, Bianca; Rodriguez, Efrain; Mateu, Dania; Coroas, Laura; Cremata, José A.; García, Reinaldo Menéndez; Pons, Tirso; Delgado, J.

doi:10.1002/(sici)1097-0061(19960930)12:12<1187::aid-yea986>3.0.co;2-u

Cited by 40 publications

(53 citation statements)

References 32 publications

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“…The deduced amino acid sequence contained an N-terminal sequence of ANQEEKQSSQAGL-RALTVS and many internal sequences (data not shown) of Lys-C peptidase-digested peptides, indicating that a signal peptide was cleaved at the typical processing site between Ala 38 and Ala 39 (26) and that the isolated gene encoded PsDex protein. A BLAST homology search revealed PsDex to be a GH-66 enzyme family member without obvious similarity to GH-49 enzymes (27)(28)(29) (30 -32). PsDex displays the highest identity with CITase (35%), whereas streptococcal Dexs display a range of only 25-26%.…”

Section: ϫ16mentioning

confidence: 99%

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Kim

Kiso

Muraki

et al. 2012

Journal of Biological Chemistry

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Background: Catalytic residues and molecular mechanism of GH-66 enzymes were hitherto unknown. Results: Novel dextranase produced isomaltotetraose and cyclo-isomaltosaccharides. Its nucleophile (Asp 340 ) and acid/base catalyst (Glu 412 ) were identified by a chemical rescue approach. Conclusion: Three GH-66 enzyme types were newly classified for the first time. Significance: This work elucidates production of isomaltotetraose and cycloisomaltosaccharides, classification of GH-66, identification of catalytic residues, and novel dextran-forming type chemical rescue.

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Section: ϫ16mentioning

confidence: 99%

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Kim

Kiso

Muraki

et al. 2012

Journal of Biological Chemistry

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“…To express the dexA gene under P ICL1 , a 1.8 Kb fragment containing the SUC2 signal peptide from S. cerevisiae fused to dextranase coding region was obtained by PCR, using plasmid pPDEX1 (Roca et al, 1996) as template and the primers: (upstream) 5 -atgctagcgcaagctttccttttc-3 ; (downstream) 5 -agctcgcgatcagctaatctgcca-3 . The PCR product was inserted into pGEM-T (Promega, USA) and the resulting plasmid named pTVDEX.…”

Section: Gene Disruptionmentioning

confidence: 99%

“…Finally, the pIV-2 plasmid was cut with EcoRI and EcoRV and the 2.4 Kb fragment was ligated into pPDEX1 vector (Roca et al, 1996), previously digested with the same enzymes. The resulting plasmid, named pPICLDEX, was used in the dextranase expression experiments.…”

Section: Gene Disruptionmentioning

confidence: 99%

The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter

Menéndez¹,

Valdés²,

Cabrera³

2003

Yeast

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We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P ICL1 is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.

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“…2) confirmed that a his-tagged protein of the expected size was present in the relevant cell extract. These findings, and the low activities of secreted dextranase in the medium, imply that processing of the heterologous dextranase constructs is not highly efficient in Saccharomyces; by contrast, Roca et al (1996) achieved more efficient secretion of the Penicillium minioluteum HIS-4 dextranase as a SUC2 (from the S. cerevisiae invertase gene) signal peptide fusion in the methylotrophic yeast Pichia pastoris. Kang et al (2005) expressed a dextranase cloned from Lipomyces starkeyi in S. cerevisiae using a very similar vector and host strain to those used here, without using an S. cerevisiae secretion leader: most dextranase activity appeared to be cell-wallassociated, and intracellular extracts were used for characterizing the enzyme.…”

Section: Discussionmentioning

confidence: 90%

Cloning and expression of Penicillium minioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins

Millson

Coker

et al. 2006

Biotechnol Lett

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A dextranase gene from Penicillium minioluteum (strain IMI068219) has been cloned, sequenced and expressed in Saccharomyces cerevisiae via fusion of the DNA segment encoding the mature dextranase protein with alpha-factor signal sequence, and insertion into the GAL1-controlled expression vector pYES2/CT. Galactose-induced expression yielded extracellular dextranase activity of 0.63 units/ml and cell-associated dextranase activity of 0.48 units/ml, after 24 h incubation. The dextranase construct was introduced into a strain of S. cerevisiae expressing the human cytochrome P450 3A4 (CYP3A4) and the cognate reductase, which was then used to develop a microplate toxicity bioassay. Toxicity was signalled as inhibition of dextranase activity, assayed fluorimetrically. This novel bioassay was assessed using six economically significant mycotoxins.

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Cloning of thePenicillium minioluteum gene encoding dextranase and its expression inPichia pastoris

Cited by 40 publications

References 32 publications

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter

Cloning and expression of Penicillium minioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins

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