Dextranase production from <i>Penicillium funiculosum</i>

Kosaric, N.; Yu, Karen; Zajic, J. E.; J, Rozanis

doi:10.1002/bit.260150407

Cited by 29 publications

(10 citation statements)

References 12 publications

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“…Dextranase activity was determined in the culture supernatant according to Kosaric et al (1973). The reducing sugars formed were determined colorimetrically by the dinitrosalicylic acid reagent method (Miller, 1959).…”

Section: Enzymatic Assaysmentioning

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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Section: Enzymatic Assaysmentioning

confidence: 99%

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

Menéndez¹,

Valdés²,

Cabrera³

2003

Yeast

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“…Fractions containing dextranase were concentrated to 15 mg ml À1 . The dextranase activity was determined according to Kosaric et al (1973) by quantifying the amount of reducing sugar released from polymeric dextran.…”

Section: Purificationmentioning

confidence: 99%

“…The use of dextranase in processing has reduced the problem by cleaving the dextran polysaccharides, thus lowering the juice viscosity (Clarke, 1997). The presence of dextranase activity has been reported in bacteria (Dewar & Walker, 1975), yeast (Koenig & Day, 1988) and other fungi (Kosaric et al, 1973), but no enzyme structure is available. Dextranase from the fungus Penicillium minioluteum is a 67 kDa glycoprotein containing three potential N-glycosylation sites.…”

Section: Introductionmentioning

confidence: 99%

Preparation and crystallization of selenomethionyl dextranase fromPenicillium minioluteumexpressed inPichia pastoris

Larsson

Ståhlberg

Jones

2002

Acta Crystallogr D Biol Cryst

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Dextranase from the fungus Penicillium minioluteum hydrolyses alpha-1,6-glycosidic bonds in dextran polymers. The enzyme has been expressed in Pichia pastoris in the presence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by amino-acid analysis to be 50%. The protein has been crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 103.6, b = 115.3, c = 49.8 A and one molecule per asymmetric unit. The crystals diffract to 2.0 A and the presence of SeMet in the crystals has been confirmed by an X-ray absorption spectrum.

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“…The reducing sugars formed were determined by the dinitrosalicylic acid (DNSA) assay. One unit of dextranase activity is defined as the amount of enzyme that liberates 1 μmol of glucose equivalents in 1 min under the conditions mentioned above [11–13].…”

Section: Methodsmentioning

confidence: 99%

Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris

Beldarraín

Acosta

Betancourt

et al. 2003

Biotech and App Biochem

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Conformational stability and structural characterization of an rDex (recombinant dextranase) expressed in Pichia pastoris were studied by enzymic assays, fluorescence, CD and DSC (differential scanning calorimetry). We also identified two disulphide bridges (Cys9-Cys14, Cys484-Cys488) and two free Cys residues (Cys336, Cys415) that are not conserved between bacterial and fungal dextranases of GH-49 (glycoside hydrolase family 49) by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS. Enzymic and fluorescence studies revealed that rDex is biological and conformationally stable at acidic pH, with maximum activity at pH 4.5-5.0, while CD spectra indicated a secondary structure basically composed of beta-sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 degrees C, but it is thermally denatured with appreciable aggregation at temperatures above 75 degrees C. DSC studies always displayed irreversible transitions and a strong dependence on the scan rate. Our combined analysis suggested that the denaturation process of rDex is under kinetic control, which is described reasonably well by the two-state kinetic scheme.

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Dextranase production from Penicillium funiculosum

Cited by 29 publications

References 12 publications

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

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

Preparation and crystallization of selenomethionyl dextranase fromPenicillium minioluteumexpressed inPichia pastoris

Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris

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