Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in <i>Pichia pastoris</i>

Beldarraín, Alejandro; Acosta, Niuris; Betancourt, Lázaro; González, Luis Javier; Pons, Tirso

doi:10.1042/ba20030063

Cited by 7 publications

(7 citation statements)

References 33 publications

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“…It seems that cysteine does not contribute to the enzyme activity of dextranase in oral streptococci. A similar finding has been made in the GH 49 family of dextranases (3). In contrast, the conserved region comprising the catalytic site and dextran-binding domain is responsible for dextranase activity (14,25).…”

Section: Discussionmentioning

confidence: 52%

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Tamura

Yamada

Kato

2007

Microbiology and Immunology

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The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene‐walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200‐amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell‐surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C‐terminus was partially purified and showed dextranase activity on blue‐dextran sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (BD‐SDS‐PAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD‐SDS‐PAGE, whereas no dextranase activity of the cells was observed on blue‐dextran brain heart infusion (BD‐BHI) agar plates. Furthermore, PCR‐based mutations of dextranase indicated that a deletion mutant of the C‐terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C‐terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp‐453 and Trp‐793 residues of S. criceti dextranase are critical to the enzyme's activity.

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Section: Discussionmentioning

confidence: 52%

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Tamura

Yamada

Kato

2007

Microbiology and Immunology

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“…Dextranases are found in a wide variety of fungi, bacteria, yeast, and Actinomyces, with fungi being the most important commercial sources 15,29 . However, only eight dextranase genes from five species of filamentous fungi plus one species of yeast have been reported to date, namely the dextranase genes from P. funiculosum (AJ272066), Penicillium marneffei ATCC 18224 (XM 002152550), P. minioluteum (L41562, DQ394070 and AF020619), Verticillium alboatrum VaMs.102 (XM 003005140), V. dahliae VdLs.17 (DS572701), and the yeast L. starkeyi (AY520537).…”

Section: Discussionmentioning

confidence: 99%

“…Sources of dextranases are widespread: the enzyme has been found in mammalian tissues and in various microorganisms, such as fungi, yeasts, bacteria, and Actinomyces 13,14 . Fungi are the most important commercial source of dextranases as they produce high levels of enzyme 15 . Although several fungi have been reported to produce dextranase, only a few nucleotide sequences of fungal dextranases are available in public databases.…”

Section: Introductionmentioning

confidence: 99%

Cloning, characterization, and heterologous expression of a dextranase gene from Penicillium pinophilum SMCU3-14

Rerngsamran¹,

Temjitpukdee²,

Assavasirijinda³

et al. 2014

ScienceAsia

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A DNA fragment encoding dextranase was cloned from Penicillium pinophilum SMCU3-14 using the genome walking approach. Sequence analysis of the gene (SMCU-DEX) revealed a putative CAAT box at −165 (CCAAT), a putative TATA box at −93 (TATAA) in the 5 -noncoding region, and a polyadenylation signal (AATAAG) in the 3 -noncoding region. A cDNA sequence analysis revealed no evidence of introns. The deduced open reading frame is 1824 bp in length and encodes a predicted protein of 608 amino acids (molecular weight (MW) of ∼66 kDa), with a putative N-terminal 20-amino acid signal peptide, giving a predicted mature protein of 588 amino acids (MW of ∼64 kDa) that belongs to glycosyl hydrolase family 49, as with other fungal dextranases. This is the first report of a dextranase gene sequence from P. pinophilum. The cDNA was cloned and expressed in Escherichia coli, and the transformants showed dextranase activity on a dextran-containing agar medium. Crude extracts from the transformants analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis containing blue dextran revealed a distinct specific band of dextranase activity at an MW of approximately 66 kDa. This recombinant dextranase is likely to have valuable and cost-effective applications in medicine and industry.

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“…The enzyme preserves its conformation even at 60°C but is then thermally denatured with aggregation at temperatures above 75°C. (12).…”

Section: Secondary and Tertiary Structures Of Dextranasesmentioning

confidence: 99%

Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications

Khalikova

Susi

Korpela

2005

Microbiol Mol Biol Rev

230

152

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Dextran is a chemically and physically complex polymer, breakdown of which is carried out by a variety of endo- and exodextranases. Enzymes in many groups can be classified as dextranases according to function: such enzymes include dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-α-glucosidases. Cycloisomalto-oligosaccharide glucanotransferase does not formally belong to the dextranases even though its side reaction produces hydrolyzed dextrans. A new classification system for glycosylhydrolases and glycosyltransferases, which is based on amino acid sequence similarities, divides the dextranases into five families. However, this classification is still incomplete since sequence information is missing for many of the enzymes that have been biochemically characterized as dextranases. Dextran-degrading enzymes have been isolated from a wide range of microorganisms. The major characteristics of these enzymes, the methods for analyzing their activities and biological roles, analysis of primary sequence data, and three-dimensional structures of dextranases have been dealt with in this review. Dextranases are promising for future use in various scientific and biotechnological applications

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

Cited by 7 publications

References 33 publications

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Identification and Characterization of a Dextranase Gene of Streptococcus criceti

Cloning, characterization, and heterologous expression of a dextranase gene from Penicillium pinophilum SMCU3-14

Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications

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