Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923

Ragon, Mélanie; Neugnot‐Roux, Virginie; Chemardin, Patrick; Moulin, G.; Boze, Hélène

doi:10.1016/j.pep.2007.12.003

Cited by 24 publications

(9 citation statements)

References 19 publications

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“…A. fumigatus WY-2 phytase , A. fumigatus ATCC 34625 phytase (Pasamontes et al, 2007) and A. niger NRRL 3135 phytase (Ullah et al, 2000) contain (Ragon et al, 2008), and a bacterium, Klebsiella pneumoniae subsp. pneumoniae XY-5, contains RHGIRYP (Wang et al, 2004).…”

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confidence: 99%

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris

et al. 2011

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A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K (m) and V (max) for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe(2+), Fe(3+), and Al(3+). When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).

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confidence: 99%

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris

et al. 2011

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“…These primers were designed using the published nucleotide sequences of the D. castellii CBS 2923 phytDc gene (GenBank accession number EF121003, Ragon et al 2008b). PCR resulted in 1.4 kb amplified DNA fragment of the whole open reading frame from genomic DNA of D. castellii CBS 2923, which underwent digestion with SmaI and XbaI, and was then inserted into the same site of pSBG with deleted b-amylase gene, generating pDCp.…”

Section: Resultsmentioning

confidence: 99%

“…SDS-PAGE (Fig. 3) showed that the recombinant phytase band (80 kDa) and a-amylase band (55 kDa) were presented in the culture supernatant from ATCC 9763/YIpdAURDpSAd (Ghang et al 2007;Ragon et al 2008b). The clone showing the highest activity among the transformants was selected (Fig.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and α-amylase

Lim

Lee

Chin³

et al. 2008

Biotechnol Lett

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Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis alpha-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the delta-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and alpha-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.

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“…5 Western blot analysis of transgenic maize seeds. M protein marker, 1 purified phytase, 2 non-transformed UMI29, 3 JB-UMI29-Z17/2, 4 JB-UMI29-Z17/3, 5 JB-UMI29-Z30/6, 6 JB-UMI29-G20/4, 7 JB-UMI29-G20/5, 8 JB-UMI29-G22/1, 9 wheat phytase castellii CBS 2923 phytase and the maximum production level obtained was 476 U/mL (Ragon et al 2008). Xiong et al (2005) and Bei et al (2001) reported 865 U/mL and 165 U/mL of enzymatic activity, respectively, with A. niger phytase in P. pastoris.…”

Section: Discussionmentioning

confidence: 99%

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger

et al. 2019

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A full-length cDNA of phyA gene of Aspergillus niger, encoding phytase enzyme, was cloned and expressed in E. coli BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404 bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33 U/mL. The phyA gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T 0 transgenic plants and their stable inheritance in the T 1 progenies. Three transgenic events expressing detectable level of A. niger phytase were characterized by western blot analysis. Phytase activity of 463.158 U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5-to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6-to 5-fold higher inorganic phosphorus content.

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Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923

Cited by 24 publications

References 19 publications

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris

Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and α-amylase

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger

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