Bacterial phytase produced in Pichia pastoris

Guerrero-Olazarán, Martha; Rodríguez-Blanco, Lilí; Carreon-Treviño, J.G.; Gallegos-López, Juan A; Castillo-Galván, Miguel; Viader-Salvadó, José M.

doi:10.1016/j.jbiotec.2007.07.425

Cited by 4 publications

(5 citation statements)

References 2 publications

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“…The use of a structure-guided consensus approach allowed for the design of a phytase with physicochemical properties and thermostability similar to those of the B. amyloliquefaciens phytase. In addition, the designed phytase is more thermostable at pH 7.5 than the PhyC-R phytase previously produced in our laboratory (12,13). Although the four recombinant phytases that were produced were highly similar (Ͼ92.6% identity), their specific activities at pH 7.5 and 37°C diverged somewhat, ranging from 11.5 to 25.7 U/mg protein.…”

Section: Discussionmentioning

confidence: 86%

“…Similarly, the KM71FBA overproducer strain yielded an extracellular phytase production level per cell that was 12.2-fold and 5.5-fold higher than the production levels per cell yielded with the KM71PhyC and GS115PhyC overproducer strains, respectively. Bacillus subtilis phytase C has sequence iden- (12,13), while the fteII and fba genes have P. pastoris-preferred codons. Therefore, the high extracellular phytase production levels per cell for the KM71FTEII and KM71FBA overproducer strains were likely due to the optimal P. pastoris codon bias used by the fteII and fba genes.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we showed the efficiency of the P. pastoris expression system for synthesizing the B. subtilis VTTE-68013 phytase (PhyC-R) and for secreting the recombinant product into the culture medium with catalytic activity at neutral pH (12,13). Although this recombinant phytase showed a high residual activity (85%) after 10 min of heat treatment at 80°C and pH 5.5 in the presence of 5 mM CaCl 2 , it maintained a residual activity of only 36% after heat treatment at pH 7.5.…”

Section: Discussionmentioning

confidence: 99%

“…The effect of using P. pastoris-preferred codons in fteII and fba was evaluated by comparing the expression levels of each overproducer strain with the expression levels under the same culture conditions for P. pastoris strains GS115 Mut ϩ (GS115PhyC) and KM71 Mut s (KM71PhyC), which are overproducers of recombinant Bacillus subtilis phytase C (PhyC-R), previously constructed in our laboratory (12,13). In addition, the influence of an F (FTEII) or K (FBA) residue at position P1Ј of the Kex2 site for the ␣-mating factor prepro-secretion signal processing was evaluated.…”

Section: Methodsmentioning

confidence: 99%

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Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris

Viader-Salvadó

Gallegos-López

Carreon-Treviño

et al. 2010

Appl Environ Microbiol

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Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

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Section: Discussionmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris

Viader-Salvadó

Gallegos-López

Carreon-Treviño

et al. 2010

Appl Environ Microbiol

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“…Although we did not include the Glu-Ala repeats after the cleavage site of the alpha-factor signal for peptide release in our heterologous gene construction, the GS115-Tg strains that were constructed in this work produce and secrete recombinant shrimp trypsinogen II into the culture medium. We previously used this type of construction in P. pastoris for the production and secretion of 22 kDa 10 and 20 kDa (unpublished results) human growth hormone, a bacterial phytase 16 and a laccase from Trametes (unpublished results). In addition, other authors have successfully used this construction, 17 and an inefficient removal of the Glu-Ala repeats from a secreted recombinant protein produced in P. pastoris was described.…”

Section: Discussionmentioning

confidence: 99%

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Guerrero-Olazarán

Escamilla‐Treviño

Castillo-Galván

et al. 2009

Biotechnology Progress

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Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.

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β-Propeller phytases: Diversity, catalytic attributes, current developments and potential biotechnological applications

Kumar

Yadav

Verma

et al. 2017

International Journal of Biological Macromolecules

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Bacterial phytase produced in Pichia pastoris

Cited by 4 publications

References 2 publications

Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris

Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

β-Propeller phytases: Diversity, catalytic attributes, current developments and potential biotechnological applications

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