Expression of a
            <i>Bacillus</i>
            Phytase C Gene in
            <i>Pichia pastoris</i>
            and Properties of the Recombinant Enzyme

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

doi:10.1128/aem.00762-10

Cited by 56 publications

(43 citation statements)

References 39 publications

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“…3A to C). Similar results have been reported with the P. pastoris-produced N-glycosylated Bacillus subtilis phytase that shows resistance to trypsin but not pepsin (41). In contrast, the N-glycosylated phytases derived from Aspergillus japonicus BCC18313, A. niger BCC18081, Eupenicillium parvum BCC17694, and Neosartorya spinosa BCC41923 are resistant to pepsin but not to trypsin (26)(27)(28).…”

Section: Discussionsupporting

confidence: 84%

“…Two main strategies have been applied to improve the protease resistance of feed enzymes. One is to explore protease-resistant feed enzymes (41,42), and the other is to screen engineered feed enzymes with improved protease resistance by rational design (43). Structure-based rational design has been less frequently used than naturally occurring protease-resistant enzymes (24,(44)(45)(46), especially with respect to pepsin resistance.…”

Section: Discussionmentioning

confidence: 99%

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N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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“…The effect on thermostability was most probably due to posttranslational modifications, such as N-glycosylation, which is present in extracellular proteins produced in Sulfolobus (17), and so it appeared for SSO1354, whereas the polypeptide produced in E. coli is surely not glycosylated. In general, the contribution of glycosylation in increasing the resistance of proteins to extreme conditions is well documented (8,28,30,33,34,44). Archeal N-linked glycans are structurally simple, being composed of 3 to 5 sugar residues that are preferentially unbranched.…”

Section: Discussionmentioning

confidence: 99%

Cellulose Degradation by Sulfolobus solfataricus Requires a Cell-Anchored Endo-β-1-4-Glucanase

2012

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A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-␤-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.

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“…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%

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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Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme

Cited by 56 publications

References 39 publications

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Cellulose Degradation by Sulfolobus solfataricus Requires a Cell-Anchored Endo-β-1-4-Glucanase

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

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