Abstract
To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ αA), and one constitutive system: P. pastoris (pGAPZαA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml−1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml−1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower K m for sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
NI Augspurger DM Webel XG Lei DH Baker (2003) ArticleTitleEfficacy of an E. coliphytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs J. Anim. Sci. 81 474–383 Occurrence Handle1:CAS:528:DC%2BD3sXhsFKhtr0%3D
H. Bisswanger (2002) Enzyme Kinetics, Principles and Methods Weinheim Willey-VCH 51–75
JL Cereghino JM Cregg (2000) ArticleTitleHeterologous protein expression in the methylotrophic yeast Pichia pastoris FEMS Microbiol. Rev. 24 45–66 Occurrence Handle1:CAS:528:DC%2BD3cXltVClug%3D%3D
PS Chen TY Toribara H Warner (1956) ArticleTitleMicrodetermination of phosphorus Anal. Chem. 28 1756–1758 Occurrence Handle1:CAS:528:DyaG2sXitFSjug%3D%3D
HP Fierobe E Mirgorodskaya TP Frandsen P Roepstorff B Svensson (1997) ArticleTitleOverexpression and characterization of Aspergillus awamoriwild-type and mutant glucoamylase secreted by the methylotrophic yeast Pichia pastoris: comparison with wild-type recombinant glucoamylase produced using Saccharomyces cerevisiaeand Aspergillus nigeras hosts Protein Exp. Purif. 9 159–170 Occurrence Handle1:CAS:528:DyaK2sXhs12jtro%3D
SL Forsburg DA Sherman (1997) ArticleTitleGeneral purpose tagging vectors for fission yeast Gene 191 191–195 Occurrence Handle1:CAS:528:DyaK2sXksVSru7c%3D
Y Han DB Wilson XG Lei (1999) ArticleTitleExpression of an Aspergillus nigerphytase gene (phyA) in Saccharomyces cerevisiae Appl. Environ. Microbiol. 65 1915–1918 Occurrence Handle1:CAS:528:DyaK1MXjtVamsro%3D
M Haraguchi S Yamashiro K Furukawa K Takamiya H Shiku K Furukawa (1995) ArticleTitleThe effects of the site-directed removal of N-glycosylation sites from beta-l,4-N-acetylgalactosaminyltransferase on its function Biochem. J. 312 273–280 Occurrence Handle1:CAS:528:DyaK2MXpsFSgsb0%3D
B Imperiali KW Rickert (1995) ArticleTitleConformational implications of asparagines linked glycosylation Proc. Natl. Acad. Sci. USA 92 97–101 Occurrence Handle1:CAS:528:DyaK2MXjtVegtbg%3D
KC Ingham SA Brew W Novokhatny (1995) ArticleTitleInfluence of carbohydrate on s tructure, stability, and function of gelatin-binding fragments of fibronectin Arch. Biochem. Biophys. 316 235–240 Occurrence Handle1:CAS:528:DyaK2MXjsV2hsL4%3D
UK Laemmli (1970) ArticleTitleCleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680–685 Occurrence Handle1:CAS:528:DC%2BD3MXlsFags7s%3D
XG Lei CH Stahl (2001) ArticleTitleBiotechnological development of effective phytases for mineral nutrition and environmental protection Appl. Microbiol. Biotechnol. 57 474–481 Occurrence Handle1:CAS:528:DC%2BD3MXptVant7s%3D
J Lin R Fido P Shewry DB Archer MJC Alcocer (2004) ArticleTitleThe expression and processing of two recombinant 2S albumins from soybean (Glycine max)in the yeast Pichia pastoris Biochim. Biophys. Acta 1698 203–212 Occurrence Handle1:CAS:528:DC%2BD2cXjvVemt7g%3D
G Miksch S Kleist K Friehs E Flaschel (2002) ArticleTitleOverexpression of the phytase from Escherichia coli and its extracellular production in bioreactors Appl. Microbiol. Biotechnol. 59 685–694 Occurrence Handle1:CAS:528:DC%2BD38XoslyktLk%3D
S Minning A Serrano P Ferrer C Sola RD Schmid F Valero (2001) ArticleTitleOptimization of the high–level production of Rhizopus oryzae lipase in Pichia pastoris J. Biotechnol. 86 59–70 Occurrence Handle1:CAS:528:DC%2BD3MXht12gtrY%3D
S Moreno A Klar P Nurse (1991) ArticleTitleMolecular genetic analysis of fission yeast Schizosaccharomyces pombe Methods Enzymol. 194 795–823 Occurrence Handle10.1016/0076-6879(91)94059-L Occurrence Handle1:CAS:528:DyaK3MXit1ahs7w%3D
S Müller T Sandal P Kamp-Hansen H Dalboge (1998) ArticleTitleComparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica Yeast 14 1267–1283
E Rodriguez Y Han XG Lei (1999) ArticleTitleCloning, sequencing, and expression of an Escherichia coliacid phosphatase/phytase gene (appA2)isolated from pig colon Biochem. Biophys. Res. Commun. 257 117–123 Occurrence Handle1:CAS:528:DyaK1MXhvF2is7k%3D
E Rodriguez EJ Mullaney XG Lei (2000) ArticleTitleExpression of the Aspergillus fumigatusphytase gene in Pichia pastorisand characterization of the recombinant enzyme Biochem. Biophys. Res. Commun. 268 373–378 Occurrence Handle1:CAS:528:DC%2BD3cXhtFGjsLY%3D
E Rodriguez EJ Mullaney XG Lei (2000) ArticleTitleSite-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia colipH 2.5 acid phosphatase/phytase expressed in Pichia pastoris Arch. Biochem. Biophys. 382 105–112 Occurrence Handle1:CAS:528:DC%2BD3cXms1eisLw%3D
PM Rudd RJ Woods MR Wormald G Opdenakker AK Downing ID Campbell RA Dwek (1995) ArticleTitleThe effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator Biochim. Biophys. Acta 1248 1–10
K Sreekrishna RG Brankamp KE Kropp DT Blankenship JT Tsay PL Smith JD Wierschke A Subramaniam LA Birkenberger (1997) ArticleTitleStrategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris Gene 190 55–62 Occurrence Handle1:CAS:528:DyaK2sXit1ehur8%3D
CH Stahl DB Wilson XG Lei (2003) ArticleTitleComparison of extracellular Escherichia coliAppA phytases expressed in Streptomyces lividans and Pichia pastoris Biotechnol. Lett. 25 827–831 Occurrence Handle1:CAS:528:DC%2BD3sXjt1Ogu78%3D
C Wang M Eufemi C Turano A Giartosio (1996) ArticleTitleInfluence of the carbohydrate moiety on the stability of glycoproteins Biochemistry 35 7299–7307 Occurrence Handle1:CAS:528:DyaK28XjtVCiurg%3D
M Wyss L Pasamontes A Friedlein R Remy M Tessier A Kronenberger A Middendorf M Lehmann L Schnoebelen U Rothlisberger E Kusznir G Wahl F Muller HW Lahm K Vogel AP Loon Particlevan (1999) ArticleTitleBiophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases, molecular size, glycosylation pattern, and engineering of proteolytic resistance Appl. Environ. Microbiol. 65 359–366 Occurrence Handle1:CAS:528:DyaK1MXpvFCntg%3D%3D
Author information
Authors and Affiliations
Corresponding author
Additional information
Revisions requested 18 November 2004; Revisions received 7 January 2005
Rights and permissions
About this article
Cite this article
Lee, S., Kim, T., Stahl, C.H. et al. Expression of Escherichia coli AppA2 phytase in four yeast systems. Biotechnol Lett 27, 327–334 (2005). https://doi.org/10.1007/s10529-005-0704-6
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s10529-005-0704-6