title: Expression of Escherichia coli AppA2 phytase in four yeast systems.
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 alphaA), and one constitutive system: P. pastoris (pGAPZalphaA). 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. pombe system produced the lowest activity (2.8 units ml(-1)). The AppA2 phytase expressed in Schizo. pombe had 60-75% lower K(m)for sodium phytate and 28% higher heat-stability at 65 degrees 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 degrees C and similar efficacy in hydrolyzing phytate-phosphate from soybean meal.
authors.affiliation: Department of Animal Science, Cornell University, Ithaca, NY 14853-4801, USA.
authors.forename: Seonho
authors.initials: S
authors.lastname: Lee
authors.forename: Taewan
authors.initials: T
authors.lastname: Kim
authors.forename: Chad H
authors.initials: CH
authors.lastname: Stahl
authors.forename: Xin Gen
authors.initials: XG
authors.lastname: Lei
mesh_terms: D010832:6-Phytase / Q000096:biosynthesis* / Q000737:chemistry*; D000135:Acid Phosphatase / Q000096:biosynthesis* / Q000737:chemistry*; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010833:Phytic Acid / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000378:metabolism*; D013025:Glycine max / Q000378:metabolism; D013045:Species Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010710:Phosphates; D011994:Recombinant Proteins; D010833:Phytic Acid; D000135:Acid Phosphatase; D010832:6-Phytase; C118716:AppA2 protein, E coli