Available online at www.sciencedirect.comEnzyme and Microbial Technology 42 (2008) 340–345Effect ofglycosylation on biochemical characterization ofrecombinant phytase expressed in Pichia pastorisMeijin Guoa,b, Haifeng Hanga, Taicheng Zhua, Yingping Zhuanga,∗,Ju Chua, Siliang ZhangaaState Key Laboratory ofBioreactor Engineering, East China University ofScience and Technology, 130 Meilong Road, Shanghai 200237, ChinabDepartment ofBiotechnology, Jiangxi Agricultural University,10 Yingshang Road, Nanchang 330045, ChinaReceived 24 July 2007; received in revised form 16 October 2007; accepted 24 October 2007AbstractRecombinant phytase expressed in Pichia pastoris FPHY34 is a typical glycoprotein, whose gene possesses 10 potential N-glycosylationsites without O-glycosylation. Effects of glycosylation on phytase’s biochemical characterization were investigated. Secreted phytase’s micro-heterogeniousglycosylationphenomenonwasobservedandverifiedbySDS–PAGE,N-terminalaminoacidsequencingandmassspectrumanalyses.Deglycosylationofrecombinantphytase reducedthe molecularweightfrom83 to63 kDa, whichindicates the presence oftotalcarbohydrate contentofapproximately 24.1%. According to image analysis of2-dimension gel electrophoresis, the range ofisoeclectric point ofphytase covers nearly1 pH value rather than a specific point, which might be due to heavy and complex glycosylation. The phytase expressed by P. pastoris still retained40% and 30% activities at 80 and 90?C for 10min, respectively. However, upon deglycosylation in vitro by peptide-N-glycohydrolase F (PNGaseF), thermostability of deglycosylated phytase significantly declined after 10min treatment at 40 and 50?C. Optimum pH of phytase tested wasshifted from pH 5.0 to 2.5 by deglycosylation, although there exhibit bi-humps pH optima at both before and after PNGase F digestion cases.© 2007 Elsevier Inc. All rights reserved.Keywords: Phytase; Glycosylation; Biochemical characterization; Thermostability; Micro-heterogeneity1. IntroductionMost of extracellular and membrane-associated proteins areglycosylated to some extent, but the role ofthe glycosylation isstill remained unclear. In higher eukaryotes, glycosylated pro-teins are ubiquitous components of extracellular matrices andcellular surfaces. Their oligosaccharide moieties are implicatedin a wide range of cell–cell and cell–matrix recognition eventsthat are required for biological processes ranging from immunerecognition to cancer development [1]. As for pharmaceuticalprotein, post-translational modification physiology is necessarytosteerthedegreeandpatternofglycosylation, whichinfluencesboth folding and secretion efficiency [2]. In lower eukaryotes,yeast glycosylation is of the high-mannose type, which con-fers a short in vivo half-life to the protein and may render itless efficacious or even immunogenic [3]. Generally, the gly-coproteins from various origins are monomeric proteins with∗Corresponding author. Tel.: +86 21 6425 3658; fax: +86 21 6425 3702.E-mail address: ypzhuang@ecust.edu.cn (Y. Zhuang).variable glycosylationpatterns whichmightaffecttheirphysicaland biochemical properties [4].Phytase(myo-inositolhexakisphosphate phosphohydrolase),effectively catalyzes the release of phosphate from phytate andphosphorylated compounds and is considered to be a uniquetype of phosphatase [5]. Addition of phytase in animal feedcan enhance the phosphorus and mineral uptake in monogas-tric animals and reduces the level of phosphorus output intheir manure. At present, the commercial phytase is mainlyproduced by A. niger. Its amino acid sequence possesses 10potential N-glycosylated sites. Although various phytases havebeencloned, over-expressedandcharacterizedbiochemicallybyseveral research groups, biochemical characteristics of an idealphytase for commercial application are still largely unknown[6–8]. Han and Lei [9] reported that glycosylation was vital tothe biosynthesis of A. niger phytase expressed in Pichia pas-toris X33 and the thermostability of the expressed enzyme asdeglycosylation of the secreted phytase resulted in reductionin the size from 95 to 55 kDa and in thermostability by 34%.Caseyetal. suggestedthatglycosylationofextracellularphytaseproduced by A. niger ATCC 9142 was an important contribut-0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.enzmictec.2007.10.013
