Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli

The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg-1, respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.


Introduction
Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6-hexakis phosphate) is the major storage form of phosphorous in cereals, legumes, oil seeds and nuts [1]. Monogastric animals are incapable of digesting phytate phosphorous. Phytate also acts as an antinutritional agent, since it forms insoluble complexes with proteins and nutritionally important metal ions, such as calcium, copper and zinc and thus decreases nutrient bioavailability. The ingested phytate is largely excreted causing nutritional deficiencies and environmental pollution [1,2].

Cloning of the phytase gene and nucleotide sequence analysis
Genomic DNA isolated from Shigella sp. CD2 [16] was partially digested with EcoRI to obtain 3 to 6 Kb fragments. The fragments were cloned in EcoRI site of pUC18 vector and transformed into E.coli XL1 Blue. The transformants were screened for phytase activity on LB-agar plates containing 100 μg mL -1 ampicillin and 1% sodium phytate. Phytase positive clones formed phytate clearance zone around the colony. The recombinant plasmid (pUCphy) was isolated from phytase positive clone with highest clearance zone; the 3.8kb insert in the plasmid was sequenced by using vector specific M13-pUC forward (5'-GTTTTCCCAGTCACGAC-3') and reverse (5'-CAGGAAACAGCTATG-3') primers and putative phytase encoding ORF was identified. The amino acid sequence encoded by the ORF was analyzed for the presence of signal peptide by SignalP 4.1 Server(http://www.cbs.dtu.dk/services/SignalP) [17] and for disulphide bond in the tertiary structure by using Softberry CYS_REC online services (www. softberry.com). Mature phytase gene without the signal sequence was amplified from pUCphy by using internal primers, PhyF (5'-ATGAATTCGCTCAGAGTGAGCCGGAG-3' with 5' EcoRI restriction site) and PhyR (5'GATGCGGCCGCCAAACTGCACGCCGGTATG-3' with 5' NotI site). The PCR product was cloned in pGEM-T vector following manufacturer's instruction and sequenced using T7 and SP6 universal primers. Homology search in GenBank was done using the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST) [18]. The amino acid sequence of the cloned gene was deduced and then aligned by ClustalW program (http://www. ebi.ac.uk/clustalW) [19]. The phylogenetic analysis of the protein was performed by neighbour joining method using MEGA 4 [20]. Bootstrap analysis was used to evaluate the tree topology of the neighbour joining data by performing 500 replicates. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The recombinant pGEM-T vector harboring the phytase gene was named pGEMT-appA S .

Construction of P. pastoris and E. coli expression plasmids and transformation
Two different plasmids were constructed for expression of appA S in P. pastoris GS115 and E. coli BL21(DE3). For P. pastoris expression, the pGEMT-appA S plasmid was cut with EcoRI and NotI. The resulting 1.2 kb DNA fragment was ligated into pPIC9 digested with EcoRI and NotI to generate pPIC9-appA S . The pPIC9-appA S linearized with BspE1 was transformed into P. pastoris GS115 by the spheroplasting protocol according to the manual of the Pichia Expression kit (Invitrogen, San Diego, CA) and transformants were selected for ability to grow on histidine-deficient medium. The his + transformants were further screened for Mut + and Mut S phenotypes. The integration of the expression cassette into the genome of P. pastoris GS115 was ascertained by PCR using the 5' AOX1 and 3' AOX1 primers. For expression in E. coli, the 1.2 kb fragment released from the pGEMT-appA S plasmid was ligated into pET-20b(+) to generate the construct pET-20b(+)-appA S , which was transformed into E. coli BL21(DE3) and transformants were selected in presence of 100 μg mL -1 ampicillin.
Expression of appA S in P. pastoris GS115 30°C and 300 rpm shaking until culture reached an OD 600 of 1. Cells were subsequently harvested by centrifugation at 2100×g for 5 min and used to inoculate 100 mL of BMMY medium containing 0.5% methanol as inducer. The culture was incubated at 30°C and 300 rpm shaking for 96 h and the induction was maintained by adding 0.5% (v/v) methanol at every 24 h intervals. Extracellular and periplasmic phytase activity and medium pH were monitored at every 12 h intervals. For isolation of extracellular fraction, the culture was centrifuged at 2100×g for 5 min and the cell free medium was concentrated and diafiltered by Vivaspin-20 (30 kDa cutoff) sample concentrator (GE Healthcare, UK). For periplasmic fraction isolation, cell pellet was submitted to 5 cycles of freezing (-20°C for 2 h) and thawing (28°C for 1 h), followed by extraction with 100 mM acetate buffer (pH 5.5) at 28°C in a rotatory shaker (100 rpm). The extracted sample served as periplasmic fraction. Induction of appA S expression was determined by 12% SDS-PAGE analysis of the extracellular fraction. P. pastoris GS115 transformed with pPIC9 vector served as control. Recombinant protein produced by appA S in P. pastoris GS115was named rAppA P .
Expression of appA S in E. coli BL21(DE3) Expression of appA S in E.coli BL21(DE3) was analysed by using MagicMedia TM E.coli Expression Medium following manufacturer's instruction. E. coli BL21 (DE3) cells transformed with pET-20b(+)-appA S was grown overnight in LB medium at 37°C and 200 rpm shaking. The culture at 1% (v/v) was inoculated into the MagicMedia (19:1, ready to use medium: IPTG solution) and grown overnight at 37°C and 300 rpm shaking. The cells were then harvested by centrifugation at 11,200×g for 10 min, suspended in 50 mM acetate buffer (pH 5.5), disrupted by sonication and centrifuged. The supernatant and the pellet dissolved in 50 mM acetate buffer (pH 5.5) served as soluble and pellet fractions, respectively. Induction of appA S expression in both the fractions was determined by 12% SDS-PAGE. Both the fractions were also checked for phytase activity. E. coli BL21 (DE3) transformed with pET-20b(+) vector was used as control. Recombinant protein produced by appA S in E.coli BL21 (DE3) was named rAppA E .

Protein estimation and SDS-PAGE analysis
Total protein concentration was determined by the dye binding assay of Bradford using bovine serum albumin (BSA) as standard [21]. SDS-PAGE analysis was performed with 12% polyacrylamide gel according to the method of Laemmli [22]. After electrophoresis, the gel was stained with CBB R-250 reagent (0.1% Coomassie Brilliant Blue R-250 in 10% acetic acid and 40% methanol) and then destained. Broad range pre-stained protein standards were used as markers.
Purification of rAppA E and rAppA P Recombinant rAppA P was purified from the cell free medium of pPIC9-appA S transformed P. pastoris GS115 culture induced with methanol for 60 h. The concentrated and diafiltered cellfree medium was loaded on to CM-cellulose column and bound proteins were eluted by 50 mM acetate buffer (pH 5.5) with linear gradient of 0-0.5 M NaCl. The active fractions were pooled for subsequent studies. For purification of rAppA E , the IPTG induced culture of pET-20b(+)-appA S transformed E.coli BL21 (DE3) was harvested by centrifugation at 11,200×g for 10 min. The cell pellet was suspended in 50 mM acetate buffer (pH 5.5), disrupted by sonication and centrifuged. The supernatant was loaded onto a Ni-Sepharose Fast Flow column (2 x 5 cm, GE Healthcare, UK) pre-equilibrated with 50 mM acetate buffer (pH 5.5) containing 10 mM imidazole. The bound proteins were eluted with 50 mM acetate buffer (pH 5.5) containing 100 mM imidazole. Fractions with phytase activity were pooled for subsequent studies.

Determination of phytase activity
Phytase activity was determined as described previously [15]. The reaction mixture in a final volume of 2 mL contained, acetate buffer (pH 5.5), 100 mM; sodium phytate, 2 mM; and 100 μL enzyme preparation. The reaction was carried out at 37°C for 30 min followed by termination of reaction by adding 2 mL of 10% trichloroacetic acid. The released Pi was measured spectrophotometrically by adding 2 mL of ammonium molybdate (0.5%), sulphuric acid (5 N) and ascorbic acid (2%) solution. One unit (U) of phytase activity represents 1 μmol of Pi released min -1 under assay conditions.

Characterization of rAppA E and rAppA P
The pH optima was determined by measuring enzymatic activity at pH 2.5-8.5 in the following buffers (50 mM): glycine-HCl (pH 2.5 and 3.5), sodium acetate (pH 4.5 and 5.5), and Tris-HCl (pH 6.5, 7.5 and 8.5). The optimum temperature for activity was determined at temperatures ranging from 10 to 80°C. Thermostability of the enzyme was determined by preincubating the purified enzyme at 10 to 80°C for 30 min followed by measuring phytase activity under standard conditions. To study the effect of metal ions and salts (2 mM), phytase activity was monitored in presence of CaCl 2 , MnSO 4 , MgSO 4 , FeSO 4 , ZnSO 4 , CuSO 4 and EDTA. To determine the susceptibility to digestive proteases, the 50 U of purified rAppA E or rAppA P was preincubated with pepsin and trypsin (30 U, Sigma) at 37°C and phytase activity was monitored 30 min later.
Substrate specificity of the enzyme was determined by replacing sodium phytate in the standard reaction mixture of various pH (pH 4.5-7.5) with an equal concentration (2 mM) of either of phosphorylated compounds, such as p-nitrophenyl phosphate (pNPP), ATP, ADP, disodium pyrophosphate (dSPP), D-glucose-6-phosphate (G6P) and D-fructose-6-phosphate (F6P). K m for phytate was determined using the Lineweaver-Burk plot. K cat values for both the enzymes were also determined.

Deglycosylation
The deglycosylation of rAppA P was carried out using Endo H deglycosylase (New England Biolabs) following manufacturer's instruction. The reaction mix containing, 50 U of purified rAp-pA P , 600 μL of 50 mM Tris buffer (pH 7.0) and 10 U of Endo H in final volume of 1 mL, was incubated at 37°C for 2 h. N-glycosylation was determined by assessing the migration shift of Endo H treated rAppA P in 12% SDS-PAGE.

Western blot analysis
For immunoblot analysis, purified rAppA E and deglycosylated rAppA P proteins separated by 12% SDS-PAGE, were transferred to polyvinylidenedifluoride (PVDF) membrane by semi-dry method using Electroblotting apparatus (Atto, Japan). Purified rabbit antibody raised against E. coli phytase, diluted 1:1000 prior to application, was the primary antibody. The reacted polypeptide was visualised with a secondary antibody, goat anti-rabbit IgG-alkaline phosphatase conjugate using colorimetric based nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (NBT/BCIP) detection kit (Invitrogen, USA). Broad range prestained standards were used as markers.

Isolation of gene encoding phytase from the genomic library
For cloning of phytase gene, a size selected genomic library of Shigella sp. CD2 was constructed in pUC18 vector using EcoRI digested genomic DNA. The library was screened for phytase activity based on formation of clearance zone in phytate-agar medium. Among the six phytase positive clones, one with highest phytate clearance zone and phytase activity in the cell lysate was selected. The clone harbouring plasmid pUC18-phy had DNA insert of 3.8 kb. Sequence analysis of the insert indicated presence of an open reading frame (ORF) of 1299 bp, encoding a protein of 432 amino acids (Fig 1).

Sequence and phylogenetic analysis
Homology analysis of deduced amino acid sequence by BLAST program revealed 98 and 62% similarity with AppA phytase of E. coli and C. braakii, respectively. Hence, Shigella sp. CD2 phytase ORF was named as appA S and the encoded protein as AppA S . The nucleotide sequence was deposited in the GenBank under accession number FR865899. AppA S contained three potential sites of N-glycosylation, a putative signal peptide of 22 amino acids at N-terminal end and 8 cysteine residues among which 99-130, 200-210, 404-413 were the most possible disulphide bond pairs. The calculated molecular mass of the protein with and without the signal sequence were about 47 and 45 kDa, respectively. Alignment of AppA S with other enteric bacterial phytases in the GenBank using ClustalW program showed presence of N-terminal RHGXRXP motif, C-terminal HDTN motif and five conserved cysteine residues. AppA S and E. coli AppA differed in sequence at six positions; AppA S contained P, Q, N, K, K, T in place of S, R, K, E, M, A in E. coli AppA at positions 102, 190, 202, 208, 298, 299, respectively (Fig 2). A phylogenetic tree was constructed based on the alignment using the neighbour joining method. The topology of the phylogram also confirmed AppA S to be closely related to AppA phytase of E. coli and C. braakii (Fig 3).
Expression of appA S in P. pastoris G115 The appA S was cloned in EcoRI and NotI sites of P. pastoris expression vector pPIC9. The recombinant plasmid pPIC9-appA S carried the appA S -expression cassette consisting of 1.2 kb appA S gene in frame with S. cerevisiae α-factor secretion signal, flanked by AOX1 promoter and terminator sequences. Transformation of linearized pPIC9-appA S into P. pastoris GS115 gave about 20 his + transformants. The integration of appA S -expression cassette into the host genome was ascertained by PCR using 5' and 3' AOX1 primers. PCR amplification products of about 0.5kb and 1.7 kb in pPIC9 transformed and pPIC9-appA S transformed P. pastoris GS115, respectively, indicated the integration of appA S -expression cassette into the genome of the later.
The pPIC9-appA S transformed P. pastoris GS115 colonies were screened for Mut phenotypes, and for extracellular and periplasmic phytase activity. A Mut + colony with highest extracellular phytase activity was selected for shake flask expression. At 60 h of methanol induction, the selected transformant showed maximum extracellular recombinant phytase (rAppA P ) production of 62 U mL -1 with specific activity 477 U mg -1 and an extracellular protein concentration of 0.13 mg mL -1 . SDS-PAGE analysis of concentrated and diafiltered cell-free extract showed two protein bands of approximate molecular mass 59 and 65 kDa (Fig 4A). Deglycosylation of rAppA P by Endo H deglycosylase resulted in single band of apparent molecular mass 45kDa (Fig 4B).

Expression of appA S in E. coli BL21(DE3)
The mature appA S was cloned into E. coli expression vector pET-20b(+) and the recombinant plasmid pET-20b(+)-appA S was transformed into E. coli BL21(DE3). The transformant was induced in MagicMedia supplemented with IPTG and after overnight induction cells were disrupted by sonication. Recombinant phytase (rAppA E ) overexpression in the soluble and pellet fractions of sonicated cells was analyzed by SDS-PAGE. As shown in the results of Fig 5A, the soluble fraction of the induced cell exhibited protein overexpression band of approximately 45kDa, which agrees with the predicted molecular weight deduced from the amino acid sequence of AppA S . Phytase activity in the soluble fraction was 176 U mL -1 (specific activity 568 U mg -1 ), whereas negligible activity was detected in the pellet fraction. The results thus indicate a correlation of rAppA E overexpression with phytase activity. Western blot analysis of rAppA E and deglycosylated rAppA P using rabbit polyclonal antibody against E. coli AppA further demonstrated that the specific band with apparent molecular mass of 45 kDa was recombinant phytase (Fig 5B).
Purification and properties of rAppA E and rAppA P Recombinant rAppA P was purified by cation exchange chromatography of diafiltered extracellular fraction of methanol induced P. pastoris GS115 culture transformed with pPIC9-appA S and rAppA E was purified from the soluble fraction of pET-20b(+)-appA S transformed E. coli BL21 (DE3) using Ni-Sepharose Fast Flow affinity chromatography. Purified rAppA P and rAp-pA E had specific activities of 967 and 2982 U mg -1 , with recovery of 75 and 83%, respectively. The results of biochemical properties of rAppA P and rAppA E are shown in Table 1. Compared with the glycosylated rAppA P , the nonglycosylated rAppA E was more active at pH 3.5-7.5. Both the enzymes had more than 50% activity in the pH range 3.5 to 6.5 with pH optima at 5.5 (Fig 6A). Both rAppA E and rAppA P had temperature optima of 60°C. Compared with rAppA P , rAppA E had 11 and 18% greater relative activity at 37 and 50°C, respectively, whereas at higher incubation temperature rAppA P was more active than rAppA E (Fig 6B). For determination of thermal stability, the purified rAppA E or rAppA P were pre-incubated at 10 to 80°C for 30 min and then assayed for enzymatic activity. Although, the two enzymes didn't differ in their thermostability in the temperature range 10 to 50°C, rAppA P was more thermotolerant at higher  temperature. Consequently, at 60 and 70°C rAppA P had 33 and 24% higher activity in comparison to rAppA E , respectively (Fig 6C). K m values for phytate as determined by Lineweaver-Burk plot were 0.18 and 0.22 mM for rAppA E and rAppA P , respectively ( Table 1). The K cat value for rAppA E was 2.23×10 3 sec -1 and for rAppA P was 0.72×10 3 sec -1 .
Both rAppA E and rAppA P were highly specific to the substrate, sodium phytate. Activity with either of phosphorylated substrates, such as ATP, ADP, pNPP, dSPP, G6P or F6P was negligible. The relative phytase activities of rAppA E and rAppA P were enhanced up to 130% in presence of Ca 2+ , Mg 2+ and Mn 2+ , whereas Cu 2+ , Fe 2+ , Zn 2+ or EDTA showed inhibitory effect. To determine the protease resistance the purified recombinant phytases (50 U) were pre-incubated separately with 30U of either pepsin or trypsin at 37°C. The rAppA E and rAppA P retained 70 and 65% activity on treatment with trypsin, and 55 and 50% of activity on treatment with pepsin, respectively, indicating greater resistance to trypsin.

Discussion
The phytase structural gene (appA S ) from Shigella sp. CD2 had an ORF of 1299 bp encoding 432 amino acid protein (AppA S ) containing N-terminal 22 amino acid signal peptide, three probable disulphide bridges and three sites of N-glycosylation. Presence of the signal peptide and disulphide bridges indicates the periplasmic localization of the native protein. AppA S showed significantly high homology with AppA phytase of E. coli and C. braakii suggesting that the proteins may have similar structure and mechanism of action. Moreover, all these AppA phytases form a separate branch in phylogenetic tree. As in the present study, phytase AppA from C. braakii was more closely related to the E. coli AppA than to other phytases [6]. AppA S contained the conserved N-terminal RHGXRXP and C-terminal HD active site motifs, and six conserved cysteine residues, which are characteristics of phytase belonging to the HAP family [23]. Till date, seven genera of family enterobacteriaceae have been reported to produce phytase, and relevant genes have been cloned and all of them belong to HAP family [6,7,11,12,23]. The expression of enzyme as secreted protein is one of the useful and important characteristics for its economical production in industry. P. pastoris has been successfully used as host organism for extracellular production of recombinant proteins at high level, including phytase [2,5,6,10,11]. Phytase appA S was expressed in P. pastoris to produce rAppA P as extracellular protein with highest activity (62 U mL -1 ) at 60 h of methanol induction, with specific activity of 477 U mg -1 . The rAppA P activity is higher than that of phyC gene encoding neutral phytase expressed in P. pastoris (12.5 U mL -1 ) [2]. However, the yield is lower than that of AppA phytase of C. braakii (197 U mL -1 ) and E. coli (112.50 U mL -1 ) [6,9]. The lower activity of rAppA P might be due to observed increase in the medium pH above 7 during cultivation of P. pastoris. This could be confirmed by significant decrease in activity of purified AppA P at pH >7 ( Fig  6A). The expression level and activity of rAppA P could be increased further by optimization of bioprocess and control of medium pH at <7. Moreover, the reduced phytase activity could also be due to the variation in codon usage between Shigella sp. and P. pastoris. Previous studies have shown the effect of codon bias on expression and activity of recombinant phytase and other enzymes [14,24]. Xiong et al. used P. pastoris preferred codons and modified signal sequences to improve the expression of heterologous phytase from Peniophora lycii by 13.6 fold [14]. Similarly, extracellular expression of phyC gene from B. subtilis WHNB02 in P. pastoris yielded 2.40 U mL -1 phytase. Synthesis of phyC according to P. pastoris codon usage without altering the protein sequence enhanced activity by about 8 folds to 18.50 U mL -1 [24]. The recombinant rAppA P was expressed as multiple proteins of higher molecular weights, which on deglycosylation produced protein of about 45 kDa, similar to that of rAppA E , indicating post translational glycosylation of the recombinant protein in Pichia system (Fig 4A and 4B). As in the present study, SDS-PAGE analysis of recombinant AppA from E. coli expressed in P. pastoris appeared as diffused band of molecular size 55 kDa, however, a sharp band was observed after the purified phytase was deglycosylated [9]. Similarly, AppA from C. braakii expressed in Saccharomyces cerevisiae migrated as a broad diffusion band (110-160 kDa) in SDS-PAGE gel due to extensive N-linked glycosylation, while the same protein expressed in E. coli had molecular size of 49 kDa [6].
To examine the effect of glycosylation on enzymatic properties of rAppA P , appA S was also expressed in E. coli to produce rAppA E . The periplasmic signal sequence was removed for targeting the enzyme to the intracellular space in order to avoid the possibility of contamination of recombinant enzyme preparation with two native periplasmic AppA phytases in the host cell [25]. Phytase activity of nonglycosylated rAppA E was 176 U mL -1 (specific activity 568 U mg -1 ). The rAppA E activity is significantly higher than that of phyA gene of O. proteus (9.6 U mg -1 ) and appA gene of E. coli (17.1 U mg -1 ) expressed in E. coli as intracellular proteins [7]. Most of the other studies on expression of recombinant phytase in E. coli have shown accumulation of phytase as inclusion body in the cell [6,26].
The purified rAppA P and rAppA E had specific activities of 967 and 2982 U mg -1 , respectively. The difference in glycosylation between the two enzymes partially affected their biochemical properties. Both the recombinant enzymes had pH optima of 5.5 and more than 50% of activity was maintained between pH 3.5 to 6.5. The pH optimum of most of the enterobacterial phytase AppA is in the range of 4.5 to 5.5. The enzyme from E. coli, O. proteus, C. braakii, Y. intermedia, and E. carotovora showed optimum pH of 4.5, 4.9, 5.0, 4.5 and 5.5, respectively [6,7,9,11,12]. Although rAppA P and rAppA E shared the same optimal temperature of 60°C, the former was more active at 70 and 80°C. As in the present study, the temperature optima of other reported bacterial AppA phytases were in the range of 40-65°C [7,9,11,12]. Glycosylated rAppA P had improved thermotolerance, especially at higher temperatures of 60 and 70°C over that of rAppA E . The K m values of 0.18 mM for rAppA E and 0.22 mM for rAppA P are less than that of the phytases from O. proteus(0.34 mM), E. coli (0.55 mM), E. carotovora (0.25 mM), K. pneumoniae (0.28 mM) [7,9,12,27], but higher than that of the phytase from Y. intermedia (0.125 mM) [11]. The catalytic efficiency of rAppA E was found to be much higher than that of rAppA P as reflected by their K cat values.
Glycosylaion is one of the most important post translational modifications that affects protein function and properties. Previous studies have shown the influence of N-glycosylation on biochemical properties of proteins, such as molecular mass, isoelectric point, surface charge distribution and thermotolerance [3,28]. As in the present study, increased level of glycosylation of phytase from A. fumigatus expressed in P. pastoris improved the thermotolerance of the protein over the deglycosylated form [28]. Similarly, phytase from C. braakii expressed in S. cerevisiae retained 50% higher activity upon heat treatment at 70°C for 30 min as compared to E. coli expressed protein [6]. Although there are very few studies on effect of glycosylation on K m , recently Yao et al. reported an alteration in K m of recombinant E. coli AppA phytase on enhancement of glycosylation. The K m values for WT, Q258N mutant and Q258N/Q349N mutant were 0.48, 0.53 and 0.43 mM, respectively [29]. Phytase in the present study was highly specific to the substrate phytate as observed for AppA phytase from E. carotovora and Y. intermedia [11,12], whereas phytase from E. coli and O. proteus also cleaved phosphorus-containing organic compounds other than phytate at a slower rate [7,9]. In contrast, phytases from Aspergillus fumigatus and Klebsiella pneumoniae showed broad specificity for phosphorylated substrates but relatively low specificity for phytate [27].
In conclusion, phytase AppA S expressed in P. pastoris (rAppA P ) had biochemical properties similar to that expressed in E. coli (rAppA E ), except for thermalstability. The enzyme has several advantageous properties, like substrate specificity, protease resistance, optimal activity at acidic pH and physiological temperature. Phytase AppA from Shigella sp. CD2 displayed 40-70% activity in the pH range 3.5 to 6.5, which can facilitate phytate degradation in salivary gland (pH 5.0-7.0), stomach (fed state pH 6.5, reducing to 3.5-4.5 upon stimulation of acid secretion) and upper part of duodenum (pH 4.0-6.0). Hence, the enzyme can be used as feed additive for improving the utilization of phytate phosphorus by monogastric animals like, swine, poultry and farm animals. Though production of rAppA P as secreted protein is advantageous for industry, its economical production requires improving its expression by using P. pastoris-preferred codons and optimization of bioprocess and scaling up when the cells are grown in a fermenter. Hence, there is a potential to increase the expression level even further, which is being pursued in the laboratory.