Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.
ABSTRACT: A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.
Project description:Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.
Project description:To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMβ1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMβ1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.
Project description:The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake flasks, a selected recombinant strain produced and secreted 0.82 U/ml (71 mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70 degrees C, had high residual activity (85% +/- 2%) after 10 min of heat treatment at 80 degrees C and pH 5.5 in the presence of 5 mM CaCl(2), and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity profiles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent.
Project description:BACKGROUND:Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. OBJECTIVES:Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . MATERIALS AND METHODS:The Y. intermedia phytase gene was optimized according to the codon usage in E. coli. The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. RESULTS:The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg-1) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. CONCLUSIONS:The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.
Project description:During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed. Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits [Laboure, Gagnon and Lescure, Biochem. J. (1993) 295, 413-419]. In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library. Several positive clones containing an insert of about 1400 bp were isolated. The nucleotide sequence of the insert of one of these clones has been established. This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids. The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established. These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11. This confirms that this cDNA codes for the maize phytase. The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected. This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate. Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase. The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds. The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings. Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize. The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.
Project description:A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37 °C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0-9.0, considerable thermostability with more than 60% residual activity at 70 °C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > ? -glycerophosphate. The phytase activity was moderately stimulated by Ca(2+), but inhibited by Al(3+), Mn(2+), Zn(2+), and Cu(2+) at a tested concentration of 5 mM.
Project description:In this study, an extracellular alkali-thermostable phytase producing bacteria, Bacillus subtilis B.S.46, were isolated and molecularly identified using 16S rRNA sequencing. Response surface methodology was applied to study the interaction effects of assay conditions to obtain optimum value for maximizing phytase activity. The optimization resulted in 137% (4.627 U/mL) increase in phytase activity under optimum condition (56.5 °C, pH 7.30 and 2.05 mM sodium phytate). The enzyme also showed 60-73% of maximum activity at wide ranges of temperature (47-68 °C), pH (6.3-8.0) and phytate concentration (1.40-2.50 mM). The partially purified phytase demonstrated high stability over a wide range of pH (6.0-10.0) after 24 h, retaining 85% of its initial activity at pH 6 and even interestingly, the phytase activity enhanced at pH 8.0-10.0. It also exhibited thermostability, retaining about 60% of its original activity after 2 h at 60 °C. Cations such as Ca(2+) and Li(+) enhanced the phytase activity by 10-46% at 1 mM concentration. The phytase activity was completely inhibited by Cu(2+), Mg(2+), Fe(2+), Zn(2+), Hg(2+) and Mn(2+) and the inhibition was in a dose dependent manner. B. subtilis B.S.46 phytase had interesting characteristics to be considered as animal feed additive, dephytinization of food ingredients, and bioremediation of phosphorous pollution in the environment.
Project description:Glycosylation affects the physical properties of proteins in a number of ways including solubility and aggregation behavior. To elucidate the mechanism underlying these effects, we have measured second virial coefficients (A2) of the heavily glycosylated pheniophora lycii phytase (Phy) and its enzymatically deglycosylated counterpart (dgPhy) in native and in denatured form by means of small angle x-ray scattering. The measured A2-values show that the native forms of Phy and dgPhy are equally repulsive at the studied pH 8 where A2 equals 10.9 +/- 0.1 x 10(4) mL mol g(-2). However, when thermally denatured, the A2 of dgPhy decreases to 9.0 +/- 0.2 x 10(4) mL mol g(-2) whereas it remained unchanged for Phy. In accord with earlier investigations, the p(r)-function measured here suggested that the glycans did not affect the peptide structure of the native protein. Conversely, glycosylation markedly changed the structure of thermally denatured protein. This was evident from the radius of gyration, which increased by 32% for Phy and only 11% for dgPhy on denaturation. We suggest that this expanding effect of the glycans on the denatured protein conformation relies on steric hindrance that limits the range of torsion angles available to the polypeptide.
Project description:BACKGROUND:Phytase supplied in feeds for monogastric animals is important for improving nutrient uptake and reducing phosphorous pollution. High-thermostability phytases are particularly desirable due to their ability to withstand transient high temperatures during feed pelleting procedures. A comparison of crystal structures of the widely used industrial Aspergillus niger PhyA phytase (AnP) with its close homolog, the thermostable Aspergillus fumigatus phytase (AfP), suggests 18 residues in three segments associated with thermostability. In this work, we aim to improve the thermostability of AnP through site-directed mutagenesis. We identified favorable mutations based on structural comparison of homologous phytases and molecular dynamics simulations. RESULTS:A recombinant phytase (AnP-M1) was created by substituting 18 residues in AnP with their AfP analogs. AnP-M1 exhibited greater thermostability than AnP at 70 °C. Molecular dynamics simulations suggested newly formed hydrogen bonding interactions with nine substituted residues give rise to the improved themostability. Thus, another recombinant phytase (AnP-M2) with just these nine point substitutions was created. AnP-M2 demonstrated superior thermostability among all AnPs at ?70 °C: AnP-M2 maintained 56% of the maximal activity after incubation at 80 °C for 1 h; AnP-M2 retained 30-percentage points greater residual activity than that of AnP and AnP-M1 after 1 h incubation at 90 °C. CONCLUSIONS:The resulting AnP-M2 is an attractive candidate in industrial applications, and the nine substitutions in AnP-M2 are advantageous for phytase thermostability. This work demonstrates that a strategy combining structural comparison of homologous enzymes and computational simulation to focus on important interactions is an effective method for obtaining a thermostable enzyme.
Project description:Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil; however, the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY-US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In the presence of PA as sole source of Pi, while the wild-type plants show hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation-induced genes (PSI, e.g. PHT1;4) and the inhibition of growth capacity, the ePHY overexpressing lines show a higher biomass production and no PSI induction. Interestingly, when co-cultured with ePHY overexpressors, wild-type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high-affinity Pi transporters, namely pht1;1 and pht1;1-1;4, both fail to accumulate Pi and to grow when co-cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants.