Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA)

The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically. Although A. fumigatus phytase shows 66.2% sequence homology with A. ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase. The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A. ficuum phytase. Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase. The cloned A. fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identically affected by cyclohexanedione. Both the thermal characterization data and kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequence difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases.     

PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases

PhyA gene products of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid residues shorter than the AF phytase. Dynamic light scattering studies indicate that the active AF phytase is a monomer while the PL phytase is a dimer. While both of the phytases retained four identical glycosylatable Asn residues, unique glycosylation sites, six for PL and seven for AF phytase, were observed. Global alignment of both the phytases has shown 38% sequence homology between the two proteins. At 58 degrees C and pH 5.0, the PL phytase gave a specific activity of 22,000 nKat/mg as opposed to about 3000 nKat/mg for AF phytase. However, the AF phytase is more thermostable than its counterpart PL phytase at 65 degrees C. Also, AF phytase is more stable at pH 7.5 than the PL phytase. The two phytases differed in K(m) for phytate, K(i) for myo-inositol hexasulfate (MIHS), and pH optima profile. Despite similarities in the active site sequences, the two phytases show remarkable differences in turnover number, pH optima profile, stability at higher temperature, and alkaline pH. These biochemical differences indicate that phytases from ascomycete and basidiomycete fungi may have evolved to degrade phytate in different environments.     

弗氏柠檬酸杆菌植酸酶的分离纯化及其酶学性质研究

从弗氏柠檬酸杆菌(Citrobacter freundii)中分离纯化了一种植酸酶并进行了酶学性质研究,其反应最适pH为4.0~4.5,最适温度为40℃,在37℃下以植酸钠为底物的Km值为0.85nmol/L,Vmax为0.53IU/(mg.min),具有较好的抗胰蛋白酶的能力。酶蛋白的分子量大小约为45kDa,成熟酶蛋白N端序列为QCAPEGYQLQQVLMM。     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5-6.0 (optimum pH 4.5) with an optimum temperature of 55 degrees C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Thermostability Engineering of Fungal Phytases Using Low-Mr Additives and Chemical Crosslinking

With the ultimate goal to develop preparations of phytase (myo-inositol hexakisphosphate phosphohydrolase) with improved thermal resistance for inclusion in animal feed, several thermostabilization approaches were investigated with a set of fungal (Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger phytase) and consensus phytases. Screening of different low-M_r additives revealed that polyethylene glycols increase the thermostability of all phytases in a molecular weight-dependent fashion. The polyols ribitol, xylitol (C₅ sugars) and sorbitol (C₆ sugar) also improved their thermostability, whereas polyols containing more or less carbon atoms, such as glycerol, erythritol and mannoheptulose, showed only minor effects. The stabilizing effects of PEGs and polyols were concentration dependent. In a second series of experiments, crosslinking of the carbohydrate chains of A. fumigatus and consensus phytase using sodium periodate and adipic acid dihydrazide resulted in the formation of oligomeric forms, which may explain the observed thermostability enhancement of 10–15d`C.     

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris . The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL⁻¹, respectively. Both recombinant phytases exhibited high affinity for phytate but not for p -nitrophenyl phosphate. Optimal phytase activity was observed at 50 °C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 °C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.     

Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.     

Phytase activity in Aspergillus fumigatus isolates

Extracellular phytase from Aspergillus fumigatus isolates was characterized and their genes were cloned and sequenced. Based on their banding pattern in SDS-PAGE all phytases were found to be glycosylated and have similar molecular mass. A correlation between lower optimum pH (4.0) and a higher optimum temperature (70 degrees C) was found in these enzymes. All enzymes characterized displayed a lower specific activity for phytic acid and were more susceptible to proteolytic degradation than the Aspergillus niger phytase that is now commercially available. DNA sequencing established almost no sequence variation in any of the genes and no correlation is evident between a specific amino acid sequence and any physicochemical and catalytic properties of the enzymes. Despite two of the isolates having identical deduced amino acid sequence, characterization of the enzymes encoded by these two identical genes revealed differences in both pH and temperature optimum. This suggests that differences in pH and temperature optimum in these four isolates of A. fumigatus may be due in part to subtle differences in posttranslational modification.     

Biophysical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.     

Aspergillus fumigatus来源植酸酶的Q23L和Q23LG272E突变研究

Aspergillus fumigatus来源的植酸酶具有热稳定性好、pH作用范围广的优点,但其比活性很低。设计的植酸酶Q23L突变能在pH4.5～7.0范围内大幅提高比活性,但pH稳定性却显著下降,为了进一步改良Q23L的pH稳定性,在Q23L分子上加入了G272E突变。将原酶、突变酶Q23L和突变酶Q23LG272E分别在毕赤酵母GS115中表达,表达酶经纯化后进行酶学性质比较分析,结果表明:突变酶Q23L的比活性比原酶显著提高,在pH5.5比活性由51u/mg提高到109u/mg,但其pH稳定性,尤其是在pH3.0～4.0酸性条件下的稳定性却显著降低,低于80%。突变酶Q23LG272E在pH3.0～4.5和pH6.5～7.0时的稳定性比Q23L有所提高,恢复到原酶的水平,而比活性基本维持在Q23L的水平。通过一级序列和三维结构比较,分析了可能影响Q23LG272E酶学性质的因素,为进一步研究植酸酶的结构与功能提供了材料。     

The effect of acid proteinases on the activity and stability of glucoamylase preparations

It was demonstrated that the presence of acid proteinases in the preparation isolated from Aspergillus awamori decreased the activity and stability of glucoamylase. Patterns of changes in the enzymatic activity and stability of glucoamylase at increased temperature and various pH values were studied over a long-term storage. A biospecific sorbent for removal of acid proteinases was synthesized, and glucoamylase preparations free of proteolytic activity were produced.     

Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60 degrees C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U. mg, of protein(-1) and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, (1)H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142

Extracellular phytase produced by Aspergillus niger ATCC 9142 was purified to homogeneity by employing an initial ultrafiltration step, followed by chromatography using ion exchange, gel filtration and chromatofocusing steps. The purified enzyme was an 84 kDa, monomeric protein. It possessed a temperature optimum of 65 degrees C, and a pH optimum of 5.0. Km and Vmax values of 100 microM and 7 nmol/s, respectively, were recorded and these values fall well within the range of those previously reported for microbial phytases. Substrate specificity studies indicated that, while the enzyme could hydrolyse a range of non-phytate-based phosphorylated substrates, its preferred substrate was phytate. Phytase activity was moderately stimulated in the presence of Mg2+, Mn2+, Cu2+, Cd2+, Hg2+, Zn2+ and F- ions. Activity was not significantly affected by Fe2- or Fe3- and was moderately inhibited by Ca2+. The enzyme displayed higher thermostability at 80 degrees C than did two commercial phytase products. Initial characterisation of the purified enzyme suggested that it could be a potential candidate for use as an animal feed supplement.     

Expression of a heat stable phytase from Aspergillus fumigatus in tobacco (Nicotiana tabacum L. cv. NC89)

Aspergillus fumigatus contains a heat-stable phytase of great potential. To determine whether this phytase could be expressed in plants as a functional enzyme, we introduced the phytase gene from A. fumigatus (fphyA) in tobacco (Nicotiana tabacum L. cv. NC89) by Agrobacterium-mediated transformation. Phytase expression was controlled by the cauliflower mosaic virus (CaMV) 35S promoter. Secretion of recombinant phytase (tfphyA) to the extracellular fluid was established by use of the signal sequence from tobacco calreticulin. Forty-one independent transgenic plants were generated. Single-copy line A was selected based on segregation of T1 seeds for kanamycin resistance, phytase expression and Southern blotting analysis for use in further study. After 4-weeks of plant growth, the phytase was accumulated in leaves up to 2.3% of total soluble protein. tfphyA was functional and shared similar profiles of pH, temperature and thermal stability to the same enzyme expressed in Pichia pastoris (pfphyA). The expressed enzyme had an apparent molecular mass of 63 kDa and showed maximum activity at pH 5.5, and temperature, 55 degrees C. It had a high thermostability and retained 28.7% of the initial activity even after incubation at 90 degrees C for 15 min. The above results showed that the thermostable A. fumigatus phytase could be expressed in tobacco as a functional enzyme and thus has the potential of overexpressing it in other crop plants also.     

Activity stabilization of Aspergillus niger and Escherichia coli phytases immobilized on allophanic synthetic compounds and montmorillonite nanoclays

The aim of this work was to study the stabilization of the activity of two commercial microbial phytases (Aspergillus niger and Escherichia coli) after immobilization on nanoclays and to establish optimal conditions for their immobilization. Synthetic allophane, synthetic iron-coated allophanes and natural montmorillonite were chosen as solid supports for phytase immobilization. Phytase immobilization patterns at different pH values were strongly dependent on both enzyme and support characteristics. After immobilization, the residual activity of both phytases was higher under acidic conditions. Immobilization of phytases increased their thermal stability and improved resistance to proteolysis, particularly on iron-coated allophane (6% iron oxide), which showed activation energy (E(a)) and activation enthalpy (ΔH(#)) similar to free enzymes. Montmorillonite as well as allophanic synthetic compounds resulted in a good support for immobilization of E. coli phytase, but caused a severe reduction of A. niger phytase activity.     

Enzymatic evaluation of Bacillus amyloliquefaciens phytase as a feed additive

Phytases from Bacillus amyloliquefaciens DS11 and Aspergillus ficuum for feed enzyme were compared on the basis of phosphate inhibition and thermal stabilities. The apparent half-life of the former enzyme at 80 °C was 42 min. The activity of B. amyloliquefaciens phytase was retained up to 5 mM phosphate while 3 mM phosphate inhibited 50% of the original activity in case of A. ficuum phytase. Addition of 5 mM CaCl₂ significantly broadened the active pH range and also increased the pH stability of DS11 phytase.