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.     

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.     

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.     

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。     

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.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Characteristics of fungal phytases from<Emphasis Type="Italic"> Aspergillus fumigatus</Emphasis> and<Emphasis Type="Italic"> Sartorya fumigata</Emphasis>

Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these phytases displayed 2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 (Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.Some of the data presented here, in particular the amino acid sequences of the phytases from different A. fumigatus and S. fumigata isolates, were first presented at the workshop on "The biochemistry of plant phytate and phytases", Copenhagen, Denmark, 25–28 October 1997     

Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.     

Structure-based fragment shuffling of two fungal phytases for combination of desirable properties

Aspergillus niger NRRL 3135 phytase (Anp) and Aspergillus fumigatus ATCC 13073 phytase (Afp) are quite different but mutually complementary in many properties. A semi-rational protein engineering strategy based on 3D structure and sequence alignment was used to take advantage of the desirable characteristics of both enzymes. Each phytase was divided into seven fragments, including regions I-VII (I, 1-47; II, 59-133; III, 139-172; IV, 178-237; V, 246-329; VI338-381; VII, 404-444). The equivalent regions were swapped to construct an array of chimeras. Among the functional chimeras expressed in the yeast Pichia pastoris, novel phytases with combinations of the most desirable properties, including heat-resistance, were obtained. Correlations of individual regions with detailed differences were established by systematic evaluation of the substitutions. Regions II and VI contributed to the difference in specific activity at pH 5.0. Regions IV and V of Anp fully accounted for its second pH optimum at pH 2.5. Most influences of substitutions were additive, except those of regions V and VI. Exchanging both regions led to different impacts upon K(m) and activity at approximately pH 4.0 compared with the replacement of either.     

Purification and physico-chemical characterisation of genetically modified phytases expressed in Aspergillus awamori

Two heterologous phytases from Aspergillus awamori and Aspergillus fumigatus obtained from submerged cultures of genetically modified fungal strains in addition to two commercially available phytase preparations (Allzyme and Natuphos phytases) were purified to homogeneity using a combination of ultrafiltration, gel filtration and ion exchange. The purified preparations were used in subsequent characterisation studies, in which Western Immunoblot analysis, pH and temperature optima, thermal stability and substrate specificity were assessed. A. fumigatus phyA phytase expressed in A. awamori exhibited activity over a broad pH range together with an increased temperature optimum, and slightly enhanced thermal stability compared to the other phytases tested, and is thus a promising candidate for animal feed applications. This particular phytase retains activity over a wide range of pH values characteristic of the digestive tract and could conceivably be more suited to the increasingly higher feed processing temperatures being utilised today, than the corresponding phytases from Aspergillus niger.     

Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.     

植酸酶产生菌黑曲霉N14的诱变选育及其基因分析

以植酸酶产生菌黑曲霉03214为出发菌株,经紫外线和亚硝基胍诱变,获得了产酶活性较出发菌株提高了22．3％,达422IU／ml发酵液的突变菌株黑曲霉N14,其最适pH值为2．5,最适温度为50℃。通过对黑曲霉N14植酸酶phyA基因进行PCR扩增,获得了一条长约1．5kb的特异性产物。以pMD18-T为载体,构建了含有目的基因片段的重组质粒。DNA序列测定表明,目的基因片段含有植酸酶phyA基因的完整序列(GenBank Accession：AY426977),phyA基因全长1506bp,其中包含一段长102bp的内含子,编码467个氨基酸,有10个潜在的糖基化位点,5’端有一编码19个氨基酸的信号肽序列。实验结果为植酸酶基因工程菌的构建奠定了基础。     

植酸酶phyAm基因结构延伸突变改善酶的热稳定性

将来源于黑曲霉N25的植酸酶基因phyA^m重组于大肠杆菌表达载体pET-30b（＋）,以重组表达载体pET30b-FphyA^e为模板经PCR扩增获得结构延伸突变植酸酶基因phyA^m（在植酸酶基因C端增加了来源于pET-30b-FphyA^m载体上13氨基酸残基）。含突变基因的重组表达载体pPIC9k-phyA^e在GS115酵母中表达。纯化的突变酶pp-NP^e与野生型酶PP-NP^m-8相比：PP-NPA^e的最适反应温度上升了3气,75℃处理10min,热稳定性提高21％,比活力略有提高。最适反应pH为5．6,有效pH范围pH4,6到pH6．6。比未突变酶扩大了0.4单位。     

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.     

Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon

Bacterial strains were isolated from the pig colon to screen for phytase and acid phosphatase activities. Among 93 colonies, Colony 88 had the highest activities for both enzymes and was identified as an Escherichia coli strain. Using primers derived from the E. coli pH 2.5 acid phosphatase appA sequence (Dassa et al. (1990), J. Bacteriol. 172, 5497-5500), we cloned a 1482 bp DNA fragment from the isolate. In spite of 95% homology between the sequenced gene and the appA, 7 amino acids were different in their deduced polypeptides. To characterize the properties and functions of the encoded protein, we expressed the coding region of the isolated DNA fragment and appA in Pichia pastoris, respectively, as r-appA2 and r-appA. The recombinant protein r-appA2, like r-appA and the r-phyA phytase expressed in Aspergillus niger, was able to hydrolyze phosphorus from sodium phytate and p-nitrophenyl phosphate. However, there were distinct differences in their pH profiles, Km and Vmax for the substrates, specific activities of the purified enzymes, and abilities to release phytate phosphorus in soybean meal. In conclusion, the DNA fragment isolated from E. coli in pig colon seems to encode for a new acid phosphatase/phytase and is designated as E. coli appA2.     

Characterization of a Chitosanase from Aspergillus fumigatus ATCC13073

Chitosanase II was purified from the culture filtrate of Aspergillus fumigatus ATCC13073. The purified enzyme had a molecular mass of 23.5 kDa. The N-terminal amino acid sequence of chitosanase II was identical to those of other Aspergillus chitosanases belonging to glycoside hydrolase family 75. The optimum pH and temperature were pH 6.0 and 40 °C. Chitosanase II hydrolyzed 70% deacetylated chitosan faster than fully deacetylated chitosan. Analysis of the degradation products generated from partially N-acetylated chitosan showed that chitosanase II split GlcN-GlcN and GlcNAc-GlcN bonds but not GlcNAc-GlcNAc or GlcN-GlcNAc, suggesting that it is a subclass I chitosanase. It degraded (GlcN)(6) to produce (GlcN)(3) as main product and small amounts of (GlcN)(2) and (GlcN)(4). Reaction rate analyses of mono-N-acetylated chitohexaose suggested that the (+3) site of chitosanase II recognizes the GlcNAc residue rather than the GlcN residue of its substrate.     

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.     

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.     

Phytase fromAspergillus niger

132 microorganisms, isolates from soil and decayed fruits, were tested for phytase production. All isolates intensively producing active extracellular phytase were of fungal origin. The most active fungal isolates with phytase activity were identified asAspergillus niger. At the end of the growth phase, the extracellular phytase activity produced byA. niger strain 92 was 132 nkat/mL, with strain 89 it was 53 nkat/mL. In both strains the extracellular enzyme activity exhibited two marked activity optima at pH 1.8 and 5.0 and a temperature optimum at 55°C.     

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.