Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg⁻¹ protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg⁻¹ of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

土壤中微生物植酸酶的活性及其提高方法与应用

磷是有限不可再生资源,土壤缺磷是植物生长和农作物生产的主要限制因子之一。无机磷肥施入土壤后,极易被土壤固相吸附或与金属阳离子形成难溶性络合物或转化为有机磷,导致其生物可利用性降低。土壤磷主要以有机磷形式存在,占比20%-80%。有机磷又以植酸(盐)为主要成分,占比约50%。植酸不可被植物直接吸收利用,需在专一性酶植酸酶作用下经脱磷酸化水解释放磷供植物吸收。土壤植酸酶主要来源于微生物,易受温度、pH、土壤吸附、钙含量及钙磷比、底物含量和有效性等影响,导致酶活降低甚至失活。如何保持或提高土壤中植酸酶活性,进而提高土壤内源植酸磷的利用率,对降低外源磷肥施加和保障农业生产具有重要意义。本文综述微生物植酸酶的来源、分类与作用机制及土壤中植酸酶活性的影响因素,重点阐述保持或提高其活性的方法及实际应用效率。针对土壤植酸酶活性低和稳定性差的问题,对通过调控最适pH范围、提高热稳定性、将植酸酶负载于纳米材料和基因工程改造等改善植酸酶性质的方法进行展望。综述内容可为理解土壤中植酸酶活性的影响因素,进而提高土壤内源植酸磷的利用效率提供理论依据和技术参考,对减少外源磷肥施用、降低磷流失和土壤面源/水体污染风险及保障农业可持续发展具有一定的现实意义。     

Functional insights revealed by the crystal structures of Escherichia coli glucose-1-phosphatase

The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.     

Diversity,abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation

A novel class of cysteine phytase showing ability to degrade phytate has recently been isolated from rumen bacteria. To expand our knowledge of this enzyme class, a total of 101 distinct cysteine phytase gene fragments were identified from the ruminal genomic DNA of Bore goats and Holstein cows, and most of them shared low identities (< 50%) with known sequences. By phylogenetic analysis, these sequences were separated into three clusters that showed substantial diversity. The two most abundant cysteine phytase genes of goat rumens were cloned and their protein products were characterized. Four findings were revealed based on our results. (i) Compared with soil and water environment, where β‐propeller phytase is the most important phytate‐degrading enzyme, cysteine phytase is the major phytate‐degrading enzyme in the anaerobic ruminal environment. (ii) Cysteine phytase fragments in the rumen contents of goat and cow have the same diversity profile, although most of the sequences and their abundance differ in the two species. (iii) Each species has their respective high‐abundance genes, which may play major roles for phytate degradation. (iv) Compared with previously reported cysteine phytases that have pH optimum at 4.5, the pH optima of the two most abundant secreted goat cysteine phytases are 6.5 and 6.0, which are within the pH range found in the rumens. This study provides valuable information about the diversity, abundance and enzymatic properties of the ruminal cysteine phytases and emphasizes the important role(s) of these cysteine phytases probably in the terrestrial cycle of phosphorus.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.     

Land‐use influences phosphatase gene microdiversity in soils

Phosphorus cycling exerts significant influence upon soil fertility and productivity – processes largely controlled by microbial activity. We adopted phenotypic and metagenomic approaches to investigate phosphatase genes within soils. Microbial communities in bare fallowed soil showed a marked capacity to utilise phytate for growth compared with arable or grassland soil communities. Bare fallowed soil contained lowest concentrations of orthophosphate. Analysis of metagenomes indicated phoA, phoD and phoX, and histidine acid and cysteine phytase genes were most abundant in grassland soil which contained the greatest amount of NaOH‐EDTA extractable orthophosphate. Beta‐propeller phytase genes were most abundant in bare fallowed soil. Phylogenetic analysis of metagenome sequences indicated the phenotypic shift observed in the capacity to mineralise phytate in bare fallow soil was accompanied by an increase in phoD, phoX and beta‐propeller phytase genes coding for exoenzymes. However, there was a remarkable degree of genetic similarity across the soils despite the differences in land‐use. Predicted extracellular ecotypes were distributed across a greater range of soil structure than predicted intracellular ecotypes, suggesting that microbial communities subject to the dual stresses of low nutrient availability and reduced access to organic material in bare fallowed soils rely upon the action of exoenzymes.     

Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

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°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.     

Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.     

Galactose-1-Phosphatase in Rat Brain

A prominent galactose-1-phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl-Sephacel, hydroxylapatite, and Sephacryl S-300 columns. The galactose-1-phosphatase was separated from alkaline phosphatase, and from two forms of glucose-1-phosphatase. The three columns gave a 10-fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose-1-phosphate was the best substrate for the purified enzyme, followed by glucose-1-phosphate, which was hydrolyzed 40% as rapidly as galactose-1-phosphate. Galactose-1-phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose-1-phosphate hydrolysis. Mg2+ was required for activity, and supported half-maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L-homoarginine, and L-phenylalanine stimulated enzyme activity. Galactose-1-phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.1S. These values were used to calculate a molecular weight of 50,200 and a frictional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose-1-phosphate concentrations in patients with galactosemia.     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.     

Cytochemical observations on glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and -glycerophosphate dehydrogenase in chick liver cell cultures infected with Trichomonas vaginalis

Cytochemical reactions specific for glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and α-glycero-phosphate dehydrogenase were observed in the epithelial cells and macrophages of chick liver cell cultures; α-glycerophosphate dehydrogenase activity was observed also in the fibroblasts. Distribution of three of the enzymes was limited to the cytoplasm, their activity being localized primarily in cytoplasmic inclusions. Weak staining of the nuclei and strong staining of the nucleoli occurred in addition to the cytoplasmic reaction in cells treated for glucose-6-phosphatase. In cell cultures inoculated with Trichomonas vaginalis, the activity of three of the enzymes decreased progressively in the course of infection, but that of α-glycerophosphate dehydrogenase increased.     

Pentamidine activates T1 the hepatic microsomal glucose 6-phosphate transport protein of the glucose-6-phosphatase system

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by pentamidine has been investigated in both intact and fully disrupted microsomes. The major effect of pentamidine is a 4.7-fold reduction in the K_m of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. The site of action of pentamidine is T₁ the hepatic microsomal glucose 6-phosphate transport protein. The activation of T₁ by pentamidine may contribute to the disturbed blood glucose homeostasis see in many patients after administration of the drug pentamidine.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg^–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.