从酶的专一性看植酸酶与酸性磷酸酶的关系

迄今为止的资料表明,大多数植酸酶与酸性磷酸酶的关系密切。通过测定Km,即可判断只有以植酸（盐）为最适底物的酶（包括酸性磷酸酶）才是严格意义上的植酸酶。     

Mobilization of Phosphorus in the Cotyledons of Young Seedlings of the Garden Pea (Pisum sativum L.)

Changes in the levels of various phosphorus fractions and ofphytase activity in the cotyledons of young pea seedlings grownin the light have been studied. It is shown that from the onsetof germination there is a lag of several days in the hydrolysisof phytic acid and that this is associated with a low levelof phytase activity in cotyledon extracts. Rapid developmentof phytase during the next few days is accompanied by a rapidincrease in the rate of phytic acid break-down and both reachmaximum levels after 6–7 days from soaking the seed. Theamount of phytic acid in the cotyledons becomes negligible afterabout 15 days and at the same time phytase activity declinesmarkedly. At this point protease activity is at a maximum andthe water content of the cotyledons begins to fall. Removal of the shoot 4 days after soaking the seed caused animmediate decrease in export of phosphorus from the cotyledonsbut did not affect the level of phytic acid for several days.Subsequently there was a small, but significant reduction inthe rate of phytic acid hydrolysis in de-shooted seedlings ascompared with intact plants in spite of the fact that phytaseactivity was not affected for several days. Similar effectswere observed when excised cotyledons were cultured on moistfilter-paper. Control mechanisms for phytic acid hydrolysis are discussedand it is concluded that regulation by the axis of the inorganicphosphate concentration at the sites of phytase activity maybe a means of controlling phytic acid hydrolysis.     

Tethered phytic acid as a probe for measuring phytase activity

A novel approach for measuring phytase activity is presented. We have developed a new chromophoric substrate analog of phytic acid, 5-O-[6-(benzoylamino)hexyl]-d-myo-inositol-1,2,3,4,6-pentakisphosphate that permits direct measurement of the phosphate ester bond-cleavage reaction using HPLC. This compound, along with its dephosphorylated T-phosphatidylinositol intermediates, are quantified using reversed phase chromatography with UV detection.     

Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate.

A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.     

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.     

Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 degrees C.     

Screening of yeast strains for phytase activity

A screening method was developed to elucidate the ability of different yeast strains to utilize phytic acid as sole phosphorus source. The growth test in liquid culture in a microtiter plate with phytic acid as sole phosphorus source was shown to be a reliable, fast and easy-to-use screening method. We tested 122 strains from 61 species with our method and observed growth differences among species and strains that were not detectable on solid medium. Specific phytase activities were measured for 10 yeasts strains, selected due to their strong growth in the liquid medium. Strains of Arxula adeninivorans and Pichia anomala reached the highest volumetric phytase activities. Arxula adeninivorans also displayed the highest intra- and extracellular specific activities. There were large differences in both extra- and intracellular phytase activities among species. Strain-specific extracellular phytase activities were detected in P. anomala . The presence of free phosphate in the media completely suppressed the extracellular phytase activity and also reduced intracellular phytase activity for all tested yeast strains.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.     

转基因植物表达植酸酶研究进展

植酸是植物体内磷的主要存在形式,其绝大部分不能被单胃动物消化吸收,而随粪便排出体外造成环境污染;同时,植酸又是一种抗营养因子,它通过络合植物体内的一些营养成分而降低植物的营养价值。通过植物转基因方法使植物自身表达足量的植酸酶,以减小植酸带来的不利影响,是提高植物性饲料营养价值和控制环境磷污染的一种经济有效的措施。就转基因植物植酸酶的优势、研究现状、存在的问题及其发展前景进行了综述。     

Specificity of hydrolysis of phytic acid by alkaline phytase from lily pollen.

Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (I-1,2,3,4,5,6-P6). A number of phytases with varying specificities, properties, and localizations hydrolyze phytic acid present in cells. The specificity of hydrolysis of phytic acid by alkaline phytase from lily (Lilium longiflorum L.) pollen is described. Structures of the intermediate inositol phosphates and the final product were established by a variety of nuclear magnetic resonance techniques (1H-, 31P-, and 31P-1H-detected multiple quantum coherence spectroscopy, and total correlation spectroscopy). On the basis of the structures identified we have proposed a scheme of hydrolysis of phytic acid. Initial hydrolysis of the phosphate ester occurs at the D-5 position of phytic acid to yield the symmetrical I-1,2,3,4,6-P5. The two subsequent dephosphorylations occur adjacent to the D-5 hydroxyl group to yield I-1,2,3-P3 as the final product. Alkaline phytase differs from other phytases in the specificity of hydrolysis of phosphate esters on the inositol ring, its high substrate specificity for phytic acid, and biochemical properties such as susceptibility to activation by calcium and inhibition by fluoride. The physiological significance of alkaline phytase and the biological role of I-1,2,3-P3 remain to be identified.     

The effect of phosphate concentration on phytase production and the reduction of phytic acid content in canola meal by Aspergillus carbonarius during a solid-state fermentation process

The use of canola meal, an abundant side-product of canola oil processing in Canada, as animal feed is hampered by high phytic acid levels that reduce metal cation availability. Aspergillus carbonarius grows well in a solid canola meal medium, produces phytase and reduces the phytic acid content to zero. Inorganic phosphate addition at a concentration of 1 mg and 5 mg/110 g solid-state culture system results in better growth of the microorganism, higher rates and levels of phytase production, and faster reduction of phytic acid content. Phosphate concentrations of 50mg and 100 mg/110 g inoculated system had a negative effect affecting primarily the initial rates of biomass and phytase production and phytic acid content reduction. Models that predict biomass production (expressed as glucosamine content) and phytase, as well as the reduction of phytic acid content in the solid-state cultures supplemented with phosphate are reported. They fit the experimental results reasonably well (with a maximum deviation of 7%).     

Influence of culture conditions on the biosynthesis of Schwanniomyces castellii phytase

Summary The influence of several factors on the biosynthesis of Schwanniomyces costellii phytase was studied in continuous culture. The level of phytase production increased with pH and dilution rate. It decreased when the phytic acid or phosphate content increased.     

植酸酶对中华绒螯蟹幼蟹生长、消化性能及物质利用率的影响

为探讨植酸酶对中华绒螯蟹(Eriocheir sinensis)幼蟹生长、消化性能及物质利用率的影响, 设计了6种配合饲料, 以不含植酸和植酸酶的组别为对照组(C), 在含有10 g/kg植酸的饲料中, 分别加入0、500、1000、1500 U/kg的植酸酶, 分别记为P0、P500、P1000、P1500和P2000。投喂初始体重为(4.34±0.05) g的幼蟹, 56d后称重并取样分析。结果发现: P0幼蟹增重率、特定生长率、蛋白质效率低于对照组, 饲料系数则高于对照组(P<0.05); 幼蟹增重率、特定生长率、蛋白质效率随着饲料中植酸酶含量的增加而升高, 在P2000达到最高, 且该组的饲料系数最低(P<0.05); P1500和P2000全蟹体磷含量显著高于P0 (P<0.05); 在P2000中, 幼蟹肝胰腺中胰蛋白酶、淀粉酶以及肠道胰蛋白酶活力达到最高(P<0.05); 中华绒螯蟹蛋白质消化率和磷透析率随着饲料中植酸酶含量的增加而逐渐升高, 其中P2000显著高于P0(P<0.05), 与对照组无显著差异(P>0.05); P2000幼蟹的氮、磷保留率最高(P<0.05)。以上结果表明, 在含有植酸的饲料中添加2000 U/kg的植酸酶, 能够显著提高幼蟹的生长和胰蛋白酶活力, 进而提高幼蟹对蛋白质的利用率, 降低饲料系数。此外, 植酸酶的添加也能有效提高幼蟹体磷含量和氮/磷保留率。     

A New Type of Phytase from Pollen of Typha latifolia L.

A phytase was isolated and partially purified from pollen of cattail, Typha latifolia. Its maximum activity was at pH 8.0 and its Km value was 1.7 × 10^?5 m for phytic acid in the presence of Ca²⁺. Among divalent cations tested only Ca²⁺ affected the activity, increasing it by about 120%, but an excess was inhibitory. The enzyme was specific for phytic acid except for 6% activity for p-nitrophenylphosphate. It seems to be a new type of phytase because it cleaved almost 50% of the total phosphate esters in phytic acid and was product-specific, yielding an inositol triphosphate as a final product.     

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.     

植酸酶的多样性及其分类

植酸酶是一类催化植酸水解逐步释放磷酸基团形成低级肌醇磷酸衍生物的正磷酸单酯磷酸水解酶。植酸酶在动物营养、资源环境保护和人类健康等领域有巨大的应用潜力。目前,人们对植酸酶的多样性及其分类的认识比较模糊甚至错误,严重影响了植酸酶的研究进程和水平。首先简要概述了基于最适pH和立体专一性的植酸酶分类,然后着重论述了基于结构和催化机理的植酸酶分类及其代表酶特征的最新研究进展,最后探讨了根据不同分类标准特别是基于结构和催化机理准确理解和全面表征各种植酸酶的重要性,以期为植酸酶的研究和应用提供参考。     

Phytase activity in Cryptococcus laurentii ABO 510

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.     

DNA生物传感器在环境污染监测中的应用

基于生物催化和免疫原理的生物传感器在环境领域中获得了广泛应用.近年来,随着分子生物学和生物技术的发展,人们开发了以核酸探针为识别元件,基于核酸相互作用原理的DNA生物传感器.该传感器可用于受感染微生物的核酸序列分析、优先控制污染物的检测以及污染物与DNA之间相互作用的研究,在环境污染监测中具有潜在的巨大应用前景.简要介绍了核酸杂交生物传感器的基本原理及其在环境微生物和优先控制污染物（priority pollutant）检测中的应用研究进展.     

Effects of dietary phytase on body weight gain, body composition and bone strength in growing rats fed a low-zinc diet

Phytic acid, a major phosphorous storage compound found in foodstuffs, is known to form insoluble complexes with nutritionally essential minerals, including zinc (Zn). Phytases are enzymes that catalyze the removal of these minerals from phytic acid, improving their bioavailability. The objective of the present study was to determine the ability of dietary phytase to affect body weight, body composition, and bone strength in growing rats fed a high phytic acid, low Zn diet. Rats (n = 20) were fed either a control (AIN-93) or phytase supplemented (Natuphos, BASF, 1,500 phytase units (FTU)/kg) diet for a period of 8 weeks. Phytase supplementation resulted in increased (P<.05) bone and plasma Zn, but no change in plasma inorganic phosphorous or bone levels of Ca, Fe, or Mg. The addition of phytase to the diets resulted in a 22.4% increase (P<.05) in body weight at the end of the study as compared with rats fed a control diet. Dual x-ray absorptiometry (DXA) revealed that phytase supplementation resulted in increase lean body mass (LBM, P<.001) and increased bone mineral content (BMC, P<.001) as compared with feeding the control diet. Bone studies indicated that femurs and tibias from phytase supplemented rats had greater mass (P<.05) and were stronger (P<.05) than rats fed the control diet. This data suggest that the addition of phytase to low Zn diets results in improved Zn status, which may be responsible for beneficial effects on growth, body composition, and bone strength.