Purification and mode of action of phytase from Phaseolus aureus

Phytase isolated from germinated mung bean cotyledons was further purified and migrated as a single protein band in polyacrylamide gel electrophoresis.     

Multiple forms of phytase in germinating cotyledons of Cucurbita maxima

Multiple forms of phytase (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) have been isolated in highly purified forms from germinating Cucurbita maxima cotyledons using acetone and ammonium sulphate fractionation, Sephadex gel filtration and ion exchange chromatography on DEAE- and CM-cellulose. Gel filtration produced two peaks of phytase activity; phytase I (high MW) and phytase II (low MW). Phytase I was further resolved into 4 distinct species on CM-cellulose and these were designated phytase IA, IB, IC and ID, according to their elution order. On the other hand, phytase II remained as a single species with a purification of 35-fold. The MWs of each phytase I species were identical (MW 66 500 ± 4000) and they were twice the MW of phytase II (MW 32 400 ± 4000) indicating that I and II may be structurally related. The properties of various molecular forms were compared. The difference in properties between phytase II and phytase I isoenzymes (IA, IB, IC and ID) was more pronounced than that observed among the isoenzymes of phytase I alone.     

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn²⁺ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- ³²P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K_m values for GDP and inositol hexaphosphate were 1·1 × 10⁻⁴ M and 1·6 × 10⁻⁶ M respectively.     

Isosojagol,a coumestan from Phaseolus coccineus

A novel coumestan isolated from Phaseolus coccineus has been characterized as 3,9-dihydroxy-10-(γ,γ-dimethylallyl)-coumestan and named isosojagol.     

Further characterization of phosphoinositol kinase isolated from germinating mung bean seeds

Phosphoinositol kinase isolated and purified from germinating mung bean seeds has been further characterized. The rate of phosphorylation varies with different inositol phosphates and this is consistent with the K_m and V_max for each of the substrates. The phosphate transfer from ATP has been found to be mediated by a phosphoprotein intermediate. In a particular step of the reaction the immediate product of the reaction has been found to be most inhibitory, other products being less or non-inhibitory. The inhibition has been found to be competitive in nature. The K_is have been found to range between 0.6 and 1 × 10^?4 M. ADP also inhibited non-competitively with respect to IP₅. K_i for this has been found to be 2.3 × 10^?4 M. The purified enzyme migrated as a single protein band on polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulphate it is dissociated into 3 subunits in the ratio 1 : 1 : 1. The MW of the three subunits are approx. 86 000, 56 000 and 35 000. The MW of the enzyme has been found to be approx. 177 000.     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Isolation and characterization of a phytase with improved properties from Citrobacter braakii

Citrobacter braakii YH-15 produced an intracellular phytase which was purified 12800 fold to homogeneity with the specific activity of 3457 units mg^–1, which is 1.9 times higher than E. coli phytase previously recorded as having the highest specific activity. Its molecular weight was 47 kDa by SDS-PAGE gel. Enzyme activity was optimal at pH 4 and at 50 °C. The K _m value for sodium phytate was 0.46 mM with a V _max 6027 U mg^–1. The phytase was resistant to proteases such as trypsin, pepsin, papain, pancreatin, and elastase.     

Isolation,purification and characterization of phytase from germinating mung beans

Phytase isolated from mung bean cotyledons was purified about 80-fold with a recovery of 28%. The enzyme is stable at 0°, has a pH optimum at 7·5 and optimal temperature of 57°. The energy of activation is approximately 8500 cal/mole between 37° and 57°. Inhibition by P_i has been found to be competitive, the K_i value being 0·40–0·43 × 10⁻³ M; the K_m value with phytate is 0·65 × 10⁻³ M. Divalent cations are not required for activity. Lower members of inositol phosphates are better substrates, as shown by their V_max and K_m values. When subjected to polyacrylamide gel electrophoresis two bands have been resolved; one (major) corresponds to phytase and the other (minor) to phosphatase and pyrophosphatase activity. Filtration through Biogel P-200 partially resolves phytase from phosphatase and pyrophosphatase. The molecular weight of phytase is approximately 160,000.     

渗透胁迫对绿豆叶圆片叶绿素荧光的影响

绿豆叶圆片用0、-0．3、-0．6、-1．2、-1．8、-2．4MPa等6个渗透梯度处理24h后,其叶绿素荧光的反应表明：光系统Ⅱ的潜在量子产量(FD／Fm)和电子传递受体(QA)的库容(pool size)均随胁迫强度的增大而减小,但QA库容的下降明显比Fg／Fm的下降缓和。结合原初荧光Fo和最大荧光Fm的变化,分析认为本实验中渗透胁迫并未导致明显的光系统Ⅱ反应中心的失活或破坏。造成光系统Ⅱ潜在量子效率降低的原因主要是通过光系统Ⅱ反应中心到Ⅱ的电子传递受到阻抑。     

New isoflavonoids related to kievitone from Phaseolus vulgaris

The new isoflavonoids 5,7,2′,4′-tetrahydroxy-8-(3,3-dimethylallyl)isoflavone (2,3-dehydrokievitone) and 7,2′,4′-trihydroxy-8-(3,3-dimethylallyl)isoflavanone (5-deoxykievitone) have been isolated from fungus-inoculated Phaseolus vulgaris pod tissue and from the inoculation droplets. A third isoflavonoid was tentatively identified as 1″,2″-dehydrocyclokievitone and appears to be a metabolite of the phytoalexin kievitone.     

Sugar-related hydroxy acids from Phaseolus and trifolium species

-Hydroxy acids isolated from leaves of French bean (Phaseolus vulgaris) and clover (Trifolium incarnatum) were analysed by GLC as trimethylsilyl derivatives and identified by MS. Large amounts of a 2-C-methyltetronic acid and appreciable amounts of gluconic acid and of a 2-C-(hydroxymethyl)pentonic acid were found from French bean. Glyceric acid was the predominant acid from clover but the presence of several other acids, e.g. threonic and malic acids, was also demonstrated.     

植酸酶产生菌的筛选与酶纯化及其性质的研究

对从土壤中分离得到的一株产植酸酶的细菌进行了生理生化鉴定,并对植酸酶进行了分离纯化,该酶反应的最适温度约为55℃,最适pH值为5．8,植酸酶蛋白分子量约为14kD。     

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.     

Alkaline phytase from Lilium longiflorum: purification and structural characterization

Phytases catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytases are of great commercial importance because their use as food and animal feed supplement has been approved by many countries to alleviate environmental and nutritional problems. Although acid phytases have been extensively studied, information regarding alkaline phytases is limited. Alkaline phytases with unique catalytic properties have been identified in plants, however, there is no report on the purification or structural properties. In this paper, we describe the purification of alkaline phytase from plant tissue. The purification was challenging because of contamination from non-specific phosphatases and acid phytases and low endogenous concentration. The purification of alkaline phytase from pollen grains of Lilium longiflorum involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified approximately 3000-fold with an overall recovery of 4.2%. The native molecular mass was estimated to be in the range of 118+/-7 kDa by Ferguson plot analysis and Mr of denatured protein in the range of 52-55 kDa by SDS-PAGE suggesting that the enzyme is a homodimer. Separation by 2-D gel and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis of separated proteins indicates the presence of multiple mass and charge isoforms with pI values between 7.3 and 8.3. To our knowledge, this is the first alkaline phytase to be purified from plant sources. The unique properties suggest that the enzyme has the potential to be useful as a feed and food supplement.     

Recombinant Pichia pastoris overexpressing bioactive phytase

Ｐｈｏｓｐｈｏｒｏｕｓｉｓａｎｅｓｓｅｎｔｉａｌｅｌｅｍｅｎｔｆｏｒｔｈｅｇｒｏｗｔｈａｎｄｄｅｖｅｌｏｐｍｅｎｔｏｆａｌｌａｎｉｍａｌｓ,ｐｌａｙｉｎｇｋｅｙｒｏｌｅｓｉｎｓｋｅｌｅｔａｌｓｔｒｕｃｔｕｒｅａｎｄｉｎｖｉｔａｌｍｅｔａｂｏｌｉｃｐａｔｈｗａｙｓ.Ｕｐｔｏ80%ｏｆｔｈｅｔｏｔａｌｐｈｏｓｐｈｏｒｏｕｓｉｎｆｅｅｄｓｔｕｆｆｓｏｆｐｌａｎｔｏｒｉｇｉｎｉｓｔｈｅｐｈｙｔａｔｅｐｈｏｓｐｈｏｒｏｕｓ.Ｉｔｉｓｐｏｏｒｌｙａｖａｉｌａｂｌｅｔｏｍｏｎｏｇａｓｔｒｉｃａｎｉｍａｌｄｕｅｔｏｔ…     

Purification and characterization of myo-inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus

myo-Inositol hexaphosphate adenosine diphosphate phosphotransferase transfers phosphate from myo-inositol hexaphosphate to adenosine diphosphate to synthesize adenosine triphosphate. This enzyme has been isolated and purified from ungerminated mungbean seeds and found to be different from guanosine diphosphate phosphotransferase. A purification of about 200-fold with 15% recovery has been obtained. The optimal pH of the reaction is 7.0 and is dependent on the presence of a divalent cation, i.e., Mg²⁺ and Mn²⁺. The K_m value for myo-inositol hexaphosphate has been found to be 0.41 × 10^?4m and V is 90.0 nmol of P_i transferred per milligram of protein per 20 min. K_m for ADP is 0.88 × 10^-4m and V is 83.3 nmol of phosphorus transferred to ADP per milligram of protein per 20 min. The ADP phosphotransferase reaction is reversible to the extent of about 50% of the forward reaction. dADP is partly effective as an acceptor but other ribonucleoside mono- and diphosphates cannot substitute for ADP. The products ATP and myo-inositol pentaphosphate have been confirmed by several criteria. It has also been shown that this enzyme transfers phosphate only from a specific phosphoryl group (C-2 position) of myo-inositol hexaphosphate for the synthesis of ATP and 1,3,4,5,6-myo-inositol pentaphosphate or pentakis (dihydrogen phosphate).     

The role of acid phosphatase activity during enzymic dephosphorylation of phytates byAspergillus niger phytase

Acid phosphatase present in preparations ofAspergillus niger phytase accelerated dephosphorylation of sodium phytate. Its influence on the reaction rate and distribution ofmyo-inositol phosphates was most apparent at low pH value (2.5) and when acid-hydrolysed substrate was de-esterified enzymatically. With partly purified phytase, the predominant inositol form was tetraphosphate but a preparation having acid phosphatase activity caused an even distribution of lower inositol phosphates after a few hours.     

青霉LQ-7植酸酶产生条件研究

筛选到一株植酸酶高产菌株 ,对其进行诱变并研究了该菌株产植酸酶的最适条件。优化产酶液体培养基组成为 :3%可溶性淀粉 ,0 5 %蛋白胨 ,0 5 %NH4NO3 ,0 0 5 %MgSO4·7H2 O ,0 0 5 %FeSO4·7H2 O ,0 0 0 1%MnSO4·4H2 O ;发酵培养基的起始pH为 6 5 ,接种量 4%的条件下 30℃ ,135r min培养 72h可获得较高的植酸酶产量。少量 (2 5mg)植酸盐的加入对植酸酶有促进作用 ,但过量 (10mg以上 )时则会产生抑制作用。     

植酸酶作用机理的初探

电喷雾电离－质谱联用仪分析结果表明,植酸酶水解植酸是以分步方式进行的,植酸酶能将植酸水解成植酸五磷酸酯至植酸一磷酸酯不同的中间产物。但最终产物主要为二磷酸肌醇,与一些同时形成的无机磷分子能与未水解的植酸以“－Ｏ－Ｏ”或“－Ｏ－”键形成多磷酸肌醇的更复杂的分子形式。     

Purification and properties of cellulase from Phaseolus vulgaris

Cotyledons of germinating kidney beans contain two forms of a carboxy methyl cellulase which can be separated by ammonium sulfate fractionation and isoelectric focusing. The two cellulases are similar in their molecular weight but differ in isoelectric points, pH and temperature optimum, pH and temperature stability and sensitivity to thiol inhibitors and metal ions. One cellulase (isoelectric point 4.8) has been purified 100-fold to give a major protein band on acrylamide gel electrophoresis.