A novel selective nucleoside phosphorylating enzyme from Morganella morganii

A selective nucleoside phosphorylating enzyme was purified to homogeneity from Morganella morganii NCIMB10466 crude extract. The enzyme appeared to consist of six subunits identical in molecular mass (M_r 25,000). It phosphorylated various nucleosides at the 5′-position to produce nucleoside-5′-monophosphates using pyrophosphate as the phosphate source. Energy-rich compounds, such as carbamylphosphate and acetylphosphate, were also very effective phosphate donors. The enzyme also exhibited phosphatase activity, and dephosphorylated various phosphate esters, but had a weak effect on nucleoside-3′-monophosphates. Based on the results of the kinetic study, the enzyme appeared to be an acid phosphatase. Its activity was partly inhibited by sulfhydryl reagents and heavy metal ions, but not by chelating reagents such as EDTA. Using the purified enzyme, 32.6 mM 5′-IMP was synthesized from inosine with a 41% molar yield, but the synthesized 5′-IMP was hydrolyzed back to inosine and phosphate as the reaction time was extended.     

Enzymatic production of 5′-inosinic acid by a newly synthesised acid phosphatase/phosphotransferase

5′-Nucleotides including 5′-inosinic acid have characteristic taste and important application in various foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase) can catalyse the synthesis of 5′-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase was purified using nickel–NTA. The optimal temperature and pH of this enzyme were 30 °C and 5.0, respectively. The activity was partially inhibited by metal ions such as Hg²⁺, Ag⁺ and Cu²⁺, but not by chelating reagents such as EDTA. The values of K_m and V_max for inosine were 40 mM and 3.5 U/mg, respectively. Using this purified enzyme, 16.83 mM of 5′-IMP was synthesised from 37 mM of inosine and the molar yield reached 45.5%. Homology modelling and docking simulation were discussed.     

Acid phosphatase/phosphotransferases from enteric bacteria

We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii. The enzymes were highly homologous in primary structure with M. morganii AP/PTase, and are classified as class A1 acid phosphatases. The synthesis of inosine-5'-monophosphate (5'-IMP) by E. coli overproducing each acid phosphatase was investigated. The P. stuartii enzyme, which is most closely related to the M. morganii enzyme, exhibited high 5'-IMP productivity, similar to the M. morganii enzyme. The 5'-IMP productivities of the E. aerogenes, E. blattae and K. planticola enzymes were inferior to those of the former two enzymes. This result underlines the importance of lower K(m) values for efficient nucleotide production. As these enzymes exhibited a very high degree of homology at the amino acid sequence level, it is likely that local sequence differences in the binding pocket are responsible for the differences in the nucleoside-PP(i) phosphotransferase reaction.     

Purification and properties of an acid phosphatase from Lactobacillus curvatus DPC2024

An acid phosphatase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-Sephacel, Phenyl Sepharose, chelating Sepharose Fast Flow and MonoQ. The purified enzyme was a tetramer with a subunit molecular mass of 26 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. Its optimum activity was at pH 4.5 and 70°C, with more than 65% of its activity retained after pre-heating for 30 min at 70°C. The enzyme was strongly inhibited by NaF (0.1 m ) and ZnCl₂ (1.0 m ); slightly inhibited by hexametaphosphate, tripolyphosphate or pyrophosphate at 1.0 m concentrations; but unaffected by 10 m ascorbic acid. The acid phosphatase hydrolysed a number of phosphate esters but not bis(p-nitrophenyl)phosphate nor uridine-5′-monophosphate. The N-terminal amino acid sequence of the first 20 residues of this enzyme showed 65% homology with an acid phosphatase from Lactobacillus plantarum DPC2739 and some homology with other phosphatases from mammals, yeasts and Escherichia coli.     

Purification and properties of an acid phosphatase from lactating bovine mammary gland

An acid phosphatase was partially purified from the cytosol of lactating bovine mammary gland by precipitation with ammonium sulfate and protamine, chromatography on carboxymethyl cellulose, and gel filtration on Sephadex G-75. The enzyme hydrolyzed aromatic phosphates but was less active toward alkyl phosphates, ATP, and phosphoproteins (casein and phosvitin). A sulfhydryl group seems to be essential for activity, since dithiothreitol and cysteine activated the enzyme; compounds that react with the sulphydryl groups in proteins were inhibitory. Orthovanadate, phosphate, and zinc ions also inhibited the phosphatase.     

PURIFICATION AND CHARACTERIZATION OF HOMOGENEOUS ACID PHOSPHATASE FROM NONGERMINATED BUCKWHEAT (FAGOPYRUM ESCULENTUM) SEEDS

A buckwheat acid phosphatase (orthophosphoric‐monoester phosphohydrolase, EC 3.1.3.2) was purified about 250‐fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p‐nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p‐nitrophenyl phosphate were determined to be K_M= 76 μmol L^?1 and k_cat= 924 s^?1 at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.     

Acid phosphatases activity in cheese and starters.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.     

Sapid Components in Carrot

SUMMARY: The relationship between various substances present in carrot to its taste was studied. Nucleic acid derivatives found in hot water extracts of carrot were adenine, adenosine, inosine, hypoxanthine, 5'-AMP, 5'-UMP, UDP, but 5'-IMP and 5'-GMP were absent. The contents of these derivatives were extremely small. Silica gel chromatography showed the presence of small quantities of succinic acid, α-ketoglutaric acid, lactic acid, pyroglutamic acid, citric acid and glycolic acid.
Amino acids in hot water extracts of carrot were detected by two-dimensional thinlayer chromatography and automatic amino acid analyzer. Identified were glutamic acid, valine, leucine, aspartic acid, lysine, and serine etc. Glutamic acid content was relatively large. Sucrose, maltose and glucose were detected in carrot and these carbohydrates were responsible for the sweetness of carrot. The taste of carrot was due mainly to the presence of glutamic acid and the buffer action of various amino acids.     

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.     

ALKALINE PHOSPHATASE FROM JAW ALA SHRIMP (ACETES INDICUS)

Alkaline phosphatase was isolated from Jawala shrimps (Acetes indicus), a tiny crustacean. The hepatopancreas (head region) is a good source of this enzyme. pH optima of the partial purified enzyme was found to he 9.5 and optimal temperature for maximal activity was 40C. Jawala phosphatase was completely inhibited by 1, 10‐phenanthroline and EDTA, indicating that it is a metalloprotein. The activity of inhibited enzyme was restored by Zn²⁺ and Mn²⁺ salts. Feed back inhibition of the enzyme by inorganic phosphate was also observed.     

5’—肌苷酸生产菌的选育及其发酵的初步研究

本文对5'-肌苷酸产生菌的选育及发酵条件对产酸的影响作了研究。 以谷氨酸棒杆菌265为出发菌,利用紫外线硫酸二乙酯以及亚硝基胍等常规诱变手段进行了反复诱变。在此基础上,又采用原生质体诱变的新技术,最后获得一株突变株519,在最佳发酵条件下积累肌苷酸量达7g/L,比原菌株提高了2倍。 本文还对影响发酵产酸的各种因素进行了研究,主要包括种子培养基和发酵培养基、pH值、种龄、接种量、通风量、温度以及添加某些成分等对发酵产酸的影响,并就其中某些因素的影响从理论上进行了探讨。     

Purification and characterization of an acid phosphatase from Lactobacillus plantarum DPC2739

An acid phosphatase was partially purified from a cell-free extract of Lactobacillus plantarum DPC2739 by a combination of anion-exchange chromatography on DEAE-Sephacel, hydrophobic interaction chromatography on Phenyl Sepharose, gel permeation chromatography on Sephacryl S200 and high performance anion-exchange chromatography on MonoQ. The native enzyme (∼110 kDa) was tetrameric with a subunit molecular mass of ∼27 kDa. The enzyme was heat-stable, retaining ∼60% of its activity after heating for 30 min at 70°C. It was optimally active in the pH range 3.5–5.0 and at 40°C. The enzyme was strongly inhibited by 0.5 mM sodium fluoride, and hexametaphosphate and by 5 mM orthophosphate, tripolyphosphate and pyrophosphate. It was insensitive to metal chelators (ethylenediaminetetraacetic acid and o-phenanthroline), ascorbic acid, sulphydryl blocking agents (e.g., N-ethylmaleimide), phenylmethylsulphonyl fluoride and divalent metal ions at 5 mM concentration. The enzyme appeared to be a non-specific phosphomonoesterase and hydrolysed a number of phosphate esters. The amino acid sequence of the first 20 residues was determined and showed some homology with mammalian, yeast and Escherichia coli acid phosphatases, phosphoglycerate mutases and phosphoglycerokinases with a common motif Arg-His-Gly. ©     

Purification and properties of alkaline phosphatase in the lactating bovine mammary gland.

Alkaline phosphatase has been purified 1400-fold from homogenates of lactating bovine mammary tissue. The purification procedure included subcellular fractionation, solubilization with butanol, fractionation with acetone, chromatography on concanavalin A-Sepharose, DEAE cellulose, DEAE-Sephadex, and gel filtration on Sephadex G-200. The enzyme activity was measured with the substrate p-nitrophenylphosphate in three buffers, and the maximum rate occurred at pH 10. For maximum activity, Mg2+ was required. Substrate specificity studies at three pH values indicated that the enzyme had broad specificity. It catalyzed the hydrolysis of aliphatic and aromatic phosphates and pyrophosphates, but the phosphoprotein beta-casein was a poor substrate. Potent inhibitors of the enzyme were levamisole and sulfhydryl reagents (2-mercaptoethanol, dithiothreitol, and cysteine).     

Purification and properties of an acid phosphoprotein phosphatase from lactating bovine mammary gland with activity toward phosphotyrosine

An acid phosphatase has been partially purified from lactating bovine mammary gland. Properties of this enzyme were compared with those of a well-characterized phosphoprotein phosphatase from bovine spleen. The two enzymes were similar in their activation by sulfhydryl reagents and inhibition by metal chelating agents. Both enzymes rapidly hydrolyze ATP and aromatic phosphates and are relatively inactive toward alkyl phosphates; both are tartrate-resistant phosphatases. The mammary enzyme has a low Michaelis constant for alpha s1-casein (42 microM), and thus, like the spleen enzyme, appears to be a phosphoprotein phosphatase. Finally, the spleen and mammary enzymes displayed reactivity toward phosphotyrosine, a model substrate for phosphotyrosyl protein phosphatase. Thus, the phosphatases from spleen and mammary gland are quite similar in reactivity and could possibly be similar in function.     

Enzymatic synthesis of fructose 1,6-diphosphate with ATP regeneration in a batch reactor and a semibatch reactor using purified enzymes of Bacillus stearothermophilus

The enzymatic synthesis of fructose 1,6-diphosphate (FDP), an important glycolytic intermediate whose applications in the field of medicine have generated a great deal of interest, was performed in a batch reactor and a semibatch reactor. Using the batch reactor, FDP was first synthesized from glucose by three enzymatic reactions and the ATP consumed was regenerated simultaneously using conjugated enzymes, all of which were purified from crude cell extract of thermophilic Bacillus stearothermophilus. The results of the experiments performed using several enzyme concentrations suggest the existence of an optimum concentration for each enzyme at which the maximum FDP yield can be attained. Since the thermal decomposition of acetyl phosphate reduced the yield of FDP in the batch reactor, the use of a semibatch reactor in which acetyl phosphate was fed continuously was examined. The yield of FDP was improved but the time required to complete the reaction was longer, resulting in a lower productivity of FDP. The yields observed in the two reactors using various enzyme and substrate concentrations were in good agreement with the theoretical predictions calculated based on differential equations derived for the system using the rate equations and the kinetic parameters determined previously. This means that these equations can be used for the analysis of the experimental results as well as for determining the optimum experimental conditions.     

Enzymic differentiation between curds of heated and raw milk. I. milk alkaline phosphatase

Alkaline phosphatase, which is present in curds made from raw milk as well as from boiled milk, does not serve to differentiate them. It was found that incubation time or acidity (lactic acid equivalent of curd acidity incubated aseptically with heated milk) does not revive heat-denatured phosphatase. The reappearance was found to be due to phosphatase of microbial origin; it was not a case of reactivation of the denatured enzyme. Low intensity, but increasing slowly, of the activity of regenerated phosphatase in curd (permeability of the microbial cells to the testing reagents is probably limiting in the rate of reaction), presence of the enzyme in the sonicated cells of curd-forming microflora and absence of activity in the supernatant of the growth medium suggest that the formed enzyme is an intracellular one.     

Purification and partial characterisation of X-prolyl dipeptidyl aminopeptidase of Lactobacillus helveticus ITG LH1

A X-prolyl dipeptidyl aminopeptidase (EC 3.4.14.5, XPDAP) from Lactobacillus helveticus ITG LH1, a strain used for Swiss-type cheese, was purified by ion exchange and affinity chromatographies. The enzyme appeared to be a 140 kDa monomer. Optimal activity occurred at pH 7 and 40°C, but it was rapidly inactivated above 50°C. The enzyme was activated by NaCl and KCl up to 50–200 mm but its activity levelled off at higher salt concentrations. Its complete inhibition was caused by 0.1 mm HgCl₂, 1 mm SnCl₂ and 2.5 mm CuCl₂. It was inactivated by reagents specific for serine proteases, such as phenylmethylsulfonyl fluoride and sulfhydryl group-blocking reagents. The enzyme hydrolysed p-nitroanilide-substituted X-Pro and X-Ala dipeptides, as well as β-casomorphin 1-4.     

Partial Purification and Characterization of an Acid Phosphatase from Papaya

A phosphatase in papaya was extracted, partially purified, and characterized. With p-nitrophenyl phosphate as substrate, the enzyme had a pH optimum of 6.0, which categorized it as an acid phosphatase, a temperature optimum of 37°C, and a Km of 1.0 mM. Heat inactivation of papaya acid phosphatase was biphasic, and the kinetics of both phases were first order reactions. D values at 60°, 65°, 70°C for the heat resistant phase were 21.0, 11.7, and 4.0 min, respectively. For the heat labile and heat resistant isozymes of papaya acid phosphatase, the activation energies, E_a, for thermal inactivation were 60.0 Kcal/mole and 37.8 Kcal/mole, respectively. The apparent molecular weight of the enzyme as determined by gel filtration was 120,000 daltons.     

嗜热拟青霉木糖苷酶的性质及其与木聚糖酶的协同作用

嗜热拟青霉(Paecilomyces thermophila)J18利用玉米芯能产胞外木糖苷酶,通过(NH_4)_2SO_4沉淀、DEAE-52离子交换层析及Q-琼脂糖凝肢-FF(QSFF)离子交换层析从上清液纯化得到了电泳纯的木糖苷酶,纯化倍数为31.9倍,回收率为2.27%。SDS-PAG E及Superdex-75凝胶过滤层析测定木糖苷酶的分子量分别为53.5 ku和51.8 ku。该酶最适温度及pH分别为55℃和pH 6.5。木糖苷酶能够水解木二糖和低聚木糖但不能水解木聚糖,水解低聚木糖的相对速率随聚合度增加而增加。该酶对木糖的抑制常数Ki值为139 mmol/L,具有很高的木糖耐受性。木糖苷酶与内源木聚糖酶一起水解木聚糖产生更多的还原糖,表现出协同作用。     

解淀粉芽孢杆菌I型耐热普鲁兰酶的纯化及酶特性分析

对来源于菌株解淀粉芽孢杆菌HxP-21的普鲁兰酶（命名为PulBa）分离纯化，研究其酶学特性，为普鲁兰酶在淀粉加工中的应用提供理论基础。通过硫酸铵沉淀、阴离子交换层析和葡聚糖凝胶过滤层析从菌株HxP-21发酵液中分离纯化出一种新型的普鲁兰酶。酶的纯化倍数20.8，回收率53.2%，比活力176.5 U/mg。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得PulBa达到电泳纯，分子质量51.2 kDa。PulBa在45～70 ℃和pH 3～6范围内具有较高酶活力，最适反应温度55 ℃、pH 4.5。PulBa有良好的pH值稳定性和热稳定性，40～70 ℃孵育120 min保留最初活性的80%以上；pH 3～7范围内具有很高的稳定性，孵育6 h后仍保留60 U/mL以上活性。PulBa对各种金属离子和化学试剂表现出不同的敏感性，Mg2＋和Ca2＋能够显著增强酶活力。PulBa最适作用底物为普鲁兰糖，对马铃薯支链淀粉、玉米支链淀粉、可溶性淀粉和糖原也有一定水解活性，但对α-环糊精和β-环糊精和直链淀粉无活性。以普鲁兰糖为底物PulBa的Km和Vmax值分别为1.34 mg/mL和24.6 μmol/（min·mg）。研究表明PulBa是典型的I型普鲁兰酶。薄层层析进一步证明，PulBa专一性水解支链淀粉α-1,6-糖苷键，产生麦芽三糖。本研究确定了一种新型的普鲁兰酶，该酶在高热稳定性和酸性环境下具有高活性，在淀粉加工等生物技术产业中有较好的应用潜力。