Purification and partial characterization of milk-clotting enzyme extracted from glutinous rice wine mash liquor

Glutinous rice wine mash liquor is a traditional food of south of China and its ability to coagulate the milk has been proved. The aim of this work was to extract milk-clotting enzyme from glutinous rice wine mash liquor. A partial purified extract of enzyme was obtained by fractional precipitation with (NH₄)₂SO₄. The fractions obtained by precipitation, 40–90% possessed the milk-clotting activity (MCA) (145.72 U/mg). The 40–90% (NH₄)₂SO₄ fraction was further purified by sephadex G-100 and DEAE-sephadex A-50 with MCA (4,360±50 U/mg), which was confirmed by SDS-PAGE that showed only one band with a molecular mass of 36.0 kDa. Highest MCA was attained at 36 °C. The enzyme was completely inactivated by heating for 20 min at 60 °C. The MCA increased with the decreasing of milk pH from 8.0 to 5.5, and it was active at the wide range of pH 1 to 7. The metal ions Mg²⁺, Ca²⁺, Ba²⁺, Mn²⁺, Al³⁺, Fe²⁺ had a very clear function to accelerate milk coagulation whereas Na⁺ and K⁺ decelerated the activity slightly. The curd effect of the milk-clotting enzyme has primarily been studied.     

Purification and characterization of a thermophilic chitinase produced by <Emphasis Type="Italic">Aeromonas</Emphasis> sp. DYU-Too7

An extracellular chitinase, produced by Aeromonas sp. DYU-Too7, was purified in the following procedures: ammonium sulfate precipitation, ultrafiltration, chromatographic separation of DEAE-sepharose CL-6B and sephacrylS-100HR. The resulting chitinase has a molecular mass of 36 kDa, an optimal reaction pH of 5.0, and an optimal reaction temperature of 70°C. It retains almost 100% activity in the pH range of 5.0–8.0. This chitinase has a high thermal tolerance and retained 90% of its activity at 50°C and 75% at 60°C. Enzyme activity was inhibited by Ba²⁺, Hg²⁺, Mg²⁺ and Ag⁺ cations, but was not substantially inhibited by the K⁺ cation nor the chelating agent EDTA. The K _m and V _mα , using colloidal chitin as a substrate, are 6.3 g/L and 18.69 μmol/min/mg-protein, respectively. The 36 kDa chitinase of Aeromonas sp. DYU-Too7 is an exo-type enzyme, because chitobiose was the main hydrolysate in hydrolysis of colloidal chitin.     

Purification and characterization of thermophilic and alkalophilic tributyrin esterase fromBacillus strain A30-1 (ATCC 53841)

An extracellular esterase (EC 3.1.1.1) from a thermophilicBacillus A30-1 (ATCC 53841) was purified 139-fold to homogeneity by sodium chloride (6 M) treatment, ammonium sulfate fractionation (30–80%) and phenyl-Sepharose CL-6B column chromatography. The native enzyme was a single polypeptide chain with a molecular weight of about 65,000 and an isoelectric point at pH 4.8. The optimum pH for esterase activity was 9.0, and its pH stability range was 5.0–10.5. The optimum temperature for its activity was 60°C. The esterase had a half-life of 28 h at 50°C, 20 h at 60°C and 16 h at 65°C. It showed the highest activity on tributyrin, with little or no activity toward long-chain (12–20 carbon) fatty acid esters. The enzyme displayed K_m and K_cat values of 0.357 mM and 8365/min, respectively, for tributyrin hydrolysis at pH 9.0 and 60°C. Cyclodextrin (α, β, and γ), Ca²⁺, Co²⁺, Mg²⁺ and Mn²⁺ enhanced the esterase activity, and Zn²⁺ and Fe²⁺ acted as inhibitors of the enzyme activity. The enzyme activity was not affected by ethylenediaminetetraacetic acid, p-chloromercuribenzoate andN-bromosuccinimide. This paper was presented in part at the 82nd Annual Meeting and Exposition of the American Oil Chemists’ Society, held May 12–15, 1991, in Chicago, Illinois.     

Kinetic,thermodynamic parameters and in vitro digestion of tannase from Aspergillus tamarii URM 7115

Tannase is an enzyme used in various industries and produced by a large number of microorganisms. The aim of this study was to evaluate tannase production to determine the biochemical, kinetic, and thermodynamic properties and to simulate tannase in vitro digestion. The tannase-producing fungal strain was isolated from “jamun” leaves and identified as Aspergillus tamarii. Temperature at 26°C for 67?h was the best combination for maximum tannase activity (6.35-fold; initial activity in Plackett–Burman design—15.53?U/mL and average final activity in Doehlert design—98.68?U/mL). The crude extract of tannase was optimally active at 40°C, pH 5.5 and 6.5. Moreover, tannase was stimulated by Na⁺, Ca²⁺, Mg²⁺, and Mn²⁺. The half-life at 40°C lasted 247.55?min. The free energy of Gibbs, enthalpy, and entropy, at 40°C, was 81.47, 16.85, and ?0.21?kJ/mol?·?K, respectively. After total digestion, 123.95% of the original activity was retained. Results suggested that tannase from A. tamarii URM 7115 is an enzyme of interest for industrial applications, such as gallic acid production, additive for feed industry, and for beverage manufacturing, due to its catalytic and thermodynamic properties.     

Purification and Biochemical Characterization of Lipase from <Emphasis Type="Italic">Ficus carica</Emphasis> Latex of Tunisian East Coast Zidi Variety

Lipase from Ficus carica L. (Moraceae) latex of the Zidi variety was purified 80.5-fold with 68.5 % recovery using silica gel chromatography. The molecular weight of the enzyme was 29 kDa as determined by SDS-PAGE. High lipolytic activity was found in the crude extract during the fruit ripening process. The activity of purified lipase (ZL) seemed to depend strongly on chain length and showed a preference to long chain triacylglycerols. Indeed, ZL specific activity was 370.3 UI/mg using olive oil as a substrate at 45 °C and pH 5.5. In contrast, activity towards short chain triacylglycerols (tributyrin) was 12-fold lower (32 UI/mg). The enzyme was quite stable in the pH range 4–8, and thermally stable at 60 °C displaying t _1/2 about 90 min using olive oil as a substrate. The values of K _m app and V _m were found to be 14.3 mM and 294.1 μmol/min/mg, respectively. ZL activity was strongly reduced by Fe²⁺, Mg²⁺ and Zn²⁺, while significantly increased by Ca²⁺ and Cu²⁺. The enzyme was stimulated by sodium dodecyl sulfate, and Tween-80, while Triton X-100 and EDTA had a slight inhibitory effect. No Effect was observed in addition of PMSF and iodoacetic acid.     

Identification and Characterization of a Novel Thermophilic,Organic Solvent Stable Lipase of <Emphasis Type="Italic">Bacillus</Emphasis> from a Hot Spring

A novel lipase gene lip256 was cloned and identified from the genomic library of hot spring strain Bacillus sp. HT19. The deduced amino acid sequence of lip256 has less than 32% identity to a predicted esterase (Cog1752) from Photobacterium leiognathi lrivu.4.1 and contains a novel motif (GTSAG) that differs from other clusters in the lipase superfamily. Following purification, a single band was obtained with a molecular mass of 33 kDa by SDS-PAGE, and the optimal temperature and pH for lipolytic activity of Lip25 were 70 °C and 9.0, respectively. Lip256 exhibited high activity at high temperatures, with 40% maximum activity at 80 °C and good stability at temperatures ranges between 50 and 80 °C. Additionally, the enzyme was highly stable in the presence of butyl-alcohol, glycerol, acetonitrile, pyridine, and urea. However, the presence of acetone, methanol, trichloromethane, petroleum ether, hexane, tert-butanol, isopropanol, dithiothreitol, ethylenediaminetetraacetic acid, polyhexamethylene biguanide, dimethyl sulfoxide, benzene, Triton X-100, Tween-20, Tween-80, and sodium dodecyl sulfate suppressed or absolutely inhibited enzyme activity. Furthermore, Ca²⁺, Mg²⁺, and Cu²⁺ suppressed enzyme activity, whereas Na⁺, Fe³⁺, K⁺, Fe²⁺, and Sr²⁺ enhanced enzyme activity. The unique characteristics of novel lipase Lip256, including its thermo-alkaliphilic performance, high tolerance toward metal ions, inhibitors, and detergents, and high stability in organic solvents, implied that this enzyme might be an interesting candidate for industrial processes.     

Biochemical Characterization of Purified Esterase from Soybean (Glycine max) Seed

Alkaline esterase (carboxylic‐ester hydrolases; EC 3.1.1.1) extracted from germinated soybean seeds (Glycine max) was purified approximately 3.6 times by chromatography in a DEAE‐cellulose anion exchange column and filtration in Sephadex G100 gel. The molecular mass of the enzyme was estimated at 45 kDa by gel electrophoresis (SDS‐PAGE). The purified enzyme showed a specific activity of 5.6 U mg^?1 using p‐nitrophenyl butyrate as substrate. The esterase showed optimal activity at 47 °C in moderately alkaline pH, low stability in temperatures higher than 50 °C, and high stability at pH values between 6 and 9.5. The Ca²⁺ and Co²⁺ ions proved to have a positive effect on enzyme activity; however, Hg²⁺ completely inhibited esterase activity. Using p‐nitrophenyl butyrate as substrate, the enzyme showed a K_m of 0.39 mM, V_max of 31.5 mM mg^?1 min^?1 and k_cat 7.60 × 10⁶ s^?1. Regarding substrate affinity, the enzyme showed greater activity for substrates containing short‐chain fatty acids, especially p‐nitrophenyl acetate. Such characteristics give the enzyme great potential for application in the production of low molecular weight esters, in the food industry, and in chemical products. This enzyme is another new member of the family of lipases and esterases from vegetable seeds with high activity and stability in alkaline pH.     

Ion—solvent interaction: viscosity of ternary electrolytes in dioxane-water mixtures at 35°

The viscosities of the chlorides, bromides and nitrates and perchlorates of Mg²⁺ and Ba²⁺, of the chlorides and nitrates of Ca²⁺ and Sr²⁺, and of the sulphates of Na⁺ and K⁺ at mass fraction of dioxane, 10, 20 and 30% have been measured at 35°. The values of the constant A and B of the viscosity equation indicate ion—ion and ion—solvent interaction respectively. The ion-solvent interaction is found to be of the order NO^?₃ > ClO^?₄ > Br^? > Cl^? and K⁺ > Na⁺.     

Factors influencing the structure of electrochemically prepared α-MnO2 and γ-MnO2 phases

The α- and γ-phases of MnO₂ prepared by electrolysis of MnSO₄ and M_xSO₄ (where M = Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺ or Mg²⁺) in aqueous solutions at various pH and voltage E_v values under ambient conditions have been systematically studied. The structures of powdery MnO₂ produced are found to depend on the radius of the M^z+ counter cation in addition to the pH and E_v conditions. In order to achieve the α-phase for MnO₂ formation under neutral pH condition, the radius of counter cation must be equal to or greater than 1.41 Å, the size of the K⁺ cation. The relative concentration ratio of [MnO₄⁻]_transient/[Mn²⁺], which is related to the pH-E_v conditions, also affects the structure of MnO₂ produced with counter ions smaller than K⁺. For samples prepared in acidified solution with the counter ions of Li⁺, Na⁺ or Mg²⁺ at 2.2 V, the electrolysis products display the γ-MnO₂ phase while those prepared at 2.8 V electrolysis produce a mixture of γ-MnO₂ and α-MnO₂ phases. Single phase of α-MnO₂ is identified in the 5 V electrolysis products. Furthermore, the valence state of manganese was found to decrease as the applied voltage was reduced from 5.0 to 2.2 V. This implies that the lower [MnO₄⁻]_transient/[Mn²⁺] ratio or the less oxidative condition is responsible for the non-stoichiometric MnO₂ structure with oxygen deficiency.     

Purification and characterization of an extracellular lipase from <Emphasis Type="Italic">Geotrichum marinum</Emphasis>

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V _max and K _m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na⁺, K⁺, Ca²⁺, and Mg²⁺ stimulated activity, but Na⁺, and K⁺ at 500 mM and Fe²⁺ and Mn²⁺ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters K_M and V_max for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0–6 M and displays no significant changes of the activity in the presence of metal ions Ni²⁺, Cu²⁺, Mg²⁺, Zn²⁺ and Ca²⁺ and EDTA added in concentrations 1 and 10 mmol/L except for Fe³⁺. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.     

Biocatalytic Properties of Lipase from Walnut Seed (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K _m and V _max values were determined as 48 mM and 23.06 × 10⁻³ U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Ni²⁺ ions, while Mg²⁺ and Zn²⁺ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.     

Production and Characteristics of an Enantioselective Lipase from Burkholderia sp. GXU56

The lipase production of Burkholderia sp. GXU56 was influenced by carbon and nitrogen sources, inorganic salts, initial pH of the medium and cultivation temperature. The maximum lipase production was 580.52 U/mL and reached 5 times the level of the basic medium in the optimum medium at pH 8.0, 32 °C, 200 rpm and 40–48 h. The lipase was purified 53.6 fold to homogeneity and the molecular weight was 35 KDa on SDS‐PAGE. The optimum pH and temperature of the lipase were 8.0 and 40 °C, respectively, and it was stable in the range of pH 7–8.5 and at temperatures below 45 °C. The lipase activity was strongly inhibited by Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Fe³⁺ ions and SDS, while it was stimulated by Li⁺ and Ca²⁺ ions and in presence of 0.1 % CTAB, 0.1 % Triton X‐100 and 10 % DMSO. K_m and V_max of the lipase were calculated to be 0.038 mmol/L, and 0.029 mmol/L min^–1, respectively, with PNPB as the substrate. The GXU56 lipase showed enantioselective hydrolysis of (R,S)‐methyl mandelate to (R)‐mandelic acid, which is an important intermediate in the pharmaceutical industry.     

Miscibility of chitosan/poly(ethylene oxide) blends and effect of doping alkali and alkali earth metal ions on chitosan/PEO interaction

The miscibility of Chitosan (CS) and poly(ethylene oxide) (PEO) in their blends and the effect of K⁺ and Ca²⁺ doping on the CS/PEO interaction have been investigated in this work. CS and PEO appeared to be miscible and the DSC analysis suggested the Flory-Huggins interaction parameter χ_AB to be −0.21. Doping of K⁺ and Ca²⁺ into the CS/PEO blend matrix enhanced the cooperative interaction between CS and PEO and this enhancement was larger for Ca²⁺ than for K⁺. The difference between Ca²⁺ and K⁺ possibly reflects a stronger multi-valence interaction of Ca²⁺ with the amino and hydroxyl groups of CS as well as the ether groups of PEO to form a stable CS/Ca²⁺/PEO complex and a less significant interaction of K⁺, as suggested by DSC, WAXD and FTIR results. MD simulations clearly indicated the correlation between the dynamic behavior and the interaction of K⁺ and Ca²⁺ in the CS/PEO blend matrix.     

Some Biochemical Properties of Lipase from Bay Laurel (<Emphasis Type="Italic">Laurus nobilis</Emphasis> L.) Seeds

Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein⁻¹ in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. K _m and V _max values were determined as 0.975 g and 1.298 U mg protein⁻¹, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, and Mg²⁺ lightly enhanced bay laurel lipase activity.     

The solubility of selenate-AFt (3CaO·Al2O3·3CaSeO4·37.5H2O) and selenate-AFm (3CaO·Al2O3·CaSeO4·xH2O)

The Se(VI)-analogues of ettringite and monosulfate, selenate-AFt (3CaO·Al₂O₃·3CaSeO₄·37.5H₂O), and selenate-AFm (3CaO·Al₂O₃·CaSeO₄·xH₂O) were synthesised and characterised by bulk chemical analysis and X-ray diffraction. Their solubility products were determined from a series of batch and resuspension experiments conducted at 25 °C. For selenate-AFt suspensions, the pH varied between 11.37 and 11.61, and a solubility product, log K_so=61.29±0.60 (I=0 M), was determined for the reaction 3CaO·Al₂O₃·3CaSeO₄·37.5H₂O+12 H⁺⇔6Ca²⁺+2Al³⁺+3SeO₄²⁻+43.5H₂O. Selenate-AFm synthesis resulted in the uptake of Na, which was leached during equilibration and resuspension. For the pH range of 11.75 to 11.90, a solubility product, log K_so=73.40±0.22 (I=0 M), was determined for the reaction 3CaO·Al₂O₃·CaSeO₄·xH₂O+12 H⁺⇔4Ca²⁺+2Al³⁺+SeO₄²⁻+(x+6)H₂O. Thermodynamic modelling suggested that both selenate-AFt and selenate-AFm are stable in the cementitious matrix; and that in a cement limited in sulfate, selenate concentration may be limited by selenate-AFm to below the millimolar range above pH 12.     

Ion-Exchange Properties of lon-Sieve-Type Manganese Oxides Prepared by Using Different Kinds of Introducing Ions

Abstract

Three ion-sieve-type manganese oxides, HMnO(Li), HMnO(Na), and HMnO(K), were prepared by acid treatments of Li⁺-, Na⁺-, and K⁺-introduced manganese oxides, respectively. Three oxides were obtained from γ-MnO² and the corresponding alkali metal hydroxides by heating at 600°C. The ion-exchange properties of the adsorbents were investigated by pH titration, K_d measurements, and the adsorption of metal ions from seawater. The selectivity sequences of alkali metal ions were Na⁺ < Cs⁺ < Rb⁺ < K⁺ < Li⁺ for HMnO(Li) and Li⁺ Na⁺ < Cs⁺ < K⁺ < Rb⁺ for HMnO(Na) and HMnO(K). The high selectivity of Li⁺ on HMnO(Li) can be ascribed to an ion-sieve effect of spinel-type manganese oxide which was produced from LiMn₂O₄ Since HMnO(Na) and HMnO(K) had [2 × 2] tunnels of edge-shared [MnO₆] octahedra, the high selectivities of K⁺ and Rb⁺ on these samples were used to explain that the sizes of the [2 × 2] tunnels were suitable for filling ions of about 1.4 Å in radius in a stable configuration. The order of metal-ion uptake from seawater was Sr²⁺ < K⁺ < Mg²⁺ < Ca²⁺ < Na⁺ < Li⁺ for HMnO(Li), Li⁺ < Sr²⁺ < Mg²⁺ < Ca²⁺ < Na⁺ < K⁺ for HMnO(Na), and Li⁺ < Sr²⁺ < Ca²⁺ < Mg²⁺ < K⁺ < Na⁺ for HMnO(K).     

Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm

Sperm-specific K⁺ ion channel (KSper) and Ca²⁺ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca²⁺ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pH_i) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na⁺/H⁺ exchangers (NHEs) have been implicated to mediate pH_i in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N,N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pH_i acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca²⁺, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.     

A Selective Na+ Aptamer Dissected by Sensitized Tb3+ Luminescence

A previous study of two RNA‐cleaving DNAzymes, NaA43 and Ce13d, revealed the possibility of a common Na⁺ aptamer motif. Because Na⁺ binding to DNA is a fundamental biochemical problem, the interaction between Ce13d and Na⁺ was studied in detail by using sensitized Tb³⁺ luminescence spectroscopy. Na⁺ displaces Tb³⁺ from the DNAzyme, and thus quenches the emission from Tb³⁺. The overall requirement for Na⁺ binding includes the hairpin and the highly conserved 16‐nucleotide loop in the enzyme strand, along with a few unpaired nucleotides in the substrate. Mutation studies indicate good correlation between Na⁺ binding and cleavage activity, thus suggesting a critical role of Na⁺ binding for the enzyme activity. Ce13d displayed a K_d of ～20 mm with Na⁺ (other monovalent cations: 40–60 mm ). The K_d values for other metal ions are mainly due to non‐specific competition. With a single nucleotide mutation, the specific Na⁺ binding was lost. Another mutant improved K_d to 8 mm with Na⁺. This study has demonstrated a Na⁺ aptamer with important biological implications and analytical applications. It has also defined the structural requirements for Na⁺ binding and produced an improved mutant.