Purification and Characterization of Thermophilic Xylanase Isolated from the Xerophytic-<Emphasis Type="Italic">Cereus pterogonus</Emphasis> sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively,. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 μmol min⁻¹ mg⁻¹. In the presence of metal ions (1 mM) such as Co²⁺,Mn²⁺, Ni²⁺, Ca²⁺ and Fe³⁺ the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg²⁺, Cd²⁺, Cu²⁺, while partial inhibition was noted with Zn²⁺ and Mg²⁺. The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.     

Characterization of trehalose synthase from <Emphasis Type="Italic">Corynebacterium nitrilophilus</Emphasis> NRC

Trehalose synthase (TSII) from Corynebacterium nitrilophilus NRC was successively purified by ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 columns. The specific activity of the trehalose synthase was increased ~200-fold, from 0.14 U mg⁻¹ protein to 28.3 U mg⁻¹ protein. TSII was found to be a monomeric protein with a molecular weight of 67–69 kDa. Characterization of the enzyme exhibited optimum pH and temperature were 7.5 and 35°C, respectively. The purified enzyme was stable from pH 6.6 to 7.8 and able to prolong its thermal stability up to 35°C. The enzyme activity was inhibited strongly by Zn²⁺, Hg²⁺ and Cu²⁺ and moderately by Ba²⁺, Fe²⁺, Pb²⁺ and Ni²⁺. Other metal ions Ca²⁺, Mg²⁺, Co²⁺, Mn²⁺ and EDTA had almost no effect.     

Probing the coordination properties of glutathione with transition metal ions (Cr2+,Mn2+,Fe2+,Co2+, Ni2+,Cu2+,Zn2+,Cd2+,Hg2+) by density functional theory

Complexes formed by reduced glutathione (GSH) with metal cations (Cr²⁺, Mn²⁺,Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺,Hg²⁺) were systematically investigated by the density functional theory (DFT). The results showed that the interactions of the metal cations with GSH resulted in nine different stable complexes and many factors had an effect on the binding energy. Generally, for the same period of metal ions, the binding energies ranked in the order of Cu²⁺>Ni²⁺>Co²⁺>Fe²⁺>Cr²⁺>Zn²⁺>Mn²⁺; and for the same group of metal ions, the general trend of binding energies was Zn²⁺>Hg²⁺>Cd²⁺. Moreover, the amounts of charge transferred from S or N to transition metal cations are greater than that of O atoms. For Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺ and Hg²⁺ complexes, the values of the Wiberg bond indices (WBIs) of M-S (M denotes metal cations) were larger than that of M-N and M-O; for Cr²⁺ complexes, most of the WBIs of M-O in complexes were higher than that of M-S and M-N. Furthermore, the changes in the electron configuration of the metal cations before and after chelate reaction revealed that Cu²⁺, Ni²⁺,Co²⁺ and Hg²⁺ had obvious tendencies to be reduced to Cu⁺,Ni⁺,Co⁺ and Hg⁺ during the coordination process.     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Luminescent sensor for Cd2+, Hg2+ and Ag+ in water based on a sulphur‐containing receptor: quantitative binding–softness relationship

A water‐soluble, high‐output fluorescent sensor, based on a lumazine ligand with a thiophene substituent for Cd²⁺, Hg²⁺ and Ag⁺ metal ions, is reported. The sensor displays fluorescence enhancement upon Cd²⁺ binding (log  β = 2.79 ± 0.08) and fluorescence quenching by chelating with Ag⁺ and Hg²⁺ (log β = 4.31 ± 0.15 and 5.42 ± 0.1, respectively). The mechanism of quenching is static and occurs by formation of a ground‐state non‐fluorescent complex followed by rapid intersystem crossing. The value of the Stern–Volmer quenching rate constant (k_q) by Ag⁺ ions is close to 6.71 × 10¹² mol/L/s at 298 K. The thermodynamic parameters (ΔG, ΔH and ΔS) were also evaluated and indicated that the complexation process is spontaneous, exothermic and entropically favourable. The quantitative linear relationship between the softness values of Klopman (σ_K) or Ahrland (σ_A) and the experimental binding constants (β) being in the order of Hg²⁺ > Ag⁺ > Cd²⁺ suggests that soft–soft interactions are the key for the observed sensitivity and selectivity in the presence of other metal ions, such as: Pb²⁺, Ni²⁺, Mn²⁺, Cu²⁺, Co²⁺, Zn²⁺ and Mg²⁺ ions. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Inhibition of Urease from Seeds of Water-melon (Citrullus vulgaris) by Heavy Metal Ions

Urease from the seeds of water melon was found to be inhibited by heavy metal ions like copper, lead, nickel and cobalt. The order of effectiveness of these metals as inhibitors was Cu²⁺ > Pb²⁺ > Ni²⁺ > Co²⁺. The inhibition by these ions was noncompetitive. Time — dependent interaction of urease with nickel and cobalt exhibited a biphasic inhibition behaviour in which approximately half of the initial activity was lost rapidly (within 2 min) and remainder in a slow phase. The inhibition was largely irreversible, hence could not be reversed by dialysis. These observations are suggestive of half-and-half distribution of — SH groups on the native enzyme resulting urease into asymmetric oligomeric molecule.     

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.     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent) : transition metal ion requirement for activity and sulfhydryl group reactivity

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phosphoenolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg²⁺ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co²⁺ > Mn²⁺ > Cd^u2+ > Ni²⁺ ⪢ Fe²⁺ > VO²⁺, while Zn²⁺ and Ca²⁺ had no activating effects. When we investigated the effect of varying the type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO₂ substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn²⁺ activated enzyme the inhibition was purely non-competitive (K_ii = K_is) towards all substrates, while for the Co²⁺-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5′-dithiobis(2-nitro-benzoate) (DTNB) lead to an almost complete inhibition of Mn²⁺-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO₃⁻ had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co²⁺-activated enzyme than for its Mn²⁺-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn²⁺ and ADP afforded almost complete protection from inactivation; in the presence of Co²⁺ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Effects of trace metals on mouse B16 melanoma cells in culture

The effects of fourteen metal ions (As³⁺, As⁵⁺, Cd²⁺, Co²⁺, Cr³⁺, Cr⁶⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Se⁴⁺, V⁵⁺, VO²⁺) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10⁻⁴ M, metal ions such as As³⁺, As⁵⁺, Cd²⁺, Cr⁶⁺, Se⁴⁺, V⁵⁺, VO²⁺, and, to a minor extent, Li⁺, Hg²⁺, and Co²⁺ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg²⁺>Cr⁶⁺=Cd²⁺>As³⁺, As⁵⁺>V⁵⁺, VO²⁺>Se⁴⁺=Ni²⁺=Co²⁺=Li⁺. An increased synthesis of melanin in B16 cells was noted after incubation with Co²⁺, Ni²⁺, Cd²⁺, and Li⁺, whereas Se⁴⁺ had, on the contrary, an inhibiting effect on melanogenesis.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Biochemical Characterization of Allantoinase from <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

Bacterial allantoinase (ALLase; EC 3.5.2.5), which catalyzes the conversion of allantoin into allantoate, possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. Here, we characterized ALLase from Escherichia coli BL21. Purified recombinant ALLase exhibited no activity but could be activated when preincubating with some metal ions before analyzing its activity, and was in the order: Mn²⁺- ≫ Co²⁺- > Zn²⁺- > Ni²⁺- > Cd²⁺- ~Mg²⁺-activated enzyme; however, activity of ALLase (Mn²⁺-activated form) was also significantly inhibited with 5 mM Co²⁺, Zn²⁺, and Cd²⁺ ions. Activity of Mn²⁺-activated ALLase was increased by adding the reducing agent dithiothreitol (DTT), but was decreased by treating with the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Inhibition of Mn²⁺-activated ALLase by chelator 8-hydroxy-5-quinolinesulfonic acid (8-HQSA), but not EDTA, was pH-dependent. Analysis of purified ALLase by gel filtration chromatography revealed a mixture of monomers, dimers, and tetramers. Substituting the putative metal binding residues His59, His61, Lys146, His186, His242, and Asp315 with Ala completely abolished the activity of ALLase, even preincubating with Mn²⁺ ions. On the basis of these results, as well as the pH-activity profile, the reaction mechanism of ALLase is discussed and compared with those of other cyclic amidohydrolases.     

Influence of temperature,pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K_m and V_max determined for substrate guaiacol were 15.4 μM and 372.9 IU mg^?1, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg^?1. Moreover, the metal ions, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺ and Cd²⁺, especially Hg²⁺, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.     

Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions

Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH₂PO₄ (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg²⁺, Mn²⁺, Ca²⁺ and Fe³⁺ ions and inhibited by Zn²⁺ and Cd²⁺ ions while Phy II activity was moderately stimulated by Fe³⁺ ions and was inhibited by Hg²⁺, Mn²⁺ and Zn²⁺ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.     

Purification and partial characterization of an extracellular alginate lyase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> isolated from brown seaweed

The extracellular enzyme alginate lyase produced from marine fungus Aspergillus oryzae isolated from brown alga Dictyota dichotoma was purified, partially characterized, and evaluated for its sodium alginate depolymerization abilities. The enzyme characterization studies have revealed that alginate lyase consisted of two polypeptides with about 45 and 50 kDa each on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed 140-fold higher activity than crude enzyme under optimized pH (6.5) and temperature (35°C) conditions. Zn²⁺, Mn²⁺, Cu²⁺, Mg²⁺, Co²⁺ and NaCl were found to enhance the enzyme activity while (Ca²⁺, Cd²⁺, Fe²⁺, Hg²⁺, Sr²⁺, Ni²⁺), glutathione, and metal chelators (ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid) suppressed the activity. Fourier transform infrared and thin-layer chromatography analysis of depolymerized sodium alginate indicated the enzyme specificity for cleaving at the β-1,4 glycosidic bond between polyM and polyG blocks of sodium alginate and therefore resulted in estimation of relatively higher polyM content than polyG. Comparison of chemical shifts in ¹³C nuclear magnetic resonance spectra of both polyM and polyG from that of sodium alginate also showed further evidence for enzymatic depolymerization of sodium alginate.     

Alkaline phosphatase conductometric biosensor for heavy-metal ions determination

Alkaline phosphatase conductometric biosensors consisting of interdigitated gold electrodes and enzyme membranes have been used for assessment of heavy-metal ions in water. These analytes act as enzyme inhibitors. Enzyme residual activity has been measured in Tris-nitrate buffer without metal preincubation in the presence of Mg²⁺ ions as activator. The results indicate that the toxicity of the various metals tested toward immobilized phosphatase is ranged as follows: Cd²⁺ > Co²⁺ > Zn²⁺ > Ni²⁺ > Pb²⁺. Detection limits were about 0.5 ppm for Cd²⁺, 2 ppm for both Zn²⁺ and Co²⁺, 5 ppm for Ni²⁺ and 40 ppm for lead ions. In addition, the responses during 10 h were stable (RSD 4%) and a drift of about 7% per day was observed. The storage stability in buffer solution at 4 °C remained stable for more than one month.