Gene Cloning,Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9

An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 °C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn²⁺, Fe²⁺, and Mg²⁺ stimulated the activity of the purified enzyme, whereas Co²⁺, Cu²⁺, and Ni²⁺ inhibited enzyme activity. The K _m and V _max values for inulin hydrolysis were 1.72 mM and 21.69 μmol min^?1 mg^?1 protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min^?1 mg^?1 protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.     

Purification and biochemical characterization of two major thermophilic xylanase isoforms (T70 and T90) from xerophytic Opuntia vulgaris plant spp.

Thermostable xylanase isoforms T₇₀ and T₉₀ were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T₇₀ and T₉₀ isoforms yielded a final specific activity 134.0 and 150.8 U mg^?1 protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T₇₀ and T₉₀ xylanase isoforms was 5.0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T₇₀ and T₉₀ enzyme isoforms was 3.49, 2.1 mg ml^?1, respectively when oat spelt xylan was used as a substrate. The T₇₀ had a Vmax of 10.4 μmol min^?1 mg^?1, and T₉₀ had a Vmax of 8.9 μmol min^?1 mg^?1, respectively. In the presence of 10 mM Co²⁺, and Mn²⁺ the activity of T₇₀ and T₉₀ isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg²⁺, Cd²⁺, Cu²⁺, Zn²⁺ while partial inhibition was observed in the presence of Fe³⁺, Ni²⁺, Ca²⁺and Mg²⁺. The T₇₀ and T₉₀ isoforms retained nearly 50 % activity in the presence of 2.0 M urea, while use of 40 mM SDS lowered the activity nearly 38–41 %. The substrate specificity of both T₇₀ and T₉₀ isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T₉₀ isoform with polyclonal anti-T₉₀ antibody raised in rabbit, as well as cross-reactivity of the antibody with the T₇₀ xylanase isoform.     

Expression,Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)⁺ to NAD(P)H. The NADP⁺-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP⁺ as the cofactor. The K _m values for l-malate and NADP⁺ were 2.19?±?0.01 and 0.38?±?0.02 mM, respectively, while the V _max values were 147?±?2 and 302?±?14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺, and low concentrations of Zn²⁺ rather than Ca²⁺, Cu²⁺, or high concentrations of Zn²⁺. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K _i values were 0.08 and 0.6 mM, respectively.     

Biochemical Characterization and Antitumor Study of l-Glutaminase from Bacillus cereus MTCC 1305

l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na⁺, K⁺) and phosphate ion activated the enzyme activity, while divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, Pb²⁺, Ca²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cu²⁺) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K _m, V _max, K _cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22?×?10² M^?1s^?1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC₅₀ value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).     

NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

NADP⁺-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg²⁺ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP⁺ and no NAD-dependent activity could be detected. The K _m values displayed for NADP⁺ and isocitrate were 59 and 31 μM (Mg²⁺), 120 μM and 58 μM (Mn²⁺), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K _m values for NADP⁺ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD⁺-dependent activity was detected for R322D mutant and the K _m and k _cat values for NAD⁺ were 47,000 μM and 0.38 s^?1, respectively. Although the R322D mutant showed low activity with NAD⁺, it revealed the feasibility of engineering an eukaryotic IDP to a NAD⁺-dependent one.     

Over-Expression of a Proline Specific Aminopeptidase from Aspergillus oryzae JN-412 and Its Application in Collagen Degradation

A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg^?1 at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7 % from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn²⁺ and Cu²⁺. DTT, β-mercaptoethanol, EDTA, and ions like Co²⁺, Mg²⁺, Mn²⁺, and Ca²⁺ had no influence on enzyme activity, whereas Ni²⁺ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.     

Purification and characterization of an extracellular lipolytic enzyme from the fermented fish-originated halotolerant bacterium, <Emphasis Type="Italic">Virgibacillus alimentarius</Emphasis> LBU20907

A halotolerant Virgibacillus alimentarius LBU20907 isolated from fermented fish (Budu) was found to be an efficient producer of extracellular halophilic lipase enzyme. The enzyme was purified 5.99-fold with a 0.15% final yield to homogeneity by ammonium sulfate precipitation, followed by dialysis, Toyopearl DEAE-650 M ion exchange chromatography, Toyopearl butyl-650 M hydrophobic interaction chromatography, and Toyopearl-HW 55 F gel filtration chromatography. SDS-PAGE of purified lipase exhibited a homogenous single band with a very high molecular weight of 100 kDa. The properties of purified lipase revealed maximum activity at pH 7.0 and 40 °C. It was also highly stable in a pH range of 6.0–7.0, retaining more than 90% activity for 24 h. It was stable at the temperature of 30–50 °C and maintained more than 80% activity for 16 h. The purified lipase performing the maximal activity in the presence of 20.0% NaCl indicated halophilic enzyme properties. Its lipolytic activity was highest against p-nitrophenyl palmitate. The lipase activity was found to be enhanced in hexane. The enzyme activity was stimulated in the presence of Zn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺; while, it was completely inhibited by Ba²⁺ and Co²⁺. The enzyme had a K _m and V _max of 108.0 mg and 79.1 U mL^?1, respectively.     

Stability and Detergent Compatibility of a Predominantly β-Sheet Serine Protease from Halotolerant B. aquimaris VITP4 Strain

The present study deals with the characterization of halotolerant protease produced by Bacillus aquimaris VITP4 strain isolated from Kumta coast, Karnataka, India. The studies were performed at 40 °C and pH 8 in Tris buffer. Metal ions such as Mn²⁺ and Ca²⁺ increased the proteolytic activity of the enzyme by 34 and 30 %, respectively, at 10 mM concentration. Cu²⁺ at 1 mM concentration was found to enhance the enzyme activity by 16 %, whereas inhibition was observed at higher concentration (>5 mM). Slight inhibition was observed even with lower (>1 mM) concentrations of Zn²⁺, Hg²⁺, Fe³⁺, Ni²⁺, and Co²⁺.The activity of protease was completely inhibited by phenylmethylsulfonyl fluoride, indicating that the VITP4 protease is a serine protease. The presence of ethylenediaminetetraacetic acid and 1,10-phenanthroline (>5 mM) moderately inhibited the activity, suggesting that the enzyme is activated by metal ions. The protease was purified to homogeneity with a purification fold of 15.7 with ammonium sulfate precipitation and 46.65 with gel filtration chromatography using Sephadex G-100, resulting in a specific activity of 424?±?2.6 U mg^?1. The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization–time of flight. Among the different substrates used (casein, egg albumin, gelatin, and bovine serum albumin), the activity was higher with casein with V _max, K _m, and k _cat values of 0.817 mg ml min^?1, 0.472 mg ml^?1, and 2.31 s^?1, respectively. Circular dichroism studies revealed that the VITP4 protease has a predominantly β-sheet structure (51.6 %) with a temperature for half denaturation of 85.8 °C in the presence of 1 mM CaCl₂. Additionally, the VITP4 protease was found to retain more than 70 % activity in the presence of 10 mM concentration of different detergents (CTAB, urea, and sodium dodecyl sulfate) and surfactants (Triton X-100, Tween-20, and Tween-80), and the results of wash performance test with various commercial detergents confirmed that it can be used in detergent formulations.     

A Novel β-Glucosidase from Humicola insolens with High Potential for Untreated Waste Paper Conversion to Sugars

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116 kDa (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and 404 kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65 °C, respectively, and the enzyme was stable for 60 min at 50 °C, maintaining 71 % residual activity after 60 min at 55 °C. BglHi2 hydrolyzed p-nitrophenyl-β-d-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (K _M?=?0.24?±?0.01 mmol L^?1) and catalytic efficiency (k _cat/K _M?=?1,304.92?±?53.32 L mmol^?1 s^?1). The activity was insensitive to Fe⁺³, Cr⁺², Mn⁺², Co⁺², and Ni²⁺, and 50–60 % residual activities were retained in the presence of Pb²⁺, Hg²⁺, and Cu²⁺. Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5 L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24–48 h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100 % glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.     

Characteristics of a High Maltose-Forming, Acid-Stable, and Ca2+-Independent α-amylase of the Acidophilic Bacillus acidicola

The purified acidic α-amylase of Bacillus acidicola is a monomer of 66.0 kDa, optimally active at pH 4.0 and 60 °C. The enzyme is Ca²⁺ independent with T _1/2 for 18 min at 80 °C. The K _m, V _max, and catalytic efficiency (k _cat/K _m) of the enzyme are 1.6 mg mL^?1, 23.8 μmol mg^?1 min^?1, and 981 μmol s^?1, respectively. Among detergents, Tween 20, 40, and 80 stimulated enzyme activity, whereas sodium dodecyl sulfate and Triton X-100 inhibited even at low concentration. EGTA has not affected the activity, whereas EDTA β-mercaptoethanol, iodoacetic acid, and Dithiothreitol exhibited a slight inhibitory action. Phenylmethanesulfonyl fluoride, N-bromosuccinimide, and Hg²⁺ strongly inhibited enzyme activity. The experimental activation energy and temperature quotient are 50.12 kJ mol^?1 and 1.37. When thermodynamic parameters (ΔH and ΔS) of the enzyme have been determined at different temperatures, ΔG is positive suggesting that the enzyme is thermostable. The enzyme hydrolyzes raw starches, and therefore, the enzyme finds application in raw starch saccharification at sub-gelatinization temperatures that saves energy needed for gelatinization of raw starch at 105 °C.     

A dual chemosensor for Cu2+ and Fe3+ based on π-extend tetrathiafulvalene derivative

A new dual chemosensor (TTF-PBA) for Fe³⁺ and Cu²⁺ in different signal pathways was designed and synthesized. The absorption spectrum, fluorescence spectrum and cyclic voltammograms changed in the presence of Cu²⁺ and Fe³⁺. The optical color changed within 5 s from yellow to orange upon the addition of Cu²⁺, and it changed to dark yellow when Fe³⁺ existed. The cyclic voltammogram of Cu²⁺/TTF-PBA changed from E_ox = 0.50 V, E_red = 0.32 V to E_ox = 0.64 V, E_red = 0.80 V (vs Ag/AgCl) upon the addition of 2.0 equiv. Cu²⁺. As for Fe³⁺/TTF-PBA, its oxidation wave disappeared, and its reduction wave appeared at E_red = ?0.59 V (vs Ag/AgCl) upon the addition of 4.0 equv. Fe³⁺. The sensor displayed high selectivity for Cu²⁺ and Fe³⁺ over other ions including Pb²⁺, Zn²⁺, Ni²⁺, Ag⁺, Cr³⁺, Mn²⁺, Al³⁺, Co²⁺, Pd²⁺, Hg²⁺, Fe^2+, Cd²⁺, Ce³⁺, Bi³⁺ and Au³⁺, the detection limits for Cu²⁺ and Fe³⁺ ion reached as low as 5.33 × 10^?7 mol/L and 5.34 × 10^?7 mol/L, respectively. Furthermore, when Fe³⁺ existed, Cu²⁺ can be detected sequentially by the sensor through the absorption spectrum and the color change observed by naked-eyes.     

Purification and Characterization of the Glucoside 3-Dehydrogenase Produced by a Newly Isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251

A soluble glucoside 3-dehydrogenase (G3DH) was purified from a newly isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251. The enzyme was purified to 35.71-fold with a yield of 41.91 % and was estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis with a molecular mass of 62 kDa. The sequences of two peptides of the enzyme were all contained in a GMC family oxidoreductase (EFK55866) by mass spectrometry analysis. The optimal pH of the enzyme was around 6.2. The enzyme was stable within a pH range of 5.0–6.6 and was sensitive to heat. G3DH from S. faecium exhibited extremely broad substrate specificity and well regioselectivity to validoxylamine A. The enzyme was completely inhibited by Hg₂Cl₂ and partly inhibited by Cu²⁺, Fe²⁺, Ca²⁺, and Cd²⁺. The apparent K _m values for D-glucose, sucrose, and validoxylamine were calculated to be 1.1, 1.7, and 2.1 mM, respectively. With this purified enzyme, 3-keto sucrose was prepared at pH 5.0, 30 °C for 10 h with a yield of 28.7 %.     

Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro

An extracellular l-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg^?1. The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified l-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K _m, V _max, and k _cat values of the enzyme were 0.257 mM, 1.537 U μg^?1, and 993.93 s^?1, respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α?+?β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified l-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the l-asparaginase has significant antineoplastic properties.     

Selective Lead Adsorption by Recombinant Escherichia coli Displaying a Lead-Binding Peptide

A highly specific lead-binding peptide ThrAsnThrLeuSerAsnAsn was displayed on Escherichia coli, and lead adsorption characteristics of the recombinant bacteria were investigated. Cell surface-displayed peptide was expressed under the control of an arabinose promoter using outer membrane protein C (OmpC_t) as an anchoring motif. The optimal induction period and arabinose concentration for the expression of peptide-fused OmpC_t were determined to be 2 h and 0.001 g/L, respectively. Selective adsorption of Pb²⁺ onto recombinant cells was verified with individual or combinatory use of four metal ions, Pb²⁺, Ni²⁺, Co²⁺, and Cu²⁺; the amount of bound Pb²⁺ onto the biosorbents was significantly higher than the other metal ions. The adsorption isotherm of recombinant cells for Pb²⁺ followed the Langmuir isotherm with a maximum adsorption loading (q _max) of 526 μmol/g dry cell weight.     

Expression and Characterization of a Novel 1,3-Propanediol Dehydrogenase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis>

1,3-Propanediol dehydrogenase (PDOR) is important in the biosynthesis of 1,3-propanediol. In the present study, the dhaT gene encoding PDOR was cloned from Lactobacillus brevis 6239 and expressed in Escherichia coli for the first time. Sequence analysis revealed that PDOR containing two Fe²⁺-binding motifs and a cofactor motif belongs to the type III alcohol dehydrogenase. The purified recombinant PDOR exhibited a single band of 42 kDa according to SDS-PAGE. Optimal temperatures and pH values of this dehydrogenase are 37 °C, 7.5 for reduction and 25 °C, 9.5 for oxidation, respectively. We found that PDOR was more stable in acid buffer than in alkaline condition, and 60 % of its relative activity still remained after a 2-h incubation at 37 °C. The activity of PDOR can be enhanced in the presence of Mn²⁺ or Fe²⁺ iron and inhibited by EDTA or PMSF by different degrees. The K _m and V _max of this dehydrogenase are 1.25 mM, 64.02 μM min^?1 mg^?1 for propionaldehyde and 2.26 mM, 35.05 μM min^?1 mg^?1 for 1,3-PD, respectively. Substrate specificity analysis showed that PDOR has a broad range of substrate specificities. The modeling superposition indicated that the structural differences may account for the diversity of PDORs’ properties. Thus, our PDOR is a potential candidate for facilitating the 1,3-PD biosynthesis.     

A Feruloyl Esterase (FAE) Characterized by Relatively High Thermostability from the Edible Mushroom Russula virescens

A monomeric feruloyl esterase (FAE) with a molecular mass of 62 kDa was acquired from fresh fruiting bodies of the edible mushroom Russula virescens. The isolation procedure involved ion exchange chromatography on CM-cellulose, Q-Sepharose, and SP-Sepharose and finally fast protein liquid chromatography–gel filtration on Superdex 75. Two amino acid sequences were obtained after tryptic digestion, and they both showed some homology with the esterase of some fungi. Maximal activity was observed at pH 5.0 and at 50 °C. The enzyme displayed relatively high thermostability as evidenced by over 70 % residual activity at 70 °C and about 34 % residual activity at 80 °C. The K _m and V _max for this enzyme on methyl ferulate were 0.19 mM and 1.65 U/mg proteins, respectively. The purified FAE prefers methyl ferulate over methyl caffeate and is least active on methyl p-coumarate. The FAE activity was not significantly affected by the presence of cations such as Mn²⁺, Ca²⁺, Cd²⁺, Zn²⁺, Mg²⁺, Cu²⁺, and K⁺ ions but inhibited by Al³⁺, Hg²⁺, Fe²⁺, and Pb²⁺ ions at a tested concentration of 2. 5 mM.     

Characterization of a Novel Organic Solvent Tolerant Protease from a Moderately Halophilic Bacterium and Its Behavior in Ionic Liquids

An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40–80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V _max and K _m of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba²⁺ and Ca²⁺ (2 mM) strongly enhanced the enzyme activity, while Fe²⁺ and Mg²⁺ activated moderately and Zn²⁺, Ni²⁺, and Hg²⁺ decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.     

Kinetics and mechanism of the oxidation of tris(2,2′-bipyridine)iron(II) and tris(1,10-phenanthroline)iron(II) complexes by nitropentacyanocobaltate(III) in acidic aqueous medium

The kinetics of the oxidation of tris(2,2′-bipyridyl)iron(II) and tris(1,10-phenanthroline)iron(II) complexes ([Fe(LL)₃]²⁺, LL = bipy, phen) by nitropentacyanocobaltate(III) complex [Co(CN)₅NO₂]^3? was investigated in acidic aqueous solutions at ionic strength of I = 0.1 mol dm^?3 (HCl/NaCl). The reactions were carried out at fixed acid concentration ([H⁺] = 0.01 mol dm^?3) and the temperature maintained at 35.0 ± 0.1 °C. Spectroscopic evidence is presented for the protonated oxidant. Protonation constants of 360.43 and 563.82 dm³ mol^?1 were obtained for the monoprotonated and diprotonated Co(III) complexes respectively. Electron transfer rates were generally faster for [Fe(bipy)₃]²⁺ than [Fe(phen)₃]²⁺. The redox complexes formed ion-pairs with the oxidant with increasing concentration of the oxidant over that of the reductant. Ion-pair constants for these reaction were 160.31 and 131.9 dm³ mol^?1 for [Fe(bipy)₃]²⁺ and [Fe(phen)₃]^2+, respectively. The activation parameters measured for these systems have values as follows: ?H ^≠ (kJ K^?1 mol^?1) = +113.4 ± 0.4 and +119 ± 0.3; ?S ^≠ (J K^?1) = +107.6 ± 1.3 and 125.0 ± 1.6; ?G ^≠ (kJ K^?1) = +81 ± 0.4 and +82.4 ± 0.4; and E _a (kJ mol^?1) = 115.9 ± 0.5 and 122.3 ± 0.6 for LL = bipy and phen, respectively. Effect of added anions (Cl^?, $ {\text{SO}}_{4}^{2 - } $ and $ {\text{ClO}}_{4}^{ - } $ ) on the systems showed decrease in the electron transfer rate constant. An outer-sphere mechanism is proposed for the reaction.     

Molecular and Biochemical Characteristics of Recombinant β-Propeller Phytase from Bacillus licheniformis Strain PB-13 with Potential Application in Aquafeed

Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase ‘rPhyPB13’ was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other β-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0–6.5 and 60 °C in the presence of 1 mM Ca²⁺ and was highly active over a wider pH range (pH 4.0–8.0) and high temperature (80 °C). It has shown maximum activity towards Na-phytate as substrate. The observed K _m , V _max and k _cat of purified rPhyPB13 were 1.064 mM, 1.32 μmol/min/mg and 27.46 s^?1, respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca²⁺ and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a β-propeller phytase that has application potential in aquaculture feed.     

Study of the gadolinium sorption on the C100 ion-exchange resin for the development of the antineutrino detector targets

Adsorption of the gadolinium from H₂O and HCl solutions on the ion-exchange resin C100 is investigated. The experiments were carried out by varying the acidity of the liquid phase, the amount of sorbent, and the temperature. The maximal sorption of the ions Gd³⁺ is observed from the solution 0–0.2 M HCl under optimal conditions, the sorption reaches more than 99.5%. Sorption of Gd³⁺ on C100 from H₂O solution occurs most intensively during the first 3 min then for 30 min the system smoothly comes to equilibrium. The maximal sorption capacity of the resin C100 amounted to 1.2 ± 0.1 mmol g^?1. The thermodynamic parameters of sorption: ΔG = ? 24.20 kJ mol^?1, ΔS = ? 90.27 J mol^?1 K^?1, ?H = ? 50.93 kJ mol^?1 were evaluated. It is shown that the sorption of gadolinium on the ion-exchange resin C100 is described by models of kinetically pseudo-first and pseudo-second order. It is established that the Gd³⁺ sorption on the C100 resin is reversible second order chemical reaction.