Isolation and characterization of an L-amino acid acylase fromAspergillus oryzae

A scheme of isolating a highly purified L-amino acylase fromAspergillus oryzae is described which excludes extraction of the enzyme from the preparation “Amilorizin,” fractionation with ethanol, chromatography on DEAE-cellulose, and gel filtration through Sephadex G-200 and Bio-Gel P-300. The enzyme, as purified 1240-fold, has a molecular weight of 118,000, apparently consists of two subunits with a molecular weight of 60,000, is stable in the pH range of 7–10 and has an optimum pH of 8.9 and a pI of 4.0. Its amino acid composition has been determined and its substrate specificity has been studied. The acylase is a metalloenzyme: Co^2+` ions in concentrations of 10^?4–5·10^?5 M increase the rate of hydrolysis of N-acetyl-L-amino acids three- to fourfold. It shows differences in its molecular and functional properties from acylase I obtained from porcine kidney.     

Purification of Peroxidase from Red Cabbage (Brassica oleracea var. capitata f. rubra) by Affinity Chromatography

Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9 % from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K _m and V _max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC₅₀ and K _i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702?±?0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.     

Neutral Serine Protease from Penicillium italicum. Purification,Biochemical Characterization,and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.     

Kinetics of immobilized pig heart fumarase

The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution.

1. An analogous pH dependence of initial activity is found for free and immobilized enzyme.
2. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent K_m values are three to eight times higher for the immobilized (2 mg/g gel) enzyme.
3. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ?g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration.
4. The activation energies for free and immobilized fumarase (50 ?g/g gel) were found to be identical between 22 and 32?C and with L-malate as substrate (E_a = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases.

Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ?g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.     

Micellar solubilization of biopolymers in hydrocarbon solvents. ii. the case of horse liver alcohol dehydrogenase

The chemical characterization of horse liver alcohol dehydrogenase solubilized in isooctane via reverse micelles formed by the anionic surfactant di (2-ethyl-hexyl) sodium sulfosuccinate (AOT) and water (0.6 to 4% v/v) is presented. The enzyme’s catalytic activity toward acetaldehyde reduction is markedly dependent upon w0 = [H2O]/[AOT], and upon the pH of the stock aqueous solution (pHst), from which the hydrocarbon enzyme solution is prepared. Kinetically, the micellar solution appears to follow a normal Michaelis-Menten behavior, with a turnover number which, under the optimal conditions (w0 = 42, pHst = 8.8), appears to be higher than in bulk water. The affinity between enzyme and NADH, as judged from direct binding studies (quenching of the protein fluorescence), is much reduced with respect to water if concentrations refer to the water pool of the micelles, and comparable to water if concentrations refer to the overall volume (hydrocarbon plus water pool). Also, the Km values are much higher if concentrations refer to the water pool. Ultraviolet absorption studies show that the aromatic chromophores are not significantly perturbed on going from a water solution to the micellar solution. The essentially aqueous environment of the protein in the reverse micelles is confirmed by fluoresence studies. Circular dichroism studies show that the enzyme’s conformation in the micelles is similar to that in water; however, under certain conditions, small but significant changes of the main chain folding seem to occur, which do not impair enzymatic activity. The spectroscopic properties of NADH in the hydrocarbon phase (fluorescence and circular dichroism) are also investigated. The potential of the LADH-NADH system for technical applications (oxidoreduction of lipophylic substrates) is discussed.     

Purification,Gene Cloning,and Characterization of a Novel Halohydrin Dehalogenase from Agromyces mediolanus ZJB120203

A novel halohydrin dehalogenase (HHDH), catalyzing the transformation of 1,3-dichloro-2-propanol (1,3-DCP) to epichlorohydrin (ECH), was purified from Agromyces mediolanus ZJB120203. The molecular mass of the enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 735-bp nucleotide fragment was obtained based on the N-terminal and internal amino acid sequences of the purified HHDH. The gene codes a protein sequence with 244 amino acid residues, and the protein sequence shows high similarity to Hhe A_AD2 (HHDH from Arthrobacter sp. AD2), defined as Hhe A_Am, which is the seventh reported HHDH. Expression of Hhe A_Am was carried out in Escherichia coli and purification was performed by nickel-affinity chromatography. The recombinant HheA_Am possessed an optimal pH of 8.5 and an optimal temperature of 50 °C and manifested a K _m of 4.58 mM and a V _max of 3.84 μmol/min^/mg. The activity of Hhe A_Am was not significantly affected by metal ions such as Zn²⁺, Ca²⁺, Cu²⁺, and EDTA, but was strongly inhibited by Hg²⁺ and Ag⁺. In particular, the Hhe A_Am exhibits an enantioselectivity for the conversion of prochiral 1,3-DCP to (S)-ECH. The applications of the Hhe A_Am as a catalyst for asymmetric synthesis are promising.     

Purification and Characterization of a Chymosin from Rhizopus microsporus var. rhizopodiformis

Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0–8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.     

Purification and properties of membrane-bound coupling factor-latent ATPase from Mycobacterium phlei.

The membrane bound coupling factor-latent ATPase was solubilized from the membrane vesicles of Mycobacterium phlei by using 0.25 M sucrose or low ionic strength buffer. Purification of the solubilized enzyme by use of Sepharose-ADP conjugate gel yielded a homogenous preparation of latent ATPase which was purified about 216-fold in a single step with an 84% yield. The enzyme exhibits a specific activity of 39 mumoles of ATP hydrolyzed per min per mg protein. The purified enzyme exhibits coupling factor activity. Electrophoresis in two dissociating solvent systems indicates that the enzyme contains at least three major polypeptides of molecular weights 56,000, 51,000, and 46,000 daltons, and two minor polypeptides of 30,000 and 17,000 daltons. Equilibrium binding studies of ADP with purified coupling factor-latent ATPase reveal the presence of two nucleotide binding sites per molecule with an apparent Ka of 8.1 X 10(-5) M. By use of affinity chromatography, another latent ATPase has been isolated from the solubilized enzyme, which does not exhibit coupling factor activity.     

Purification and some properties of tryptophan-5-monooxygenase from rabbit hindbrain

An affinity chromatography procedure for the rapid purification of tryptophan-5-monooxygenase from rabbit hindbrains was developed using e-aminocaproyl-D-tryptophan methyl ester-Sepharose-4B gels. The precise requirements for the optimal biospecific interaction between the affinity ligand and the ligate (enzyme) was established from a study of the effects of the variation in the length of the "spacer’’ on the affinity properties of the gel. The enzyme preparation isolated by this procedure was found to be essentially homogeneous and was characterized by a molecular weight of 200,000 ±20,000. SDS-polyacrylamide gel electrophoresis of the enzyme revealed it to be a dimer, the molecular weight of each subunit being approximately 90,000. The specific activity of the enzyme preparation is approxi-mately 7-10 times that of the crude homogenate, but a further fivefold enhancement in the specific activity could be obtained by limited proteolysis with trypsin. The extreme lability of the enzyme could be circumvented by its immobilization on activated Sepharose or by cross-linking with dimethyl suberimidate. The kinetic properties as well as the advantages of such stabilized enzyme preparations are presented.     

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.     

Purification and Characterization of Paraoxonase 1 (PON1) from Swiss Black,Holstein, and Montofon Bovines

Paraoxonase 1 (PON1: EC 3.1.8.1) is a calcium-dependent enzyme associated with high-density lipoproteins (HDLs) and has a protective effect against oxidation of low-density lipoproteins (LDLs) in mammals. PON1 is the best-studied member of a family of enzymes called serum paraoxonases, or PONs, identified in mammals and other vertebrates as well as in invertebrates. PONs exhibit a range of important activities, including drug metabolism and detoxification of organophosphates such as nerve agents. This study reports, for the first time, purification and biochemical characterization of serum PON1 from different bovine breeds namely Swiss Black, Holstein, and Montofon. Bovine serum PON1s were purified using ammonium sulfate precipitation followed by Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS–polyacrylamide gel electrophoresis of the purified enzymes indicates a single band with an apparent MW of 43 kDa. The purified enzymes had a specific activity of 10.78, 27.00, and 22.38 U/mg for Swiss Black, Holstein, and Montofon bovines, respectively. The overall purification rates of our method were 262.47-, 2,476.90-, and 538.06-fold for Swiss Black, Holstein, and Montofon bovines, respectively. Furthermore, using phenyl acetate as a substrate, we determined the K _M and V _max values of the purified enzymes, as 0.80 mM, 1428.5 U/ml for Swiss Black; 0.40 mM, 714.3 U/ml for Holstein; and 0.50 mM, 1,111.1 U/ml for Montofon bovine. The present study has revealed that there is no substantial difference in PON1 activities among the studied bovine breeds.     

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.     

Properties of human parotid amylase immobilized by covalent binding to glass or sepharose or by reaction with immobilized antibody

Human parotid amylase was immobilized by covalent binding to CNBr-activated Sepharose, to Corning GAO-3940 silica glass biomaterial support by the diazonium reaction or reaction with glutaraldehyde, or as a result of the antigen-antibody reaction between rabbit antihuman parotid amylase IgG that was covalently bonded to GAO glass and soluble amylase. The amylase directly bonded to the supports showed constant activity at flow rates of 3-15 ml/min through a 1.76-cm³ (8-mm diameter) support bed, did not lose enzyme into a circulating starch solution, retained its activity in the presence of soluble antiamylase IgG, was optimally active at 35°-40°C, and lost activity at 40°-45°C. When the enzyme was bound by interaction with immobilized antibody, full activity was expressed, but some enzyme was solubilized by a circulating starch solution. Immobilization of either amylase or antiamylase IgG makes dissolution of the antigen-antibody bond difficult.     

On synthesis of Fe2SiO4/SiO2 and Fe2O3/SiO2 composites through sol–gel and solid-state reactions

Major processing factors in forming Fe₂SiO₄/SiO₂ and Fe₂O₃/SiO₂ powders via sol–gel synthesis followed by solid-state reactions are investigated. The results clearly indicate that the chemical compositions of the precursors, the ratio of the precursors, the nature of the catalyst used, and the gas atmosphere during solid-state reactions can all affect the outcome of the reaction product(s). The formation of Fe₂SiO₄/SiO₂ is enhanced by using the precursor iron(III) acetylacetonate as the Fe source with the precursor ratio of iron(III) acetylacetonate to tetraethyl orthosilicate being 1:1 and the addition of formic acid. Otherwise, crystalline Fe and Fe₃C are formed in place of Fe₂SiO₄. By altering the gas atmosphere during solid-state reactions from argon to oxygen, the reaction products change from Fe₂SiO₄/SiO₂ to Fe₂O₃/SiO₂. All of the observed phenomena can be rationalized via the degree of mixing of the Fe–O and Si–O domains at the molecular level in the gel network during sol–gel reactions and the presence of a reducing or oxidizing atmosphere during the solid-state reaction.     

Isolation of protease B from cotton seeds

Protease B has been isolated from dormant cotton seeds by fractionation with ammonium sulfate, ion-exchange chromatography on CM-cellulose, and gel filtration through Acrilex P-10 and Sephadex G-75, with 128-fold purification. The enzyme exists in dimeric and monomeric forms. According to the results of gel filtration, their molecular weights are 72,000 and 36,000, respectively. The enzyme consists of a single polypeptide chain including sugars. The N-terminal amino acid of protease B is alanine. The enzyme possesses proteolytic activity in the pH range from 4 to 6.     

TiO2 and TiO2/SiO2 nanoparticles obtained by sol–gel method and applied on dye sensitized solar cells

In this work we show the synthesis and characterization of TiO₂ and TiO₂/SiO₂ nanoparticles synthesized by sol–gel method using HF and HCl as catalysts. The obtained nanoparticles were analyzed by N₂ adsorption–desorption isotherms, transmission electronic microscopy, Ultraviolet–visible spectroscopy and X-ray diffractometry. Mesoporous, homogeneously polycondensed TiO₂/SiO₂ materials, containing nanocrystalline anatase phase with band gap similar to pure titania were obtained. Films of the powdered oxides were applied to assemble dye sensitized solar cells that presented electrical parameters, Fill Factor and efficiencies similar to devices obtained by only TiO₂. The sol–gel route arises as an alternative way to prepare TiO₂/SiO₂ materials for solar cells.     

Some improvements in the estimation of137Cs in urine by the AMP-chlorostannate method

An accurate and reliable method has been developed for the estimation of radiocesium in urine. Initially cesium is adsorbed on ammonium phosphomolybdaste (AMP) precipitate and separated by ion exchange from other contaminants. Cesium thus separated is estimated as cesium chlorostannate, Cs₂SnCl₆, from a 50 (v/v)% solution of concentrated HCl in ethyl alcohol. While the results are in good agreement with the values obtained by γ-spectrometry using a Marinelli beaker in its range of applicability, the present method has a much lower detection limit. It is observed that the method has significant advantages over the methods available with respect to analysis time, accuracy and detection limits.     

Affinity chromatography ofd-lactate dehydrogenases fromLimulus polyphemus (horseshoe crab) andHaliotus sp. (abalone) voluntary muscle on 8-(6-aminohexyl)-amino-adenine nucleotide-sepharose

An improved method of purification employing sequential isoenzyme elution on DEAE-cellulose at pH 6.5 and biologically specific elution with the reduced NAD-pyruvate adduct from 8-(6-aminohexyl)-amino-NAD⊕-Sepharose is described forLimulus (horseshoe crab) muscle D- lactate dehydrogenase. The protein is judged as being at least 98%pure by its constant specific activity in the terminal purification steps, a molar extinction coefficient (280nm) identical with that previously reported for the purified enzyme, and its protein and enzyme electrophoretic patterns on starch and polyacrylamide gel electrophoresis at three pHs. The binding of ammonium sulfate fractionated D- lactate dehydrogenase from crude cell- free Limulus andHaliotus (abalone) muscle homogenates to 8-(6-aminohexyl)-amino-AMP-and 8-(6-aminohexyl)-amino-NAD®-Sepharose columns is demonstrated. A comparison of the binding properties of these two enzymes with those of vertebrate L-lactate dehydrogenases suggests that they may be significantly different in terms of their binding sites for the adenine portion of the coenzyme.     

Undariase,a Direct-Acting Fibrin(ogen)olytic Enzyme from Undaria pinnatifida,Inhibits Thrombosis In Vivo and Exhibits In Vitro Thrombolytic Properties

A direct-acting fibrinolytic serine protease named undariase possessing anticoagulant and antiplatelet properties was purified from Undaria pinnatifida. Undariase showed a molecular weight of 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. It displayed a strong fibrin zymogram lysis band corresponding to the same molecular mass. The N-terminal sequence of undariase, LTATTCEELAAAPTD, does not match with any known fibrinolytic enzyme. The enzyme was stable and active at high temperatures (35–70 °C). The fibrinolytic activity of undariase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and 4-(amidinophenyl) methanesulfonyl fluoride (APMSF). The K _m and V _max values for substrate S-2251 were determined as 6.15 mM and 90.91 mM/min/ml, respectively. Undariase resulted in clot lysis by directly cleaving α and β chains of fibrin. Similarly, it preferentially acted on the Aα chain of fibrinogen followed by cleavage of the Bβ chain. It significantly prolonged the PFA-100 closure times of citrated whole human blood. In addition, undariase delayed the coagulation time and increased activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Undariase exerted a significant protective effect against collagen plus epinephrine-induced pulmonary thromboembolism in mice. It prevented carrageenan-induced thrombus formation in the tail of mice. It also resulted in prolongation of APTT ex vivo. In conclusion, these results suggested a therapeutic potential of undariase for thrombosis.