Artificial cell-microencapsulated phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) is immobilized in collodion artificial cells. Once technical problems associated with the encepsulation of this enzyme were solved, the enzyme kinetics were compared to PAL in free solution. Microencapsulated PAL has an apparent enzyme activity that is 20% of the activity of enzyme in free solution. TheK _m for both free and immobilized PAL is 475 μM. TheV _m for the microen-capsulated PAL is 9 μM/min, whereas that of PAL in free solution is 55 μM/min.     

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.     

Synthesis and characterization of carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrid for amylase immobilization

Carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrids have been synthesized in a modified Stöber process. The hybrid synthesis was optimized to obtain an efficient immobilization matrix for diastase alpha amylase, a multimeric enzyme of high technological significance. The synthesized hybrids were characterized using FTIR, XRD, SEM, TGA and BET studies. The enzyme immobilization was done by adsorption and using the immobilized enzyme, the hydrolysis of soluble starch has been optimized in comparison to free enzyme. The optimum usable pH for the immobilized enzyme ranged from pH 4 to 5, while pH 5 was optimum pH for the free enzyme activity. The kinetic parameters for the immobilized, (K _M = 3.4610 mg ml^?1; V _max = 6.3540 mg ml^?1 min^?1) and free enzyme (K _M = 4.1664 mg ml^?1; V _max = 4.291 mg ml^?1 min^?1) hydrolysis indicated that the immobilization at the nanohybrid has significantly improved the catalytic property of the enzyme. In the immobilized state, the enzyme remained usable for many repeated cycles like our previous material, gum acacia-gelatin-AgNp-silica. Storage experiments indicated that the immobilization has increased the stability of the enzyme and also that AgNps play a role in stabilizing the immobilized enzyme.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

An activated enzyme electrode for creatinine

A highly selective enzyme electrode for creatinine, based on tripolyphosphate-activated creatininase enzyme, is described and evaluated. Kinetic studies comparing purified creatininase enzyme in the activated and non-activated forms show that the activation mechanism involves an increase in V_max but no change in K_m. The analytical effect of enzyme activation is to extend the sensitivity of the electrode to lower limits and to improve the response slope of calibration curves. As a result, this activated creatininase enzyme electrode shows promise as a sensor for urine and serum samples.     

Water Miscible Mono Alcohols’ Effect on the Proteolytic Performance of Bacillus clausii Serine Alkaline Protease

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V _m, k _cat and k _cat/K _m values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K _m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K _I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG ^≠ and ΔG ^≠ _E???T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ^≠ _ES value of the enzyme remained almost the same. The constant K _m and ΔG ^≠ _ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k _cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG ^≠ and ΔG ^≠ _E???T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.     

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 %.     

Application of a magnetic immobilizedβ-glucosidase in the enzymatic saccharification of steam-exploded lignocellulosic residues

Aβ-glucosidase preparation derived fromAspergillus niger was immobilized onto a magnetic support and used in the enzymatic saccharification of a lignocellulosic material. The enzyme was immobilized onto polyethyleneimine-glutaraldehyde activated magnetite (PAM) and also onto titanium (IV) oxide (TiO₂)-coated magnetite (TAM). Although > 80% of the protein applied was immobilized, only 15–27% of the enzyme activity was recovered after immobilization. Theβ-glucosidase immobilized onto TiCO₂-coated magnetite suffered from enzyme being removed from the matrix under hydrolysis-use conditions, whereas the PAM enzyme remained attached to the matrix. The physicochemical properties of the immobilizedβ-glucosidase preparations are described. Both immobilizedβ-glucosidase preparations were capable of completely hydrolyzing cellobiose. Recycling of the immobilized enzymes (IME) resulted in reduced rates of hydrolysis with each recycling of the enzyme, although cellobiose was still capable of being completely hydrolyzed. The reduced hydrolysis performance was attributable to physical losses of IME during recovery and, in the case of TAM, enzyme loss from the matrix. Supplementing cellulase digests of steam-explosion pretreatedEucalyptus regnons pulps with immobilizedβ-glucosidase resulted in enhanced hydrolysis. Cellulose-to-glucose yields of 80% of theoretical predictions resulted within 24 h. The magnetically immobilizedβ-glucosidase could easily be recovered from the lignocellulose solids suspension in a stirred batch reactor by applying a magnetic field. The recycled immobilized enzyme continued to convert cellobiose into glucose in 80% yields over a 24-h period. This is the first report of a magnetically immobilizedβ-glucosidase preparation used in the enzymatic saccharification of a lignocellulosic material.     

Ultrasound Irradiation Promoted Enzymatic Transesterification of (<em>R/S</em>)-1-Chloro-3-(1-naphthyloxy)-2-propanol

(R)-1-Chloro-3-(1-naphthyloxy)-2-propanol (3), which is the key intermediate of (S)-propranolol, was successfully prepared via enantioselective transesterification catalyzed by lipase under ultrasound irradiation. Compared with conventional shaking, the enzyme activity and enantioselectivity were dramatically increased under ultrasound exposure. Effects of various reaction conditions on the synthetic activity of enzyme as well as enantioselectivity, including the type of enzyme, ultrasound power, solvent, acyl donor, temperature and substrate molar ratio, were investigated. Pseudomonas sp. lipase (PSL) showed an excellent catalytic performance under optimum conditions (enzyme activity: 78.3 ± 3.2 μmol·g^-1·min^-1, E value: 98 ± 6).     

Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications

This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214?U/ml) under optimized conditions: carbon source, citric acid??1.5?% (w/w); inducer, soyabean meal??2?% (w/w); pH?11.0; shaking condition 37?°C for 48?h. The enzyme had pH and temperature optima of 9.0 and 60?°C, respectively. The enzyme displayed the molecular mass of 36?kDa in sodium dodecyl sulphate?Cpolyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0?C11.0) and thermostability (20?C70?°C). More than 70?% residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H₂O₂ for 30?min. The protease activity was also enhanced by divalent cations such as Ba²⁺, Ca²⁺ and Mg²⁺ and was strongly inhibited by Fe²⁺, Zn²⁺, Sr²⁺, Hg²⁺ and urea. The enzyme retained more than 50?% of its initial activity after pre-incubation for 1?h in the presence of 5?% (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1?%?w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the eco-friendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.     

Enhancement of thermostability of the Luciola mingrelica firefly luciferase by site-directed mutagenesis of nonconservative cysteine residues Cys62 and Cys146

Mutant forms of the firefly (Luciola mingrela) luciferase with point mutations Cys62Ser and Cys146Ser were obtained by site-directed mutagenesis. The mutations did not affect the catalytic activity and fluorescence spectra of the enzyme. The rate constants of the fast (k ₁) and slow (k ₂) stages of thermoinactivation of the wild-type and mutant enzymes were determined at 37°C in the absence and presence of 12 mM dithiothreitol (DTT). The thermostability of the mutant forms of luciferase increased several times compared to the wild-type enzyme. In the presence of DTT, k ₂ of the wild-type enzyme decreased three times whereas neither k ₁ nor k ₂ of the mutant forms changed. It was concluded that amino acid residues Cys62 and Cys146 play a major role in luciferase inactivation and that their substitution with Ser stabilizes the enzyme.     

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.     

Characterization of the DNA repair spore photoproduct lyase enzyme from Clostridium acetobutylicum: A radical-SAM enzyme

Spore photoproduct lyase (SPL) is a “Radical-SAM” repair enzyme which catalyzes the cleavage of spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine), a specific lesion found in bacterial spore DNA, to thymine monomers by a free-radical mechanism. The enzyme requires S-adenosyl-l-methionine (SAM) and a [4Fe–4S] cluster for activity. SPL from Bacillus subtilis has been difficult to isolate and characterize due to problems with the solubility and stability of the overexpressed protein in Escherichia coli and the lability of the [Fe–S] cluster, even if the protein was purified under strictly anaerobic conditions. In order to overcome these problems we searched for another SPL enzyme and we found that the recombinant SPL enzyme from Clostridium acetobutylicum, isolated either aerobically or anaerobically from overexpressing E. coli, behaves more stably than the B. subtilis one. We report here a complete spectroscopic and biochemical characterization of this enzyme. In particular we show for the first time that, using HYSCORE spectroscopy, SAM binds to the cluster as observed in the case of other members of the “Radical-SAM” enzyme family such as the activases of pyruvate formate lyase and ribonucleotide reductase.     

Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8

During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl - PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) - PHAs. The optimum pH range was 7.5-9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.     

Behavior of angiotensin-converting enzyme containing 67Zn

Magnetic nuclei of isotope ⁶⁷Zn (S ?5/2) were introduced into the active center of an angiotensin-converting enzyme instead of the zinc of usual isotope composition in order to consider their possible influence on the enzyme activity toward peptide substrates. No changes were observed in vitro, that gives us a possibility to study the influence of the zinc magnetic isotope on lymphocytes in various blood cell lines in vitro and ex vivo, neglecting contribution of this enzyme.     

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.     

Extracellular Enzymes of the White-Rot Fungus Fomes fomentarius and Purification of 1,4-β-Glucosidase

Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U?ml^?1), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U?ml^?1). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5–5.0, and K _M and V _max values were 62 μM and 15.8 μmol?min^?1?l^?1, respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K _i of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0–6.0, respectively. The combination of relatively low molecular mass and low K _M value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.     

p-aminophenyl phosphate: an improved substrate for electrochemical enzyme immnoassay

An alternative substrate is described for enzyme immunosaasay with electrochemical detection. Alkaline phosphatase (EC.3.1.3.1) activity is determined by using p-aminophenyl phosphate as the enzyme substrate. Enzyme-generated p-aminophenol is detected amperometrically at a glassy carbon electrody by liquid chromatography with electrochemical detection. The oxidation potential obtained for the detectionof p-aminophenol is lower than that for phenol, the previously used substrate product. The detection limit for p-aminophenol is 0.20pmol. A detection limit of 30 pg ml^-1 for digoxin and a 5-min incubationtime for the enzyme reaction were obtained with the new system.     

Partial Purification and Characterization of a Novel Extracellular Tyrosinase from Auricularia auricula

Extracellular tyrosinase from Auricularia auricula RF201 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 12.6 kDa on SDS-PAGE. The optimum pH for tyrosinase activity was 7, and the enzyme was stable between pH 6 and 9. Tyrosinase has optimal activity at 40 °C and retained most of its activity between 4 and 50 °C. A. auricula tyrosinase could oxidize l-tyrosine, l-DOPA, catechol, and caffeic acid and displayed dark brown or peach color. However, the enzyme was unable to catalyze l-phenylalanine and ferulic acid. In comparison with other substrates, l-tyrosine displayed the highest affinity (K _m of 0.11 mM) and the maximal reaction velocity (V _max of 102.58 μmol/min). Tyrosinase activity was reduced in the presence of numerous tested compounds. Particularly SDS, it significantly inhibited enzyme activity. CuSO₄ and NaCl showed an activation effect on enzyme activity, with the maximum activation found in the presence of CuSO₄.     

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme^®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme^®435 (L435) (lipase B from Candida antarctica), Lipozyme^®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.