Enzyme Activity Stability Research Articles

A catalytic membrane used for H2O2 decomposition

The present work describes the results of the preliminary characterization of a novel enzyme with catalase activity isolated from psychrotolerant bacteria. Catalases decompose hydrogen peroxide (H2O2) to water and oxygen. Chemical immobilization is the best solution in case of reactions in which a gaseous product is created hence immobilization via divinyl sulfone having used a polymeric regenerated cellulose membrane was proposed. The preparations were checked according to their activity and stability.In the textile industry, a catalase is used to decompose the hydrogen peroxide waste after bleaching fabrics. During photocatalytic oxidation, H2O2 is added at relatively high concentration app. 20–80g/L and the pH of these solutions is in the range between 7 and 10.The psychrophilic catalase described in this work presents high activity up to 40g/L at pH 7–10, while a commercial alternative isolated from bovine liver only displays activity up to 14g/L at pH not higher than 9. The stability of enzymatic activity is strongly dependent on temperature and is also a function of substrate concentration.The results obtained with this psychrophilic catalase are promising for textile effluence treatment.

A disposable biosensor based on immobilization of laccase with silica spheres on the MWCNTs-doped screen-printed electrode

BackgroundBiosensors have attracted increasing attention as reliable analytical instruments in in situ monitoring of public health and environmental pollution. For enzyme-based biosensors, the stabilization of enzymatic activity on the biological recognition element is of great importance. It is generally acknowledged that an effective immobilization technique is a key step to achieve the construction quality of biosensors.ResultsA novel disposable biosensor was constructed by immobilizing laccase (Lac) with silica spheres on the surface of multi-walled carbon nanotubes (MWCNTs)-doped screen-printed electrode (SPE). Then, it was characterized in morphology and electrochemical properties by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The characterization results indicated that a high loading of Lac and a good electrocatalytic activity could be obtained, attributing to the porous structure, large specific area and good biocompatibility of silica spheres and MWCNTs. Furthermore, the electrochemical sensing properties of the constructed biosensor were investigated by choosing dopamine (DA) as the typical model of phenolic compounds. It was shown that the biosensor displays a good linearity in the range from 1.3 to 85.5 μM with a detection limit of 0.42 μM (S/N = 3), and the Michaelis-Menten constant (Kmapp) was calculated to be 3.78 μM.ConclusionThe immobilization of Lac was successfully achieved with silica spheres to construct a disposable biosensor on the MWCNTs-doped SPE (MWCNTs/SPE). This biosensor could determine DA based on a non-oxidative mechanism in a rapid, selective and sensitive way. Besides, the developed biosensor could retain high enzymatic activity and possess good stability without cross-linking reagents. The proposed immobilization approach and the constructed biosensor offer a great potential for the fabrication of the enzyme-based biosensors and the analysis of phenolic compounds.

β-GALACTOSIDASE ACTIVITY OF MEIOTHERMUS RUBER CELLS

Freeze-dried cells of Meiothermus ruber catalyses cleavage of o-nitrophenyl-β-D-galactopiranoside (oNPβ-gal) and conversion of lactose into glucose and galactose. The permeabilization with 2% toluene, 20% ethanol and 20% acetone increased enzymatic activity from 74.87 U/g of lyophilized cells up to 129.44, 114.38 and 90.19 U/g, respectively. Ethanol was an effective permeabilizing agent and its efficiency was dependent on the concentration, the incubation time and incubation temperature. The Km values for the untreated and permeabilized cells were 2.94 mM and 2.26 mM but Vmax values were 122 µmol/min and 193 µmol/min, respectively. The optimum pH for the β-galactosidase activity in the untreated and permeabilized cells were 6.5 and optimum of temperatures 65C. The stability of enzymatic activity in M. ruber cells incubated for 1 h at pH 6.5 was almost unchanged at temperatures below 65C. PRACTICAL APPLICATIONS The current study shows that freeze-dried permeabilized cells of Meiothermus ruber can be used instead of isolated β-galactosidase. It leads to elimination of expensive purification procedures and assures longer half-life time of enzyme activity. Application of such biocatalyst for lactose hydrolysis in milk products for human consumption is limited until designation of the Generally Recognized As Safe status of Meiothermus ruber. It can be employed, however, for other purposes, e.g., for production of alkyl glycosides, a group of non-ionic surfactants with a variety of applications in detergents, cleaning agents, personal care products and pharmaceuticals.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, β-cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5°C and 40% at 25°C after 8 months. The lipase dried with 10% of β-cyclodextrin retained 72% of activity at 5°C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.

Effects of cations on Helicobacter pylori urease activity, release, and stability.

The urease of Helicobacter pylori is an important antigen and appears critical for colonization and virulence. Several studies have indicated a superficial localization for the H. pylori urease, and the purpose of this study was to determine the effects of cations on the release and stability of urease activity from H. pylori cells. Incubation of partially purified H. pylori urease in water containing 1, 5, or 10 mM Ca2+, Mg2+, K+, Na+, EDTA, or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] had little effect on activity. In contrast, 1 mM Fe3+, Cu2+, Co2+, or Zn2+ substantially (> 80%) inhibited activity, and 10 mM Fe2+, Mn2+, and Ni2+ inhibited about 30% of the activity. Addition of Ca2+ or Mg2+ markedly decreased extraction of urease from intact H. pylori cells by water, but 1 mM Na+, K+, EGTA, or EDTA each had minimal effects on release, suggesting that divalent cations have a role in attachment of urease to H. pylori cells. The stability of enzymatic activity at 4 degrees C was enhanced by addition of glycerol or 2-mercaptoethanol; however, even after loss of activity, full antigenicity for human serum was retained.

Characterization of L-Histidine Ammonia-lyase Immobilized by Microencapsulation in Artificial Cells: Preparation, Kinetics, Stability, and in Vitro Depletion of Histidine

L-histidine ammonia-lyase (histidase) was encapsulated within cellulose nitrate artificial cells, and its kinetic parameters were evaluated. Microencapsulated histidase had an apparent activity of histidase in solution. Encapsulation did not alter the Km of histidase. The Km of histidase solution and the Km apparent of micro-encapsulated histidase were both 20 mM. Encapsulation of histidase resulted in increased stability of enzymatic activity at storage temperatures of 4 degrees C and 37 degrees C. At 37 degrees C histidase solution reached 50% of its original activity after 9.5 days of storage, while microencapsulated histidase reached the same level after 15 days. At 4 degrees C histidase solution had 63% of its original activity after 21 days of storage, while encapsulated histidase had 95%. In vitro experiments to evaluate the feasibility of microencapsulated histidase for possible experimental therapy in histidinemia were carried out. These experiments evaluated the effectiveness of encapsulated histidase in depleting histidine. Three different volume ratios of histidase loaded artificial cells to substrate solution were tested. A ratio of 1: 100 allowed 25% histidine depletion after 120 hours. A 1: 50 ratio allowed 35% histidine depletion after 72 hours. A 1: 25 ratio allowed 40% histidine depletion after 24 hours (Int J Artif Organs 1990; 13: 189-95).

Partial purification of hepatic UDP-glucuronyltransferase: Studies of some of its properties

1. 1. Purification of UDP-glucuronyltransferase (UDP-glucuronate glucuronyltransferase, EC 2.4.1.17) from guinea pig liver, achieved by ultrasonication, sucrose gradient fractionation and Sephadex G-200 elution, increased the capacity to conjugate bilirubin in vitro 20-fold and o- aminophenol 50-fold. The stability of enzymatic activity in this preparation was greater than previously described for this enzyme in preparations with a comparable increase in initial activity and was strikingly similar with both substrates. The purification procedure did not separate conjugating ability for any of the substrates tested. 2. 2. The inadequacy of gravitational criteria alone in indicating solubilization of a membrane-bound enzyme was demonstrated by electron microscopy. Methods used unsuccessfully in attempting true solubilization are listed. 3. 3. The purified enzyme preparation was shown to contain a number of proteins including microsomal enzymes and lipids. The possible effect of these on kinetic studies is critically discussed. 4. 4. Application of the purification procedure to livers of cat, Gunn rat and hypophysectomized or thyroidectomized rats also yielded a stable purified enzyme preparation, without separating conjugating ability for the substrates tested. 5. 5. The results of these investigations are discussed and their relationship to possible multiplicity of UDP-glucuronyltransferase considered.