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Results for “Enzyme stabilization”

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At least 811 records · Page 45Linked to original sources
PubMed1986

A stable liquid control material for urinary enzyme assays.

A stable liquid control material for urinary enzyme assays was prepared by addition of ethylene glycol (30% v/v) or glycerol (30% v/v) to centrifuged urine, and pH adjustment to 7.0. Tested for 375 days, the material exhibited stable beta-N-acetylglucosaminidase, alanine aminopeptidase, gamma-glutamyltransferase and lactate dehydrogenase activities, at both 4 and -20 degrees C. The material further has been successfully evaluated in an external quality control program.

Enzyme Stability↗
PubMed1995

Multi-enzyme reference material from established human cell lines and human sources.

A multi-enzyme reference material was prepared from seven enzymes of asparatate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), alkaline phosphatase (ALP, EC 3.1.3.1), lactate dehydrogenase (LD, EC 1.1.1.27), creatine kinase (CK, EC 2.7.2.2), gamma-glutamyltranspeptidase (gamma-GT, EC 2.3.2.2) and amylase (AMY, EC 3.2.1.1) which were purified from human sources including established human cell lines. The enzymatic properties of the material closely resembled those of human serum. In lyophilized form the preparation was stable for at least 200 days when stored at 40 degrees C. Intermethod comparisons of the enzyme activities in 80 clinical specimens were done by correcting the mean values with calibration constants for different assay methods resulting from use of a human serum, the multi-enzyme reference and a commercial control serum. The results from the comparison for the six enzymes of AST, ALT, LD, CK, gamma-GT and AMY in use of the multi-enzyme reference were almost the same as those with use of a human serum as a calibrator, but were not satisfactory for ALP. Even though further search for more reliable material for ALP is required the multi-enzyme reference material can be used for standardization in clinical chemistry.

Alanine Transaminase↗
PubMed1978

The depression of thalamic nociceptive neurones by D-ala2,D-leu5-enkephalin.

The activity of single thalamic nociceptive neurones was recorded extracellularly. BW 180C, (D-ala2,D-leu5-enkephalin) applied locally, by iontophoresis (10--40 nA) or systemically by i.v. injection (1.5--6 mg/kg) markedly depressed the evoked excitation of these neurones following peripheral noxious stimulation. This effect could be antagonised by naloxone. Enhanced enzymic stability did not appear to prolong the duration of action of BW 180C iontrophoretically applied to these neurones.

Animals↗
PubMed1989

Stabilisation and immobilisation of penicillin amidase.

Penicillin amidase was coupled to a periodate-oxidised dextran by reductive alkylation in the presence of sodium cyanoborohydride. A loss of activity (25%) was observed but the conjugate enzyme dextran was more thermostable than the native enzyme. Native and dextran-conjugated penicillin amidase were immobilised on amino activated silica (Promaxon, Spherosil, Aerosil) by a classical method using glutaraldehyde for the native enzyme and reductive alkylation for the modified enzyme. Good relative activity of the enzymes was obtained after insolubilisation. Immobilisation of both native and modified enzymes resulted in the thermostabilisation of the penicillin amidase.

Enzyme Stability↗
PubMed1994

Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation.

In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro. Without nucleotides the refolding reactions were arrested by GroEL. Addition of GroES and ADP to the reaction mixture initiated the refolding reactions and the enzyme activities were regained efficiently. The presence of GroE (GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures. These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL-facilitated protein folding.

Adenosine Diphosphate↗
PubMed1994

Biochemical approaches of increasing thermostability of beta-amylase from Bacillus megaterium B6.

Studies on the irreversible thermoinactivation of beta-amylase from Bacillus megaterium B6 exposed to 60 degrees C revealed that the deactivation mechanism probably results from the oxidation of thiols present at the active site of the enzyme. Several attempts were made to increase its thermostability, which indicated that Mn2+ played a key role in determining thermostability and partially reactivating the inactivated enzyme. Immobilization of beta-amylase through gel-entrapment and covalent crosslinking brought about a remarkable increase in thermotolerance with about a 14-fold increase in catalytic half-life.

Bacillus megaterium↗
PubMed1996

Oligomerization of sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle.

Although the primary structure and catalytic cycle of the sarcoplasmic reticulum Ca2+-ATPase has been revealed, it is not well understood whether functional Ca2+ pump proteins exist in a monomeric or an oligomeric state in native skeletal muscle membranes. Here, we show that the Ca2+-ATPase tends to form high molecular weight complexes, estimated to be dimers and tetramers using immunoblotting of two-dimensionally separated microsomal membranes following crosslinking. This agrees with both electron microscopical and biochemical findings which demonstrate that Ca2+-ATPase clusters are the predominant molecular species in native membranes and that oligomerization may play a role in cooperative kinetics and enzyme stabilization.

Animals↗
PubMed1991

Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor: enzymes in thin liquid films.

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures of substrate and water vapor over a range of relative humidities up to 100%. Evidence was obtained for operation of the enzyme in a thin liquid film of concentrated buffer on the surface of the glass beads. Formation of the thin film was associated with hygroscopicity of the buffer used to suspend the enzyme in preparation of the enzyme-coated beads. At some buffer concentrations estimated to be on the bead surface, the enzyme was partially soluble and both soluble and insoluble forms were enzymatically active. The vapor mode of operation over a range of relative humidities had comparatively small effects on kinetic constants for hydrolysis of ethyl acetate, which were also similar to those in phosphate buffer. The extent of reaction occurred in the order hydrolysis greater than alcoholysis greater than ester interchange greater than esterification. Reaction preference between alcoholysis and hydrolysis changed as acyl chain length of substrate increased with C. rugosa lipase, as well as with Rhizopus arrhizus lipase and porcine liver esterase, with details depending on the enzyme. The vapor mode approach has the potential of being used with a wide variety of substrates, as shown by the ability to obtain hydrolysis at 30 degrees C with substrate vapor pressures as low as 0.08 mm Hg and with substrates with boiling points as high as 206 degrees C.

Candida↗
PubMed1992

Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids.

The production of D-aminoacylase by Alcaligenes faecalis DA1 was induced 5- to 50-fold by N-acetyl-D-amino acids. This strain produced about 443 units of D-aminoacylase and 52 units of L-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-DL-leucine as the carbon source. The D-aminoacylase was partially purified by Fractogel DEAE 650 column chromatography and then immobilized on another Fractogel DEAE 650 column. The catalytic activity of the immobilized D-aminoacylase was 2,650 units per milliliter of gel. The Km values for the free and the immobilized enzymes were found to be 1.00 and 0.22 mM, respectively, using N-acetyl-D-methionine as a substrate. The optimal reaction pH and temperature for both soluble and immobilized enzyme were around 8.0 and 45 degrees C, respectively. The free enzyme was stable in the pH range from 5.0 to 11.0, whereas the immobilized enzyme tended to detach from the gel at pH values higher than 9.0. Both forms of enzyme were stable up to 40 degrees C. When used for the optical resolution of N-acetyl-DL-methionine, the immobilized enzyme maintained 90% initial activity after 17 days of continuous operation at 45 degrees C. The process of purification and immobilization of D-aminoacylase described in this report is very effective and easy to scale up.

Alcaligenes↗
PubMed1992

Screening of whole-cell immobilization procedures for the delta 1-dehydrogenation of steroids in organic medium.

Different whole-cell immobilization methods and conditions were tested for the delta 1-dehydrogenation of 6-alpha-methyl-hydrocortisone-21-acetate with Arthrobacter simplex cells, in n-decane-1-ol and chloroform. Among the entrapment methods, polyurethane foams gave the best productivity of n-decane-1-ol, but could not be used in chloroform. Partition and diffusional barriers appeared to be the factors limiting productivity in entrapped-cell systems. The coentrapment of enzyme-stabilizing (glycerol, sucrose, sodium sulfate, monosodium glutamate) or hydrophobic additives did not significantly improve dehydrogenation rates in chloroform, although in some cases higher activities resulted in n-decane-1-ol. Dehydration of kappa-carrageenan-immobilized cells lowered the productivity in both solvents. The use of cell adsorption methods on silica-based carriers produced biocatalysts that in some cases were more active in chloroform than the entrapped cells. However, the surface-attached cells appeared to be more sensitive to solvent toxicity, more hydrophilic supports generally giving higher dehydrogenation rates. The estimated partition coefficients for the substrate between several of the tested supports and the two solvents are also presented. High partition ratios followed high measured activities in entrapment matrices but not in Celite (cell adsorption matrix). Activities in chloroform were always very low (less than 1 mumol product per hour and gram of cell dry weight). Polyurethane entrapment with n-decane-1-ol as a reaction medium was the most promising system, with measured dehydrogenation rates up to 40 times higher.

Agar↗
PubMed1993

Applications of immobilized lipases to transesterification and esterification reactions in nonaqueous systems.

Lipases from Candida cylindracea, Aspergillus niger, and Pseudomonas fluorescens were immobilized by adsorption on anion-exchange resin and diatomaceous earth using buffer or hexane as a reaction medium. The enzyme preparations were tested in the transesterification of triolein with lauric acid and the esterification of lauric acid with different alcohols. Immobilized C. cylindracea preparations were more active when hexane was used as the reaction medium, and anion-exchange resin was a better support than diatomaceous earth. Hexane was also a better immobilization medium for A. niger lipase. No difference was observed in the lipase activity of P. fluorescens lipase immobilized in different ways. The synthetic activities of the immobilized enzymes could be predicted from their hydrolytic activities: the higher the hydrolytic activity, the higher the synthetic activity. There was no direct correlation, however, between the lipolytic and the transesterification activities.

Anion Exchange Resins↗
PubMed1993

DNase activity of micrococcal endonuclease covalently immobilized on nylon and polystyrene.

Water-insoluble nucleases were prepared by immobilizing the endonuclease from S. aureus onto the surface of nylon-66 and polystyrene spheres. The activation phase of the synthetic supports was optimized to define optimal conditions of pH, temperature, and Ca2+ concentration for using immobilized enzymes. The activity, evaluated by hydrolysis of high-molecular-weight and supercoiled DNA, indicates that both derivatives are highly stable for storage and further use. Immobilization of the enzyme is much more effective when the covalent binding is performed on polystyrene. By using different activation methods with these matrices, a set of immobilized nucleases with various levels of enzymatic activity can be prepared. The possibility of working in a wide range of enzymatic activity and at low temperature and Ca2+ concentrations in different buffers makes these immobilized nucleases very useful for investigating accessible DNA regions in chromatin structure.

Biotechnology↗