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At least 991 records · Page 55Linked to original sources
PubMed2004

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.

Cellulase↗
PubMed2004

Unexpected reaction profile observed in the synthesis of propyl laurate when using Candida rugosa lipases immobilized in microemulsions based organogels.

1-Propyl laurate synthesis should not be used as standard reaction test of immobilized enzymes in microemulsion-based organogels (MBGs) prepared using lecithin/1-propanol as surfactant when extremely active enzymes with high load are used. In these cases, an anomalous kinetic reaction constant value is observed over short reaction times. Such an anomalous profile is strongly dependent on the concentration of catalyst in the crude powder and, consequently, is not appreciated when either commercial or low activity lipase samples are employed.

Candida↗
PubMed2004

Protein-based biosensors for diabetic patients.

In this article we show the recent progress in the field of glucose sensing based on the utilization of enzymes and proteins as probes for stable and non-consuming fluorescence biosensors. We developed a new methodology for glucose sensing using inactive forms of enzymes such as the glucose oxidase from Aspergillus niger, the glucose dehydrogenase from the thermophilic microorganism Thermoplasma acidophilum, and the glucokinase from the thermophilic eubacterium Bacillus stearothermophilus. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8-Anilino-1-naphthalene sulfonic acid was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the KD of holo-glucose oxidase. The ANS-labeled apo-glucose dehydrogenase from Thermoplasma acidophilum also displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be also used to measure the glucose concentration. The thermophilic apo-glucose dehydrogenase was also stable in the presence of organic solvents, allowing determination of glucose in the presence of acetone. The third enzyme used for glucose sensing was the glucokinase from Bacillus stearothermophilus. A fluorescence competitive assay for the determination of glucose was developed based on the utilization of this thermostable enzyme. Taken together, our results show that enzymes which use glucose as their substrate can be used as reversible and non-consuming glucose biosensors in the absence of required co-factors. Moreover, the possibility of using inactive apo-enzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.

Anilino Naphthalenesulfonates↗
PubMed2002

Structural changes in the calcium pump accompanying the dissociation of calcium.

In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca(2+) ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 A resolution in a Ca(2+)-free (E2) state, and compare it with that determined previously for the Ca(2+)-bound (E1Ca(2+)) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca(2+). Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions.

Animals↗
PubMed2005

Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding.

Thermodynamic stability is fundamental to the biology of proteins. Information on protein stability is essential for studying protein structure and folding and can also be used indirectly to monitor protein-ligand or protein-protein interactions. While clearly valuable, the experimental determination of a protein's stability typically requires biophysical instrumentation and substantial quantities of purified protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining protein stability by using pulse proteolysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The DeltaG(unf) degrees values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding protein to maltose, as well as to rapidly screen cognate ligands for this protein. The simplicity of pulse proteolysis suggests that it is an excellent strategy for the high-throughput determination of protein stability in protein engineering and drug discovery applications.

Drug Stability↗
PubMed2003

Enzyme-dispersed carbon-nanotube electrodes: a needle microsensor for monitoring glucose.

The preparation of an enzyme-dispersed carbon-nanotube (CNT) electrode, based on mixing glucose oxidase (GOx) within CNT, is described. The new binderless biocomposite was packed within a 21-gauge needle and used for amperometric monitoring of glucose. The resulting microsensor offers a low-potential highly selective and sensitive detection of glucose. The high sensitivity and selectivity are coupled to a wide linear range, prolonged lifetime and oxygen independence. About 80% of the GOx activity is retained during a 24 h thermal stress at 90 degrees C, reflecting the enzyme-stabilization action of CNT. The marked electrocatalytic action towards hydrogen peroxide allows highly selective detection of the glucose substrate at -0.1 V (vs. Ag/AgCl) with no interferences from coexisting ascorbic acid, acetaminophen or uric acid. Linearity prevails up to 40 mM glucose (with analytically useful signals observed up to 0.1 M). Factors affecting the performance of the CNT-based glucose biosensor were assessed and optimized. The attractive performance of the new needle electrode offers great promise for continuous monitoring of glucose in connection to the management of diabetes, and for the biosensing of other metabolites.

Electrochemistry↗
PubMed2003

Colloidal stable silica encapsulated nano-magnetic composite as a novel bio-catalyst carrier.

A colloidal stable silica-encapsulated magnetic nano-composite of a controlled dimension is, for the first time, employed to carry beta-lactamase via chemical linkage on the silica overlayer: activity study reflects that this new type of immobilisation allows site (enzyme) isolation, accessibility as good as free enzyme and recovery & reusability upon application of magnetic separation.

Biotechnology↗
PubMed2001

Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221-->Met mutant.

A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221-->Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pK(a) of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by (1)H-NMR data, and the loss of the pi-pi interaction between His-170 and Phe-221.

Arabidopsis↗
PubMed2001

Papain immobilization on a nitrilon fibre carrier containing primary amine groups.

A carrier containing primary amine groups was synthesized by the reaction between nitrilon fibre and diethylenetriamine, and papain was immobilized on this kind of carrier by using glutaraldehyde as a coupling agent. The factors involved with the activity recovery of the immobilized papain and the enzymic properties of the resulting immobilized papain were studied in comparison with free papain, for which casein was chosen as a substrate. The results show that the activity recovery of immobilized papain can reach 47.3%, and that the immobilized papain exhibits better environmental adaptability and reusability than free papain.

Amines↗
PubMed2000

Immobilization of Pseudomonas cepacia lipase in a phyllosilicate sol-gel matrix: effectiveness as a biocatalyst.

A novel procedure is described for immobilizing a lipase from Pseudomonas cepacia (PS-30) within a phyllosilicate sol-gel matrix. The method is based on cross-linking a phyllosilicate clay with silicate polymers produced by the controlled hydrolysis of tetramethyl orthosilicate (TMOS). The activity of the phyllosilicate sol-gel-immobilized lipase was dependent upon the type of alkylammonium salt, inorganic catalyst and volume ratio of phyllosilicate clay to TMOS used. Lipase PS-30 immobilized in this way was more stable and had higher activity compared with the free lipase. Studies on the lipase-catalysed esterification of lauric acid with octan-1-ol in iso-octane showed that under controlled water activity conditions the phyllosilicate sol-gel-immobilized lipase had better activity compared with other supported lipase preparations. In addition, the phyllosilicate sol-gel-immobilized lipase was reusable for at least five esterification cycles without significant loss of activity.

Catalysis↗
PubMed2002

Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel.

Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l. and gran- C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30). All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system. Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran- T.l. and gran- C.a. lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.

Ascomycota↗
PubMed2002

Modified high amylose starch for immobilization of uricase for therapeutic application.

Urate oxidase (uricase) was immobilized on carboxymethyl high amylose starch cross-linked 35 (CM-HASCL-35), on aminoethyl high amylose starch cross-linked 35, as well as on commercial supports, CNBr-activated Sepharose and diaminodipropylamine agarose. The N -ethyl-5-phenylisoxazolium-3'-sulphonate (Woodward reagent K) gave a high binding but totally inhibited the enzyme activity. Best results were obtained with CM-HASCL-35 using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide as a coupling agent. The immobilized enzyme retained 88% of its initial limit rate [V (max)(app)=16 EU/mg for immobilized uricase versus V (max)=18 EU/mg for the free enzyme], with an apparent decrease of affinity for urate substrate [K (m)(app)=0.17 mM versus K (m)=0.03 mM for the free enzyme]. The coupling yield was 60% and the modified uricase was found more resistant to proteolysis than the free enzyme. The immobilized uricase retained 25% of its initial activity after 60 min in pancreatic proteolysis medium (pancreatin), whereas the free enzyme retained only 5% of its initial activity. The best immobilization yield was obtained with the polymeric support based on CM-HASCL-35 (53%), which gave better results than commercial supports based on agarose.

Amylose↗
PubMed2004

A kinetic study of bovine haemoglobin hydrolysis by pepsin immobilized on a functionalized alumina to prepare hydrolysates containing bioactive peptides.

The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2-ethanolamine- O -phosphate (2-EAOP)-modified acidic alumina. Surface charge of the two supports was determined as a function of pH. Kinetic studies were performed at 23 degrees C in 0.1 M acetate buffer, pH 4.5. At this pH, the surface charge of the two supports was almost the same. The coating of alumina by 2-EAOP only introduced a two carbon spacer between alumina surface and the reaction medium. Adsorption on the two supports of haemoglobin, haem and peptides produced in the course of hydrolyses were compared. Fixation of 2-EAOP on a pepsin-alumina complex gave hydrolysis kinetics of urea-denatured haemoglobin close to that obtained with the same amount of pepsin in solution, but with comparatively less adsorption of peptides and complete adsorption of haem. Heterogeneous hydrolyses of haemoglobin with pepsin, immobilized on functionalized alumina, resulted in the presence of VV-haemorphin-4, VV-haemorphin-7 and neokyotorphin in the supernatants without haem, the presence of which makes further purification difficult.

Aluminum Oxide↗
PubMed2006

Reduction of flatus-inducing factors in soymilk by immobilized alpha-galactosidase.

Alpha-galactosidase from Aspergillus oryzae was immobilized on chitosan beads using glutaraldehyde as a cross-linking agent. The general properties of free and immobilized enzymes were determined. The optimum pH for the free and immobilized enzymes was 4.8 and 4.6 respectively. The optimum temperature for the free enzyme was 50 degrees C, whereas that of immobilized enzyme was increased to 56 degrees C. Kinetic parameters were determined with synthetic substrate (p-nitrophenyl alpha-D-galactopyranoside) and raffinose. Immobilized enzyme showed a higher Km and a lower Vmax than the free enzyme. The immobilized enzymes were used in batch, repeated and continuous mode. A level of 92% hydrolysis was observed at a flow rate of 60 ml/h. The immobilized enzyme was used repeatedly ten times without any change in the performance of the immobilized enzyme in fluidized-bed reactor. The results obtained are of considerable interest for industrial purposes.

Aspergillus oryzae↗
PubMed2006

Interaction of human GTP cyclohydrolase I with its splice variants.

Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.

Alternative Splicing↗