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PubMedDate not supplied

Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk.

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.

Animals↗
PubMedDate not supplied

Study on mesophilic and thermophilic alcohol dehydrogenases in gas-phase reaction.

The initial reaction rate and the thermostability of the mesophilic alcohol dehydrogenase (ADH) from Lactobacillus brevis (LBADH), and the thermophilic ADH from Thermoanaerobacter sp. (ADH T) in gas-phase reaction were compared. The effects of water activity, cofactor-to-protein molar ratio, and reaction temperature on the reduction of acetophenone to 1-phenylethanol were studied. An optimal water activity of 0.55 in terms of productivity was found for both ADHs. The cofactor-to-protein molar ratio was chosen slightly higher than equimolar to increase both activity and thermostability. An excellent optimal productivity of 1,000 g x L(-1) x d(-1) for LBADH and 600 g x L(-1) x d(-1)for ADH T was found at 60 degrees C, while the highest total turnover numbers with respect to the enzyme were achieved at 30 degrees C and amounted to 4.2 million for LBADH and 1.7 million for ADH T, respectively. Interestingly, the ADH from the mesophilic L. brevisshowed the higher thermostability in the nonconventional medium gas phase.

Alcohol Dehydrogenase↗
PubMed2005

How the Co-C bond is cleaved in coenzyme B12 enzymes: a theoretical study.

The homolytic cleavage of the organometallic Co-C bond in vitamin B12-dependent enzymes is accelerated by a factor of approximately 10(12) in the protein compared to that of the isolated cofactor in aqueous solution. To understand this much debated effect, we have studied the Co-C bond cleavage in the enzyme glutamate mutase with combined quantum and molecular mechanics methods. We show that the calculated bond dissociation energy (BDE) of the Co-C bond in adenosyl cobalamin is reduced by 135 kJ/mol in the enzyme. This catalytic effect can be divided into four terms. First, the adenosine radical is kept within 4.2 angstroms of the Co ion in the enzyme, which decreases the BDE by 20 kJ/mol. Second, the surrounding enzyme stabilizes the dissociated state by 42 kJ/mol using electrostatic and van der Waals interactions. Third, the protein itself is stabilized by 11 kJ/mol in the dissociated state. Finally, the coenzyme is geometrically distorted by the protein, and this distortion is 61 kJ/mol larger in the Co(III) state. This deformation of the coenzyme is caused mainly by steric interactions, and it is especially the ribose moiety and the Co-C5'-C4' angle that are distorted. Without the polar ribose group, the catalytic effect is much smaller, e.g. only 42 kJ/mol for methyl cobalamin. The deformation of the coenzyme is caused mainly by the substrate, a side chain of the coenzyme itself, and a few residues around the adenosine part of the coenzyme.

Carbon↗
PubMed2005

Enzymatic interesterification of butterfat with rapeseed oil in a continuous packed bed reactor.

Lipase-catalyzed interesterification of butterfat blended with rapeseed oil (70/30, w/w) was investigated both in batch and in continuous reactions. Six commercially available immobilized lipases were screened in batch experiments, and the lipases, Lipozyme TL IM and Lipozyme RM IM, were chosen for further studies in a continuous packed bed reactor. TL IM gave a fast reaction and had almost reached equilibrium with a residence time of 30 min, whereas RM IM required 60 min. The effect of reaction temperature was more pronounced for RM IM. TL IM showed little effect on the interesterification degree when the temperature was raised from 60 degrees C to 90 degrees C, whereas RM IM had a positive effect when the temperature was increased from 40 degrees C to 80 degrees C. Even though TL IM is an sn-1,3 specific lipase, small changes in the sn-2 position of the triacylglycerol could be seen. The tendency was toward a reduction of the saturated fatty acid C14:0 and C16:0 and an increase of the long-chain saturated and unsaturated fatty acids (C18:0 and C18:1), especially at longer residence times (90 min). In prolonged continuous operation the activity of TL IM was high for the first 5 days, whereafter it dramatically decreased over the next 10 days to an activity level of 40%. In general, the study shows no significant difference for butterfat interesterification in terms of enzyme behavior from normal vegetable oils and fats even though it contains short-chain fatty acids and cholesterol. However, the release of short-chain fatty acids from enzymatic reactions makes the sensory quality unacceptable for direct edible applications.

Butter↗
PubMed2001

Facile enzymatic aldol reactions with dihydroxyacetone in the presence of arsenate.

Aldol reactions of in situ formed dihydroxyacetone arsenate with different aldehydes were catalyzed by bacterial D-fructose-1,6-bisphosphate aldolase (FruA). Aldolases from bacteria were found to be much more stable and active than FruA from rabbit muscle. Arsenate acts as a phosphate mimic and can, in principle, be used in catalytic amounts. The use of inorganic arsenate and dihydroxyacetone afforded high yields with hydrophobic aldehydes. Cosolvents increased the solubility of hydrophobic aldehydes and afforded higher reaction rates and enzyme stability. Insight is given, for the first time, in the influence of arsenate on the stereoselectivity of the aldol reaction.

Arsenates↗
PubMed2006

Silica-encapsulated nanomagnetic particle as a new recoverable biocatalyst carrier.

Enzymes are versatile biocatalysts with major advantages of ultrahigh reaction selectivity and specificity under mild conditions, which currently find increasing applications. However, their applications are often hampered by difficulties in recovery and recycling. As a result, we carried out detailed investigations on the synthesis and characterization of silica-encapsulated iron oxide magnetic nanoparticles of controlled dimension as an enzyme carrier. It is shown that the relatively smaller sized silica-coated magnetic nanoparticle prepared by the microemlusion technique can a carry bulky enzyme, beta-lactamase, via chemical linkages on the silica overlayer without severely blocking the enzymatic active center (which is commonly encountered in conventional solid supports). An activity study by Michalis-Menten kinetics reflects that this new type of immobilization allows enzyme isolation with accessibility as good as free enzyme. The recovery and reusability of the nanoparticle-supported enzyme upon application of magnetic separation are also demonstrated.

Catalysis↗
PubMed2004

Effect of the addition of a nonaqueous polar solvent (glycerol) on enzymatic catalysis in reverse micelles. Hydrolysis of 2-naphthyl acetate by alpha-chymotrypsin.

The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by alpha-chymotrypsin (alpha-CT), in reverse micellar solutions formed by glycerol (GY)-water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of this reaction with that observed in micelles with water in the core, as well as in the corresponding homogeneous media, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in both homogeneous media (water and GY-water, 38% v/v mixture). In every media, alpha-CT was characterized by the absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of its tryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellar systems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover, the data obtained allowed the evaluation of the 2-NA partition constant between the organic and the micellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increase in the catalytic properties of alpha-CT. The fluorescence anisotropy studies in the different media show that the addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GY addition to the reverse micellar aggregates results in a decrease of the conformational mobility of alpha-CT, which leads to an increase of the enzyme stability and activity.

Catalysis↗
PubMed2005

Immobilization of active hydrogenases by encapsulation in polymeric porous gels.

Hydrogenases encapsulated in porous polymeric silica gels retain significant levels of hydrogen production activity when compared to hydrogenases in solution using reduced methyl viologen as an electron donor. Encapsulated hydrogenases remain active after storage at room temperature for longer than four weeks and are less sensitive to proteolytic digestion. Nanoscopic confinement of active hydrogenases in solids paves the way for their potential use in hydrogen producing catalytic materials applications.

Capsules↗
PubMed2006

Proteolytic surface functionalization enhances in vitro magnetic nanoparticle mobility through extracellular matrix.

Steric barriers such as collagen I sharply limit interstitial delivery of macromolecular and nanoparticle (NP) based therapeutic agents. Collagenase-linked superparamagnetic NPs overcame these barriers and moved through in vitro extracellular matrix (ECM) at 90 microm h(-1), a rate similar to invasive cells, under the influence of a magnetic field. NP migration in ECM diminished linearly over 5 days. The collagenase-NP construct overcame two of the most significant barriers to nano- and microscale therapeutics deployment: proteolytic enzyme stability was maintained during a clinically useful time frame by immobilization on the NP surface and degradation of interstitial barriers to tissue biodistribution was enabled by the conjugated microbial protease.

Animals↗
PubMed2005

Cross-linked aggregates of (R)-oxynitrilase: a stable, recyclable biocatalyst for enantioselective hydrocyanation.

[Reaction: see text] The (R)-oxynitrilase from almonds was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with 1,2-dimethoxyethane and subsequent cross-linking using glutaraldehyde. The resulting preparation was a highly effective hydrocyanation catalyst under microaqueous conditions, which suppress the nonenzymatic background reaction. The beneficial effect of these latter conditions on the hydrocyanation of slow-reacting aldehydes is demonstrated. The oxynitrilase CLEA was recycled 10 times without loss of activity.

Aldehyde-Lyases↗
PubMed1996

Esterase-sensitive cyclic prodrugs of peptides: evaluation of an acyloxyalkoxy promoiety in a model hexapeptide.

PURPOSE: To evaluate a cyclic acyloxyalkoxycarbamate prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of the peptide and stabilize it to metabolism. METHODS: Conversion to the linear hexapeptide was studied at 37 degrees C in aqueous buffered solutions and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. RESULTS: In buffered solutions the cyclic prodrug degraded chemically to the linear hexapeptide in stoichiometric amounts. Maximum stability was observed between pH 3-4. In 90% human plasma (t1/2 = 100 +/- 4 min) and in homogenates of the rat intestinal mucosa (t1/2 = 136 +/- 4 min) and rat liver (t1/2 = 65 +/- 3 min), the cyclic prodrug disappeared faster than in buffered solution, pH 7.4 (t1/2 = 206 +/- 11 min). Pretreatment of these media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug (t1/2 = 282 +/- 25 min) was significantly more stable than the hexapeptide (t1/2 = 14 min) and at least 76-fold more able to permeate (Papp = 1.30 +/- 0.15 x 10(-7) cm/s) than the parent peptide (Papp < or = 0.17 x 10(-8) cm/s). CONCLUSIONS: Preparation of a cyclic peptide using an acyloxyalkoxy promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.

Animals↗
PubMed1999

Is the difference in alpha-amylase activity in the strains of Drosophila melanogaster with different allozymes due to transcriptional or posttranscriptional control?

The alpha-amylase in Drosophila melanogaster is a highly polymorphic enzyme, at both the allozyme level and the specific activity level. This enzyme changes its specific activity drastically depending on both food conditions and developmental stages, and it has been suggested that the ability to change its activity depending on the source of food has positive correlation with fitness. But the cause of the difference of specific activity among strains and food compositions is not known. In order to investigate the cause of these differences, we measured both the specific activity of amylase and the relative amount of Amy mRNA in eight strains of D. melanogaster with different electromorphs, in two food environments and two developmental stages. We found the following. First, the food-dependent activity change is regulated at the transcription level. Second, there was a significant correlation between specific activity and mRNA level among strains. So 80 to 90% of the specific activity difference can be explained by differences in the level of mRNAs, but the remaining part cannot. Finally, there were significant differences in specific activity per mRNA both among strains and between developmental stages. This suggests that there are differences in the catalytic efficiency of each allozyme, strain- or stage-specific translation rate, enzyme stability, or differential use of two Amy loci.

Animals↗
PubMed2003

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens.

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4 degrees C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40 degrees C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50 degrees C.

Agrobacterium tumefaciens↗
PubMed2003

Continuous isomerization of D-fructose to D-mannose by immobilized Agrobacterium radiobacter cells.

D-Fructose was isomerized to D-mannose using immobilized Agrobacterium radiobacter that produces a thermostable mannose isomerase. The cells were immobilized by adsorption on chitosan or by glutaraldehyde crosslinking in the presence of albumin. Optimum conditions for mannose isomerase activity were 60 degrees C and pH 7.5. Continuous reaction at 55 degrees C was achieved with immobilized cells packed in a column to produce D-mannose.

Albumins↗
PubMed2003

A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium.

A new enzymatic route for biodiesel production from soybean oil was developed using methyl acetate as a novel acyl acceptor. Novozym 435 (immobilized Candida antarctica lipase) gave the highest methyl ester (ME) yield of 92%. The optimum conditions of the transesterification were 30% enzyme based on oil weight; a molar ratio of methyl acetate/oil of 12:1; temperature 40 degrees C and reaction time 10 h. Since no glycerol was produced in the process, this method is very convenient for recycling the catalyst and by-product triacetylglycerol showed no negative effect on the fuel property.

Acetates↗
PubMed2003

Investigations into the stabilization of drugs by sugar glasses: III. The influence of various high-pH buffers.

PURPOSE: To study the effect of the high-pH buffers ammediol, borax, CHES, TRIS, and Tricine on the glass transition temperature of the freeze concentrated fraction (Tg') of trehalose/buffer and inulin/buffer solutions at pH 6.0 and pH 9.8. Also, the glass transition temperature (Tg) of sugar glasses obtained after freeze drying of these solutions was elucidated. Additionally, the effect occurring during the freezing process on the pH of the various buffers was investigated. Furthermore, the stability of alkaline phosphatase (AP) incorporated in these sugar glasses prepared from solutions at pH 9.8 was evaluated. METHODS: The Tg' and Tg were measured using differential scanning calorimetry (DSC), and the change of pH during freezing was estimated by using an indicator solution added to the respective solutions. The enzymatic activity of AP after freeze drying and storage at 60 degrees C was evaluated by an enzymatic activity assay. RESULTS: It was found that the Tg' and Tg of the samples investigated are strongly influenced by the presence of the buffer. On freezing, only minor changes of the pH were observed. The samples with the lowest Tg and the samples containing buffers that formed complexes with the sugars showed the poorest stability of the AP. CONCLUSIONS: The stabilizing capacities of sugars that are currently recognized as excellent stabilizers for proteins during drying and storage can be completely lost if certain high-pH buffers such as ammediol, borax, and TRIS are used at high concentrations. Loss of stabilizing capacities can be ascribed to strong depression of the Tg' and Tg or to complex formation.

Alkaline Phosphatase↗
PubMed2004

A novel hydrophilic support, CoFoam, for enzyme immobilization.

Lipases from different sources (porcine pancreas, Mucor miehei and Candida antarctica B) were covalently immobilized on a hydrophilic polyurethane composite (CoFoam). Their hydrolytic activities assayed with tributyrin were 0.55, 2.1 and 447 U g(-1), respectively. The activity of the C. antarctica B composite in the synthesis of methyl oleate in hexane was 8.8 U g(-1) compared to 60.6 U g(-1) for commercial Novozyme 435. The advantage of the CoFoam composite lies in the low pressure drop in a packed-bed reactor at fairly large flow rates. For example, at flow rates of 10-12 l min(-1), the pressure drop over 15 cm is typically 3 kPa.

Animals↗
PubMed2004

Hydrolysis of rice bran oil using an immobilized lipase from Candida rugosa in isooctane.

The kinetics of enzymatic hydrolysis of rice bran oil in isooctane by immobilized Candida rugosa lipase in a batch reactor showed competitive inhibition by isooctane with a dissociation constant, K1, of 0.92 M. Continuous hydrolysis of rice bran oil was performed in recycling, packed bed reactor with 4352 U of immobilized lipase; the optimum recycle ratio was 9 and the operational half-life was 360 h without isooctane but 288 h with 25% (v/v) isooctane in rice bran oil.

Bioreactors↗