Higher Relative Activity Research Articles

Immobilization of lipase on hydrogels: Structural aspects of polymeric matrices as determinants of enzyme activity in different physical environments

AbstractWell‐defined and characterized polymeric matrices showing close chemical similarities but wide differences in water uptake and swellability in aqueous medium were used for lipase immobilization. Biphasic networks of 2‐hydroxypropylcellulose (HPC) were synthesized with acrylamide (AAm), methacrylamide (MAAm),N‐isopropylacrylamide (N‐i‐PAAm), and 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPSA) and simultaneously crosslinked withN,N‐methylene bisacrylamide in aqueous medium by using simultaneous γ‐radiation technique. Lipase enzyme was produced from a mesophilic bacterial isolate (HBK‐8) and was immobilized onto all the matrices by adsorption method. The activity of the immobilized enzyme was optimized for pH, temperature, and amount of crude enzyme and effect of dehydration. High relative activity for the immobilized enzymes was observed and loss of activity with time was minimal; reusability was found to be good. The activity of the immobilized enzyme was also observed to be good in both esterification and hydrolysis of esters. In the present study, lipase immobilization, hydrolysis ofp‐nitrophenyl palmitate, and optimum pH and temperature for substrate hydrolysis were evaluated for different matrices to study polymer structure and enzyme activity relationship in diverse physical environments. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3135–3143, 2004

Bacillus agaradhaerens LS-3C cyclodextrin glycosyltransferase: activity and stability features

Activity and stability characteristics of an alkaline active cyclodextrin glycosyltransferase (CGTase) enzyme from the alkaliphilic Bacillus agaradhaerens LS-3C strain are reported. The enzyme displays unusually high amylolytic activity in relation to the cyclization activity. Disproportionation activity of the CGTase was optimal with maltose as the acceptor substrate. The product of cyclization reaction was predominantly β-cyclodextrin (CD) along with α-CD as a minor product, however, the CDs profile was influenced by the reaction conditions. At pH 10, α-CD was replaced by γ-CD formation. Cyclization reaction and β-CD formation were significantly promoted in the presence of CaCl 2. The salt allowed the cyclization reaction to be performed at higher temperature. Increase in CDs production was also seen in the presence of 1 M sorbitol and 10% (w/v) PEG 3000, respectively. The enzyme stability was greatly enhanced with sorbitol, while alcohols had a highly negative effect on enzyme stability and hence on CDs production. β-CD and maltose brought about significant inhibition of the cyclization reaction.

Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria.

Ervatamia coronaria, a flowering plant (family Apocynaceae) indigenous to India, has medicinally important applications. A search for biochemical constituents of the latex of the plant yielded at least three thiol proteases with distinctly different properties. One of them, a highly active protease (ervatamin A), was purified to homogeneity by ion exchange and gel filtration chromatography. The enzyme exhibited high proteolytic activity toward natural substrates and amidolytic activity toward synthetic substrates. The pH and temperature optima for proteolytic activity were 8-8.5 and 50-55 degrees C, respectively. Proteolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. The estimated molecular mass of the enzyme was 27.6 kDa. The extinction coefficient (epsilon(1%) 280) of the enzyme was estimated as 21.9, and the protein molecule consists of 8 tryptophan, 11 tyrosine and 7 cysteine residues. Isoelectric point of the purified enzyme was 8.37. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and a typical color in ELISA. The N-terminal sequence of the enzyme showed conserved amino acid residues to other plant cysteine proteases. Ervatamin A shows high activity in relation to the other thiol proteases isolated from the same source.

NADPH-dependent metabolism of estrone by human liver microsomes.

We characterized the NADPH-dependent metabolism of estrone (E1) by liver microsomes of 21 male and 12 female human subjects. The structures of 11 hydroxylated or keto metabolites of E1 formed by human liver microsomes were identified by chromatographic and mass spectrometric analyses. 2-Hydroxylation of E1 was the dominant metabolic pathway with all human liver microsomes tested. E1 is more prone to form catechol estrogens (particularly 4-OH-E1) than 17beta-estradiol (E2) and the average ratio of E1 4-hydroxylation to 2-hydroxylation (0.24) was slightly higher than the ratio of E2 4- to 2-hydroxylation (0.20, P < 0.001). An unidentified monohydroxylated E1 metabolite (y-OH-E1) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6beta-OH-E1, 16alpha-OH-E1, and 16beta-OH-E1 were also formed in significant quantities. 16alpha-hydroxylation was not a major pathway for E1 metabolism. The overall profiles for the E1 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. Hepatic CYP3A4/5 activity in both male and female liver microsomes correlated strongly with the rates of formation of several hydroxyestrogen metabolites. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of human CYP3A4 or CYP3A5 with [3H]E1 and NADPH. Notably, human CYP3A5 has very high relative activity for E1 4-hydroxylation, exceeding its activity for E1 2-hydroxylation by approximately 100%. It will be of interest to determine the potential biological functions associated with any of the E1 metabolites identified in our present study.

Biological reprocessing of mixed office waste (mow) using modified cellulase by production of functional copolymer

Copolymer containing functional groups such as polyethylene oxide (PEO) and maleic anhydride (MA) was synthesized to modify the cellulase. MA was attached to PEO allyl ester, which was the product of the reaction between PEO allyl alcohol and lauric acid. The number of ethylene oxide (EO) units in one PEO chain was varied from 10 to 40. MA groups of the copolymer formed the chemical bond with the amino acid groups of the cellulase in the modification reaction. When cellulase was modified with synthesized copolymer, modified enzyme showed high relative activity regardless of high degree of modification compared with other modification methods. In mixed office waste reprocessing, modified cellulase improved many physical properties of the paper including freeness, optical properties, and strengths compared to the conventional process. Even though native cellulase improved the physical properties, modified cellulase showed an increased tensile strength and internal bond over those of unmodified cellulase. From these results, modified cellulase method is an effective biological treatment that would save pulp resources in the reprocessing.

G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues.

A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system. The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE. The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degrees C instead of 42.5 degrees C). Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose [4-MU-(GlcNAc)3] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest that the active region of A. caviae ChiA is located in the region before G561 of the protein molecule.

The involvement of glutamatergic transmission in the mechanism of movement disorders induced by reversive rotation of white mice.

The ability of the selective non-competitive NMDA receptor blocker MK-801 and a series of new glutamate antagonists --the adamantane derivatives IEM-1754 and IEM-1857 and phencyclidine (IEM-1925)--to prevent movement disorders induced by reversive rotation in mice was studied. l.p. MK-801 at a dose of 0.15 ml and IEM-1754 at a dose of 5.0 mg/kg prevented the development of akinesia in response to reversive rotation as effectively as scopolamine, a known agent which provides effective prophylaxis for movement diseases. IEM-1857, the quaternary analog of IEM-1754, was not effective. IEM-1925 significantly increased the responses of mice to reversive rotation, possibly because of its high activity in relation to other subtypes of glutamate receptors. These data provide evidence for the involvement of glutamatergic transmission in the mechanism of movement disorders of vestibular origin.

Behavior of glucose oxidase immobilized on ultrafiltration membranes obtained by copolymerizing acrylonitrile and N-vinylimidazol

Ultrafiltration membranes were prepared from a copolymer of acrylonitrile and N-vinylimidazol containing 25.4 mol% N-vinylimidazol. The water permeability and membrane selectivity were measured. The asymmetric structure of the membranes was proved by SEM. The membranes obtained were used as carriers for immobilization of glucose oxidase (GOD). The bound glucose oxidase was found to have high relative activity (91%). The temperature and pH optima, as well as thermal stability of the bound GOD were studied and compared with these for free enzyme. The kinetic parameters of the enzyme reaction — Michaelis constant ( k m) and maximum reaction rate ( V max) were investigated. The results obtained show that ultrafiltration membranes prepared from copolymer of acrylonitrile and N-vinylimidazol are suitable for use as carriers for immobilization of glucose oxidase.

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

DEVELOPMENT OF A BIOSENSOR WITH IMMOBILIZED L-AMINO ACID OXIDASE FOR DETERMINATION OF L-AMINO ACIDS

The objectives of this research were to investigate conditions for immobilization of L-amino acid oxidase (LAAO) on a preactivated nylon membrane, and to develop an enzyme sensor which can detect ammonia. LAAO was immobilized onto a preactivated nylon membrane. Optimal immobilization conditions were 0.3% glutaraldehyde, 1 mg bovine serum albumin, and 1.6 units of enzyme with 2 h coupling time. The enzymic membrane and an ammonium selective nonactin membrane were attached to an ammonia electrode to fabricate an L-amino acid sensor. L-amino acids were enzymatically degraded by immobilized LAAO, and the ammonia generated was measured by a potentiometric electrode at pH 8.5 and 45C. The sensor showed high relative activities for L-amino acids, and the responses of the sensor for phenylalanine and isoleucine were linear to 10 mM with a detection limit of 0.05 mM. The L-amino acid sensor was applied for monitoring increases in amino acids levels, expressed as L-isoleucine equivalents, during yeast autolysis. Determination of L-amino acids was complete in 3 min. Moreover, the activity of the enzymic membrane was stable for at least 260 assays, and did not noticeably decline for 2 months.

Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction—based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastasess = 54.5, lymph node metastasess = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Myocardial sestamibi uptake in healthy subjects is related to age, gender and habitus.

The object of this study was to evaluate the effect of gender, age and anthropometric data on regional isotope uptake in myocardial perfusion scintigraphy using 99mTc-MIBI-SPECT (myocardial sestamibi single-photon emission technique). Seventy-one healthy, non-smoking subjects, 42 men and 29 women between 40 and 80 years of age, with less than 5% likelihood of having coronary artery disease were studied. All subjects underwent a maximal exercise and rest MIBI-SPECT using a 2-day protocol and a 180 degrees anterior circular rotation. No correction for scatter or attenuation was made. Normalized regional activity was different in men and women in the anterior and inferior regions, with higher values anteriorly in men and inferiorly in women. Regional activity also differed with age in both women and men with higher relative activity in the anterior regions in the oldest age groups. Higher activities were seen in the basal parts of the left ventricle at rest compared with stress in both men and women. Regional isotope uptake was significantly affected by habitus expressed as body mass index (BMI) and thoracic circumference. Different protocols for stress and rest seem to be needed for men and women in different age groups, and for stress and rest when performing semiquantitative MIBI-SPECT and comparing data with a normal file of healthy subjects. Furthermore, such anthropometric data as BMI and/or thoracic circumference should be considered in order to minimize the risk for false-positive or false-negative scintigraphic results.

Ubiquitin-Dependent Pathway is Up-Regulated in Differentiating Lens Cells

The mammalian eye lens is composed of two distinct types of cells, epithelial cells and fiber cells. The fiber cells are generated throughout life via continuous differentiation of epithelial cells. Differentiation of lens cells involves dramatic changes in cellular components including altered activity of the ubiquitin dependent pathway. The concentration of high mass ubiquitin conjugates in the mitotically active-, differentiating-equatorial epithelial cells was 5–10 fold higher than that observed in mitotically quiescent, non-differentiated, central epithelial cells, even though there was a significant dilution of non-crystallin proteins due to an increase in level of crystallins in the differentiating cells. Similar observations were made when differentiation was modeled by exposure of lens epithelial explants to bFGF in culture. Activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) in the differentiating equatorial epithelial cells were also up to 100% higher than those noted in non-differentiated central epithelial cells and E1 appears to be rate controlling for ubiquitinylation. Consistent with the higher concentrations of high mass ubiquitin conjugates, there was a trend of enhanced ability to execute ATP-dependent protein degradation in the differentiating equatorial epithelial cells as compared with degradation in the non-differentiated central epithelial cells. These data indicate that the ubiquitin dependent pathway is up-regulated during differentiation of lens cells. In the differentiated fibers, the concentration of high mass ubiquitin conjugates and relative activities of E1 and E2s were 50% lower than in the non-differentiated central epithelial cells. In comparison, the concentration of the 110 kDa E1 was unchanged in differentiated fibers. However, if the factor of dilution by the significant increase in the level of crystallins was taken into account, the level or activities of the components of ubiquitin pathway in the differentiated cells was higher than the level noted in non-differentiated cells. These data indicate that, as compared with other non-crystallin proteins, there is differential stabilization and/or synthesis of the 110 kDa E1 and some other components of the ubiquitin dependent pathway in differentiated fibers.

A method of computationally enhanced detection of chromatogram regions with apparent high relative agonist activity

An occasional but difficult problem arises in drug discovery during a chromatographic analysis in which high background activity is associated with the presence of most eluting molecular species. This makes the isolation of material of high relative activity difficult. A computational method is shown that clarifies the identification of regions of the chromatogram of interest. The data for bioactivity and absorbance are normalized to percent of maximal response, filtered to raise very small or zero values to a minimal level, and the activity/absorbance ratio is plotted per fraction. The fractions with relatively high activity become evident. This technique is a helpful adjunct to existing graphical methods and provides an objective relationship between the data sets. It is simple to implement with Visual Basic and spreadsheet data, making it widely accessible.

Cloning and Characterization of Vitis viniferaUDP-Glucose:Flavonoid 3-O-Glucosyltransferase, a Homologue of the Enzyme Encoded by the Maize Bronze-1Locus That May Primarily Serve to Glucosylate Anthocyanidins in Vivo

We report here the cloning and optimized expression at 16 degrees C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1 locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereas Km values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases.

Effects of anticonvulsant agents and ethanol on the complexes of epileptic foci created by applications of bicuculline, strychnine, and penicillin in the rat brain cortex

Anticonvulsive effects of direct agonists of the GABA receptor system, medinal and phenazepam, as well as ethanol, on the complexes of epileptic foci created in the rat brain cortex by applications of various convulsant agents (bicuculline, strychnine, or penicillin) were studied. Ethanol, as well as phenazepam and medinal, were shown to possess high anti-epileptic activity in relation to the complexes. It was shown, using an i.v. infusion technique, that threshold doses of strychnine and bicuculline depend, in a linear and a nonlinear fashion, respectively, on the alcohol doses administered. A specific feature was found in the pharmacolgoical effect of ethanol: anticonvulsive activity of ethanol was pronounced within the postinjection interval from 5 min to 4 h, while within the following interval, from 4 to 24 h, the animals turned into a seizure-ready state. The subseizure phase was observed under the conditions when the penicillin complex was induced, and also following i.v. injections of strychnine and bicuculline.

Effect of elevated CO2 on rhizosphere carbon flow and soil microbial processes

Direct effects of increased above‐ground CO2 concentration on soil microbial processes are unlikely, due to the high pCO2 of the soil atmosphere in most terrestrial ecosystems. However, below‐ ground microbial processes are likely to be affected through altered plant inputs at elevated CO2. A major component of plant input is derived from litter fall and root turnover. Inputs also derive from rhizodeposition (loss of C‐compounds from active root systems) which may account for up to 40% of photoassimilate. This input fuels the activity of complex microbial communities around roots. These communities are centrally important not only to plant–microbe interactions and consequent effects on plant growth, but also, through their high relative activity and abundance, to microbially mediated processes in soil generally. This review focuses on approaches to measure C‐flow from roots, in particular, as affected by increased atmospheric CO2 concentration. The available evidence for impacts on microbial communities inhabiting this niche, which constitutes an interface for possible perturbations on terrestrial ecosystems through the influence of environmental change, will also be discussed. While methodologies for measuring effects of increased CO2 concentration on plant growth, physiology and C‐partitioning are abundant and widely reported, there is relatively little information on plant‐mediated effects on soil microbial communities and processes. Importantly, many studies have also neglected to recognize that any secondary effects on microbial communities may have profound effects on plant parameters measured in relation to environmental change. We critically review approaches which have been used to measure rhizodeposition under conditions of increased atmospheric CO2 concentration, and then consider evidence for changes in microbial communities and processes, and the methodologies which have been recently developed, and are appropriate to study such changes.

Nitrite accumulation in submerged biofilters - combined effects

The combined effect of temperature, pH and ammonium concentration over the nitrite accumulation phenomena in situations of free ammonia inhibition, their effect over the ammonia and nitrite oxidizer microorganiisms influence over the nitrification, have been studied in an Up-flow Biological Aerated Filter (UBAF). The free ammonia inhibition effect highly depends on the values of pH, temperature and ammonium concentration. For the same specific free ammonia concentration different values of temperature, pH and ammonium concentration bring about different nitrite accumulations. In conditions of no free ammonia inhibition and low values of temperature and pH, high ammonium concentrations bring about a higher relative activity of ammonia oxidizer microorganisms of the filter increases the nitratation efficiency in zones close to the outlet and will favour the nitrite accumulation in situations of free ammonia inhibition.

The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase.

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent. The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin. Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data. It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate. The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate. Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used. In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase. A reduction of both the acylation and deacylation reaction was consistent with the data.

Grafting of acrylonitrile copolymer membranes with hydrophilic monomers for immobilization of glucose oxidase

AbstractPoly(acrylonitrile‐methylmethacrylate‐sodium vinylsulphonate) membranes were subjected to modification by grafting of 2‐dimethylaminoethyl methacrylate (DMAEM) and 2‐acrylamido‐2‐methylpropanesulphonic acid (AMPSA). An Fe2+ H2O2 system was used to initiate the polymerization. Reaction time, temperature, pH, and concentration of an initiator and modifying agent were the variable parameters. Tertiary amino groups, quaternary ammonium groups, and sulfo groups were determined quantitatively by potentiometric titration. The degree of grafting and hydrophilicity and charge density of the modified membranes were studied. Glucose oxidase was immobilized onto modified acrylonitrile copolymer membranes with DMAEM and AMPSA and showed high relative activity. The amount of bound protein and storage of the activity of the immobilized enzyme were determined. © 1995 John Wiley &amp; Sons, Inc.