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PubMedDate not supplied

Cefmetazole (CS-1170), a "new" cephamycin with a decade of clinical experience.

In vitro and in vivo study results were reviewed from cefmetazole, a "new" parenteral cephamycin. Cefmetazole's spectrum of activity was comparable to that of second-generation cephalosporins, which includes clinical coverage of many Enterobacteriaceae, Staphylococcus spp., streptococci, Haemophilus spp., pathogenic Neisseria, Branhamella catarrhalis, and anaerobic bacteria. Cefmetazole was generally more potent (two- to eightfold) than cefoxitin against organisms within their spectrums and was particularly active for staphylococci (MIC90, 2.0 micrograms/ml). Methicillin-resistant S. aureus strains were more susceptible to cefmetazole alone or in combination (fosfomycin) than any other cephamycin. Cefmetazole has demonstrated excellent stability to aerobic and anaerobic organism-produced beta-lactamases. It also inhibits Type I cephalosporinases and, uniquely, some other cephalosporinases produced by the Bacteroides. This superior stability, enzyme interaction, and better penetration into bacterial cells results in a sustained bactericidal effect and a capacity for more infrequent dosing. The cefmetazole serum elimination half-life was 1.5 hr, also justifying use at greater than or equal to 8-hr intervals. Clinical trials in the United States and Japan demonstrated an acceptably high cefmetazole infection cure rate (88% to 100%), especially in direct comparative studies with cefoxitin. Cefmetazole was also proven very effective in minimizing infectious wound morbidity (prophylaxis) using 2 g single- or multidose regimens. Adverse drug reactions were usually minor; in the Japanese surveillance trial (118,318 patients) the rate was only 2.2% (8.8% in United States). Cefmetazole has been extensively and safely used in Japan since 1980.

Bacteria↗
PubMed1992

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.

Animals↗
PubMed1992

Monitoring glutamine in mammalian cell cultures using an amperometric biosensor.

An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.

Amino Acid Oxidoreductases↗
PubMed1991

Alteration of enzyme specificity and catalysis by protein engineering.

New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds. The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase. Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.

Amino Acid Sequence↗
PubMed1995

Engineering thermostability: lessons from thermophilic proteins.

As several groups begin to tap the rich pickings found in the Archaea--a vast kingdom that stretches the concept of life as we know it--the structures of proteins from hyperthermophiles are being elucidated. Certain features are beginning to emerge, such as compactness and hydrophobic clustering, but the ability to engineer these features into temperature-intolerant proteins is still some way off.

Amino Acids↗
PubMed1994

Synthetic polymers as solubilizing vehicles for enzymes in water-poor media.

A recent method for exposing enzymes to organic solvents is reviewed. By complex formation between the enzyme and polymers that per se are soluble in organic solvents it is possible to disperse the enzyme in the organic medium in such a way that an optically transparent (in the visible region) solution is obtained. After reaction, the separation of the enzyme from the organic medium can be obtained simply by addition of water. The enzyme can be recovered from the water phase. Physicochemical studies have revealed that the enzyme is more stable in the complex-bound form.

Cellulose↗
PubMed1990

Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent.

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins.

Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using crude mouse liver homogenates, four distinct glutathione S-transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The "digital" images generated resemble Western blots without the use of antibodies or signal amplification techniques.

Amino Acid Sequence↗
PubMed2003

A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii.

A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii. These two genes were cloned and the corresponding expressed proteins were characterized. The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity. The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178). The protein from PH1426 was a typical, homodimeric flavoprotein. These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P. horikoshii. The redox protein and thioredoxin reductase retained their full activities for over 1h at 100 degrees C. The redox potential of the redox protein was similar to that of thioredoxin from E. coli and lower than that of glutathione. Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence.

Amino Acid Sequence↗
PubMed2005

Effects of extremely low frequency electromagnetic fields on membrane-associated enzymes.

The effects of extremely low frequency electromagnetic fields of 75 Hz were studied on different membrane-associated enzymes. Only the activities of three enzymes out of seven exposed to the field decreased approximately of about 54-61% with field amplitudes above a threshold of 73-151 microT depending on the enzyme. The same field had no effect on the activities of either integral membrane enzymes such as Ca,ATPase, Na/K,ATPase, and succinic dehydrogenase or peripheral membrane enzymes such as photoreceptor PDE. The decrease in enzymatic activity of the field-sensitive enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton, no effect of the field was obtained on the enzymatic activity, suggesting the crucial role of the membrane in determining the conditions for enzyme inactivation. The role of the particular linkage of the field-sensitive enzymes to the membranes is also discussed.

Cell Membrane↗
PubMed2006

A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency.

A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/Km value (Vmax/Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 degrees C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.

Adsorption↗
PubMed2004

Redox-triggered events in cytochrome c nitrite reductase.

Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.

Coated Materials, Biocompatible↗
PubMed2005

Directed evolution of biocatalytic processes.

The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.

Animals↗
PubMed2006

Bioaffinity-based an inexpensive and high yield procedure for the immobilization of turnip (Brassica rapa) peroxidase.

This study demonstrates the immobilization of carbohydrate containing turnip peroxidase on an inexpensive bioaffinity adsorbent, Concanavalin A-cellulose support. The bioaffinity support was prepared simply by incubating cellulose powder with jack bean extract at 4 degrees C. Cellulose powder adsorbed 30 mg concanavalin A/g of the matrix. Concanavalin A adsorbed cellulose has been employed for the simultaneous purification and immobilization of glycoenzymes directly from ammonium sulphate fractionated proteins of turnip. The obtained bioaffinity support was quite effective in high yield immobilization of peroxidase from turnip and it retained 672 U/g. Turnip peroxidase immobilized on concanavalin A-cellulose support retained 80% of the initial activity. Immobilized turnip peroxidase preparation was quite resistant against the denaturation mediated by pH, heat, urea, guanidinium-HCl, Surf Excel, cetyltrimethylammonium bromide and water-miscible organic solvents; dimethyl formamide, dioxane and n-propanol. Low concentration of detergents like Surf Excel and cetyltrimethylammonium bromide enhanced the activity of soluble and immobilized turnip peroxidase.

Brassica rapa↗
PubMed2005

Highly active, lipase silicone elastomers.

Lipase Candida rugosa was entrapped in silicone rubber via condensation-cure room temperature vulcanization of silanol-terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, to give a highly active silicone-enzyme elastomer. The effect on enzyme activity of addition of water and hydrophilic polymeric moieties based on poly(ethylene oxide) 2 was examined, as were crosslinker concentration, enzyme concentration, and elastomer thickness. It was demonstrated that lipase is most active in silicone elastomers and more active in silicone oils than simple hydrocarbons. Crosslink density in these elastomers was not an important factor in the reactivity of the rubber. However, the addition of hydrophilic species prior to elastomer formation decreased the efficiency both of the dispersion of the enzyme and the resulting activity of the elastomer. This effect could be moderated by prior exposure of the lipase to silicone oil. Thus, hydrophobic silicones play a protective/activating role for lipase.

Biocompatible Materials↗
PubMed2006

Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor.

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12h at 50 degrees C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.

Acetates↗
PubMed2004

Urea biosensor based on PANi(urease)-Nafion/Au composite electrode.

The polyaniline (PANi)-Nafion composite film was prepared onto the ceramic plate by the cyclic voltammetry (CV) method with the various cycle numbers. When the PANi-Nafion/Au/ceramic plate with the preparing cycle number of 5 was as working electrode, the cathodic peak current was achieved as 84.0 microA in 60 mg dl(-1) NH4Cl buffer solution. On the other hand, the small cathodic peak currents for buffer solution in the presence of 60 mg dl(-1) LiOH, NaCl and KCl, respectively, were found with the same composite electrode as working electrode. The cathodic peak current decreased from 84.0 to 16.3 microA in the 60 mg dl(-1) NH4Cl buffer solution when the cycle number for preparing PANi-Nafion/Au/ceramic plate composite electrode with the CV method increased from 5 to 15. The enzyme of urease was immobilized onto the PANi-Nafion/Au/ceramic plate composite film by the electrochemical immobilization and the casting methods and used as sensing electrode to detect the concentration of urea in the buffer solution. The sensitivity of composite electrode immobilized with the casting method was greater than that of electrochemical immobilization method. The sensitivity and the detecting limit of the urea sensor were found to be 0.7 and 5.27 microA (mg dl(-1))(-1)cm(-2), as well as 6 and 0.3 mg dl(-1), respectively, when urease was immobilized by glutaraldehyde (GA) cross-linker and Nafion network, respectively.

Biosensing Techniques↗