The immobilization of adenine nucleotides on polysaccharides by using glutaraldehyde coupling and borohydride reduction.
ABSTRACT: Adenine nucleotides were immobilized on modified Sepharose 4B or Dextran T40 with glutaraldehyde and reduced with KBH4. Binding was dependent on pH and the nature of the amino group on the modified polysaccharide. ATP bound to soluble dextran retained coenzyme activity with glycerol kinase. Binding is proposed to occur via a Schiff base.
Project description:We demonstrate an enzyme stabilization approach whereby a model enzyme is PEGylated, followed by controlled chemical modification with glutaraldehyde. Using this stabilization strategy, size increases and aggregation due to intermolecular crosslinking are avoided. Immediately following synthesis, the PEGylated enzyme with and without glutaraldehyde modification possessed specific activities of 372.9 ± 20.68 U/mg and 373.9 ± 15.14 U/mg, respectively (vs. 317.7 ± 19.31 U/mg for the native enzyme). The glutaraldehyde-modified PEGylated enzyme retains 73% original activity after 4 weeks at 37 °C (vs. 2% retention for control).
Project description:Although glutaraldehyde is known to be bactericidal in solution, its potential use to create novel antibacterial polymers suitable for use in healthcare environments has not been evaluated. Here, novel materials were prepared in which glutaraldehyde was either incorporated into polyurethane using a simple "swell-encapsulation-shrink" method (hereafter referred to as "glutaraldehyde-impregnated polyurethane"), or simply applied to the polymer surface (hereafter referred to as "glutaraldehyde-coated polyurethane"). The antibacterial activity of glutaraldehyde-impregnated and glutaraldehyde-coated polyurethane samples was tested against Escherichia coli and Staphylococcus aureus. Glutaraldehyde-impregnated polyurethane resulted in a 99.9% reduction in the numbers of E. coli within 2 h and a similar reduction of S. aureus within 1 h, whereas only a minimal reduction in bacterial numbers was observed when the biocide was bound to the polymer surface. After 15 days, however, the bactericidal activity of the impregnated material was substantially reduced presumably due to polymerization of glutaraldehyde. Thus, although glutaraldehyde retains antibacterial activity when impregnated into polyurethane, activity is not maintained for extended periods of time. Future work should examine the potential of chemical modification of glutaraldehyde and/or polyurethane to improve the useful lifespan of this novel antibacterial polymer.
Project description:The objective of the current study was to understand the glutaraldehyde resistance mechanisms in P. fluorescens and P. aeruginosa biofilms. Glutaraldehyde is a common biocide used in various industries to control the microbial growth. Recent reports of emergence of glutaraldehyde resistance in several bacterial species motivated this study to understand the genetic factors responsible got glutaraldehyde resistance. Using a combination of phenotypic assays, chemical genetic assays and RNA-seq, we demonstrate that novel efflux pump, polyamine biosynthesis, lipid biosynthesis and phosphonate degradation play significant role in glutaraldehyde resistance and post-glutaraldehyde recovery of Psudomonad biofilms. Examination of P. fluorescens 72 h biofilm transcriptome was elucidated upon exposure to glutaraldehyde. The results were confirmed using qRT--PCR and chemical genetic appraoches in P. fluorescens and P. aeruginosa.
Project description:N6-(6-Aminohexylcarbamoylmethyl)-NAD+ was coupled to lactate dehydrogenase by using glutaraldehyde to form an active complex. The stability of this complex could be considerably improved by reduction with KBH4, although this treatment caused a partial decrease in specific activity. NAD+ was also coupled directly to the enzyme by this method. All of these complexes exhibited an intrinsic activity in the absence of exogenous NAD+.
Project description:Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc-l-alanine 4-nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50 °C, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60 °C, where the activities of immobilized and free enzyme became similar. The chemical amination of the immobilized enzyme or the treatment of the enzyme with glutaraldehyde did not produce any significant stabilization (a factor of 2) with high costs in terms of activity. However, the modification with glutaraldehyde of the previously aminated enzyme permitted to give a jump in Alcalase stability (e.g., with most than 80% of enzyme activity retention for the modified enzyme and less than 30% for the just immobilized enzyme in stress inactivation at pH 7 or 9). This preparation could be used in the hydrolysis of casein at pH 9 even at 67 °C, retaining around 50% of the activity after 5 hydrolytic cycles when the just immobilized preparation was almost inactive after 3 cycles. The modified enzyme can be reused in hydrolysis of casein at 45 °C and pH 9 for 6 cycles (6 h) without any decrease in enzyme activity.
Project description:The effect of glutaraldehyde stress on Desulfovibrio vulgaris Hildenborough (DvH) gene expression was determined by comparing the gene expression profiles of DvH with glutaraldehyde added at mid-log phase against DvH grown without glutaraldehdyde. Low concentration glutaraldehyde had little influence over instantaneous growth rate constant and final cell density of the culture. Changes in gene expression caused by exposure to glutaraldehyde were determined using full-genome DvH microarrays. A lot of gene expressions were altered by glutaraldehyde. Keywords: Glutaraldehyde Stress Overall design: For each condition 2 unique biological samples were hybridized to 4 arrays that each contained duplicate spots. Genomic DNA was used as universal reference.
Project description:A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms.
Project description:There is a significant need for fixed biological tissues with desired structural and material constituents for tissue engineering applications. Here, we introduce the lung ligament as a fixed biological material that may have clinical utility for tissue engineering. To characterize the lung tissue for potential clinical applications, we studied glutaraldehyde-treated porcine pulmonary ligament (n = 11) with multiphoton microscopy (MPM) and conducted biaxial planar experiments to characterize the mechanical property of the tissue. The MPM imaging revealed that there are generally two families of collagen fibers distributed in two distinct layers: The first family largely aligns along the longitudinal direction with a mean angle of ? = 10.7 ± 9.3 deg, while the second one exhibits a random distribution with a mean ? = 36.6 ± 27.4. Elastin fibers appear in some intermediate sublayers with a random orientation distribution with a mean ? = 39.6 ± 23 deg. Based on the microstructural observation, a microstructure-based constitutive law was proposed to model the elastic property of the tissue. The material parameters were identified by fitting the model to the biaxial stress-strain data of specimens, and good fitting quality was achieved. The parameter e0 (which denotes the strain beyond which the collagen can withstand tension) of glutaraldehyde-treated tissues demonstrated low variability implying a relatively consistent collagen undulation in different samples, while the stiffness parameters for elastin and collagen fibers showed relatively greater variability. The fixed tissues presented a smaller e0 than that of fresh specimen, confirming that glutaraldehyde crosslinking increases the mechanical strength of collagen-based biomaterials. The present study sheds light on the biomechanics of glutaraldehyde-treated porcine pulmonary ligament that may be a candidate for tissue engineering.
Project description:Riemerella anatipestifer is a gram-negative bacterium that causes disease in ducks and other birds. Despite being an important pathogen in poultry, the pathogenesis and drug resistance mechanisms of this bacterium are poorly understood. An analysis of our unpublished RNA-Seq data showed that lptD, a gene encoding one of the lipopolysaccharide transport components, is transcribed at higher levels in strain CH-1 than in strain ATCC11845. In addition, strain CH-1 has been shown to display broader drug resistance than strain ATCC11845. Since LptD is involved in LPS biogenesis and drug resistance, we wondered if lptD is associated with increased R. anatipestifer resistance to glutaraldehyde, a disinfectant used in the production industry. In this study, the minimal inhibitory concentration (MIC) of glutaraldehyde for strain CH-1 was determined to be 0.125% (vol/vol), whereas an MIC of 0.05% (vol/vol) was observed for strain ATCC11845. Furthermore, the level of lptD transcription in strain CH-1 was consistently 2-fold higher than that observed in strain ATCC11845. Moreover, lptD transcription was upregulated in both strains at a subinhibitory concentration of glutaraldehyde. The role of lptD in R. anatipestifer was further assessed by constructing an ATCC11845 mutant strain with low lptD expression, R. anatipestifer ATCC11845 lptD -. The growth of R. anatipestifer ATCC11845 lptD - was severely impaired, and this strain was more susceptible than the wild-type strain to glutaraldehyde. Moreover, compared to the wild-type strain, R. anatipestifer ATCC11845 lptD - exhibited decreased biofilm formation and was more sensitive to duck serum. Finally, low lptD expression led to decreased colonization in ducklings. These results suggest that LptD is involved in R. anatipestifer glutaraldehyde resistance and pathogenicity.