Cross-linked esterase aggregates (CLEAs) using nanoparticles as immobilization matrix

Abstract

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100?mM glutaraldehyde for 4?h at 30?°C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30?°C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations.     

Rat plasma proteomics: effects of abundant protein depletion on proteomic analysis

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and alpha1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2-3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7-8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.     

In vivo stereological assessment of caudate volume in man: effect of normal aging

Using intermediate weighted magnetic resonance imaging (MRI) and a systematic sampling stereological method in 39 normal volunteers aged 24-79 years old, we demonstrated a marked age-associated decline in caudate nuclei volume (r = -0.69, p less than 0.0001). The mean absolute volume of the caudate nuclei in this study (9.4 cm3) was almost identical to that reported in a previous autopsy study and further confirms the validity of this stereological technique for use with MR images. This technique will provide a method for measuring the caudate and other nuclei in vivo, from brain images and, as such, a research tool to correlate age-associated changes in cognitive, sensory and motor function with caudate nucleus volume and other brain regions.     

Dissecting the Gene Dose-Effects of the APOE ε4 and ε2 Alleles on Hippocampal Volumes in Aging and Alzheimer’s Disease

Objective

To investigate whether there is a specific dose-dependent effect of the Apolipoprotein E (APOE) ε4 and ε2 alleles on hippocampal volume, across the cognitive spectrum, from normal aging to Alzheimer’s Disease (AD).

Materials and Methods

We analyzed MR and genetic data on 662 patients from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database–198 cognitively normal controls (CN), 321 mild-cognitive impairment (MCI) subjects, and 143 AD subjects–looking for dose-dependent effects of the ε4 and ε2 alleles on hippocampal volumes. Volumes were measured using a fully-automated algorithm applied to high resolution T1-weighted MR images. Statistical analysis consisted of a multivariate regression with repeated-measures model.

Results

There was a dose-dependent effect of the ε4 allele on hippocampal volume in AD (p = 0.04) and MCI (p = 0.02)–in both cases, each allele accounted for loss of >150 mm³ (approximately 4%) of hippocampal volume below the mean volume for AD and MCI subjects with no such alleles (Cohen’s d = −0.16 and −0.19 for AD and MCI, respectively). There was also a dose-dependent, main effect of the ε2 allele (p<0.0001), suggestive of a moderate protective effect on hippocampal volume–an approximately 20% per allele volume increase as compared to CN with no ε2 alleles (Cohen’s d = 0.23).

Conclusion

Though no effect of ε4 was seen in CN subjects, our findings confirm and extend prior data on the opposing effects of the APOE ε4 and ε2 alleles on hippocampal morphology across the spectrum of cognitive aging.     

Enhanced stability of immobilized keratinolytic protease on electrospun nanofibers

Abstract

Feathers from poultry industries are considered a major pollutant and its degradation is a challenging problem due to its recalcitrant nature. The high cost of energy and loss of essential amino acids by conventional methods have paved a way for an environmentally benign approach using microbial keratinolytic proteases. The widespread application of keratinolytic proteases is limited due to autolysis and denaturation of the enzyme upon storage. Immobilization overcomes these disadvantages by adsorbing the enzyme onto a solid support. Recently, electrospun nanofibers have been used due to their increased surface area and porous structure. The biocompatible and hydrophilic polyvinyl alcohol (PVA) has been blended with biodegradable chitosan for immobilization in electrospinning. The present study focuses on feather degradation by immobilized keratinolytic proteases on electrospun nanofibers. The keratinolytic protease production was enhanced by using a media containing hydrolyzed feather under optimized conditions. The immobilized keratinolytic protease on electrospun PVA chitosan (PVA-Ch) nanofibers (100–150?nm diameter) degraded the chicken feathers with 88% efficiency at the end of 72?hr.