Enhanced Enzyme Reuse through the Bioconjugation of L-Asparaginase and Silica-Based Supported Ionic Liquid-like Phase Materials.

L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme's single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase-SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10-3 mg of ASNase per mg of SSILLP material, and 60 min). The ASNase-SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.

Superior operational stability of immobilized L-asparaginase over surface-modified carbon nanotubes.

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes the L-asparagine hydrolysis into L-aspartic acid and ammonia, being mainly applied in pharmaceutical and food industries. However, some disadvantages are associated with its free form, such as the ASNase short half-life, which may be overcome by enzyme immobilization. In this work, the immobilization of ASNase by adsorption over pristine and modified multi-walled carbon nanotubes (MWCNTs) was investigated, the latter corresponding to functionalized MWCNTs through a hydrothermal oxidation treatment. Different operating conditions, including pH, contact time and ASNase/MWCNT mass ratio, as well as the operational stability of the immobilized ASNase, were evaluated. For comparison purposes, data regarding the ASNase immobilization with pristine MWCNT was detailed. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA) and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95% under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5 × 10-3 g mL-1 of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), paving the way for its use in industrial processes.

Comparing Hospital and Primary Care Physicians' Attitudes and Knowledge Regarding Antibiotic Prescribing: A Survey within the Centre Region of Portugal.

BACKGROUND: Antibiotic resistance is a worldwide public health problem, leading to longer hospital stays, raising medical costs and mortality levels. As physicians' attitudes are key factors to antibiotic prescribing, this study sought to explore their differences between primary care and hospital settings. METHODS: A survey was conducted between September 2011 and February 2012 in the center region of Portugal in the form of a questionnaire to compare hospital (n = 154) and primary care (n = 421) physicians' attitudes and knowledge regarding antibiotic prescribing. RESULTS: More than 70% of the attitudes were statistically different (p < 0.05) between hospital physicians (HPs) and primary care physicians (PCPs). When compared to PCPs, HPs showed higher agreement with antibiotic resistances being a public health problem and ascribed more importance to microbiological tests and to the influence of prescription on the development of resistances. On the other hand, PCPs tended to agree more regarding the negative impact of self-medication with antibiotics dispensed without medical prescription and the need for rapid diagnostic tests. Seven out of nine sources of knowledge's usefulness were statistically different between both settings, with HPs considering most of the knowledge sources to be more useful than PCPs. CONCLUSIONS: Besides the efforts made to improve both antibiotic prescribing and use, there are differences in the opinions between physicians working in different settings that might impact the quality of antibiotic prescribing. In the future, these differences must be considered to develop more appropriate interventions.

Recent Strategies and Applications for l-Asparaginase Confinement.

l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.

Development and characterization of a novel l-asparaginase/MWCNT nanobioconjugate.

The enzyme l-asparaginase (ASNase) presents effective antineoplastic properties used for acute lymphoblastic leukemia treatment besides their potential use in the food sector to decrease the acrylamide formation. Considering their applications, the improvement of this enzyme's properties by efficient immobilization techniques is in high demand. Carbon nanotubes are promising enzyme immobilization supports, since these materials have increased surface area and effective capacity for enzyme loading. Accordingly, in this study, multi-walled carbon nanotubes (MWCNTs) were explored as novel supports for ASNase immobilization by a simple adsorption method. The effect of pH and contact time of immobilization, as well as the ASNase to nanoparticles mass ratio, were optimized according to the enzyme immobilization yield and relative recovered activity. The enzyme-MWCNTs bioconjugation was confirmed by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), Raman and transmission electron microscopy (TEM) studies. MWCNTs have a high ASNase loading capacity, with a maximum immobilization yield of 90%. The adsorbed ASNase retains 90% of the initial enzyme activity at the optimized conditions (pH 8.0, 60 min, and 1.5 × 10-3 g mL-1 of ASNase). According to these results, ASNase immobilized onto MWCNTs can find improved applications in several areas, namely biosensors, medicine and food industry.