J.L. Gómez

A comparison of different methods of β-galactosidase immobilization

Abstract β-Galactosidase was immobilized in a range of supports showing suitable physico-chemical characteristics for use in fluidized bed reactors. Uncoated porous glass, alginate and κ-carrageenan beads and chromosorb-W were used as carriers. The intrinsic kinetic constants (V max and K M ) and coupling parameters for the immobilization were calculated. The highest immobilized protein percentages and activity yields were obtained when β-galactosidase was attached through its amine groups to aldehyde-glass. The final choice of derivative for use in fluidized bed reactors should be based not only on the enzymatic activity shown by the derivatives but also on the hydrodynamic behaviour of the supports.

Fluidized bed reactors operating with immobilized enzyme systems: Design model and its experimental verification

Abstract A general model for the design of fluidized bed reactors operating with immobilized enzymes in spherical porous particles is presented. It was solved for monosubstrate reactions following reversible Michaelis-Menten kinetics, as well as competitive inhibition by the product. The general model also allows the evaluation of irreversible and/or non-inhibited kinetics. To check the model, experimental conversions obtained in a fluidized bed reactor were compared to those predicted theoretically. The chemical process used for the validation of the model was the hydrolysis of o -nitrophenyl-β, d -galactopyranoside catalyzed by β-galactosidase covalently immobilized in Chromosorb-W. The model was tested in 160 different experimental conditions.

Frailty is a short-term prognostic marker in acute coronary syndrome of elderly patients:

Background: Frailty is a biological condition that reflects a state of decreased physiological reserve and vulnerability to stressors. The role of frailty in acute coronary syndrome patients has not been fully explored. Our study aims to assess the prevalence of frailty and its impact on in-hospital adverse outcomes of patients aged ⩾75 years admitted for acute coronary syndrome. Methods: This prospective, observational study included patients aged ⩾75 years admitted due to type 1 myocardial infarction in four tertiary hospitals. Frailty was assessed by the SHARE-FI index. The primary endpoint was the combination of in-hospital death or non-fatal myocardial (re)infarction. Secondary endpoints included the assessment of individual rates of (re)infarction, mortality, stroke, major bleeding and the combination of in-hospital death, (re)infarction and mortality. Results: A total of 202 patients were analysed. Frail patients (n=71, 35.1%) were older, more often women, had higher rates of comorbidities, and a higher risk profile according to GRACE, TIMI and CRUSADE scores at admission. The primary endpoint was significantly more frequent among frail patients (9.9% vs. 1.5%; P=0.006), as well as the combination of death, myocardial infarction and stroke (11.3% vs. 1.5%; P=0.002), driven mainly by a higher mortality rate (8.5% vs 0.8%; P=0.004). On multivariate analysis, frailty phenotype was an independent predictor of major adverse cardiac events (odds ratio 7.13; 95% confidence interval 1.43–35.42). Conclusions: Over one third of elderly patients with high-risk acute coronary syndrome are frail. Frailty phenotype is an important and independent prognostic marker in these patients.

Analysis of diffusion effects on immobilized enzymes on porous supports with reversible Michaelis-Menten kinetics

Abstract A mathematical analysis for the evaluation of the effectiveness factor and concentration profiles for immobilized enzymes into porous spherical particles is presented. It has been solved for monosubstrate reactions following reversible Michaelis-Menten kinetics. It also allows the evaluation of simple Michaelis-Menten and product competitive inhibition kinetics. The mathematical model for such systems was solved by numerical calculus. It showed that the diffusion and concentration moduli were the parameters that affected the effectiveness factor to a greater extent. The influence of the modulus associated with the reaction reversibility degree was negligible. The model is operative at high diffusion modulus values at which previous reported models were not.

Frailty predicts major bleeding within 30 days in elderly patients with Acute Coronary Syndrome

OBJECTIVE Bleeding in ACS patients is an independent marker of adverse outcomes. Its prognostic impact is even worse in elderly population. Current bleeding risk scores include chronological age but do not consider biologic vulnerability. No studies have assessed the effect of frailty on major bleeding. The aim of this study is to determine whether frailty status increases bleeding risk in patients with ACS. METHODS This prospective and observational study included patients aged ≥75years admitted due to type 1 myocardial infarction. Exclusion criteria were severe cognitive impairment, impossibility to measure handgrip strength, cardiogenic shock and limited life expectancy due to oncologic diseases. The primary endpoint was 30-day major bleeding defined as a decrease of ≥3g/dl of haemoglobin or need of transfusion. RESULTS A total of 190 patients were included. Frail patients (72, 37.9%) were older, with higher comorbidity features and with a higher CRUSADE score at admission. On univariate analysis, frailty predicted major bleeding during 30-day follow-up despite less frequent use of a P2Y12 inhibitor (66.2% vs 83.6%, p=0.007) and decreased catheterisation rate (69.4% vs 94.1%, p<0.001). Major bleeding was associated with increased all-cause mortality at day 30 (18.2% vs 2.5%, p<0.001). On multivariate analysis, frailty was an independent predictor for major bleeding. CONCLUSION Frailty phenotype, as a marker of biological vulnerability, is an independent predictor of major bleeding in elderly patients with ACS. Frailty can play an important role in bleeding risk stratification and objective indices should be integrated into routine initial evaluation of these patients.

Ultrafiltration membrane reactors for enzymatic resolution of amino acids: design model and optimization.

In this paper the possibility of continuous resolution of DL-phenylalanine, catalyzed by L-aminoacylase in a ultrafiltration membrane reactor (UFMR) is presented. A simple design model, based on previous kinetic studies, has been demonstrated to be capable of describing the behavior of the experimental system. The model has been used to determine the optimal experimental conditions to carry out the asymmetrical hydrolysis of N-acetyl-DL-phenylalanine.

Muscular dystrophy alters the processing of light acetylcholinesterase but not butyrylcholinesterase forms in liver of Lama2dy mice

In order to know whether the histopathological changes of liver, which accompany muscular dystrophy, affect the synthesis of cholinesterases, the distribution and glycosylation of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) forms in normal (NL) and dystrophic Lama2dy mouse liver (DL) were investigated. About half of liver AChE, and 25% of BuChE were released with a saline buffer (fraction S1), and the rest with a saline‐Brij 96 buffer (S2). Abundant light (G2A and G1A) AChE (87%) and BuChE (93%) forms, and a few G4H and G4A ChE species were identified in liver. The dystrophic syndrome had no effect on solubilization or composition of ChE forms. Most of the light AChE and BuChE species (>95%) were bound by octyl‐Sepharose, while most light AChE forms (80%), but not BuChE isoforms (15%), were retained in phenyl‐agarose. About half of the AChE dimers lost their amphiphilic anchor with phosphatidylinositol‐specific phospholipase C (PIPLC), and the fraction of PIPLC‐resistant species increased in DL. AChE T and R transcripts were detected by reverse transcriptase‐polymerase chain reaction (RT‐PCR) of liver RNA. ChE components of liver, erythrocyte, and plasma were distinguished by their amphiphilic properties and interaction with lectins. The dystrophic syndrome increased the liver content of the light AChE forms with Lens culinaris agglutinin (LCA) reactivity. The abundance of ChE tetramers in plasma and their small amount in liver suggest that after their assembly in liver they are rapidly secreted, while the light species remain associated to hepatic membranes. J. Neurosci. Res. 62:134–145, 2000.

Stabilization studies of L-aminoacylase-producing Pseudomonas sp. BA2 immobilized in calcium alginate gel

Abstract Pseudomonas sp. BA2 showing l -aminoacylase activity was immobilized by entrapment in calcium alginate gel. To enhance operational stability, the immobilized cells were treated with hardening reagents using four different methods: treatment of the cells with glutaraldehyde before immobilization, stabilization of the immobilized cell preparation with glutaraldehyde or glutaraldehyde and hexamethylenediamine, and finally, activation of the alginate solution with periodate followed by treatment with polyethyleneimine. Very active and stable immobilized cell preparations were obtained when Pseudomonas sp. BA2 cells immobilized in calcium alginate were treated with 20 m m glutaraldehyde. Compared with the untreated preparation, the enzymatic activity was markedly increased (132%) and the half-life period was increased by 4 h. Moreover, the suitability of the biocatalyst for reuse in batch processes was noticeably improved (only 60% of the initial l -aminoacylase activity was lost after four runs). When the immobilized and stabilized cell preparation was used for l -alanine production in a continuous stirred-tank reactor, substrate conversion at the reactor outlet was higher than that obtained with the untreated beads. The reactor production after 33 h was 61 of 17 m m l -alanine solution (9 g optically pure amino acid) more than double that obtained using the untreated immobilized cells, and significantly higher than those previously reported in the literature.

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.

Removal efficiency and toxicity reduction of 4-chlorophenol with physical, chemical and biochemical methods

Chlorophenols are well-known priority pollutants and many different treatments have been assessed to facilitate their removal from industrial wastewater. However, an absolute and optimum solution still has to be practically implemented in an industrial setting. In this work, a series of physical, chemical and biochemical treatments have been systematically tested for the removal of 4-chlorophenol, and their results have been compared in order to determine the most effective treatment based on removal efficiency and residual by-product formation. Chemical treatments based on advanced oxidation processes (AOP) produced the best results on rate and extent of pollutant removal. The non-chemical technologies showed advantages in terms of complete (in the case of adsorption) or easy (enzymatic treatments) removal of toxic treatment by-products. The AOP methods led to the production of different photoproducts depending on the chosen treatment. Toxic products remained in most cases following treatment, though the toxicity level is significantly reduced with combination treatments. Among the treatments, a photochemical method combining UV, produced with a KrCl excilamp, and hydrogen peroxide achieved total removal of chlorophenol and all by-products and is considered the best treatment for chlorophenol removal.