Álvaro Sánchez-Ferrer

Phase Separation of Biomolecules in Polyoxyethylene Glycol Nonionic Detergents

The advantage of aqueous two-phase systems based on polyoxyethylene detergents over other liquid-liquid two-phase systems lies in their capacity to fractionate membrane proteins simply by heating the solution over a biocompatible range of temperatures (20 to 37 degrees C). This permits the peripheral membrane proteins to be effectively separated from the integral membrane proteins, which remain in the detergent-rich phase due to the interaction of their hydrophobic domains with detergent micelles. Since the first reports of this special characteristic of polyoxyethylene glycol detergents in 1981, numerous reports have consolidated this procedure as a fundamental technique in membrane biochemistry and molecular biology. As examples of their use in these two fields, this review summarizes the studies carried out on the topology, diversity, and anomalous behavior of transmembrane proteins on the distribution of glycosyl-phosphatidylinositol-anchored membrane proteins, and on a mechanism to describe the pH-induced translocation of viruses, bacterial endotoxins, and soluble cytoplasmic proteins related to membrane fusion. In addition, the phase separation capacity of these polyoxyethylene glycol detergents has been used to develop quick fractionation methods with high recoveries, on both a micro- and macroscale, and to speed up or increase the efficiency of bioanalytical assays.

Partial purification of a thylakoid-bound enzyme using temperature-induced phase partitioning.

Triton X-114 was used to partially purify broad bean polyphenol oxidase, a thylakoid membrane-bound enzyme, in latent form, free of phenolic compounds and chlorophylls, with a high recovery rate. The activation of the latent enzyme by detergents or trypsin was 10 times higher than that obtained when the enzyme was purified by other methods used in plant biochemistry, such as acetone powders and ammonium sulfate fractionation. The kinetic parameters of the latent and activated enzyme are also given.

Improved cross-linked enzyme aggregates for the production of desacetyl β-lactam antibiotics intermediates

Cross-linked enzyme aggregates (CLEAs) are reported for the first time for a recombinant acetyl xylan esterase (AXE) from Bacillus pumilus. With this enzyme, CLEAs production was most effective using 3.2M (80%-saturation) ammonium sulfate, followed by cross-linking for 3h with 1% (v/v) glutaraldehyde. Particle size was a key determinant of the CLEAs activity. The usual method for generating particles, by short-time vortexing was highly inefficient in terms of enzyme activity yields. In contrast, the use of long-time vortexing increased activity recovery, and a novel approach consisting in the utilization of a commercial mechanical cell disruptor which is based on a reciprocating movement recovered all the enzyme activity in few seconds. In the CLEAs thus produced, the enzyme was much more resistant to shear, heat and extreme pH values than the soluble enzyme. The CLEAs were highly effective in transforming fully 7-amino cephalosporanic acid and cephalosporin C into their corresponding desacetyl derivatives, which are important advanced intermediates in the production of semisynthetic beta-lactam antibiotics. The operational stability of such CLEAs was remarkable, with a half life of 45 cycles. Therefore, the new methodology used here should decrease the industrial cost of the CLEAs, both in terms of biocatalyst production and reusability.

Triton X-114 phase partitioning in plant protein purification

Abstract A brief overview is given of how Triton X-114 can be used not only to solubilize plant membranes but also as an excellent reagent in a bulk fractionation method to purify enzymes compared with the classical drastic methods using acetone powder or ammonium sulphate fractionation. Triton X-114 removes the tenacious phenols and chlorophylls on centrifugation. There is no need to use insoluble synthetic resins or organic solvents as Triton X-114 is so mild that the enzymes can be extracted in their natural form without activating them. The methods developed with Triton X-114 are easily reproducible and sufficiently cheap to be used in large-scale purification procedures. The classical topological use of Triton X-114 in plant membranes is also discussed.

Characterization of a Novel Thermostable Carboxylesterase from Geobacillus kaustophilus HTA426 Shows the Existence of a New Carboxylesterase Family

The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65 degrees C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new alpha/beta hydrolase family different from IV and VI.

Triton ×-114 as a tool for purifying spinach polyphenol oxidase

Abstract Spinach polyphenol oxidase was partially purified by using phase partitioning in a solution of Triton X-114 in a latent form, free of phenolics and chlorophylls, with a high recovery rate. The enzyme had both catecholase and cresolase activity. The latter, never before described, presented a lag period which was affected by enzyme and substrate concentration. The K m for p -cresol was 2 mM. Substrate inhibition was seen for catecholase activity when 4-methylcatechol was used. The values found for V max , K m and K si were 244 μM/min, 4.2 mM and 104 mM, respectively. The latent enzyme was activated by trypsin (4.5-fold), by SDS and 1-decanesulphonic acid at detergent concentration of 1 mM, but it was strongly inhibited by CTAB, frost, and SDS at a detergent concentration of 10 mM. Non-ionic detergents did not alter significantly the activity of latent enzyme.

Molecular Characterization of a Novel N-Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

ABSTRACT N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-d-mannosamine (ManNAc). In nature, N-acetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how an N-acetylneuraminate lyase was cloned from the human gut commensal Lactobacillus plantarum WCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter−1 culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70°C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed.

Biocatalysis in reverse self-assembling structures : reverse micelles and reverse vesicles

The use of two reverse self-assembling systems, such as reverse micelles and reverse vesicles, to model the enzymatic function of biological membranes is discussed. They permit direct measurement of enzyme kinetics since these ternary systems form optically transparent solutions. The physicochemical characteristics of both systems are differentiated since they clearly affect enzyme behavior. The four enzymatic profiles that have been described in reverse micelles as a function of micelle size (omega 0) and the kinetic models developed to explain them are discussed. Reverse vesicles, first described in 1991, are also presented as a new system that shares properties with reverse micelles and liposomes, and in which enzymes show unexpected behavior. Finally, the potential use of these systems in protein extraction, hydrophobic protein stabilization, and biotechnology are noted, although a better physicochemical characterization is needed in order to explore their full potential.

Triton X-114-aided purification of latent tyrosinase.

Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre- and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.

Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins

Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4), an IFNγ-inducible GTPase protein harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Despite the presence of the CARD domain, Gbp4 requires the universal inflammasome adaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4 is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophils are recruited to the infection site through the inflammasome-independent production of the chemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearance through the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our results point to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis and bacterial clearance by neutrophils and suggest that transient activation of the inflammasome may be used to treat bacterial infections.