Garcia-Carmona F

References (7)

Title : An improved method to measure lipase activity in aqueous media - Hernandez-Garcia_2017_Anal.Biochem_530_104
Author(s) : Hernandez-Garcia S , Garcia-Garcia MI , Garcia-Carmona F
Ref : Analytical Biochemistry , 530 :104 , 2017
Abstract : An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca+2 to be studied, while avoiding turbidity in the reaction medium.
ESTHER : Hernandez-Garcia_2017_Anal.Biochem_530_104
PubMedSearch : Hernandez-Garcia_2017_Anal.Biochem_530_104
PubMedID: 28502711

Title : The crystal structure of the cephalosporin deacetylating enzyme acetyl xylan esterase bound to paraoxon explains the low sensitivity of this serine hydrolase to organophosphate inactivation - Montoro-Garcia_2011_Biochem.J_436_321
Author(s) : Montoro-Garcia S , Gil-Ortiz F , Garcia-Carmona F , Polo LM , Rubio V , Sanchez-Ferrer A
Ref : Biochemical Journal , 436 :321 , 2011
Abstract : Organophosphorus insecticides and nerve agents irreversibly inhibit serine hydrolase superfamily enzymes. One enzyme of this superfamily, the industrially important (for beta-lactam antibiotic synthesis) AXE/CAH (acetyl xylan esterase/cephalosporin acetyl hydrolase) from the biotechnologically valuable organism Bacillus pumilus, exhibits low sensitivity to the organophosphate paraoxon (diethyl-p-nitrophenyl phosphate, also called paraoxon-ethyl), reflected in a high K(i) for it (~5 mM) and in a slow formation (t((1/2))~1 min) of the covalent adduct of the enzyme and for DEP (E-DEP, enzyme-diethyl phosphate, i.e. enzyme-paraoxon). The crystal structure of the E-DEP complex determined at 2.7 A resolution (1 A=0.1 nm) reveals strain in the active Ser(1)(8)(1)-bound organophosphate as a likely cause for the limited paraoxon sensitivity. The strain results from active-site-size limitation imposed by bulky conserved aromatic residues that may exclude as substrates esters having acyl groups larger than acetate. Interestingly, in the doughnut-like homohexamer of the enzyme, the six active sites are confined within a central chamber formed between two 60 degrees -staggered trimers. The exclusive access to this chamber through a hole around the three-fold axis possibly limits the size of the xylan natural substrates. The enzyme provides a rigid scaffold for catalysis, as reflected in the lack of movement associated with paraoxon adduct formation, as revealed by comparing this adduct structure with that also determined in the present study at 1.9 A resolution for the paraoxon-free enzyme.
ESTHER : Montoro-Garcia_2011_Biochem.J_436_321
PubMedSearch : Montoro-Garcia_2011_Biochem.J_436_321
PubMedID: 21382014
Gene_locus related to this paper: bacpu-AXE

Title : Characterization of a novel thermostable carboxylesterase from Geobacillus kaustophilus HTA426 shows the existence of a new carboxylesterase family - Montoro-Garcia_2009_J.Bacteriol_191_3076
Author(s) : Montoro-Garcia S , Martinez-Martinez I , Navarro-Fernandez J , Takami H , Garcia-Carmona F , Sanchez-Ferrer A
Ref : Journal of Bacteriology , 191 :3076 , 2009
Abstract : 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.
ESTHER : Montoro-Garcia_2009_J.Bacteriol_191_3076
PubMedSearch : Montoro-Garcia_2009_J.Bacteriol_191_3076
PubMedID: 19304850
Gene_locus related to this paper: geotn-a4isp0

Title : A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate - Martinez-Martinez_2007_Anal.Biochem_369_210
Author(s) : Martinez-Martinez I , Montoro-Garcia S , Lozada-Ramirez JD , Sanchez-Ferrer A , Garcia-Carmona F
Ref : Analytical Biochemistry , 369 :210 , 2007
Abstract : A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.
ESTHER : Martinez-Martinez_2007_Anal.Biochem_369_210
PubMedSearch : Martinez-Martinez_2007_Anal.Biochem_369_210
PubMedID: 17651681

Title : Candida rugosa lipase-catalyzed intramolecular O- to N- transacylation of butyryl propranolol in the presence of cyclodextrins - Avila-Gonzalez_2005_Biotechnol.Prog_21_338
Author(s) : Avila-Gonzalez R , Perez-Gilabert M , Lopez-Lopez MA , Garcia-Carmona F
Ref : Biotechnol Prog , 21 :338 , 2005
Abstract : In the present paper, a novel enzymatic reaction between (R,S)-O-butyryl propranolol (O-BP) and lipase from Candida rugosa in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is described. Under the used condition, lipase catalyzed the intramolecular transacylation of O-BP into N-butyryl propranolol (N-BP). Propranolol, the product of the expected hydrolysis reaction, was not detected in the reaction medium. A chiral analysis of the reaction product indicated that lipase showed a preference for (R)-O-butyryl propranolol since it first transformed the (R)-enantiomer and then the corresponding (S)-enantiomer. The influence of different reaction conditions on the initial rate is also studied.
ESTHER : Avila-Gonzalez_2005_Biotechnol.Prog_21_338
PubMedSearch : Avila-Gonzalez_2005_Biotechnol.Prog_21_338
PubMedID: 15801768

Title : Lipase-catalyzed preparation of S-propranolol in presence of hydroxypropyl beta-cyclodextrins - Avila-Gonzalez_2005_J.Biosci.Bioeng_100_423
Author(s) : Avila-Gonzalez R , Perez-Gilabert M , Garcia-Carmona F
Ref : J Biosci Bioeng , 100 :423 , 2005
Abstract : A simple method for the preparation of S-propranolol catalyzed by a Rhizopus niveus lipase in an aqueous medium is described. Hydroxypropyl-beta-cyclodextrin was used for the first time to increase the solubility of (R,S)-O-butyryl propranolol thus permitting the reaction to be carried out in water. The formation of an inclusion complex between (R,S)-O-butyryl propranolol and hydroxypropyl-beta-cyclodextrin was studied and a stoichiometry of 1:1 was determined. The influences of the hydroxypropyl-beta-cyclodextrin concentration, pH and percentage of ethanol on the enzymatic activity were also investigated. Under the conditions presented in this paper, values of ee(s) of 90% and E=48 were obtained.
ESTHER : Avila-Gonzalez_2005_J.Biosci.Bioeng_100_423
PubMedSearch : Avila-Gonzalez_2005_J.Biosci.Bioeng_100_423
PubMedID: 16310732

Title : Characterization and histochemical localization of nonspecific esterase from ascocarps of desert truffle (Terfezia claveryi Chatin) - Perez-Gilabert_2005_J.Agric.Food.Chem_53_5754
Author(s) : Perez-Gilabert M , Morte A , Avila-Gonzalez R , Garcia-Carmona F
Ref : Journal of Agricultural and Food Chemistry , 53 :5754 , 2005
Abstract : An esterase activity from Terfezia claveryi Chatin ascocarps, a mycorrhizal hypogeous fungus, is described for the first time. The enzyme was partially purified using phase partitioning in Triton X-114 (TX-114), achieving a reduction of 87% in the triglyceride content and the removal of 63% of phenols. The enzyme showed maximum activity toward short-chain p-nitrophenyl esters, and no interfacial activation was observed, indicating that the enzyme responsible for this activity is an esterase and not a lipase. This esterase presented its maximum activity at pH 7.4 and 60 degrees C. The values obtained for Km at pH 7.4 were 0.3 mM for p-nitrophenyl butyrate and 0.6 mM for p-nitrophenyl acetate with catalytic efficiencies (Vmax/Km) of 0.23 and 0.32, respectively. T. claveryi esterase was inhibited by phenylboric acid, indicating that serine residues were involved in the enzyme activity. This activity was localized only in the hypothecium and was absent from the peridium and gleba.
ESTHER : Perez-Gilabert_2005_J.Agric.Food.Chem_53_5754
PubMedSearch : Perez-Gilabert_2005_J.Agric.Food.Chem_53_5754
PubMedID: 15998144