Substrate Report for: Ketoprofen-Ethyl-Ester

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 +/- 0.0) was improved in the mutants F72G (E = 1.9 +/- 0.2), L255W (E = 16.1 +/- 1.1), and F72G/L255W (E = 60.1 +/- 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 +/- 0.2 and E = 0.7 +/- 0.0): V138G (ee% = 35.9 +/- 1.0 and E = 3.0 +/- 0.1) and V138G/L200R (ee% = 89.2 +/- 0.2 and E = 19.5 +/- 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 A using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 A, beta = 97.1 degrees.

Esterases are important biocatalysts for chemical synthesis. Several bHSL family esterases have been used to prepare (S)-2-arylpropionic acids with stronger anti-inflammatory effects via kinetic resolution. Here, we presented the discovery of key residues that controlled the enantioselectivity of bHSL family esterases to ethyl 2-arylpropionates, through careful analysis of the structural information and molecular docking. A new bHSL family esterase, Est924, was identified as a promising catalyst for kinetic resolution of racemic ethyl 2-arylpropionates with slight (R)-stereopreference. Using Est924 as the starting enzyme, protein engineering was conducted at hotspots, and the substitution of A203 was proved to enhance the enantioselectivity. The stereopreference of the mutant M1 (A203W) was inverted to ethyl (S)-2-arylpropionates, and this stereopreference was further improved in variant M3 (I202F/A203W/G208F). In addition, the optimal variant, M3, was also suitable for the resolution of ibuprofen ethyl ester and ketoprofen ethyl ester, and their efficient (S)-isomers were synthesized. Next, the whole-cell catalyst harboring M3 was used to prepare (S)-ketoprofen. (S)-ketoprofen with 86%ee was produced by whole-cell catalyst with a single freeze-thaw cycle, and the cells could be reused for at least five cycles. Our results suggested that Est924 variants could kinetically resolve economically important racemates for industrial production and further offer the opportunity for the rational design of enzyme enantioselectivity. Moreover, it is an economical process to prepare optically pure (S)-ketoprofen and (S)-naproxen by using an engineered strain harboring M3 as the catalyst.

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 +/- 0.0) was improved in the mutants F72G (E = 1.9 +/- 0.2), L255W (E = 16.1 +/- 1.1), and F72G/L255W (E = 60.1 +/- 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 +/- 0.2 and E = 0.7 +/- 0.0): V138G (ee% = 35.9 +/- 1.0 and E = 3.0 +/- 0.1) and V138G/L200R (ee% = 89.2 +/- 0.2 and E = 19.5 +/- 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix alpha5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha5 and alpha10. The simulation also confirmed the function of helix alpha5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C(alpha) branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family.

Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 A using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 A, beta = 97.1 degrees.

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000-6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an eep (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% eep and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10 mM (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10 U ml(-1) and, at 30 degrees C and pH 7 over 48 h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.

A biotransformation process was developed for the production of (S)-ketoprofen by enantioselective hydrolysis of racemic ketoprofen ester using the mutant Trichosporon laibacchii strain CBS 5791. A satisfactory result was obtained, in which the E was 82.5, with an ee of 0.94 and a conversion of 0.47 under the optimum hydrolysis conditions [E is enantiomeric ratio, E=ln[1-X(1+ee)]/ln[1-X(1-ee)]; ee is enantiomeric excess, ee=(CS-CR)/(CS+CR): temperature of hydrolysis was 23 degrees C]. The medium used in biotransformation was a mixture of growth broth and biotransformation broth at a ratio of 1:9, the concentration of Tween 80 was 15 g/l, the time of hydrolysis, 72 h. These results are promising for further scale-up. Tween 80 significantly improved lipase enantioselectivity and activity at the optimum concentration.

The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially. With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail. Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus. The expression level of the enzyme was more than 20% of the total protein in E. coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step. As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (99% ee(p)). The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.

For the production of optically active ketoprofen, enzymatic resolution of racemic ketoprofen in an organic solvent has been accomplished via enantioselective esterification. Pharmacologically inactive (R)-ketoprofen is converted into the corresponding (R)-ester by this method. Enantioselectivity in lipase-catalyzed resolution of racemic ketoprofen was mainly dependent on the sources of lipase, alcohol moiety, organic solvent, and water content. Ethanol was used as the alkyl donor and the optimum water content required for highly efficient enzymatic resolution was determined to be 0.1-0.15% (v/v), which was maintained using salt hydrates such as Na2SO4 x 10H2O. (S)-Ketoprofen could be obtained with high enantioselectivity (E=15) in n-hexane supplemented with ethylene dichloride (20% (v/v)) using commercially available Candida antarctica lipase (Novozym 435).