Substrate Report for: IPG-butyrate

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of beta-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 degrees C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 A, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (\<= C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE).

AIMS: To identify microbial strains with esterase activity able to enantioselectively hydrolyse esters of (R,S)-1,2-O-isopropylidene glycerol. METHODS AND RESULTS: The microbial hydrolysis of various racemic esters of 1,2-O-isopropylidene glycerol (IPG) was attempted by screening among Streptomyces spp. previously selected on the basis of their carboxylesterase activity. The best results were observed in the hydrolysis of butyrate ester and two strains appeared promising as they showed opposite enantioselectivity: Streptomyces sp. 90852 gave predominantly (S)-IPG, while strain 90930 mostly gave the R-alcohol. Streptomyces sp. 90930 was identified as Streptomyces violaceusniger, whereas Streptomyces sp. 90852 is a new species belonging to the Streptomyces violaceus taxon. The carboxylesterase belonging to strain 90852 gave a maximum value of enantiomeric ratio (E) of 14-16. This strain was lyophilized and used as dry mycelium for catalysing the synthesis of isopropylidene glycerol butyrate in heptane showing reaction rate and enantioselectivity (E = 6.6) lower than what observed for the hydrolysis. CONCLUSIONS: A new esterase with enantioselective activity towards (R,S)-IPG butyrate has been selected. The best enantioselectivity is similar or even better than the highest reported value in the literature with commercial enzymes. The enzyme is produced by a new species belonging to the S. violaceus taxon. SIGNIFICANCE AND IMPACT OF THE STUDY: New esterases from streptomycetes can be employed for the enantioselective hydrolysis of chiral esters derived from primary alcohols, not efficiently resolved with commercial enzymes.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of beta-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 degrees C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 A, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (\<= C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE).

The Escherichia coli esterase YbfF displays high activity towards 1,2-O-isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R-enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2-fold higher enantioselectivity than wild-type YbfF, with an E=38 for IPG butyrate and an E=77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.

Previously studied Bacillus subtilis carboxylesterases (CesA and CesB) have potential for the kinetic resolution of racemic esters of 1,2-O-isopropylideneglycerol (IPG). CesA exhibits high activity but low enantioselectivity towards IPG-butyrate and IPG-caprylate, while the more enantioselective CesB does not process IPG-butyrate and exhibits several-fold lower activity than CesA towards IPG-caprylate. A sequence and structure comparison allowed us to identify active site residues that may cause the difference in (enantio)selectivities of CesA and CesB towards these IPG esters. This structure-based approach led to the identification of two active site residues in CesA (F166 and F182), as promising candidates for mutagenesis in order to enhance its enantioselectivity. Mutagenesis of positions 166 and 182 in CesA yielded novel variants with enhanced enantioselectivity and without significant loss of catalytic activity. For IPG-butyrate, a CesA double mutant F166V/F182C (ER=13) was generated showing a approximately 13-fold increased enantioselectivity as compared to wild-type CesA (E=1). For IPG-caprylate, we designed a CesA double mutant F166V/F182Y (ER=9) displaying a approximately 5-fold increased enantioselectivity as compared to the wild-type enzyme (ER=2). These findings, combined with the results of molecular docking experiments, demonstrate the importance of residues at positions 166 and 182 for the enantioselectivity of CesA, and may contribute to the development of efficient biocatalysts.

Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E = 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E >/= 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.

Enzymatic hydrolysis of racemic mixtures may provide an attractive method for the enantiopure production of chiral pharmaceuticals. For example, the carboxylesterase NP of Bacillus subtilis Thai I-8 is an excellent biocatalyst in the kinetic resolution of NSAID esters, such as naproxen and ibuprofen methyl esters. Two homologues of this enzyme were identified when the genome sequence of B. subtilis 168 was revealed in 1997. We characterised one of the homologous, YbfK, as a very enantioselective 1,2-O-isopropylidene-sn-glycerol caprylate esterase, while only modest enantioselectivity towards the naproxen ester was observed. The other homologue, the carboxylesterase NA has not been characterised yet. The purpose of the present study was to fully characterise these three highly homologous esterases with respect to their applicability towards the enantiospecific hydrolysis of a wide range of compounds. The esterase genes were cloned and expressed in B. subtilis using a combination of two strong promotors in a multi-copy vector. After purification of the enzymes from the cytoplasm of B. subtilis, the biochemical and enantioselective properties of the enzymes were determined. Although all carboxylesterases have similar physico-chemical properties, comparison of their specific activities and enantioselectivities towards several compounds revealed rather different substrate specificities. We conclude that carboxylesterase NP and carboxylesterase NA are particularly suited for the enzymatic conversion of naproxen esters, while YbfK offers enantiopure (+)-IPG from its caprylate ester. Given the carboxylesterase activities of the esterases it has been proposed to rename the nap gene of B. subtilis 168 into cesA and the ybfK gene into cesB.

AIMS: To identify microbial strains with esterase activity able to enantioselectively hydrolyse esters of (R,S)-1,2-O-isopropylidene glycerol. METHODS AND RESULTS: The microbial hydrolysis of various racemic esters of 1,2-O-isopropylidene glycerol (IPG) was attempted by screening among Streptomyces spp. previously selected on the basis of their carboxylesterase activity. The best results were observed in the hydrolysis of butyrate ester and two strains appeared promising as they showed opposite enantioselectivity: Streptomyces sp. 90852 gave predominantly (S)-IPG, while strain 90930 mostly gave the R-alcohol. Streptomyces sp. 90930 was identified as Streptomyces violaceusniger, whereas Streptomyces sp. 90852 is a new species belonging to the Streptomyces violaceus taxon. The carboxylesterase belonging to strain 90852 gave a maximum value of enantiomeric ratio (E) of 14-16. This strain was lyophilized and used as dry mycelium for catalysing the synthesis of isopropylidene glycerol butyrate in heptane showing reaction rate and enantioselectivity (E = 6.6) lower than what observed for the hydrolysis. CONCLUSIONS: A new esterase with enantioselective activity towards (R,S)-IPG butyrate has been selected. The best enantioselectivity is similar or even better than the highest reported value in the literature with commercial enzymes. The enzyme is produced by a new species belonging to the S. violaceus taxon. SIGNIFICANCE AND IMPACT OF THE STUDY: New esterases from streptomycetes can be employed for the enantioselective hydrolysis of chiral esters derived from primary alcohols, not efficiently resolved with commercial enzymes.

The enantioselective hydrolysis of different (RS)-1,2-O-isopropylidene glycerol esters has been achieved with whole cells of Bacillus coagulans NCIMB 9365 furnishing the (S)-alcohol as the major enantiomer. The reaction is catalysed by a thermostable cell-bound carboxylesterase and improvement of the enantioselectivity has been achieved by heat treatment of the whole cells, which causes the knock-outs a non-enantioselective competing enzyme. Thermally-treated cells hydrolysed (RS)-1,2-O-isopropylidene glycerol esters with high enantioselectivity, the highest enantiomeric ratio (80100) being observed for the benzoate. The biocatalyst displayed good stability and could be re-used after filtration for 12 cycles before showing significant loss of activity; repeated biotransformation batches allowed the recovery of 9.55 g/L of enantiomerically pure (S)-isopropylideneglycerol benzoate starting from 24.0 g/L of the racemic mixture.

The hydrolysis of (RS)-isopropylideneglycerol acetate with whole cells of the yeast Kluyveromyces marxianus is reported. The biotransformation furnished (R)-isopropylideneglycerol as major enantiomer with good enantioselectivity (E=28) under optimised conditions. The reaction can be performed in an ultrafiltration-membrane reactor allowing for the obtainment of 19.2 g/L of enantiomerically pure (R)-isopropylideneglycerol acetate starting from 60 g/L of racemic mixture.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.