Burkholderia Cepacia Lipase Research Articles

Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition state

Secondary alcohols having bulky substituents on both sides of the hydroxy group are inherently poor substrates for most lipases. In view of this weakness, we redesigned a Burkholderia cepacia lipase to create a variant with improved enzymatic characteristics. The I287F/I290A double mutant showed a high conversion and a high E value (>200) for a poor substrate for which the wild-type enzyme showed a low conversion and a low E value (5). This enhancement of catalytic activity and enantioselectivity of the variant resulted from the cooperative action of two mutations: Phe287 contributed to both enhancement of the (R)-enantiomer reactivity and suppression of the (S)-enantiomer reactivity, while Ala290 created a space to facilitate the acylation of the (R)-enantiomer. The kinetic constants indicated that the mutations effectively altered the transition state. Substrate mapping analysis strongly suggested that the CH/π interaction partly enhanced the (R)-enantiomer reactivity, the estimated energy of the CH/π interaction being -0.4 kcal mol(-1). The substrate scope of the I287F/I290A double mutant was broad. This biocatalyst was useful for the dynamic kinetic resolution of a variety of bulky secondary alcohols for which the wild-type enzyme shows little or no activity.

Resolution of 2-nitroalcohols by Burkholderia cepacia lipase-catalyzed enantioselective acylation

Racemic 2-nitro-1-phenylethanol was resolved by via enantioselective transesterification catalyzed by Burkholderia cepacia lipase. The reaction afforded excellent E values (E > 200) and enantioselectivity (up to >99% enantiomeric excesses [ee]) of both remaining substrates and acetylated product. Moreover, the lipase displayed high enantioselectivity in the resolution of additional 2-nitroalcohols (E up to >200). This method provides an efficient alternative for obtaining enantiopure 2-nitroalcohols.

Ionic‐Surfactant‐Coated Burkholderia cepacia Lipase as a Highly Active and Enantioselective Catalyst for the Dynamic Kinetic Resolution of Secondary Alcohols

The widerapplications of these DKR procedures in organic synthesis,however, can be hampered by the narrow specificity, moder-ate enantioselectivity, and low activity of the enzymeemployed. Therefore, it is of great importance to develop ahighly active enzyme having high enantioselectivity toward awide range of substrates for DKR. We herein report thationic-surfactant-coated Burkholderia cepacia lipase(ISCBCL) has great potential as such an excellent enzyme.Commercially available Burkholderia cepacia lipase(BCL; brand names: Lipase PS and Lipase PS-C) displayssignificantly lower activity than Candida antarctica lipase B(CALB; brand name: Novozym 435) (Table 1, entries 1–3);the latter has been most frequently employed in the metallo-enzymatic DKR. Fortunately, we have developed a practicalapproach for enhancing its activity dramatically. In a typicalprocedure, buffer-free soluble BCL (24% protein),

Ionic-Surfactant-Coated Burkholderia cepacia Lipase as a Highly Active and Enantioselective Catalyst for the Dynamic Kinetic Resolution of Secondary Alcohols

Aktiv durch Umhüllung: Das auf Burkholderia-cepacia-Lipase basierende Titelsystem ist hoch aktiv in der dynamischen kinetischen Racematspaltung (DKR). Seine wichtigsten Eigenschaften sind: die schnellste DKR von 1-Phenylethanol, die hoch enantioselektive DKR einer Vielzahl sekundärer Alkohole (RCH(OH)Ar) und das Schalten der Lipase-DKR-Enantioselektivität durch die Form der aliphatischen Kette (R). Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents

Biodiesel synthesis and conformation of Burkholderia cepacia lipase (BCL) were studied in 19 different room temperature ionic liquids (RTLLs) with a range of cation and anion structures. Overall, anion selection had a greater influence on biodiesel conversion than cation choice. RTILs containing Tf2N−and PF6- anions were suitable reaction media, while RTIL of [OmPy][BF4] was the best reaction medium with a biodiesel yield of 82.2±1.2%. RTILs with strong water miscible properties showed very low biodiesel yields. Conformational analysis by FT-IR revealed that higher biodiesel conversion in RTILs was correlated with a low tendency in α-helix content of BCL. An ultrasound-assisted biocatalysis process in RTILs was used to improve mass transfer rate, leading to 83% reduction of the reaction time for biodiesel production.

Solvent Dielectric Effect and Side Chain Mutation on the Structural Stability of Burkholderia cepacia Lipase Active Site: A Quantum Mechanical/Molecular Mechanics Study

Quantum mechanical and molecular dynamics methods were used to analyze the structure and stability of neutral and zwitterionic configurations of the extracted active site sequence from a Burkholderia cepacia lipase, histidyl-seryl-glutamin (His86-Ser87-Gln88) and its mutated form, histidyl-cysteyl-glutamin (His86-Cys87-Gln88) in vacuum and different solvents. The effects of solvent dielectric constant, explicit and implicit water molecules and side chain mutation on the structure and stability of this sequence in both neutral and zwitterionic forms are represented. The quantum mechanics computations represent that the relative stability of zwitterionic and neutral configurations depends on the solvent structure and its dielectric constant. Therefore, in vacuum and the considered non-polar solvents, the neutral form of the interested sequences is more stable than the zwitterionic form, while their zwitterionic form is more stable than the neutral form in the aqueous solution and the investigated polar solvents in most cases. However, on the potential energy surfaces calculated, there is a barrier to proton transfer from the positively charged ammonium group to the negatively charged carboxylat group or from the ammonium group to the adjacent carbonyl oxygen and or from side chain oxygen and sulfur to negatively charged carboxylat group. Molecular dynamics simulations (MD) were also performed by using periodic boundary conditions for the zwitterionic configuration of the hydrated molecules in a box of water molecules. The obtained results demonstrated that the presence of explicit water molecules provides the more compact structures of the studied molecules. These simulations also indicated that side chain mutation and replacement of sulfur with oxygen leads to reduction of molecular flexibility and packing.

Highly active lipase immobilized on biogenous iron oxide via an organic bridging group: the dramatic effect of the immobilization support on enzymatic function

Iron-oxidizing bacteria, Leptothrix ochracea, produce biogenous iron oxide (BIO), while the heat treatment of BIO gives its magnetized material (m-BIO). In this study, they were chemically modified with silane coupling agents to give organic–inorganic hybrid materials, which were then used as immobilization supports for enzymes. BIO-M, which was prepared by modifying BIO with 3-methacryloxypropyltrimethoxysilane, was found to be the best immobilization support. The immobilized lipases showed remarkably high catalytic activity; for example, Burkholderia cepacialipase (BCL) immobilized on BIO-M showed a turnover frequency of 33 000 h−1 for 1-phenylethanol (1a). m-BIO-M, which was prepared by modifying m-BIO with 3-methacryloxypropyltrimethoxysilane, was also used as an immobilization support. Although the lipases immobilized on m-BIO-M showed lower catalytic activity than those immobilized on BIO-M, the former could be separated and reused easily by using a magnet. These immobilized enzymes could be recycled at least five times and are environmentally benign biocatalysts.

Chemoenzymatic dynamic kinetic resolution as a key step in the enantioselective synthesis of (S)-salbutamol

The synthesis of (S)-salbutamol is described. By utilizing DKR in the enantiodetermining step, employing Burkholderia cepacia lipase (PS-IM), (S)-acetate ((S)-6) was obtained in excellent enantiomeric excess (98%). The subsequent transformations yielded the salt of (S)-salbutamol with retained stereochemistry.

Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil

BackgroundThe enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel.ResultsThe Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield.ConclusionsThe presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow-through bioreactor system.

Applying Biocatalysis to the Synthesis of Diastereomerically Enriched Cyanohydrin Mannosides

AbstractFully acetylated D‐ and L‐α‐mannosylacetaldehydes have been prepared and used as substrates to produce the corresponding cyanohydrins or cyanohydrin acetates with (2S) or (2R) configuration, respectively, at the cyanohydrin moiety. The (R)‐oxynitrilase‐catalysed synthesis and lipase‐catalysed diastereomeric kinetic and dynamic kinetic resolutions were investigated. Sequential catalysis with almond meal [an economic source of (R)‐oxynitrilase] and Burkholderia cepacia lipase was shown to be a straightforward method that yielded the four diastereomeric target cyanohydrins, the absolute configurations of which were confirmed by 1H NMR analysis.

Kinetic resolution of a drug precursor by Burkholderia cepacia lipase immobilized by different methodologies on superparamagnetic nanoparticles

Burkholderia cepacia lipase was immobilized on superparamagnetic nanoparticles using three different methodologies (adsorption, chemisorption with carboxibenzaldehyde and chemisorption with glutaraldehyde) and employed in the kinetic resolution of a chiral drug precursor, (RS)-2-bromo-1-(phenyl)ethanol, via enantioselective acetylation reaction. An excellent improvement of lipase catalytical performance was observed. Free B. cepacia lipase gave the ester (S)-2 with poor E-value 200. The effect of several reaction parameters in the kinetic resolution was studied. The best results for kinetic resolution were obtained using vinyl acetate as acetyl donor and toluene as solvent, typically yielding the ester in high enantiomeric excess (>99%) and E-value (E > 200). Of the three tested immobilization methods, chemisorption with glutaraldehyde was the best one in terms of temperature stability and yield product.

Metadynamics Simulations of Enantioselective Acylation Give Insights into the Catalytic Mechanism of Burkholderia cepacia Lipase

The catalytic mechanism of Burkholderia cepacia lipase (BCL), which catalyzes the enantioselective hydrolysis of racemic esters of primary alcohols, was investigated by modeling the first stage of ...

Enzymatic synthesis of carnosine derivatives catalysed by Burkholderia cepacia lipase

A new enzymatic synthesis of α,β-dipeptides has been developed with particular focus on the preparation of carnosine (β-alanyl-α-histidine) and analogues. The lipase PS-D from Burkholderia cepacia has been used as a catalyst for the formation of the peptide bond starting from a β-lactam and a protected α-amino acid.

Influence of temperature on the activity and enantioselectivity of Burkholderia cepacia lipase in the kinetic resolution of mandelic acid enantiomers

The effect of temperature on Burkholderia cepacia lipase (Amano PS) activity and enantioselectivity in the kinetic resolution of mandelic acid has been investigated. Experiments were carried out at temperatures between 25 and 60 °C. The Bi Bi Ping Pong model has been used to calculate kinetic parameters of the transesterification of mandelic acid by vinyl acetate. The rate of transesterification of R enantiomer was found to be substantially lower than that of S enantiomer. A decrease in the reaction rates was observed in temperature above 55 °C. The equilibrium model of enzyme deactivation was used to describe the dependence of mandelic acid transesterification rate on temperature. The enthalpy and entropy of the enzyme inactivation have been estimated. The enantioselectivity, expressed as enantiomeric ratio ( η E), was very high and decreased a little with the temperature increase. The transition state theory was used to determine the thermodynamic activation parameters. The ( S)-mandelic acid transesterification has been found to be enthalpically and entropically favoured. The differentiation of mandelic acid enantiomers in the reaction conditions is driven by the difference in activation enthalpy and entropy as well as by the energy of the substrate binding.

Lipase-catalyzed kinetic resolution of 1,2,3,4-tetrahydroisoquinoline-1-acetic acid esters

The enantiomers of 1,2,3,4-tetrahydroisoquinoline- and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-acetic acids were prepared via Burkholderia cepacia lipase (lipase PS-D)-catalyzed kinetic resolution of the corresponding ethyl and 2-methoxyethyl esters using enantioselective ( E > 200) hydrolysis in DIPE. The ( S)-acids were produced enzymatically, whereas the ( R)-acids were obtained via the chemical hydrolysis of the unreacted ( R)-esters. The solvent and its water content had major effects on both reactivity and enantioselectivity. The methoxyethyl moiety of the ester had a key role concerning the reactivity of the substrates.

Chemoenzymatic preparation of optically active anthracene derivatives

Syntheses and lipase-catalyzed resolutions of a trans-2-aminocyclopentanol rac- 2 and trans-cyclopentane-1,2-diamine rac- 3, bearers of an anthracene unit, have been effectively carried out. Burkholderia cepacia lipase catalyzed the transesterification of the β-amino alcohol with very high enantioselectivity ( E > 200). Lipase B from Candida antarctica showed moderate enantioselectivity in the acetylation of the diamine when 1-phenylethyl acetate was used as an acyl donor. In addition, the treatment of the optically active diamine (1 S,2 S)- 3 (ee = 95%) with pyridine-2,6-dicarbonyl dichloride yielded the bis(aminoamide) 6, which was tested as a chiral solvating agent (CSA) of carboxylic acids.

Chemoenzymatic method to enantiopure sulphur heterocyclic β-hydroxy nitriles

Sulphur heterocyclic β-hydroxy nitriles are prepared from the suitable aldehyde and (trimethylsilyl)acetonitrile in the presence of lithium acetate at good yields. Burkholderia cepacia lipase allowed the formation of the both enantiomers of β-hydroxy nitriles in highly enantiopure forms (ee 99%) through acylation of a racemic mixture and alcoholysis of the acylated enantiomer in tert-butyl methyl ether.

A Structure‐Controlled Investigation of Lipase Enantioselectivity by a Path‐Planning Approach

A novel approach based on efficient path-planning algorithms was applied to investigate the influence of substrate access on Burkholderia cepacia lipase enantioselectivity. The system studied was the transesterification of 2-substituted racemic acid derivatives catalysed by B. cepacia lipase. In silico data provided by this approach showed a fair qualitative agreement with experimental results, and hence the potential of this computational method for fast screening of racemates. In addition, a collision detector algorithm used during the pathway searches enabled the rapid identification of amino acid residues hindering the displacement of substrates along the deep, narrow active-site pocket of B. cepacia lipase and thus provided valuable information to guide the molecular engineering of lipase enantioselectivity.

Combined X-ray Diffraction and QM/MM Study of theBurkholderiacepaciaLipase-Catalyzed Secondary Alcohol Esterification

To understand the origin of high enantioselectivity of Burkholderia cepacia lipase (BCL) toward secondary alcohol, (R,S)-1-phenoxy-2-hydroxybutane (1), and its ester (E1), we determined the crystal structure of BCL complexed with phosphonate analogue of S-E1 and accomplished a series of MM, MC, and QM/MM studies. We have found that the inhibitor in the S configuration binds into the BCL active site in the same manner as the R isomer, with an important difference: while in case of the R-inhibitor the H-bond between its alcohol oxygen and catalytic His286 can be formed, in the case of the S-inhibitor this is not possible. Molecular modeling for both E1 enantiomers revealed orientations in which all hydrogen bonds characteristic of productive binding are formed. To check the possibility of chemical transformation, four different orientations of the substrate (two for each enantiomer) were chosen, and a series of ab initio QM/MM calculations were accomplished. Starting from the covalent complex, we modeled the ester (E1) hydrolysis and the alcohol (1) esterification. The calculations revealed that ester release is possible starting with all four covalent complexes. Alcohol release from the BCL-E1 complex in which the S-substrate is bound in the same manner as the S-inhibitor in the crystal structure however is not possible. These results show that the crystallographically determined binding modes should be taken with caution when modeling chemical reactions.

Optical Resolution of 2-Alkanol by Lipase-Catalyzed Acetylation with Vinyl Acetate in Packed-Bed Reactor with Recycling System

The lipase-catalyzed acetylation of 2-alkanol with vinyl acetate was studied using Burkholderia cepacia lipase (BCL), three alcohol and three organic solvents in a packed-bet reactor with a recycling system (flow method). The optical resolution data were found in agreement with those of the batch method in which BCL was suspended in the substrate solution. Repeated reaction results clearly indicated BCL in the packed-bed to be quite stable and to be usable for at least 50 reaction runs or to remain effective for as long as two months in the water-insoluble solvents such as hexane and 1,2-dichloroethane. In the reaction using a water-soluble solvent such as acetonitrile, the catalytic power of BCL showed only a 1% decrease of conversion per run or solvent recycling possibly owing to compression of BCL in the bed although enantioselectivity was independent of the number of reaction repetitions. The present method showed thus be applicable to kinetic resolution by enzyme-catalyzed acylation in hydrophobic organic solvents with no waste of enzyme.