Burkholderia cepacia lipase: A versatile catalyst in synthesis reactions

The lipase from Burkholderia cepacia, formerly known as Pseudomonas cepacia lipase, is a commercial enzyme in both soluble and immobilized forms widely recognized for its thermal resistance and tolerance to a large number of solvents and short‐chain alcohols. The main applications of this lipase are in transesterification reactions and in the synthesis of drugs (because of the properties mentioned above). This review intends to show the features of this enzyme and some of the most relevant aspects of its use in different synthesis reactions. Also, different immobilization techniques together with the effect of various compounds on lipase activity are presented. This lipase shows important advantages over other lipases, especially in reaction media including solvents or reactions involving short‐chain alcohols.     

Spectroscopic investigation of lipase from Pseudomonas cepacia solubilized in 1,4‐dioxane by non‐covalent complexation with methoxypoly(ethylene glycol)

Lipase from Pseudomonas cepacia was made soluble in 1,4‐dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme–PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far‐ and near‐UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near‐UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase–PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme–substrate interactions. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 624–629, 1999.     

Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so‐called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position‐restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Poly(ethylene glycol)-lipase complex that is catalytically active for alcoholysis reactions in ionic liquids

Lipase-catalyzed alcoholysis was investigated in three different ionic liquids. Lyophilized native lipase had a low activity in all the ionic liquids but a poly(ethylene glycol) (PEG)-lipase complex (with a molar ratio of the polymer/enzyme of 10:1) had an increased activity of over 14-fold. Of several lipases tested, PEG-lipase PS (from Pseudomonas cepacia) exhibited the highest activity (1.07 mmol/(h g^–1 protein)) in 1-octyl-3-methylimidazolium hexafluorophosphate.     

Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase),Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20–50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20–68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media

Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl-propionylamino)-pentanedioic acid didodecyl ester (d- and l-BIG2C₁₂CA); d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (d- and l-2C₁₂GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19–48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the l-BIG2C₁₂CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68–78 fold.     

Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support

The purified lipase from Pseudomonas cepacia was used as free and immobilized enzyme preparation for hydrolysis of p-nitrophenyl palmitate (pNPP) and p-nitrophenyl acetate (pNPA) in organic media. The free enzyme was mixed with bovine serum albumin and lyophilized. Immobilization was on porous polypropylene. Conditions where diffusional limitations of the substrate were not limiting the reaction rate were defined. The specific activity of the lipase was greatly enhanced upon immobilization: 16.5- and 7.8-fold for pNPP and pNPA respectively. Both the free and immobilized lipases followed Michaelis–Menten kinetics in organic solvent despite the heterogeneity (solid/liquid) of the reaction mixture. For pNPP, the activation factor upon immobilization came mainly from a reduction in K _m, app while k _cat was increased for pNPA. Received: 30 January 1997 / Accepted: 14 February 1997     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Acidic lipase Lip I.3 from a Pseudomonas fluorescens‐like strain displays unusual properties and shows activity on secondary alcohols

Aims

Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR‐611.

Methods and Results

The corresponding gene was identified and isolated by PCR‐amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0‐1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C‐terminal type I secretion signal and several putative Ca²⁺ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca²⁺ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols.

Conclusions

A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts.

Significance and Impact of the Study

The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.     

Chemoenzymatic Route for the Synthesis of (S)‐Moprolol,a Potential β‐Blocker

A biocatalytic route for the synthesis of a potential β‐blocker, (S)‐moprolol is reported here. Enantiopure synthesis of moprolol is mainly dependent on the chiral intermediate, 3‐(2‐methoxyphenoxy)‐propane‐1,2‐diol. Various commercial lipases were screened for the enantioselective resolution of (RS)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol to produce the desired enantiomer. Among them, Aspergillus niger lipase (ANL) was selected on the basis of both stereo‐ and regioselectivity. The optimized values of various reaction parameters were determined such as enzyme (15 mg/mL), substrate concentration (10 mM), organic solvent (toluene), reaction temperature (30 °C), and time (18 h).The optimized conditions led to achieving >49% yield with high enantiomeric excess of (S)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol. The lipase‐mediated catalysis showed regioselective acylation with dual stereoselectivity. Further, the enantiopure intermediate was used for the synthesis of (S)‐moprolol, which afforded the desired β‐blocker. Chirality 28:313–318, 2016. © 2016 Wiley Periodicals, Inc.     

Chemical modification of lipases with various hydrophobic groups improves their enantioselectivity in hydrolytic reactions

Semi-purified lipases from Candida rugosa, Pseudomonas cepacia and Alcaligenes sp. were chemically modified with a wide range of hydrophobic groups such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl, lauroyl and acetyl moieties. The Candida rugosa lipase MY modified with the benzyloxycarbonyl group (modification ratio = 84%) brought about a 15-fold increase in enantioselectivity (E value) towards the hydrolysis of racemic butyl 2-(4-ethylphenoxy)propionate in an aqueous buffer solution, although the enzymatic activity was decreased. The origin of the enantioselectivity enhancement by chemical modification of the lipase is attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer.     

Production of biodiesel by Burkholderia cepacia lipase as a function of process parameters

Despite the already established route of chemically catalyzed transesterification reaction in biodiesel production, due to some of its shortcomings, biocatalysts such as lipases present a vital alternative. Namely, it was noticed that one of the key shortcomings for the optimization of the enzyme catalyzed biodiesel synthesis process is the information on the lipase activity in the reaction mixture. In addition to making optimization difficult, it also makes it impossible to compare the results of the independent research. This article shows how lipase intended for use in biodiesel synthesis can be easily and accurately characterized and what is the enzyme concentration that enables achievement of the desired level of fatty acid methyl esters (FAME) in the final product mixture. Therefore, this study investigated the effect of two different activity loads of Burkholderia cepacia lipase on the biodiesel synthesis varying the pH and temperature optimal for lipase activity. The optimal lipase pH and temperature were determined by two different enzyme assays: spectrophotometric and titrimetric. The B. cepacia lipase pH optimum differentiated between assays, while the lipase optimally hydrolyzed substrates at 50°C. The analysis of FAME during 24 hr of biodiesel synthesis, at two different enzyme concentrations, pH 7, 8, and 10, and using two different buffers, revealed that the transesterification reaction at optimal pH, 1 hr reaction time and lipase activity load of 250 U per gram of reaction mixture was sufficient to produce more than 99% FAME.     

Lipase-Catalyzed Resolution of Isopropylidene Glycerol: Effect of Co-Solvents on Enantioselectivity

The resolution of 1,2-O-isopropylidene glycerol via enzyme catalyzed hydrolysis of the corresponding benzoic ester was investigated. Using lipase PS from Pseudomonas cepacia, we determined the influence of organic co-solvents on the activity and enantioselectivity of the enzyme. The performance of the lipase was correlated to the nature (logP, ?,μ and the percentage of the organic media. The highest enzymatic activity was found in solvents completely miscible or completely immiscible in water. The enzyme stereoselectivity was inversely related to the logP of the solvent.     

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.     

Lipase-catalyzed regioselective protection/deprotection of hydroxyl groups of the isoflavone irilone isolated from Iris germanica

The regioselective acylation of irilone, isolated from Iris germanica, with vinylacetate and propenylacetate and deacylation of irilone diacetate with n-butanol were studied using lipases from Aspergillus niger, Mucor miehei, Pseudomonas cepacia, Candida cylindracea, porcine pancreas and Candida antarctica. Significant conversion of irilone to 4′-O-acetylirilone was achieved using P. cepacia lipase, while irilone diacetate was converted to 5-O-acetylirilone by the enzymatic action of lipases from M. miechei, P. cepacia and porcine pancreas under different experimental conditions. This preferential protection/deprotection furnishes an opportunity to modify the structure of irilone by selective derivatization that may help to change its biological activities by modifying its amphiphilic/lipophilic balance.     

Proteolytic Activity in Commercial Triacylglycerol Hydrolase Preparations

The proteolytic activity of 34 commercial lipase preparations (CLP) was determined using a labeled casein substrate. Only three CLP were free from proteolytic activity. Porcine pancreatic lipases exhibited levels of proteolytic activity comparable to or greater than that of a reference porcine trypsin. Bacterial lipases contained up to 10% of the proteolytic activity of commercial trypsin. Proteolytic activities in lipases from fungal species were present at low levels (<1% of the activity in trypsin). Among preparations of fungal origin, lipases from Aspergillus niger and Mucor javanicus were highest in proteolytic activity; Aspergillus oryzae and Pseudomonas cepacia lipases were lowest. Proteins in CLP were separated by non-denaturing PAGE; between 4 and 17 protein bands in the range &#104 6.5- &#83 200 kDa were observed. With the exception of a single pair of Rhizomucor miehei lipases, the distribution of apparent molecular weights (AMW) was unique to each preparation. Bands of caseinolytic activity in commercial lipases were visualized by applying a zymographic technique. CLP contained between 0 (P. cepacia lipases) and 6 (porcine pancreas lipase and Rhizopus oryzae lipase) discrete proteolytic bands. Common themes of proteolytic AMW emerged, including 21-23 kDa and 30-35 kDa bands.     

Synthetic activity enhancement of membrane-bound lipase from <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> by pretreatment with isooctane

The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase™ firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4°C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.     

Pseudomonas cepacia lipase: Esterification reactions in AOT microemulsion systems

Summary The activity of purifiedPseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis(2ethylhexyl)sulfosuccinate sodium salt (AOT). The optima pH, T and water content (w_o) for the enzyme activity in this type of microemulsions have been determined. Studies on the effect of various fatty acids and alcohols on the enzyme specificity have shown a preference of this lipase for palmitic and caprylic acid as well as for propanol, while reactions involving cyclic alcohols can not be catalyzed at all. The differences on the behavior of this lipase as compared to other lipases studied in microemulsion systems as well as in other systems are discussed.     

Mono- and disaccharides enhance the activity and enantioselectivity of Burkholderia cepacia lipase in organic solvent but do not significantly affect its conformation

Sucrose, trehalose, and mannitol were colyophilized with lipase from Burkholderia cepacia and their effects on the activity and enantioselectitivity of the enzyme evaluated using as model reactions the transesterification between n-octanol or 6-methyl-5-hepten-2-ol with vinyl acetate. The lipase co-lyophilized with sugars showed an activity which was up to 4.7-fold higher (at a sugar/lipase ratio >or= 20) than that observed without sugar. Analogously, lipase enantioselectivity, expressed as the enantiomeric ratio, increased up to 2.8-fold in the presence of sugars. The conformation of the lipase was investigated by means of Fourier transform infrared spectroscopy (FT/IR) in water and as lyophilized powder. The infrared spectra of lyophilized lipase in the presence and, even more so, in the absence of sugars were different from that of the enzyme in water. In particular, the band at around 1,654/cm, typically assigned to alpha-helix, was less intense in the lyophilized samples. Nevertheless, the enzyme in the presence of sugars showed a decrease of the bands at 1,614-1,620/cm and at 1,680-1,695/cm that indicates a lower content of intermolecular beta-sheets (typical of protein aggregates). Additionally the increase of the component at 1,546/cm in the amide II region is consistent with a hydrogen bond pattern of the enzyme more similar to that shown in water. These results suggest that although sugars are not able to fully preserve the native secondary structure, they might contribute to reduce the conformational changes caused by protein/protein interactions. These factors in combinations with others (e.g., ability to reduce deleterious interactions between the enzyme and inert supports) make sugars (both mono- and disaccharides) an interesting class of additives for improving the performance of biocatalysts in organic solvents.     

Thermal stability and conformational structure of salmon calcitonin in the solid and liquid states

Salmon calcitonin (sCT) was selected as a model protein drug for investigating its intrinsic thermal stability and conformational structure in the solid and liquid states by using a Fourier transform infrared (FT‐IR) microspectroscopy with or without utilizing thermal analyzer. The spectral correlation coefficient (r) analysis between two second‐derivative IR spectra was applied to quantitatively estimate the structural similarity of sCT in the solid state before and after different treatments. The thermal FT‐IR microspectroscopic data clearly evidenced that sCT in the solid state was not effected by temperature and had a thermal reversible property during heating–cooling process. Moreover, the high r value of 0.973 or 0.988 also evidenced the structural similarity of solid‐state sCT samples before and after treatments. However, sCT in H₂O exhibited protein instability and thermal irreversibility after incubation at 40°C. The temperature‐induced conformational changes of sCT in H₂O was occurred to transform the α‐helix/random coil structures to β‐sheet structure and also resulted in the formation of intramolecular and intermolecular β‐sheet structures. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 200–207, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com