Burkholderia Cepacia Lipase Research Articles

Immobilized Burkholderia cepacia lipase and its application in selective enrichment of EPA by hydrolysis of fish oil

Eicosapentaenoic acid (EPA), an orally active Omega-3 long-chain polyunsaturated fatty acid, has attracted much attention for its medical and healthcare value. Due to a large amount of polyunsaturated fatty acids (PUFA) including docosahexanoic acid (DHA) in fish oil, the highly specific enrichment of EPA from fish oil is difficult to achieve. In our study, we conducted immobilization studies on Burkholderia cepacia lipase (BCL) and its application in the selective enrichment of EPA in fish oil. BCL was immobilized on resin LX201A by covalent crosslinking, and immobilization conditions including glutaraldehyde concentration, pH, time were optimized. The optimal immobilization conditions were 0.5% glutaraldehyde, 4 h, and pH 8.0. The loading rate of BCL reached to 93.5%, and its activity was up to 68263 U/g under the optimal immobilization conditions. After hydrolysis reaction of fish oil for 15h catalyzed by immobilized BCL, EPA content showed a more increase by 33.2%, while a slight increase by 0.78% in DHA content. Moreover, after recycling use for 10 times, the catalytic efficiency of immobilized BCL remained at 93.0%. These indicated that immobilized BCL possessed good selectivity, reusability and stability, providing great application potential in EPA selective enrichment in fish oil industry.

Eggshell waste transformation to calcium chloride anhydride as food-grade additive and eggshell membranes as enzyme immobilization carrier

Abstract In continuation of our efforts to fully utilize eggshell waste (ESW), here we report the possibility of ESW transformation to calcium chloride (CaCl2) anhydride of food-grade additive purity and eggshell membranes (ESMs) as potential enzyme immobilization carriers. ESW chemical transformation by 5% (w/v) hydrochloric acid to CaCl2 solution and ESM completely devoid of the remnants of ESW calcium carbonate was performed in the constructed 15 L batch reactor during 4 h at room temperature, followed by separation of ESM from CaCl2 solution by filtration. ESW-derived CaCl2 solution containing the excess hydrochloric acid was neutralized by adding calcium hydroxide, concentrated to approximately 1/8th of volume, and spray dried. Separated ESM was washed with water and acetone, dried, and ground to a size of less than 0.5 mm. The ESW transformation process produced 102.42 ± 3.31 g of CaCl2 anhydrous and 2.48 ± 0.28 g of ESM per 100 g of ESW dry matter. ESW-derived CaCl2 fulfilled all criteria for food-grade additive, while obtained ESM showed their suitability for Burkholderia cepacia lipase immobilization by adsorption.

Novel Hybrid Gel-Fiber Membranes as Carriers for Lipase Catalysis Based on Electrospinning and Gelation Technology.

An excellent oil-water interface is one of the prerequisites for effective lipase catalysis. Therefore, this study aimed to improve lipase activity in terms of catalytic interface optimization. A novel approach for constructing oil-water interfaces was proposed. The structural similarity and the hydrophilic differences between polyvinyl pyrrolidone gel-fiber membranes (GFMs) and poly(lauryl methacrylate) (PLMA) organogel inspired us to hybridize the two to form PVP/PLMA hybrid gel-fiber membranes (HGFMs) based on electrospinning and gelation. The prepared PVP/PLMA-HGFMs were capable of being adopted as novel carriers for lipase catalysis due to their ability to swell both in the aqueous phase (swelling ratio = 187.5%) and the organic phase (swelling ratio = 40.5%). Additionally, Confocal laser scanning microscopy (CLSM) results showed that abundant network pores inside the carriers enabled numerous effective microscopic oil-water interfaces. The catalytic activity of Burkholderia cepacia lipase (BCL) in PVP/PLMA-HGFMs ranged between 1.21 and 8.70 times that of the control ("oil-up/water-down" system) under different experimental conditions. Meanwhile, PVP/PLMA-HGFMs increased lipase activity by about eight times at -20 °C and had good application characteristics at extreme pH conditions.

The Conformational Transitions and Dynamics of Burkholderia cepacia Lipase Regulated by Water-Oil Interfaces.

Structural dynamics and conformational transitions are crucial for the activities of enzymes. As one of the most widely used industrial biocatalysts, lipase could be activated by the water-oil interfaces. The interface activations were believed to be dominated by the close-to-open transitions of the lid subdomains. However, the detailed mechanism and the roles of structure transitions are still under debate. In this study, the dynamic structures and conformational transitions of Burkholderia cepacia lipase (LipA) were investigated by combining all-atom molecular dynamics simulations, enhanced sampling simulation, and spectrophotometric assay experiments. The conformational transitions between the lid-open and lid-closed states of LipA in aqueous solution are directly observed by the computational simulation methods. The interactions between the hydrophobic residues on the two lid-subdomains are the driven forces for the LipA closing. Meanwhile, the hydrophobic environment provided by the oil interfaces would separate the interactions between the lid-subdomains and promote the structure opening of LipA. Moreover, our studies demonstrate the opening of the lids structure is insufficient to initiate the interfacial activation, providing explanations for the inability of interfacial activation of many lipases with lid structures.

Production of Human Milk Fat Substitutes by Lipase-Catalyzed Acidolysis: Immobilization, Synthesis, Molecular Docking and Optimization Studies

Human milk fat (HMF) triacylglycerols (TAGs) mainly contain palmitic acid esterified at the sn-2 position while oleic and other unsaturated fatty acids are located at positions sn-1,3. This study aimed at the production of HMF substitutes (HMFS) by lipase-catalyzed acidolysis of tripalmitin with oleic acid, in a solvent-free medium. Burkholderia cepacia lipase (BCL) was immobilized in silica (prepared with protic or aprotic ionic liquids) by covalent binding or encapsulation and used as biocatalyst. The supports and immobilized biocatalysts were characterized by FTIR, TGA, and SEM. Molecular docking analysis showed that BCL preferentially attacks oleic acid rather than tripalmitin, due to the lower free energy of hydrophobic binding with this acid (−6.5 kcal·mol−1) than with tripalmitin (5.4 kcal·mol−1). Therefore, the tripalmitin attack by BCL and subsequent HMFS production only occurs after the binding to most of the oleic acid molecules. The highest acidolysis activity was obtained with BCL immobilized by covalent binding in prepared silica with aprotic ionic liquid. A central composite rotatable design, as a function of temperature (58–72 °C) and oleic acid/tripalmitin molar ratio (MR = 2:1–6.8:1), was performed for acidolysis optimization. Under optimized conditions (58 °C and MR = 4:1 or 60 °C and MR = 2:1), the oleic acid incorporation of 28 mol.% was achieved after 48 h.

Co-Immobilization of Lipases with Different Specificities for Efficient and Recyclable Biodiesel Production from Waste Oils: Optimization Using Response Surface Methodology.

Lipase-catalyzed transesterification is a promising and sustainable approach to producing biodiesel. To achieve highly efficient conversion of heterogeneous oils, combining the specificities and advantages of different lipases is an attractive strategy. To this end, highly active Thermomyces lanuginosus lipase (1,3-specific) and stable Burkholderia cepacia lipase (non-specific) were covalently co-immobilized on 3-glycidyloxypropyltrimethoxysilane (3-GPTMS) modified Fe3O4 magnetic nanoparticles (co-BCL-TLL@Fe3O4). The co-immobilization process was optimized using response surface methodology (RSM). The obtained co-BCL-TLL@Fe3O4 exhibited a significant improvement in activity and reaction rate compared with mono and combined-use lipases, achieving 92.9% yield after 6 h under optimal conditions, while individually immobilized TLL, immobilized BCL and their combinations exhibited yields of 63.3%, 74.2% and 70.6%, respectively. Notably, co-BCL-TLL@Fe3O4 achieved 90-98% biodiesel yields after 12 h using six different feedstocks, demonstrating the perfect synergistic effect of BCL and TLL remarkably motivated in co-immobilization. Furthermore, co-BCL-TLL@Fe3O4 could maintain 77% of initial activity after nine cycles by removing methanol and glycerol from catalyst surface, accomplished by washing with t-butanol. The high catalytic efficiency, wide substrate adaptability and favorable reusability of co-BCL-TLL@Fe3O4 suggest that it will be an economical and effective biocatalyst for further applications.

Use of Bioprinted Lipases in Microwave-Assisted Esterification Reactions

In this study, a comparative evaluation was performed in batch esterification reactions under conventional heating (CH) and assisted by microwave irradiation (MW) using bioprinted lipases. Microwave-irradiation-assisted reactions generally provide higher productivities and improve synthesis performance in terms of increased rate and reduced reaction times, resulting in higher interest yields in less time. Productivity was calculated with the enzymes: Burkholderia cepacia lipase (BCL), Candida rugosa lipase (CRL), and porcine pancreas lipase (PPL) using different fatty acids (lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1)) and alcohols at a molar ratio of 1:8. The microwave reactor was operated at a temperature of 45 °C, and power varied between 50 W and 200 W. Bioprinted BCL (bBCL) showed the highest productivity among the tested lipases. In the reaction with the best result, bBCL with lauric acid under MW, the reaction time decreased from 24 h (CH) to 25 min (MW) and the productivity increased 33 times compared with the reactions under CH. The increase in productivity demonstrates its activation that occurred as a result of conformational changes of the enzyme in the bioprinting process, confirmed by Fourier transform infrared (FTIR) spectrometric analysis, which reduces the content of bBCL α-helix with lauric acid. The biocatalyst showed high operational stability over eight cycles, while losing only 19% of its initial activity with half-life times of 12.8 batches. The storage time was five weeks, maintaining ≈80% activity. The results demonstrate the prospect of a new enzymatic route to obtain hyperactive catalysts, with the use of bioprinted lipases in esterification reactions under microwave irradiation, for the synthesis of esters with a view to large-scale industrial application.

Development of Carbon-Based Support Using Biochar from Guava Seeds for Lipase Immobilization

Enzymes are promising tools for achieving an environmentally benign process. However, enzymes are required to be immobilized on economically competent supports to be reusable and maintain their activity. In this work, the aim was to evaluate the application of the biochar for immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption (PA) and covalent binding (CB). Additionally, it was observed that regarding the biochemical properties, the optimal pH was 4.0 for the BCL immobilized by PA and pH 7.0 for the BCL free and immobilized by CB. Among the kinetic parameters, the maximal velocity (Vmax) for the free enzyme was 2500 µmol g−1·min−1, and for the PA- and CB-immobilized biocatalyst the values of Vmax were 2000 and 3333 µmol g−1·min−1, respectively. The Michaelis-Menten constant (Km) value for the free lipase was 665 mM and for the biocatalysts immobilized by PA and CB the Km values were 219 and 369 mM, respectively. Immobilized LBC exhibited superior thermal stability. The reusability tests showed that the LBC immobilized by PA preserved 50% of the initial activity after 16 cycles. Thus, biochar is a by-product of a renewable source; therefore, it is a promising alternative for lipase immobilization demonstrating its potential for use in a wide range of greener industrial processes.

Whole-Genome Sequencing of a Potential Ester-Synthesizing Bacterium Isolated from Fermented Golden Pomfret and Identification of Its Lipase Encoding Genes.

Microbial ester synthases are regarded as valuable catalysts in the food industry. Here, one strain of Acinetobacter venetianus with ester synthase-production capacity, SCSMX-3, was isolated from traditional fermented golden pomfret. It exhibited good growth in mesophilic, low salt, and slightly alkaline environments. The ester synthase produced by SCSMX-3 displayed maximum activity at pH 8.0 and 35 °C. Genome sequencing revealed that the strain contains one circular chromosome of 336313 bp and two circular plasmids (plasmid A-14424 bp and plasmid B-11249 bp). Six CRISPR structures enhance the genomic stability of SCSMX-3 and provide the opportunity to create new functional strains. Gene function analysis indicated that SCSMX-3 produces the necessary enzymes for survival under different conditions and for flavor substance synthesis. Furthermore, 49 genes encoding enzymes associated with lipid metabolism, including three triacylglycerol lipases and two esterases, were identified through the NCBI Non-Redundant Protein Database. The lipase encoded by gene0302 belongs to the GX group and the abH15.02 (Burkholderia cepacia lipase) homolog of the abH15 superfamily. Our results shed light on the genomic diversity of and lipid metabolism in A. venetianus isolated from fermented golden pomfret, laying a foundation for the exploration of new ester synthases to improve the flavor of fermented fish products.

The first example of an on-surface enzymatic reaction evaluated by paper spray mass spectrometry (PS-MS): Deacetylation of 1,2,3,4-tetra-O-acetyl-β-D-xylopyranose

Paper spray mass spectrometry (PS-MS) is a recent and innovative ambient ionization technique conveniently applied to investigate on-surface chemical reactions. The easy, fast, and efficient PS-MS technique has shown to be a convenient platform to investigate organic reactions, yielding promising results. Lipases are a group of enzymes responsible for esterification and hydrolysis reactions of organic analytes, such as carbohydrates and lipids. Hence, this work describes for the first time an on-surface enzymatic reaction monitored by PS-MS: the deacetylation of 1,2,3,4-tetra-O-acetyl-β-D-xylopyranose (1) induced by the enzyme Burkholderia cepacia lipase (BCL). The reaction was followed over 60 min when the sodium monodeacetylated product (including the respective sodium-bound dimer) and the oxonium intermediate were detected at high intensities. Besides, the influence of BCL concentration and the presence of water and hydrochloric acid were evaluated. Finally, the results described herein demonstrated the feasibility of applying PS-MS to evaluate and get information on on-surface enzymatic reactions, a field still underexplored. These results also open up a new and exciting perspective concerning studying other and perhaps more complex enzymatic reactions in a straightforward manner.

Evaluation of lipase access tunnels and analysis of substance transport in comparison with experimental data.

Lipases (E.C. 3.1.1.3) have buried active sites and used access tunnels in the transport of substrates and products for biotransformation processes. Computational methods are used to predict the trajectory and energy profile of ligands through these tunnels, and they complement the experimental methodologies because they filter data, optimizing laboratory time and experimental costs. Access tunnels of Burkholderia cepacia lipase (BCL), Candida rugosa lipase (CRL), and porcine pancreas lipase (PPL) and the transport of fatty acids, alcohols and esters through the tunnels were evaluated using the online server CaverWeb V1.0, and server calculation results were compared with experimental data (productivity). BCL showed higher productivity with palmitic acid-C16:0 (4029.95µmol/hmg); CRL obtained productivity for oleic acid-C18:1 (380.80µmol/hmg), and PPL achieved productivity for lauric acid-C12:0 (71.27µmol/hmg). The highest probability of transport for BCL is through the tunnels 1 and 2, for CRL through the tunnel 1, and for PPL through the tunnels 1, 2, 3 and 4. Thus, the best in silico result was the transport of the substrates palmitic acid and ethanol and product ethyl palmitate in tunnel 1 of BCL. This result corroborates with the best result for the productivity data (higher productivity for BCL with palmitic acid-4029.95µmol/hmg). The combination of in silico evaluation and experimental data gave similar results, demonstrating that in silico approaches are a promising alternative for reducing screening tests and minimizing laboratory time in the bio-catalysis area by identifying the lipases with the greatest reaction potential, as in the case of this proposal.

Synthesis of SBA-15 and pore-expanded SBA-15 and surface modification with tin for covalent lipase immobilization

The mesoporous silicas were extensively used in lipase immobilization due to the high specific area, pore diameter, and the possibility of surface modification. Due to this, the present work aimed to synthesize the SBA-15 with different pore diameters (S8 with 9.1 nm and S23 with 23 nm) and modify the silica surfaces with tin (SnS8 and SnS23), a little-explored technique for immobilization, for Burkholderia cepacia lipase immobilization by covalently bond between the functional group in the enzyme and the metal. The tin-modified silica (SnS23) derived from the pore expansion of the SBA-15 was the support with the largest pore size (25 nm) and led to the highest specific activity of the immobilized enzyme (SnS23B, 268 ± 12 U/mg). In the leaching tests, no lipase was detected in the buffer solution used for lipase desorption, indicating the strong interaction between lipases and modified silicas. • Immobilization enzyme was carried out in mesoporous silica with different pore diameters and surfaces modified with tin. • The interaction between lipase and support was strong according to the leaching test. • Pore diameter and pore volume were the parameters that most influence biocatalyst activity.

Enhanced hydrogen fuel production using synergistic combination of solar radiation and TiO2 photocatalyst coupled with Burkholderia cepacia lipase

Promising and sustainable alternatives for hydrogen production have been investigated. Among these, the use of enzymes may represent an efficient alternative. In this work, an increase in hydrogen production under simulated sunlight by combining TiO 2 and TiO 2 /MgCl 2 with Burkholderia cepacia lipase (BCL) was reported. The samples (TiO 2 , TiO 2 /MgCl 2 , and TiO 2 /MgCl 2 /BCL) were characterized by X-ray diffraction (XRD), thermo-gravimetric (TGA), N 2 adsorption-desorption isotherms (BET), scanning electron microscopy (SEM), and UV–Visible absorption spectra. Hydrogen production tests were performed in aqueous methanol solutions under simulated sunlight. The results demonstrated that 130-times greater increase in hydrogen production rate was observed by the addition of BCL than individual TiO 2 . This better hydrogen production rate can be attributed to the increase in the amount of reduction equivalent of the system due to the oxidation of protein peptides caused by TiO 2 . In conclusion, the results indicate that hydrogen production efficiency can be significantly improved when integrating TiO 2 /MgCl 2 with BCL. • TiO 2 /MgCl 2 and Burkholderia cepacia lipase improved hydrogen production using sunlight. • Characterization of photobiocatalytic system by an array of advanced techniques. • Inclusion of lipase increased the hydrogen production rate. • TiO 2 increase the amount of system reduction equivalent by oxidizing protein peptides.

Continuous Synthesis of Biodiesel from Outstanding Kernel Oil in a Packed Bed Reactor Using Burkholderia cepacia Lipase Immobilized on Magnetic Nanosupport

Continuous Synthesis of Biodiesel from Outstanding Kernel Oil in a Packed Bed Reactor Using Burkholderia cepacia Lipase Immobilized on Magnetic Nanosupport

Miscibility and enzymatic degradability of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-based polyester blends by PHB depolymerase and lipase

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) containing 5 and 17 mol% 3-hydroxyhexanoate has been blended with atactic poly(3-hydroxybutyrate) (PHB), polycaprolactone (PCL), and polybutylene succinate (PBS) at different ratios by the solvent-casting method. PHBH-5%/atactic PHB and PHBH-17%/atactic PHB showed a single glass-transition temperature (Tg), indicating miscibility in the amorphous state. Immiscibility was observed in the PHBH-5%/PCL and PHBH-17%/PCL blends based on the non-transitioning Tg of the PHBH component, presence of two distinct Tg values for some of the blend films, and individual crystallization manner. The thermal properties and spherulitic morphologies of the PHBH-5%/PBS blend films suggested partial miscibility. The enzymatic degradability of the three types of blends by Ralstonia pickettii T1 PHB depolymerase and Burkholderia cepacia lipase was investigated. Depending on the enzyme–substrate specificity, miscibility, and phase structure of the blend, the variation of the enzymatic degradation rate with the blend ratio was different among the three types of blends. The weight loss rate of PHBH-5%/atactic PHB was higher than that of PHBH-17%/atactic PHB when degraded by PHB depolymerase. The PCL component accelerated the weight loss rate of PHBH-17%/PCL but decreased that of PHBH-5%/PCL. Lipase barely eroded PHBH-5%/PBS, but the weight loss rate increased as the PCL content increased. From quantitative analysis of the water-soluble products liberated during degradation, the degradation manners of the blends by PHB depolymerase and lipase are discussed.

Continuous production of monoacylglycerol via glycerolysis of babassu oil by immobilized Burkholderia cepacia lipase in a packed bed reactor.

This study investigated the glycerolysis of babassu oil by Burkholderia cepacia lipase immobilized on SiO2-PVA particles in a continuous packed bed reactor. Experiments were conducted in a solvent-free system at 273.15K either in an inert atmosphere or in the presence of cocoa butter to prevent lipid oxidation. The reactor (15 × 55mm) was run at a fixed space time of 9.8h using different molar ratios of babassu oil to glycerol (1:3, 1:6, 1:9, 1:12, and 1:15) to assess the effects of reactant molar ratio on monoacylglycerol productivity and selectivity. Nitrogen atmosphere and cocoa butter were equally effective in inhibiting lipid oxidation, indicating that addition of cocoa butter to glycerolysis reactions may be an interesting cost-reduction strategy. An oil/glycerol molar ratio of 1:9 resulted in the highest productivity (52.3 ± 2.9mgg-1h-1) and selectivity (31.5 ± 1.8%). Residence time distribution data were fitted to an axial dispersion model for closed-vessel boundary conditions, giving a mass transfer coefficient (kc) of 3.4229 × 10-6ms-1. A kinetic model based on elementary steps of the studied reaction was written in Scilab and compared with experimental data, providing standard deviations in the range of 5.5-7.5%.

Computational and experimental analysis on the preferential selectivity of lipases for triglycerides in Licuri oil.

In the present study, we demonstrated the use of molecular docking as an efficient in silico screening tool for lipase-triglyceride interactions. Computational simulations using the crystal structures from Burkholderia cepacia lipase (BCL), Thermomyces lanuginosus lipase (TLL), and pancreatic porcine lipase (PPL) were performed to elucidate the catalytic behavior with the majority triglycerides present in Licuri oil, as follows: caprilyl-dilauryl-glycerol (CyLaLa), capryl-dilauryl-glycerol (CaLaLa), capryl-lauryl-myristoyl-glycerol (CaLaM), and dilauryl-myristoyl-glycerol (LaLaM). The computational simulation results showed that BCL has the potential to preferentially catalyze the major triglycerides present in Licuri oil, demonstrating that CyLaLa, (≈25.75% oil composition) interacts directly with two of the three amino acid residues in its catalytic triad (Ser87 and His286) with the lowest energy (-5.9kcal/mol), while other triglycerides (CaLaLa, CaLaM, and LaLaM) interact with only one amino acid (His286). In one hard, TLL showed a preference for catalyzing the triglyceride CaLaLa also interacting with His286 residue, but, achieving higher binding energies (-5.3kcal/mol) than found in BCL (-5.7kcal/mol). On the other hand, PPL prefers to catalyze only with LaLaM triglyceride by His264 residue interaction. When comparing the computational simulations with the experimental results, it was possible to understand how BCL and TLL display more stable binding with the majority triglycerides present in the Licuri oil, achieving conversions of 50.86 and 49.01%, respectively. These results indicate the production of fatty acid concentrates from Licuri oil with high lauric acid content. Meanwhile, this study also demonstrates the application of molecular docking as an important tool for lipase screening to reach a more sustainable production of fatty acid concentrates from vegetable oils.

Physical adsorption used to the immobilization of Burkholderia cepacia lipase into powder polymeric supports

This study aims to combine distinctive supports with a feasible, easy and cleaning method to lipase immobilization. Commercial lipase from Burkholderia cepacia is physically adsorbed using polymeric powders such as polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and copolymer styrene-divinylbenzene (St-DVB). It is found that the immobilization on PHB has a high amount of the lipase per support mass (1065 U g−1). The activation energy also shows a value higher than the lipase immobilized onto both PHBV (348 U g−1) and St-DVB (355 U g−1). FTIR, TG, DSC and XRD techniques are used to polymer powders and immobilized lipase characterization. Morphological arrays, loss masses, enthalpy values and crystallinity degrees are evaluated. In order to analyze the morphological aspects of the raw polymers after alcohol treatment (before immobilization) and the immobilized lipase, the optical microscopy analysis is used. The immobilized Burkholderia cepacia lipase provides distinctive behavior in the polymer powders.

Lipase activation by molecular bioimprinting: The role of interactions between fatty acids and enzyme active site.

Bioimprinting is an easy, sustainable and low-cost technique that promotes a printing of potential substrates on enzyme structure, inducing a more selective and stable conformation. Bioimprinting promotes conformational changes in enzymes, resulting in better catalytic performance. In this work, the effect of bioimprinting of Burkholderia cepacia lipase (BCL) and porcine pancreatic extracts (PPE) with four different fatty acids (lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0)) was investigated. The results demonstrated that the better bioimprinting effect was in BCL with lauric acid in esterification reaction, promoting BCL activation in which relative enzyme activity was 70 times greater than nonimprinted BCL. Bioimprinting results were influenced by the carbon chain length of fatty acids imprinted in the BCL, in which the effects were weaker with the chain increase. Molecular docking was performed to better understand the bioimprinting method. The results of these simulations showed that indeed all fatty acids were imprinted in the active site of BCL. However, lauric acid presented the highest imprinting preference in the active site of BCL, resulting in the highest relative activity. Furthermore, Fourier transform infrared (FTIR) analysis confirmed important variations in secondary structure of bioimprinting BCL with lauric acid, in which there was a reduction in the α-helix content and an increase in the β-sheet content that facilitated substrate access to the active site of BCL and led higher rigidity, resulting in high activity. Bioimprinted BCL with lauric acid showed excellent operational stability in esterification reaction, maintaining its original relative activity after five successive cycles. Thus, the results show that bioimprinting of BCL with lauric acid is a successful strategy due to its high catalytic activity and reusability.

Enzymatic transesterification of coconut oil by using immobilized lipase on biochar: An experimental and molecular docking study.

Guava seed biochar appears as a new alternative of the effective support to the immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption. The objective of this work was to evaluate the potential of this immobilized biocatalyst in the transesterification reaction of crude coconut oil and ethanol and to understand the mechanism of the reaction through the study of molecular docking. The best loading of BCL was determined to be 0.15genzyme /gsupport having a hydrolytic activity of 260U/g and 54% immobilization yield. The products of transesterification reaction produced a maximum yield at 40°C under different reaction conditions. The monoacylglycerols (MAGs) conversion of 59% was using substrate molar ratio oil:ethanol of 1:7 with the reaction time of 24H. In addition, the highest ethyl esters yield (48%) had the molar ratio of 1:7 with the reaction time of 96H and maximum conversion of diacylglycerols (DAGs) was 30% with the molar ratio of 1:6 with the reaction time of 24H. Molecular Docking was applied to clarify the mechanisms of transesterification reaction at the molecular level. MAGs and DAGs are compounds with excellent emulsifying properties used in industrial production of several bioproducts such as cosmetic, pharmaceuticals, foods, and lubricants.