Theoretical calculation on degradation mechanism of novel copolyesters under CALB enzyme.

Poly(butylene succinate-co-furandicarboxylate) (PBSF) and poly(butylene adipate-co-furandicarboxylate) (PBAF) are novel furandicarboxylic acid-based biodegradable copolyesters with great potential to replace fossil-derived terephthalic acid-based copolyesters such as poly(butylene succinate-co-terephthalate) (PBST) and poly(butylene adipate-co-terephthalate) (PBAT). In this study, quantum chemistry techniques after molecular dynamics simulations are employed to investigate the degradation mechanism of PBSF and PBAF catalyzed by Candida antarctica lipase B (CALB). Computational analysis indicates that the catalytic reaction follows a four-step mechanism resembling the ping-pong bibi mechanism, with the initial two steps being acylation reactions and the subsequent two being hydrolysis reactions. Notably, the first step of the hydrolysis is identified as the rate-determining step. Moreover, by introducing single-point mutations to expand the substrate entrance tunnel, the catalytic distance of the first acylation step decreases. Additionally, energy barrier of the rate-determining step is decreased in the PBSF system by site-directed mutations on key residues increasing hydrophobicity of the enzyme's active site. This study unprecedently show the substrate binding pocket and hydrophobicity of the enzyme's active site have the potential to be engineered to enhance the degradation of copolyesters catalyzed by CALB.

Preparation of highly stable immobilized Candida antarctica lipase B (CALB) through adjusting the surface properties of carrier: Preparation, characterization and performance evaluation.

The stability of the immobilized lipase is the key factor that determines the economy and feasibility of its industrial application. Here, two robust immobilized Candida antarctica lipase B (CALB) were prepared through adjusting the surface properties of ECR1030 resin. Silane coupling agent (SCA) and dialdehyde cellulose (DAC) were employed to modify the carrier surface. Contact angle measurement showed that the hydrophobicity of the modified carrier increased first, and then decreased with the increase of the chain length of SCA. FTIR results showed that Si-O-Si bond and aldehyde group were attached to ECR1030, respectively, indicating that the ECR1030 resin was successfully modified. Meanwhile, the NH and CN bond were observed in the corresponding immobilized CALB, suggesting CALB was immobilized onto the modified carriers. The effects of immobilization conditions on CALB immobilization was further investigated, and the C8-ECR1030-CALB and DAC-ECR1030-CALB with the activity of 12,736 U/g and 11,962 U/g were obtained. Moreover, the stability of the immobilized lipases was evaluated and compared with the commercial Novozym 435. The C8-ECR1030-CALB and DAC-ECR1030-CALB exhibited comparable or superior stability to Novozym 435 and showed better deacidification effect than Novozym 435. This study paves road for further study involving preparation of highly stable immobilized lipase.

Synthesis of Enantiopure (S)-Atenolol by Utilization of Lipase-Catalyzed Kinetic Resolution of a Key Intermediate.

(S)-Atenolol ((S)-2-(4-(2-Hydroxy-3-(isopropylamino)propoxy)phenyl)acetamide) has been synthesized in >99% enantiomeric excess (ee) with the use of Candida antarctica lipase B from Syncozymes (Shanghai, China), in a kinetic resolution of the corresponding racemic chlorohydrin. A catalytic amount of base was used in deprotonation of the phenol building block. The enantiopurity of the chlorohydrin building block remained unchanged upon subsequent amination to yield the final drug. All four steps in the synthesis protocol have been optimized compared to previously reported methods, which makes this new protocol more sustainable and in accordance with green chemistry principles. The overall yield of (S)-atenolol was 9.9%, which will be further optimized.

Molecular insight into the enhanced performance of CALB toward PBDF degradation.

Poly(butylene diglycolate-co-furandicarboxylate) (PBDF) is a newly developed biodegradable copolyester. Candida antarctica lipase B (CALB) has been identified as an effective catalyst for PBDF degradation. The mechanism is elucidated using a combination of molecular dynamics simulations and quantum chemistry approaches. The findings unveil a four-step catalytic reaction pathway. Furthermore, bond analysis, charge and interaction analysis are conducted to gain a more comprehensive understanding of the PBDF degradation process. Additionally, through the introduction of single-point mutations to crucial residues in CALB's active sites, two mutants, T138I and D134I, are discovered to exhibit improved catalytic efficiency. These significant findings contribute to the advancement of our comprehension concerning the molecular mechanism of underlying copolyesters degradation, while also presenting a novel approach for expediting the degradation rate by the CALB enzyme modification.

Immobilized Candida antarctica lipase B as an sn-1,3 regiospecific biocatalyst for the interesterification of triacylglycerols with fatty acid ethyl esters.

Candida antarctica lipase B (CALB) is regarded as non-regiospecific. This study aimed to investigate the regiospecificity of CALB in the solvent-free interesterification of high-oleic sunflower oil with stearic acid ethyl ester for 1,3-distearoyl-2-oleoylglycerol (SOS)-rich fat preparation using a packed bed reactor. The content ratio of 1,2-distearoyl-3-oleoylglycerol (SSO) to SOS (denoted by SSO/SOS content) obtained using Lipozyme 435 (a commercially immobilized CALB; 0-4.1%), at residence times (1-32 min) was similar to that obtained using Lipozyme RM IM (0-3.0%), but lower than that obtained using Lipozyme TL IM (6.0-39.4%). When immobilized on Lewatit VP OC 1600, Lipozyme CALB had an SSO/SOS content of 0-10.4%, which was greater than that of Palatase 20,000 L (0-1.1%) but was lower than that of Lipozyme TL 100 L (8.8-97.7%). Our findings suggest that immobilized CALB shows distinct sn-1,3 regiospecificity in the interesterification of triacylglycerol with fatty acid ethyl esters.

Regiospecificity of Immobilized Candida antarctica Lipase B (CAL-B) towards 2,3-Diacyl-1-O-alkyl Glyceryl Ether in Ethanol.

Highly pure 2,3-dioleoyl-1-O-alkyl glyceryl ether (DOGE), whose 1-position is a lipase-tolerant ether bond, was chemically synthesized and its detailed regioselectivity and acyl transfer were confirmed. During ethanolysis using immobilized Candida antarctica lipase B (CAL-B) with DOGE as the substrate, monooleoyl-1-O-alkyl glyceryl ethers (MOGEs) and a few 1-alkyl glyceryl ethers were formed upon consumption of the substrate. The structure of MOGE was confirmed using nuclear magnetic resonance spectroscopy and only the isomer of 2-MOGE was formed, indicating that CAL-B has complete α- regiospecificity. During ethanolysis, 3-MOGE was formed via acyl migration. These results indicate that the formation of 1-alkyl glyceryl ethers is not due to the imperfect regiospecificity of CAL-B, but rather due to ethanolysis of the formed 3-MOGE. The ethanolysis rate at the 3-α-position of DOGE was faster and the rate of acyl transfer was slightly slower for chain lengths greater than 14. These results show for the first time that both deacylation at the 3-position and acyl migration from the 2- to 3-position are affected by the structure of 1-position.

Long chain capsaicin analogues synthetized by CALB-CLEAs show cytotoxicity on glioblastoma cell lines.

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives. KEY POINTS: • This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs. • The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil. • Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells.

Candida antarctica lipase B performance in organic solvent at varying water activities studied by molecular dynamics simulations.

Applications of lipases in low-water environments are found across a broad range of industries, including the pharmaceutical and oleochemical sectors. This includes condensation reactions in organic solvents where the enzyme activity has been found to depend strongly on both the solvent and the water activity (aw). Despite several experimental and computational studies, knowledge is largely empirical, and a general predictive approach is much needed. To close this gap, we chose native Candida antarctica lipase B (CALB) and two mutants thereof and used molecular dynamics (MD) simulations to gain a molecular understanding of the effect of aw on the specific activity of CALB in hexane. Based on the simulations, we propose four criteria to understand the performance of CALB in organic media, which is supported by enzyme kinetics experiments. First, the lipase must be stable in the organic solvent, which was the case for native CALB and the two mutants studied here. Secondly, water clusters that form and grow close to the active site must not block the path of substrate molecules into the active site. Thirdly, the lipase's lid must not cover the active site. Finally, mutations and changes in aw must not disrupt the geometry of the active site. We show that mutating specific residues close to the active site can hinder water cluster formation and growth, making the lipase resistant to changes in aw. Our computational screening criteria could potentially be used to screen in-silico designed variants, so only promising candidates could be pushed forward to characterisation.

Biocatalytic selective acylation of technical lignins: a new route for the design of new biobased additives for industrial formulations.

In this article, we describe a proof of concept of the potential use of a biocatalytic process for the functionalization of technical soda lignins from wheat straw through the selective acylation of primary hydroxy groups of lignin oligomers by acetate or hexanoate, thus preserving their free, unreacted phenols. The selectivity and efficiency of the method, although they depend on the structural complexity of the starting material, have been proven on model compounds. Applied to technical lignins, the acylation yield is only moderate, due to structural and chemical features induced by the industrial mode of preparation of the lignins rather than to the lack of efficiency of the method. However, most of the physicochemical properties of the lignins, including their antioxidant potential, are preserved, advocating the potential use of these modified lignins for industrial applications.

Immobilization of Candida antarctica lipase B on ILs modified CNTs with different chain lengths: Regulation of substrate tunnel "Leucine gating".

Ionic liquids (ILs) have been widely used as chemical modifiers to modify the carriers and thus improve the efficiency, activity and stability of the enzymes. However, as thousands of ILs have been found up to date, it's a huge work for screening and designing suitable ILs for immobilization of enzymes. Moreover, the mechanism of improving enzymes catalytic performance is still remain ambiguous. Thus, this study investigated the impact of ILs with different chain lengths on the enzymatic properties of Candida antarctica lipase B (CALB). Molecular dynamics simulations were employed to examine the interaction between ILs modified CNTs and CALB, as well as their effects on CALB's structure. The results revealed that ILs with different chain lengths significantly influenced the absorption orientation of CALB. Tunnel analysis identified a key role for Leu278 in regulating the open or closed state of Tunnel 2 during CALB's catalytic cycle. The weak interaction analysis demonstrated that ILs with suitable chain lengths provided spatial freedom and formed strong interactions with CNTs and ILs (vdW and hbond). This led to a conformational flip of Leu278, stabilizing the open state of Tunnel 2 and improving the activity and stability of immobilized CALB. This study provides novel insights into the design of new green modifiers to modulate carrier performance and obtain immobilized enzymes with better performance, and establishes a theoretical basis for the design and selection of modifiers for ILs in future work.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine.

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Activity and stability of lipase from Candida Antarctica after treatment in pressurized fluids.

Lipase B from Candida antarctica (CalB) is one of the biocatalysts most used in organic synthesis due to its ability to act in several medium, wide substrate specificity and enantioselectivity, tolerance to non-aqueous environment, and resistance to thermal deactivation. Thus, the objective of this work was to treat CalB in supercritical carbon dioxide (SC-CO2) and liquefied petroleum gas (LPG), and measure its activity before and after high-pressure treatment. Residual specific hydrolytic activities of 132% and 142% were observed when CalB was exposed to SC-CO2 at 35 â„ƒ, 75 bar and 1 h and to LPG at 65 â„ƒ, 30 bar and 1 h, respectively. Residual activity of the enzyme treated at high pressure was still above 100% until the 20th day of storage at low temperatures. There was no difference on the residual activity loss of CalB treated with LPG and stored at different temperatures over time. Greater difference was observed between CalB treated with CO2 and flash-frozen in liquid nitrogen (- 196 â„ƒ) followed by storage in freezer (- 10 â„ƒ) and CalB stored in freezer at - 10 â„ƒ. Such findings encourage deeper studies on CalB as well as other enzymes behavior under different types of pressurized fluids aiming at industrial application.

Evidence for a high pKa of an aspartic acid residue in the active site of CALB by a fully atomistic multiscale approach.

Candida antarctica Lipase B (CALB) is a paradigm for the family of lipases. At pH 7, the optimal pH for catalysis, the protonation state of an aspartic acid of the active site (Asp134) could not be conclusively assigned. In fact, the pKa estimate provided by a widely used computational tool, namely PropKa, that predicts pKa values of ionizable groups in proteins based on the crystallographic structure, is only slightly above 7 (pKa = 7.25). This, along with the lack of an experimental evaluation, makes the assignment of its protonation state at neutral pH challenging. Here, we calculate the pKa of Asp134 by means of a fully atomistic multiscale computational approach based on classical molecular dynamics (MD) simulation and the perturbed matrix method (PMM), namely the MD-PMM approach. MD-PMM is able to take into account the dynamics of the system and, at the same time, to treat the deprotonation step at the quantum level. The calculations provide a pKa value of 8.9 ± 1.1, hence suggesting that Asp134 in CALB should be protonated at neutral, and even at slightly basic, pH.Communicated by Ramaswamy H. Sarma.

Enzymatic PCL-grafting to NH2-end grouped silica and development of microspheres for pH-stimulated release of a hydrophobic model drug.

This study set out to evaluate novel PCL-based silica containing nanohybrids as the polymer matrix in a hydrophobic drug-loaded microsphere system. Nanohybrids were synthesized by PCL-grafting to NH2-end grouped silica by in situ enzymatic ring opening polymerization of ε-caprolactone. Molecular weight and monomer conversion, PCL grafting percentage, thermal properties and crystallinity of the nanohybrids were determined by 1H NMR, TGA, DSC and XRD. Synthesized nanohybrids had low crystallinity percentage (32 and 39 %) and molecular weight (4800 and 8700 g/mol), promising for controlled drug release applications. The nanohybrids were used for fabrication of trans-chalcone-loaded microspheres by O/W single emulsion solvent evaporation. Mean particle diameter of the microspheres were between 15 and 30 µm. The result of release studies showed that optimum microsphere formulations (AP4 and A2, respectively) had 61 and 64 % encapsulation efficiency. One of the more significant findings to emerge from this investigation is that TC release was extended to 16 and 37 days, in a controlled manner. TC release was significantly enhanced in acidic pH media (pH 3.6 and 5.6) indicating pH-dependent release from nanohybrid microspheres; releasing 80-100 % of the loaded drug in 4-14 days. Drug/polymer interactions and molecular structures were investigated by FT-IR spectroscopy and DSC analysis. According to the results obtained, enzymatically synthesized nanohybrids have potential for pH-dependent release of the model drug, trans-chalcone.

Bioinspired Lipase Immobilized Membrane for Improving Hesperidin Lipophilization.

Lipophilization is a promising way to improve the bioavailability of flavonoids. However, the traditional enzymatic esterification methods are time-consuming, and present low yields and purity. Herein, a novel membrane-based lipophilization technology-bioinspired lipase immobilized membranes (BLIMs), including CAL-B@PES, CAL-B@PDA/PES and GA/CAL-B@PDA/PES- were fabricated to improve the antioxidant flavanone glycoside hesperidin lipophilization. Via reverse filtration, PDA coating and GA crosslinking, Candida antarctica lipase B (CAL-B) was stably immobilized on membrane to fabricate BLIMs. Among the three BLIMs, GA/CAL-B@PDA/PES had the greatest enzyme activity and enzyme loading, the strongest tolerance of changes in external environmental conditions (temperatures, pH, heating time, storage time and numbers of cycles) and the highest hesperidin esterification efficiency. Moreover, the optimal operating condition for GA/CAL-B@PDA/PES fabrication was the CAL-B concentration of 0.36 mg/mL, operation pressure of 2 bar, GA concentration of 5% and crosslinking time of 1 h. Afterwards, the hesperidin esterification process did not affect the micromorphology of BLIM, but clearly improved the BLIM permeability and esterified product efficiency. The present study reveals the fabrication mechanism of BLIMs and offers insights into the optimizing strategy that governs the membrane-based lipophilization technology process.

Amphiphilic Pentablock Copolymers Prepared from Pluronic and ε-Caprolactone by Enzymatic Ring Opening Polymerization.

Amphiphilic copolymers are appealing materials because of their interesting architecture and tunable properties. In view of their application in the biomedical field, the preparation of these materials should avoid the use of toxic compounds as catalysts. Therefore, enzymatic catalysis is a suitable alternative to common synthetic routes. Pentablock copolymers (CUC) were synthesized with high yields by ring-opening polymerization of ε-caprolactone (ε-CL) initiated by Pluronic (EPE) and catalyzed by Candida antarctica lipase B enzyme. The variables to study the structure-property relationship were EPEs' molecular weight and molar ratios between ε-CL monomer and EPE macro-initiator (M/In). The obtained copolymers were chemically characterized, the molecular weight determined, and morphologies evaluated. The results suggest an interaction between the reaction time and M/In variables. There was a correlation between the differential scanning calorimetry data with those of X-ray diffraction (WAXD). The length of the central block of CUC copolymers may have an important role in the crystal formation. WAXD analyses indicated that a micro-phase separation takes place in all the prepared copolymers. Preliminary cytotoxicity experiments on the extracts of the polymer confirmed that these materials are nontoxic.

Short-chain alcohols inactivate an immobilized industrial lipase through two different mechanisms.

Broadly used in biocatalysis as acyl acceptors or (co)-solvents, short-chain alcohols often cause irreversible loss of enzyme activity. Understanding the mechanisms of inactivation is a necessary step toward the optimization of biocatalytic reactions and the design of enzyme-based sustainable processes. The functional and structural responses of an immobilized enzyme, Novozym 435 (N-435), exposed to methanol, ethanol, and tert-butanol, are explored in this work. N-435 consists of Candida antarctica lipase B (CALB) adsorbed on polymethacrylate beads and finds application in a variety of processes involving the presence of short-chain alcohols. The nature of the N-435 material required the development of an ad hoc method of structural analysis, based on Fourier transform infrared microspectroscopy, which was complemented by catalytic activity assays and by morphological observation by transmission electron microscopy. The inactivation of N-435 was found to be highly dependent on alcohol concentration and occurs through two different mechanisms. Short-chain alcohols induce conformational changes leading to CALB aggregation, which is only partially prevented by immobilization. Moreover, alcohol modifies the texture of the solid support promoting the enzyme release. Overall, knowledge of the molecular mechanisms underlying N-435 inactivation induced by short-chain alcohols promises to overcome the limitations that usually occur during industrial processes.

Bioconjugation Strategy for Ceramic Membranes Decorated with Candida Antarctica Lipase B-Impact of Immobilization Process on Material Features.

A strategy for the bioconjugation of the enzyme Candida antarctica lipase B onto titania ceramic membranes with varied pore sizes (15, 50, 150, and 300 kDa) was successfully performed. The relationship between the membrane morphology, i.e.,the pore size of the ceramic support, and bioconjugation performance was considered. Owing to the dimension of the enzyme (~33 kDa), the morphology of the ceramics allowed (50, 150, and 300 kDa) or did not allow (15 kDa) the entrance of the enzyme molecules into the porous structure. Such a strategy made it possible to better understand the changes in the material (morphology) and physicochemical features (wettability, adhesiveness, and surface charge) of the samples, which were systematically examined. The silane functionalization and enzyme immobilization were accomplished via the covalent route. The samples were characterized after each stage of the modification, which was very informative from the material point of view. As a consequence of the modification, significant changes in the contact angle, roughness, adhesion, and zeta potential were observed. For instance, for the 50 kDa membrane, the contact angle increased from 29.1 ± 1.5° for the pristine sample to 72.3 ± 1.5° after silane attachment; subsequently, it was reduced to 57.2 ± 1.5° after the enzyme immobilization. Finally, the contact angle of the bioconjugated membrane used in the enzymatic process rose to 92.9 ± 1.5°. By roughness (Sq) controlling, the following amendments were noticed: for the pristine 50 kDa membrane, Sq = 1.87 ± 0.21 µm; after silanization, Sq = 2.33 ± 0.30 µm; after enzyme immobilization, Sq = 2.74 ± 0.26 µm; and eventually, after the enzymatic process, Sq = 2.37 ± 0.27 µm. The adhesion work of the 50 kDa samples was equal to 136.41 ± 2.20 mN m-1 (pristine membrane), 94.93 ± 2.00 mN m-1 (with silane), 112.24 ± 1.90 mN m-1 (with silane and enzyme), and finally, 69.12 ± 1.40 mN m-1 (after the enzymatic process). The materials and physicochemical features changed substantially, particularly after the application of the membrane in the enzymatic process. Moreover, the impact of ceramic material morphology on the zeta potential value is here presented for the first time. With an increase in the ceramic support cut-off, the amount of immobilized lipase rose, but the specific productivity was higher for membranes possessing smaller pores, owing to the higher grafting density. For the enzymatic process, two modes of accomplishment were selected, i.e., stirred-tank and cross-flow. The latter method was characterized by a much higher effectiveness, with a resulting productivity equal to 99.7 and 60.3 µmol h-1 for the 300 and 15 kD membranes, respectively.

Encapsulation of CALB by nucleotide/metal ions coordination nanoparticles: highly selective catalysis of esterification while poor performance in glycerolysis reaction.

BACKGROUND: Enzymatic esterification is attracting for particular high-acid oil deacidification. In this study, Candida antarctica lipase B (CALB) was encapsulated into a series of nucleotide-hybrid metal coordination polymers (CPs), which were constructed by guanosine 5'-monophosphate (GMP) and various metals. RESULTS: We here found that, most of the present CPs encapsulated CALB (CALB@CPs) samples were highly selective for esterification while poor in glycerolysis reaction. They exhibited quite poor performance in glycerolysis, with triacylglycerols (TAGs) conversion lower than 5%, despite this considerable enzymatic hydrolysis activities were observed. However, they (most of them) showed good performance in esterification of fatty acids and glycerol for TAG synthesis. In addition, the GMP/Tb (CPs constructed by GMP and Tb3+ ) encapsulated CALB (CALB@GMP/Tb) transformed over 98% of oleic acid into glycerides in the high-acid oil deacidification process, and TAG content from 87 to 89% was obtained. Moreover, the CALB@GMP/Tb showed good reusability in the esterification system. CONCLUSION: The present CALB@CPs samples are selective for esterification and suitable for high-acid oils deacidification. This work provides a new system for enzymatic selectivity improvement study. © 2021 Society of Chemical Industry.

The influence of calcium ions (Ca2+) on the enzymatic hydrolysis of lipopolysaccharide aggregates to liberate free fatty acids (FFA) in aqueous solution.

The chemical environment in aqueous solutions greatly influences the ability of amphiphilic molecules such as lipopolysaccharides (LPS) to aggregate into different structural phases in aqueous solutions. Understanding the substrate's morphology and conditions of aqueous solution that favor both enzymatic activity and the disruption of LPS aggregates are crucial in developing agents that can counteract the new trend of multidrug resistance by gram-negative bacteria. In this study, we developed two LPS morphologies using LPS from Escherichia coli as a model to study the in vitro hydrolytic response when using a lipase treatment. The hydrolysis was performed using lipase b from Candida antarctica to understand the catalytic effect in removing fatty acids from its lipid A moiety on different LPS aggregates. Physical and chemical characterizations of the products included dynamic light scattering, small angle X-ray scattering, Fourier transform infrared spectroscopy, thin-layer chromatography, and gas chromatography. Our results suggest a trend of prominent hydrolytic response (72% enhancement) upon the addition of calcium ions to induce LPS aggregates into bilayer formations. Moreover, our results revealed the detection of myristic acid (C14:0) as the product of the hydrolysis when using RaLPS in its aggregate forms.