Lipase-catalyzed production of pyridoxine monolaurate in solvent-free bioreactor system.

This study demonstrated that solvent-free gas bubbling system enhanced production efficiency of pyridoxine monolaurate in the esterification catalyzed by immobilized Candida antarctica lipase B (Lipozyme 435). Volumetric productivity in solvent-free gas bubbling system (41.24 mmol/L/h) was 3.7 and 2.1-fold higher than those in conventional organic solvent system (11.10 mmol/L/h) and solvent-free system (19.86 mmol/L/h) using magnetic stirring, respectively. Among the three bioreactor systems, solvent-free gas bubbling system provided the best reusability of the lipase retaining 94.45 % of initial activity for six batch reactions. In the bioreactor system, 5-O-lauroyl-pyridoxine was regioselectively produced with maximum production of 371.17 mmol/L at 70 °C and 0.10 of substrate molar ratio ([pyridoxine]/[lauric acid]) for 9 h. Pyridoxine monolaurate exhibited interfacial activity at oil-water interfaces, suggesting it had emulsifying properties. Pyridoxine monolaurate is expected to be applied as a multi-functional emulsifier with nutritional values to replace both small molecule emulsifiers and pyridoxine hydrochloride in fortified beverages.

Lipase-catalyzed synthesis of antibacterial and antioxidative erythorbyl ricinoleate with high emulsifying activity.

Erythorbyl ricinoleate (ERO) was synthesized as a novel multi-functional emulsifier with antibacterial and antioxidative activities via lipase-catalyzed esterification between erythorbic acid and ricinoleic acid. Esterification regioselectively produced ERO (6-O-ricinoleoyl-erythorbate) of 238.67 mM at 48 h. ERO effectively reduced interfacial tension to 2.66 mN/m at its critical micelle concentration (0.73 mM), compared with other erythorbyl fatty acid esters (EFEs). Oil-in-water (O/W) emulsion stabilized by ERO remained stable for 15 days with a droplet size of 256.3 nm and polydispersity index of 0.22, whereas the emulsion stabilized by the other EFEs became unstable within six days. ERO had antibacterial activity against Gram-positive bacteria with minimum inhibitory concentrations from 0.2 to 0.6 mM. In O/W emulsion, ERO exhibited higher antioxidative activity than erythorbic acid against lipid oxidation. These findings suggest that ERO has high potential as multi-functional food additive to control lipid oxidation and bacterial contamination for O/W emulsion foods.

A jacalin-related lectin domain-containing lipase from chestnut (Castanea crenata): Purification, characterization, and protein identification.

A novel lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was discovered from Korean chestnut (Castanea crenata). The lipase was isolated and purified by ammonium sulfate precipitation and a fast protein liquid chromatography system equipped with HiTrap DEAE-Sepharose Fast Flow, HiTrap Q-Sepharose Fast Flow, and HiPrep Sephacryl S-100 Hi-Resolution columns. The purified C. crenata lipase showed a 15.8% yield, purification fold number of 465.8, and specific activity against triolein of 88.5 mU/mg. The enzyme exhibited hydrolytic activity toward tributyrin, trilaurin, and triolein, and was maximally active at pH 8.0 and 35 °C, with triolein used as the substrate. The activation energy (Ea) and deactivation energy (Ed) of triolein hydrolysis were 38.41 and 83.35 kJ/mol, respectively. In the enzyme kinetic study, Vmax, Km, and k cat were 110.58 mU/mg, 0.11 mM, and 0.221 min-1, respectively. The relatively low Km value indicated that the lipase has high affinity for its substrate. Moreover, Mg2+ and Ca2+ increased the lipase activity to 115.4% and 108.3%, respectively. The results of peptide fingerprinting revealed that the C. crenata lipase with a molecular weight of 33.3 kDa was structurally similar to the mannose-binding lectin of the jacalin-related lectin domain superfamily, implying that it has potential as a therapeutic agent for use in the biomedical industry.

Divergent substrate specificities and regioselectivities of three lipase isoforms from Cordyceps militaris: Combinatorial advantages for entomopathogenicity and prospects as biocatalysts.

Cordyceps militaris, an entomopathogenic Cordyceps mushroom, is a crucial ethnopharmacological agricultural product with applications in traditional oriental remedies in East Asia. Since lipases are reported to serve as key enzymatic equipment for entomopathogenic fungi during the host infection, the presence of various lipases with different biochemical features in C. militaris was elucidated. Three lipases from C. militaris (CML) of 60-70 kDa were isolated according to protein hydrophobicity; isoform relationships were identified by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry. The CML isoforms exhibited distinct substrate specificities, which were related to the hydrophobicity of each isoform. Furthermore, the integral stereoselectivity of each lipase towards trioleoylglycerol diverged into two classes (sn-1,3 and sn-2 regioselectivity) that are rare in canonical fungal lipases. Overall, our results demonstrate that C. militaris secretes lipase isoforms with cocktail-like enzyme functions that may contribute to the entomopathogenic life cycle of C. militaris. Each CML isoform has distinct advantages for biocatalyst applications in the food and oleochemical industries.

Lipase-catalyzed two-step esterification for solvent-free production of mixed lauric acid esters with antibacterial and antioxidative activities.

Mixed lauric acid esters (MLE) with antibacterial and antioxidative activities were produced through lipase-catalyzed two-step esterification in solvent-free system without purification. In the first reaction, erythorbyl laurate was synthesized for 72 h. Successive reaction for 6 h at molar ratio of 1.0 (lauric acid to glycerol) produced MLE containing erythorbyl laurate and glyceryl laurate with small amounts of residual substrates, by converting 99.52% of lauric acid. MLE addition (0.5-2.0%, w/w) to Tween 20-stabilized emulsions decreased droplet size, polydispersity index, and zeta-potential, possibly enhancing the emulsion stability. In the emulsions, MLE at 0.5 and 2.0% (w/w) caused 4.4-4.6 and 5.9-6.1 log reductions of Gram-positive (Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), respectively, within 12 h. Lipid hydroperoxide concentrations decreased to 50.8-98.3% in the presence of 0.5-2.0% (w/w) MLE. These findings support a novel approach without needing purification to produce multi-functional food additives for emulsion foods.

Synergistic inactivation of Listeria and E. coli using a combination of erythorbyl laurate and mild heating and its application in decontamination of peas as a model fresh produce.

We investigated the synergistic antimicrobial activity of erythorbyl laurate (EL) and mild heating co-treatment on the Gram-positive Listeria innocua and Gram-negative Escherichia coli O157:H7 bacteria. EL (2 mM) and mild heating (55 °C for 3 min) resulted in 3.1 and 0.5 log colony forming units (CFU)/mL reductions in the number of L. innocua, respectively, compared to a 6.4 log CFU/mL reduction induced by the combined treatment of EL and mild heating in saline. EL (10 mM) and mild heating (55 °C for 3 min) resulted in 1.3 and 0.7 log CFU/mL reductions in the number of E. coli O157:H7, respectively, compared to a 6.2 log CFU/mL reduction with the combined treatment in saline. EL, a membrane-active compound, showed a strong synergistic effect with mild heating, possibly due to enhanced disruption of the bacterial cell membrane. The synergistic antibacterial effect was evaluated using inoculated English peas (Pisum sativum) and this combined treatment (2 mM EL and mild heating against L. innocua and 10 mM EL and mild heating against E. coli O157:H7) resulted in more than 7 log reductions in the numbers of L. innocua and E. coli O157:H7, inoculated on the surface of fresh peas. The treatments did not show significant difference in the color or texture of treated peas compared to the non-treated controls. This is the first report illustrating synergistic activity of EL and mild heating for both the gram positive (L. innocua) and the gram negative (E. coli O157:H7) bacteria on food. Overall, this research will illustrate the development of more effective and rapid antibacterial surface disinfection method for application in the processing of minimally processed foods.

Controlled rate slow freezing with lyoprotective agent to retain the integrity of lipid nanovesicles during lyophilization.

We designed a novel lyophilization method using controlled rate slow freezing (CSF) with lyoprotective agent (LPA) to achieve intact lipid nanovesicles after lyophilization. During the freezing step, LPA prevented water supercooling, and the freezing rate was controlled by CSF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933 °C/min) was the optimal medium for the CSF system. Lyophilized lipid nanovesicle using both CSF and LPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 133.4 nm, polydispersity index = 0.144), similar to intact vesicles (120.7 nm and 0.159, respectively), after rehydration. Only lyophilized lipid nanovesicle using both CSF and LPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs while retaining their original activity.

Synergistic Inactivation of Bacteria Using a Combination of Erythorbyl Laurate and UV Type-A Light Treatment.

This study evaluated the synergistic antimicrobial activity of erythorbyl laurate (EL) and UV type-A (UVA). To investigate the mode of synergism, changes in gene expression and bacterial inactivation activity were examined. Individual treatments with EL (10 mM) or UVA caused a 1.9- or 0.5-log CFU/ml reduction respectively, whereas EL/UVA co-treatment resulted in a 5.5-log CFU/ml reduction in Escherichia coli viable cell numbers. Similarly, treatment with either EL (2 mM) or UVA for 30 min resulted in a 2.8- or 0.1-log CFU/ml reduction in Listeria innocua, respectively, whereas combined treatment with both EL and UVA resulted in a 5.4-log CFU/ml reduction. Measurements of gene expression levels showed that EL and UVA treatment synergistically altered the gene expression of genes related to bacterial membrane synthesis/stress response. However, addition of 10-50-fold excess concentration of exogenous antioxidant compared to EL reduced the synergistic effect of EL and UVA by approximately 1 log. In summary, the results illustrate that synergistic combination of EL and UVA enhanced membrane damage independent of the oxidative stress damage induced by UVA and thus illustrate a novel photo-activated synergistic antimicrobial approach for the inactivation of both the Gram-positive and Gram-negative bacteria. Overall, this study illustrates mechanistic evaluation of a novel photochemical approach for food and environmental applications.

Erythorbyl fatty acid ester as a multi-functional food emulsifier: Enzymatic synthesis, chemical identification, and functional characterization of erythorbyl myristate.

Erythorbyl myristate (EM), a potential multi-functional food emulsifier, was newly synthesized by immobilized lipase-catalyzed esterification between antioxidative erythorbic acid and antibacterial myristic acid. The yield and productivity of EM were 56.13 ± 2.51 mg EM/g myristic acid and 1.76 ± 0.08 mM/h, respectively. The molecular structure of EM was identified as (R)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl tetradecanoate using HPLC-ESI/MS and 2D [1H-1H] NMR COSY. The hydrophilic-lipophilic balance of EM was 11.5, suggesting that EM could be proper to stabilize oil-in-water emulsions. Moreover, isothermal titration calorimetry demonstrated the micellar thermodynamic behavior of EM and determined its critical micelle concentration (0.36 mM). In terms of antioxidative property, EM exhibited the radical scavenging activity against DPPH (EC50: 35.47 ± 0.13 µM) and ABTS (EC50: 36.45 ± 1.98 µM) radicals. Finally, EM showed bacteriostatic and bactericidal activities against Gram-positive foodborne pathogens (minimum inhibitory concentration: 0.06-0.60 mM; minimum bactericidal concentration: 0.07-0.93 mM).

Lipase-catalyzed synthesis of lauroyl tripeptide-KHA with multi-functionalities: Its surface-active, antibacterial, and antioxidant properties.

Lipase-catalyzed acylation of a hydrophilic tripeptide-KHA (TP-KHA; amino acid sequence Lys-His-Ala) with a lipophilic lauric acid was performed to produce a multi-functional compound, lauroyl tripeptide-KHA (TPL-KHA), with surface, antibacterial, and antioxidant activities. The significant acylation reaction parameters were optimized as follows: organic solvent of 2-methyl-2-butanol, reaction temperature at 55 °C, substrate molar ratio (lauric acid:TP-KHA) of 4.0, and reaction time for 72 h. Structural analyses by LC-ESI-MS and 1H NMR identified that Nε-lauroyl tripeptide-KHA was chemo-selectively synthesized by the acylation reaction under the optimum conditions. TPL-KHA showed the surface activity at the air-water interface with critical micelle concentration (CMC) of 2.71 mM and γCMC of 30.44 mN/m. TPL-KHA exhibited bacteriostatic and bactericidal effects on Gram-positive and Gram-negative foodborne pathogens (minimum inhibitory concentrations: 2.83-4.00 mM, minimum bactericidal concentrations: 3.17-5.83 mM). Moreover, it was demonstrated that TPL-KHA had the ability to scavenge ABTS+ radicals and inhibit the lipid oxidation.

Effect of intense pulsed light on the deactivation of lipase: Enzyme-deactivation kinetics and tertiary structural changes by fragmentation.

The effect of intense pulsed light (IPL) irradiation on Chromobacterium viscosum lipase was investigated with a primary focus on catalytic activity and molecular structure. During IPL irradiation, lipase activity decreased significantly with increasing pulse fluence (Fp) and exposure time (te). IPL-induced deactivation kinetics were further elucidated based on a two-step series-type deactivation model (constant deactivation rate k1 >k2). Fp was found to be the dominant variable affecting the degree of lipase deactivation, and residual activity was not associated with increasing te below a certain Fp energy density (2.66 mJ/cm2), implying a critical threshold for IPL-induced deactivation of lipase. From the results of fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), we determined that IPL-induced deactivation was caused by fragmentation, leading to lipase tertiary structural changes. Furthermore, the results of FindPept analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the internal sensitive bonds of lipase were cleaved preferentially by IPL, such that IPL irradiation induced site-sensitive fragmentation and peptide bond cleavage.

Lipase-catalyzed solvent-free synthesis of erythorbyl laurate in a gas-solid-liquid multiphase system.

Erythorbyl laurate is a potential food additive as a multi-functional emulsifier having antioxidant and antimicrobial activities. In this study, a gas-solid-liquid multiphase system (GSL-MPS) was established to enhance the production yield of erythorbyl laurate in a lipase-catalyzed solvent-free synthesis. The significant reaction variables were optimized as follows: substrate molar ratio of 2:1 (lauric acid:erythorbic acid) and enzyme concentration of 120 mg/mL (840 PLU/mL). Under these conditions, the maximum production yield in GSL-MPS was 13.974 mg/mL, which is 8.60- and 4.26-fold higher than the yields obtained in an organic solvent monophase system (OS-MPS) and a solid-liquid biphase system (SL-BPS), respectively. Moreover, the operational stability of the immobilized lipase was significantly improved in GSL-MPS compared with OS-MPS. These results indicate that GSL-MPS can be an enzymatic reaction system facilitating efficient production of ester compounds as a means of increasing production yields and the reusability of the immobilized lipase.

Catalytic characteristics of a sn-1(3) regioselective lipase from Cordyceps militaris.

A total of 39 agricultural products were screened for natural sources of lipases with distinctive positional specificity. Based on this, Cordyceps militaris lipase (CML) was selected and subsequently purified by sequential chromatography involving anion-exchange, hydrophobic-interaction, and gel-permeation columns. As a result of the overall purification procedure, a remarkable increase in the specific activity of the CML (4.733 U/mg protein) was achieved, with a yield of 2.47% (purification fold of 94.54). The purified CML has a monomeric structure with a molecular mass of approximately 62 kDa. It was further identified as a putative extracellular lipase from C. militaris by the partial sequence analysis using ESI-Q-TOF MS. In a kinetic study of the CML-catalyzed hydrolysis, the values of Vmax , Km , and kcat were determined to be 4.86 µmol·min-1 ·mg-1 , 0.07 mM, and 0.29 min-1 , respectively. In particular, the relatively low Km value indicated that CML has a high affinity for its substrate. With regard to positional specificity, CML selectively cleaved triolein at the sn-1 or 3 positions of glycerol backbone, releasing 1,2(2,3)-diolein as the major products. Therefore, CML can be considered a distinctive biocatalyst with sn-1(3) regioselectivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2744, 2019.

Hydrophilic and lipophilic characteristics of non-fatty acid moieties: significant factors affecting antibacterial activity of lauric acid esters.

It has been proposed that the hydrophilic and/or lipophilic characteristics of fatty acid derivatives affect their antibacterial activities according to their ability to incorporate into the bacterial cell membrane. To verify this hypothesis, six kinds of lauric acid derivatives esterified with different non-fatty acid moieties were selected to confirm whether antibacterial activity from their precursor (i.e., lauric acid) is retained or lost. Three compounds, monolaurin, sucrose laurate, and erythorbyl laurate, exerted bacteriostatic and bactericidal effects against Gram-positive bacteria, while the others showed no inhibitory activity. Interestingly, the calculated log P (octanol-water partition coefficient) values of monolaurin, sucrose laurate, and erythorbyl laurate were - 4.122, - 0.686, and 3.670, respectively, relatively lower than those of the other compounds without antibacterial activity. Moreover, the hydrophilic-lipophilic balance values of the three compounds with antibacterial activity were higher than those of the other compounds, corresponding to the log P result.

Amperometric Detection of Conformational Change of Proteins Using Immobilized-Liposome Sensor System.

An immobilized liposome electrode (ILE)-based sensor was developed to quantify conformational changes of the proteins under various stress conditions. The ILE surface was characterized by using a tapping-mode atomic force microscopy (TM-AFM) to confirm surface immobilization of liposome. The uniform layer of liposome was formed on the electrode. The current deviations generated based on the status of the proteins under different stress were then measured. Bovine carbonic anhydrase (CAB) and lysozyme were tested with three different conditions: native, reduced and partially denatured. For both proteins, a linear dynamic range formed between denatured concentrations and output electric current signals was able to quantify conformational changes of the proteins. The pattern recognition (PARC) technique was integrated with ILE-based sensor to perform data analysis and provided an effective method to improve the prediction of protein structural changes. The ILE-based stress sensor showed potential of leveraging the amperometric technique to manifest activity of proteins based on various external conditions.