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International Journal of Molecular... Jul 2023Green leaf volatiles (GLVs), including short chain volatile aldehydes, are widely used in the flavor and food industries because of their fresh aroma. To meet the...
Green leaf volatiles (GLVs), including short chain volatile aldehydes, are widely used in the flavor and food industries because of their fresh aroma. To meet the growing demand for natural GLVs with high added value, the use of biocatalytic processes appears as a relevant application. In such processes, vegetable oils are bioconverted into GLVs. First, the triacylglycerols of the oils are hydrolyzed by a lipase. Then, the free polyunsaturated fatty acids are converted by a lipoxygenase. Finally, volatile C6 or C9 aldehydes and 9- or 12-oxoacids are produced with a hydroperoxide lyase. Optimization of each biocatalytic step must be achieved to consider a scale-up. In this study, three oils (sunflower, hempseed, and linseed oils) and three lipases (, , and lipases) have been tested to optimize the first step of the process. The experimental design and response surface methodology (RSM) were used to determine the optimal hydrolysis conditions for each oil. Five factors were considered, i.e., pH, temperature, reaction duration, enzyme load, and oil/aqueous ratio of the reaction mixture. lipase was selected as the most efficient enzyme to achieve conversion of 96 ± 1.7%, 97.2 ± 3.8%, and 91.8 ± 3.2%, respectively, for sunflower, hempseed, and linseed oils under the defined optimized reaction conditions.
Topics: Lipase; Hydrolysis; Plant Oils; Biocatalysis; Linseed Oil; Fatty Acids, Nonesterified; Aldehydes
PubMed: 37569649
DOI: 10.3390/ijms241512274 -
Journal of the Science of Food and... Dec 2023Pine sterol ester is a type of novel food source nutrient with great advantages in lowering blood cholesterol levels, inhibiting tumors, preventing prostate enlargement,...
BACKGROUND
Pine sterol ester is a type of novel food source nutrient with great advantages in lowering blood cholesterol levels, inhibiting tumors, preventing prostate enlargement, and regulating immunity. Macroporous resins with large specific surface area, stable structures, and various functional groups (epoxy, amino, and octadecyl groups) have been selected for immobilization of Candida rugosa lipase (CRL) to improve its stability and efficiency in the synthesis of pine sterol esters. A solvent-free strategy using oleic acid (substrate) as an esterification reaction medium is an important alternative for avoiding the use of organic solvents.
RESULTS
The immobilization conditions of CRL immobilized on several types of commercial macroporous resins were optimized. Fortunately, by adsorption (hydrophobic interaction), a high immobilization efficiency of CRL was obtained using macroporous resins with hydrophobic octadecyl groups with an immobilization efficiency of 86.5%, enzyme loading of 138.5 mg g and enzyme activity of 34.7 U g . The results showed that a 95.1% yield could be obtained with a molar ratio of oleic acid to pine sterol of 5:1, an enzyme amount of 6.0 U g (relative to pine sterol mass) at 50 °C for 48 h.
CONCLUSION
The hydrophobic macroporous resin (ECR8806M) with a large specific surface area and abundant functional groups was used to achieve efficient immobilization of CRL. CRL@ECR8806M is an efficient catalyst for the synthesis of phytosterol esters and has the potential for further large-scale applications. Therefore, this simple, green, and low-cost strategy for lipase immobilization provides new possibilities for the high-efficiency production of pine sterol esters and other food source nutrients. © 2023 Society of Chemical Industry.
Topics: Lipase; Solvents; Enzymes, Immobilized; Oleic Acid; Biocatalysis; Candida; Sterols; Hydrophobic and Hydrophilic Interactions; Enzyme Stability; Esters
PubMed: 37467367
DOI: 10.1002/jsfa.12869 -
International Journal of Molecular... Jul 2023Clopidogrel is a chiral compound widely used as an antiplatelet medication that lowers the risk of blood clots, strokes, and heart attacks. The main aim of the study...
Clopidogrel is a chiral compound widely used as an antiplatelet medication that lowers the risk of blood clots, strokes, and heart attacks. The main aim of the study presented herein was to obtain ()-clopidogrel, which is commercially available in treatments, via the kinetic resolution of racemic clopidogrel carboxylic acid with the use of lipase from and a two-phase reaction medium containing an ionic liquid. For this purpose, the enantioselective biotransformation of clopidogrel carboxylic acid and chiral chromatographic separation with the use of a UPLC-MS/MS system were optimized. The best kinetic resolution parameters were obtained by using a catalytic system containing lipase from OF as a biocatalyst, cyclohexane and [EMIM][BF4] as a two-phase reaction medium, and methanol as an acyl acceptor. The enantiomeric excess of the product was ee = 94.21% ± 1.07 and the conversion was c = 49.60% ± 0.57%, whereas the enantioselectivity was E = 113.40 ± 1.29. The performed study proved the possibility of obtaining ()-clopidogrel with the use of lipase as a biocatalyst and a two-phase reaction medium containing an ionic liquid, which is in parallel with green chemistry methodology and does not require environmentally harmful conditions.
Topics: Ionic Liquids; Clopidogrel; Chromatography, Liquid; Tandem Mass Spectrometry; Lipase; Stereoisomerism
PubMed: 37446300
DOI: 10.3390/ijms241311124 -
Spectrochimica Acta. Part A, Molecular... Dec 2023Candida rugosa lipase (CRL, EC3.1.1.3) is one of the main enzymes synthesizing esters, and ZIF-8 was chosen as an immobilization carrier for lipase. Enzyme activity...
Candida rugosa lipase (CRL, EC3.1.1.3) is one of the main enzymes synthesizing esters, and ZIF-8 was chosen as an immobilization carrier for lipase. Enzyme activity testing often requires expensive reagents as substrates, and the experiment processes are time-consuming and inconvenient. As a result, a novel approach based on near-infrared spectroscopy (NIRs) was developed for predicting CRL/ZIF-8 enzyme activity. The absorbance of the immobilized enzyme catalytic system was evaluated using UV-Vis spectroscopy to investigate the amount of CRL/ZIF-8 enzyme activity. The powdered samples' near-infrared spectra were obtained. The sample's enzyme activity data were linked with each sample's original NIR spectra to establish the NIR model. A partial least squares (PLS) model of immobilized enzyme activity was developed by coupling spectral preprocessing with a variable screening technique. The experiments were completed within 48 h to eliminate inaccuracies between the reduction in enzyme activity with increasing laying-aside time throughout the test and the NIRs modeling. The root-mean-square error of cross-validation (RMSECV), the correlation coefficient of validation set (R) value, and the ratio of prediction to deviation (RPD) value were employed as assessment model indicators. The near-infrared spectrum model was developed by merging the best 2nd derivative spectral preprocessing with the Competitive Adaptive Reweighted Sampling (CARS) variable screening method. This model's root-mean-square error of cross-validation (RMSECV) was 0.368 U/g, the correlation coefficient of calibration set (R_cv) value was 0.943, the root-mean-square error of prediction (RMSEP) set was 0.414 U/g, the correlation coefficient of validation set (R) value was 0.952, and the ratio of prediction to deviation (RPD) was 3.0. The model demonstrates that the fitting relationship between the predicted and the reference enzyme activity value of the NIRs is satisfactory. The findings revealed a strong relationship between NIRs and CRL/ZIF-8 enzyme activity. As a result, the established model could be implemented to quantify the enzyme activity of CRL/ZIF-8 quickly by including more variations of natural samples. The prediction method is simple, rapid, and adaptable to be the theoretical and practical basis for further studying other interdisciplinary research work in enzymology and spectroscopy.
Topics: Spectroscopy, Near-Infrared; Enzymes, Immobilized; Least-Squares Analysis; Calibration
PubMed: 37390722
DOI: 10.1016/j.saa.2023.123072 -
Journal of Biotechnology Jul 2023Metal-organic frameworks (MOFs) are used as ideal support materials thanks to their unique properties and have become the focus of interest in enzyme immobilization...
Metal-organic frameworks (MOFs) are used as ideal support materials thanks to their unique properties and have become the focus of interest in enzyme immobilization studies, especially in recent years. In order to increase the catalytic activity and stability of Candida rugosa lipase (CRL), a new fluorescence-based MOF (UiO-66-Nap) derived from UiO-66 was synthesized. The structures of the materials were confirmed by spectroscopic techniques such as FTIR, H NMR, SEM, and PXRD. CRL was immobilized on UiO-66-NH and UiO-66-Nap by adsorption technique and immobilization and stability parameters of UiO-66-Nap@CRL were examined. Immobilized lipases UiO-66-Nap@CRL exhibited higher catalytic activity (204 U/g) than UiO-66-NH @CRL (168 U/g), which indicates that the immobilized lipase (UiO-66-Nap@CRL) carries sulfonate groups, this is due to strong ionic interactions between the surfactant's polar groups and certain charged locations on the protein surface. The Free CRL lost its catalytic activity completely at 60 °C after 100 min, while UiO-66-NH @CRL and UiO-66-Nap@CRL retained 45 % and 56 % of their catalytic activity at the end of 120 min, respectively. After 5 cycles, the activity of UiO-66-Nap@CRL remained 50 %, while the activity of UiO-66-NH @CRL was about 40 %. This difference is due to the surfactant groups (Nap) in UiO-66-Nap@CRL. These results show that the newly synthesized fluorescence-based MOF derivative (UiO-66-Nap) can be an ideal support material for enzyme immobilization and can be used successfully to protect and increase the activities of enzymes.
Topics: Biocatalysis; Metal-Organic Frameworks; Surface-Active Agents; Candida; Enzymes, Immobilized; Lipase
PubMed: 37301292
DOI: 10.1016/j.jbiotec.2023.06.003 -
Enzyme and Microbial Technology Aug 2023Multiple sequence alignments of three lipase isoforms from the filamentous fungus, Cordyceps militaris, have revealed that the deduced protein from their common sequence...
Multiple sequence alignments of three lipase isoforms from the filamentous fungus, Cordyceps militaris, have revealed that the deduced protein from their common sequence belongs to the Candida rugosa lipase-like group. To express the protein in its active form, recombinant lipase from C. militaris (rCML) was extra cellularly expressed in Pichia pastoris X-33 after removing its signal peptide. Purified rCML was a stable monomeric protein with a molecular mass of 90 kDa, and was highly N-mannosylated compared to the native protein (69 kDa). The catalytic efficiency (k/K) of rCML was greater than the native protein (1244.35 ± 50.88 and 1067.17 ± 29.07 mM·min, respectively), yet they had similar optimal pH values and temperatures (40 °C and pH 7.0-7.5), and showed preferences for Tween esters and short-chain triacylglycerols. Despite its monomeric conformation, interfacial activation was not observed for rCML, unlike the classical lipases. From the structural model of rCML, the binding pocket of rCML was predicted as a funnel-like structure consisting of a hollow space and an intramolecular tunnel, which is typical of C. rugosa lipase-like lipases. However, a blockage shortened the tunnel to 12-15 Å, which endows strict short-chain selectivity towards triacylglycerols and a perfect match for tricaproin (C6:0). The limited depth of the tunnel may enable accommodation of triacylglycerols with medium-to-long-chain fatty acids, which differentiates rCML from other C. rugosa lipase-like lipases with broad substrate specificities.
Topics: Lipase; Cordyceps; Recombinant Proteins; Pichia; Triglycerides; Substrate Specificity
PubMed: 37201411
DOI: 10.1016/j.enzmictec.2023.110254 -
Journal of Agricultural and Food... Feb 2023Rationally designing carriers to obtain efficient and stable immobilized enzymes for the production of food raw materials is always a challenge. In this work, hollow...
Rationally designing carriers to obtain efficient and stable immobilized enzymes for the production of food raw materials is always a challenge. In this work, hollow cube carbon (HMC) as a carrier of lipase (CRL) was prepared to construct a Pickering interfacial biocatalysis system, which was applied to biphasic biocatalysis. For comparison, the nonporous carbon (HC) and porous MoS (HMoS) were also designed. On these grounds, -NPP and linolenic acid were selected as the representative substrates for hydrolysis and esterification reactions. Under the optimal conditions, the protein loading amount, specific activity, and expressed activity of CRL immobilized on HMC (HMC@CRL) were 167.2 mg g, 5.41 U mg, and 32.34 U/mg protein, respectively. In the "oil-water" biphase, the relative hydrolytic activity of HMC@CRL was higher than that of HC@CRL, HMoS@CRL, and CRL by 50, 68, and 80%, respectively, as well as itself in one phase. Compared to other reports (1.13%), HMC@CRL demonstrated a satisfactory hydrolysis rate (3.02%) and was the fastest among all other biocatalysts in the biphase. Moreover, compared with the free CRL in one-phase system, the Pickering interfacial biphasic biocatalyst, HMC@CRL, exhibited a higher esterification rate (85%, 2.7-fold enhancement). Therefore, the HMC@CRL nanoreactors had more optimal performance in the field of biomanufacturing and food industry.
Topics: Biocatalysis; Enzyme Stability; Enzymes, Immobilized; Lipase; Nanotechnology; Phytosterols; Esters
PubMed: 36688464
DOI: 10.1021/acs.jafc.2c06756 -
Open Forum Infectious Diseases Jan 2023Fungal species have undergone and continue to undergo significant nomenclatural change, primarily due to the abandonment of dual species nomenclature in 2013 and the... (Review)
Review
Fungal species have undergone and continue to undergo significant nomenclatural change, primarily due to the abandonment of dual species nomenclature in 2013 and the widespread application of molecular technologies in taxonomy allowing correction of past classification errors. These have effected numerous name changes concerning medically important species, but by far the group causing most concern are the yeasts. Among common species, , , , , and have been changed to , , , , and , respectively. There are currently no guidelines for microbiology laboratories on implementing changes, and there is ongoing concern that clinicians will dismiss or misinterpret laboratory reports using unfamiliar species names. Here, we have outlined the rationale for name changes across the major groups of clinically important fungi and have provided practical recommendations for managing change.
PubMed: 36632423
DOI: 10.1093/ofid/ofac559 -
Probiotics and Antimicrobial Proteins Feb 2024Probiotic microorganisms are incorporated in foods due to their numerous health benefits. We investigated lactic acid bacteria (LAB) and yeasts isolated from goat milk...
Probiotic microorganisms are incorporated in foods due to their numerous health benefits. We investigated lactic acid bacteria (LAB) and yeasts isolated from goat milk in Nigeria for novel probiotic strains. In this study, a total of 27 LAB and 23 yeast strains were assessed for their probiotic potentials. Only six LAB strains (Weissella cibaria GM 93m3, Weissella confusa GM 92m1, Pediococcus acidilactici GM 18a, Pediococcus pentosaceus GM 23d, Lactiplantibacillus pentosus GM 102s4, Limosilactobacillus fermentum GM 30m1) and four yeast strains (Candida tropicalis 12a, C. tropicalis 33d, Diutina rugosa 53b, and D. rugosa 77a) identified using partial 16S and 26S rDNA sequencing, respectively, showed survival at pH 2.5, 0.3% bile salt, and simulated gastrointestinal conditions and possessed auto-aggregative and hydrophobic properties, thus satisfying key in vitro criteria as probiotics. All LAB strains showed coaggregation properties and antimicrobial activities against pathogens. Pediococcus pentosaceus GM 23d recorded the strongest coaggregation percentage (34-94%) against 14 pathogens, while W. cibaria GM 93m3 showed the least (6-57%) against eight of the 14 pathogens. The whole cell and extracellular extracts of LAB and yeast strains, with the exception of D. rugosa 77a, had either 2,2-diphenyl-1-picryl-hydrazyl and/or hydroxyl radical scavenging activity. In conclusion, all six LAB and four yeast strains are important probiotic candidates that can be further investigated for use as functional starter cultures.
Topics: Animals; Lactobacillales; Milk; Nigeria; Goats; Probiotics; Yeasts; Pediococcus pentosaceus
PubMed: 36520357
DOI: 10.1007/s12602-022-10022-w -
Applied Biochemistry and Biotechnology Nov 2022This study aimed to (i) prepare functionalized maghemite nanoparticles for immobilization of Candida rugosa lipase (CRL) by covalent binding, (ii) evaluate the...
This study aimed to (i) prepare functionalized maghemite nanoparticles for immobilization of Candida rugosa lipase (CRL) by covalent binding, (ii) evaluate the application of the immobilized derivative in the hydrolysis of waste cooking oil (WCO) to fatty acids, and (iii) assess the potential of the hydrolyzed material for biodiesel production by hydroesterification. Maghemite (γFeO) obtained by precipitation of FeCl with NHOH served as an efficient support for covalent immobilization of CRL. Fourier-transform infrared spectroscopy and hydrolytic activity analysis indicated that CRL was covalently immobilized on the surface of the maghemite support. The derivative showed an activity of 166.62 ± 8 U g in WCO hydrolysis at 40 °C and pH 6. Scanning electron microscopy revealed that, after lipase immobilization, nanoparticles became more dispersed, which is advantageous for biocatalysis reactions, as it increases the contact area with the substrate. WCO hydrolysis afforded 96 ± 0.2 wt% free fatty acids. In the second step, free fatty acids were subjected to chemical esterification with sulfuric acid, affording 94.4 ± 0.02 wt% fatty acid methyl esters (biodiesel). The findings of this study contribute to the field of biotechnology and may promote the development of enzymatic technologies for the synthesis of products of economic and social interest.
Topics: Lipase; Enzymes, Immobilized; Biofuels; Enzyme Stability; Magnetite Nanoparticles; Fatty Acids, Nonesterified; Candida; Temperature; Esterification; Biocatalysis; Sulfuric Acids
PubMed: 35789983
DOI: 10.1007/s12010-022-04046-9