Acyl Acceptor Research Articles

Glycerol-Free Biodiesel via Catalytic Interesterification: A Pathway to a NetZero Biodiesel Industry

Conventional biodiesel manufacturing uses alcohol as an acyl acceptor, resulting in glycerol as a side product. The increased demand for biodiesel has led to the production of a substantial surplus of glycerol, exceeding the market need. Consequently, glycerol is now being regarded as a byproduct, and in some cases, even as waste. The present study aims to suggest an economically viable and ecologically friendly approach for maintaining the viability of the biodiesel sector. This involves generating an alternative byproduct of higher value, rather than glycerol. Triacetin is produced through the interesterification of triglycerides with methyl acetate, and is a beneficial ingredient to biodiesel, reducing the need for extensive product separation. The primary objective of this research is to improve the interesterification reaction by optimising process parameters to maximise biodiesel production while using sulphuric acid as an economically viable catalyst. The study utilised the Box–Behnken design (BBD) to investigate the influence of various process variables on biodiesel yield, such as reaction time, methyl acetate to oil molar ratio, and catalyst concentration. An optimisation study using Response Surface Methodology (RSM) focused on key process reaction parameters, including the methyl acetate to oil (MA:O) molar ratio, catalyst concentration, and residence time. The best conditions produced a biodiesel blend with a 142% yield at a 12:1 MA:O molar ratio, with 0.1 wt% of catalyst loading within 1.7 h. The established technique is deemed to be undeniably effective, resulting in an efficient biodiesel production process.

Turning noncovalent interactions via halogen substituents for improving activity and enantioselectivity of Candida antarctica lipase B

Noncovalent interactions associated with the enzyme, enzyme-substrate complexes, and transition states have profound influences on varying the biocatalytical activity and enantioselectivity. The recent rediscovery of halogen bonds incorporating in biocatalysis has induced our motivation on introducing halogen substituents into the substrate for improving the enzyme performance. By selecting Candida antarctica lipase B (CALB)-catalyzed kinetic hydrolysis (or butanolysis) of trans-2-(p-halophenyl)cyclopropyl 1,2,4-triazolide in methyl tert-butyl ether (MTBE) as the model reaction system, (R, R)-2-(p-halophenyl)cyclopropane-1-carboxylic acid (or ester) of high-optical purity that acts as the key building block for synthesizing the corresponding (1 R, 2S)-2-(p-halophenyl)cyclopropylamine derivatives was obtainable. The thermodynamic and kinetic analysis was then employed for elucidating effects of the halogen substituent and acyl acceptor on enhancing the noncovalent interactions of the rate-limiting tetrahedral intermediate and hence CALB activity and enantioselectivity. A putative model was moreover proposed for interpreting the possible hydrogen and halogen bonding involving the halogen substituent in turning the noncovalent interactions.

Systematic characterization of gene families and functional analysis of PvRAS3 and PvRAS4 involved in rosmarinic acid biosynthesis in Prunella vulgaris.

Prunella vulgaris is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses showed that 17 of them could be involved in RA biosynthesis. In vitro enzymatic assay showed that PvRAS3 catalyzed the condensation of p-coumaroyl-CoA and caffeoyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was higher than caffeoyl-CoA. PvRAS4 catalyzed the condensation of p-coumaroyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was lower than PvRAS3. UPLC and LC-MS/MS analyses showed the existence of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid in P. vulgaris. Generation and analysis of pvras3 homozygous mutants showed significant decrease of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid and significant increase of DHPL and pHPL. It suggests that PvRAS3 is the main enzyme catalyzing the condensation of acyl donors and acceptors during RA biosynthesis. The role of PvRAS4 appears minor. The results provide significant information for quality control of P. vulgaris medicinal materials.

Genome-wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid.

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

Twisted 8-Acyl-1-dialkyl-amino-naphthalenes Emit from a Planar Intramolecular Charge Transfer Excited State

Fluorescence from dialkylamino donor–acyl acceptor substituted 1,8-naphthalene derivatives can occur either from a planar (PICT) or a twisted (TICT) intramolecular charge transfer excited state. The photophysical properties of 8-acetyl-1-(dimethyl-amino)naphthalene (3) and 8-pivaloyl-1-(dimethyl-amino)naphthalene (4) are compared with 1-methyl-2,3-dihydronaphtho[1,8-bc]azepin-4(1H)-one (5). In 3 and 4, both the carbonyl and amino groups are forced to twist out of the plane of the naphthalene ring. In 5, these groups are nearly coplanar with the naphthalene. Neither 3 nor 4 fluoresce as strongly as 5, but all three show similar degrees of solvato-chromism and all are strongly quenched by alcohol solvents. Nitrile 6, 8-cyano-1-(dimethyl-amino)naphthalene, does not show the same degree of solvato-chromism as 3–5, nor is it as affected by alcohols. Calculations corroborate the experimental results, indicating that 3–5 emit from a PICT excited state.

In situ Transesterification of Crambe Seeds with Dimethyl Carbonate Using an Enzymatic Catalyst

In situ transesterification of crambe seeds oil was investigated to produce fatty acid methyl esters using dimethyl carbonate as acyl acceptor and lipase Novozyme 435® as the catalyst. First, the effects of solvent-to-seeds ratio, seed pretreatment, and extraction time were evaluated on the seed oil removal, comparing the results with conventional Soxhlet extraction. Then, the effects of enzyme loading (5 to 30 wt.% based on the oil mass) and reaction time (60 to 540 min) were evaluated on the simultaneous extraction and reaction. The highest oil extraction yield (ca. 26 wt.%) was achieved at the solvent-to-seeds ratio of 6 mL g-1 and 360 min. The oil obtained from seeds that received thermal pretreatment showed a higher concentration of minor compounds. The highest ester content (76.71 wt.%) was obtained with an enzyme loading of 20 wt.%, solvent-to-seeds ratio of 6 mL g-1, for 480 min. The reaction samples showed a predominance of erucate and oleate esters, with identification of the co-products formation (glycerol carbonate and glycerol dicarbonate), and ca. 5 wt.% of acylglycerols.

Comparative Analysis of Shikonin and Alkannin Acyltransferases Reveals Their Functional Conservation in Boraginaceae.

Shikonin and its enantiomer, alkannin, are bioactive naphthoquinones produced in several plants of the family Boraginaceae. The structures of these acylated derivatives, which have various short-chain acyl moieties, differ among plant species. The acylation of shikonin and alkannin in Lithospermum erythrorhizon was previously reported to be catalyzed by two enantioselective BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1). However, the mechanisms by which various shikonin and alkannin derivatives are produced in Boraginaceae plants remain to be determined. In the present study, evaluation of six Boraginaceae plants identified 23 homologs of LeSAT1 and LeAAT1, with 15 of these enzymes found to catalyze the acylation of shikonin or alkannin, utilizing acetyl-CoA, isobutyryl-CoA or isovaleryl-CoA as an acyl donor. Analyses of substrate specificities of these enzymes for both acyl donors and acyl acceptors and determination of their subcellular localization using Nicotiana benthamiana revealed a distinct functional differentiation of BAHD acyltransferases in Boraginaceae plants. Gene expression of these acyltransferases correlated with the enantiomeric ratio of produced shikonin/alkannin derivatives in L. erythrorhizon and Echium plantagineum. These enzymes showed conserved substrate specificities for acyl donors among plant species, indicating that the diversity in acyl moieties of shikonin/alkannin derivatives involved factors other than the differentiation of acyltransferases. These findings provide insight into the chemical diversification and evolutionary processes of shikonin/alkannin derivatives.

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris.

Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol-3-phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC-like activity was supported by the functional complementation of a temperature-sensitive plsC-deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC-like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent-solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes.

Structural identification of catalytic His158 of PtMAC2p from Pseudozyma tsukubaensis, an acyltransferase involved in mannosylerythritol lipids formation.

Mannosylerythritol lipids (MELs) are extracellular glycolipids produced by the basidiomycetous yeast strains. MELs consist of the disaccharide mannosylerythritol, which is acylated with fatty acids and acetylated at the mannose moiety. In the MEL biosynthesis pathway, an acyltransferase from Pseudozyma tsukubaensis, PtMAC2p, a known excellent MEL producer, has been identified to catalyze the acyl-transfer of fatty acid to the C3'-hydroxyl group of mono-acylated MEL; however, its structure remains unclear. Here, we performed X-ray crystallography of recombinant PtMAC2p produced in Escherichia coli and homogeneously purified it with catalytic activity in vitro. The crystal structure of PtMAC2p was determined by single-wavelength anomalous dispersion using iodide ions. The crystal structure shows that PtMAC2p possesses a large putative catalytic tunnel at the center of the molecule. The structural comparison demonstrated that PtMAC2p is homologous to BAHD acyltransferases, although its amino acid-sequence identity was low (<15%). Interestingly, the HXXXD motif, which is a conserved catalytic motif in the BAHD acyltransferase superfamily, is partially conserved as His158-Thr159-Leu160-Asn161-Gly162 in PtMAC2p, i.e., D in the HXXXD motif is replaced by G in PtMAC2p. Site-directed mutagenesis of His158 to Ala resulted in more than 1,000-fold decrease in the catalytic activity of PtMAC2p. These findings suggested that His158 in PtMAC2p is the catalytic residue. Moreover, in the putative catalytic tunnel, hydrophobic amino acid residues are concentrated near His158, suggesting that this region is a binding site for the fatty acid side chain of MEL (acyl acceptor) and/or acyl-coenzyme A (acyl donor). To our knowledge, this is the first study to provide structural insight into the catalytic activity of an enzyme involved in MEL biosynthesis.

Enzymatic Interesterification of Crude Palm Oil with Methyl acetate: Effect of Pre-treatment, Enzyme’s Dosage and Stability

In the present study, biodiesel was produced via the enzymatic interesterification of Crude Palm Oil (CPO) and methyl acetate within ultrasonic condition. In contrast to alcohol, methyl acetate as an acyl acceptor does not inhibit lipase activity and can create triacetin as a useful byproduct. In this work, Immobilized lipase from Candida Antartica A (CaLA) was utilized as biocatalyst and the effect of using non-pretreated CPO and pre-treated CPO as feedstock were explored. The pre-treatment of CPO involves degumming with acid, washing with water, and bleaching. The enzymatic interesterification was conducted in three-neck flasks using an ultrasonic water bath at 45o C. Few parameter effects on biodiesel production were also investigated, including the effect of molar ratio of CPO to methyl acetate, the effect amount of lipase, and the reusability of immobilized lipase (CaLA) in the interesterification reaction. The highest average Biodiesel yield of 80.6% was obtained from pretreated CPO at a molar ratio of 1:9 with 100 mg (1% w/v) of Immobilized CaLA, after three hours of reaction. Further research on the reusability of immobilized CaLA revealed that the yield of biodiesel reduced significantly after the second run. The results of the present study also demonstrated that Immobilized CaLA performed well at low concentrations but had low stability, with productivity decreasing to 92% upon reuse after the initial run. In order to make Immobilized lipase economically viable, further research must be conducted to overcome its low stability in the reaction.

Transglutaminase in Foods and Biotechnology.

Stabilization and reusability of enzyme transglutaminase (TGM) are important goals for the enzymatic process since immobilizing TGM plays an important role in different technologies and industries. TGM can be used in many applications. In the food industry, it plays a role as a protein-modifying enzyme, while, in biotechnology and pharmaceutical applications, it is used in mediated bioconjugation due to its extraordinary crosslinking ability. TGMs (EC 2.3.2.13) are enzymes that catalyze the formation of a covalent bond between a free amino group of protein-bound or peptide-bound lysine, which acts as an acyl acceptor, and the γ-carboxamide group of protein-bound or peptide-bound glutamine, which acts as an acyl donor. This results in the modification of proteins through either intramolecular or intermolecular crosslinking, which improves the use of the respective proteins significantly.

Shifts in catalyst deactivation mechanisms as a function of surface coverage during Friedel-Crafts acylation in zeolites

The production of commodity chemicals from renewable resources is often accompanied by rapid catalyst deactivation rates. An important example is direct Friedel Crafts acylation, which can yield a range of valuable products from surfactants to renewable transportation fuels. Furanic acyl acceptors are among the most intriguing and promising possible acceptors; however, they undergo self-coupling reactions in the presence of Brønsted acid surface sites leading to rapid catalyst deactivation. In this contribution, we study 2-methylfuran acylation with acetic acid over HZSM-5 catalysts at temperatures ranging from 160 to 240 °C, revealing the remarkable shift in deactivation behavior as a function of surface coverage of acetic acid. We reveal that a surface saturated with acetic acid lowers the deactivation rate by blocking the direct access of furanic species to surface sites. However, on surfaces fully saturated with carboxylic acids, deactivation is revealed to proceed via reactions between adsorbed products with gas phase methylfuran reactants.

Bio-Approach for Obtaining Enantiomerically Pure Clopidogrel with the Use of Ionic Liquids.

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 (S)-clopidogrel, which is commercially available in treatments, via the kinetic resolution of racemic clopidogrel carboxylic acid with the use of lipase from Candida rugosa 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 Candida rugosa 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 eep = 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 (S)-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.

Characterization of three succinyl-CoA acyltransferases involved in polyketide chain assembly

Polyketides are a class of natural products with astonishing structural diversities, fascinating biological activities, and a versatile of applications. In polyketides biosynthesis, acyltransferases (ATs) are the 'gatekeeping' enzymes selecting the specific CoA-activated acyl groups as building blocks and transferring them onto the phosphopantetheine arm of acyl carrier proteins (ACPs) to enable the following condensation reactions to assemble the polyketide chain. Herein, the Art2 protein from aurantinins, a group of the antibacterial polyketides, is characterized in vitro as an AT that can load a CoA-activated succinyl unit onto the first ACP domain of Art17 (ACPArt17-1). In addition, another two proteins, GbnB and EtnB, involved in the biosynthesis of gladiolin and etnangien respectively, were traced by literature mining, homologous searching, and product structure analysis and then identified as functional succinyl-CoA ATs by the ACPArt17-1 assays. Taken together, by the assay method employing ACPArt17-1 as an acyl acceptor, we identified three ATs that can introduce a succinyl unit into the polyketide assembly line, which enriches the toolbox of polyketide biosynthetic enzymes and sets a stage for incorporating a succinyl unit into polyketide backbones in synthetic biological manners. KEY POINTS: • Three acyltransferases that are able to load ACP with a succinyl unit were characterized in vitro. • ACPArt17-1 can be used as an acceptor to assay succinyl-CoA AT from different polyketides. • The succinyl unit can be incorporated into polyketides assembly process.

A Cascade Enzymatic Reaction Scheme for Irreversible Transpeptidative Protein Ligation.

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1' position of the Asn-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.

Enzymatic Transesterification of Atlantic Salmon (Salmo salar) Oil with Isoamyl Alcohol.

In this experimental study, biodiesel was synthesized from the salmon oil using the Lipozyme®RM IM (Bagsværd, Denmark) as a biocatalyst. Isoamyl alcohol was used as an acyl acceptor in the transesterification process. The aim of this study is to select the best process conditions, aiming to obtain the highest transesterification degree that meets the requirements of the EN 14214 standard. Response surface methodology (RSM) was used for statistical analysis and optimization of process parameters. A four-factor experimental design was modelled by central compositional design (CCD) to investigate the effects of biocatalyst concentration, isoamyl alcohol-to-oil molar ratio, temperature, and duration on transesterification degree. It was determined that the optimal parameters for biodiesel synthesis were the following: an enzyme concentration of 11% (wt. of oil mass); a process temperature of 45 °C; a process duration of 4 h; and an alcohol-to-oil molar ratio of 6:1. The transesterification degree of biodiesel reached 87.23%. The stepwise addition of isoamyl alcohol during the transesterification process further increased the degree of transesterification to 96.5%.

Fusel Oil: Chemical Composition and an Overview of Its Potential Application

Fusel oil (FO) is one of the by-products of the sugar-alcohol industry, which has application as an additional source of higher alcohols for production of esters with flavoring, fuels and lubricants properties, as well as additives in petroleum-based fuels and in pesticide formulations. Thus, this review is focused on the characteristics of FO and its applications as an acyl acceptor in transesterification and esterification reactions for esters production, its addition to fossil fuels, and the herbicidal and fungicidal potential of the compounds presented in its chemical composition. The operating conditions of the process, results obtained, and patented applications in the food, cosmetic, transportation and agricultural sectors are also reported and discussed. Furthermore, main trends, challenges and recommendations are proposed in order to overcome the disadvantages of applying FO in the reported sectors, as well as alternatives for the development of new lines of research are suggested.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity invitro and invivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-β-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

Transesterification under high pressure as a sequential step from pressurized liquid extraction: Effect of operational parameters and characterization

In this study, the sequential step of high pressure transesterification to obtain fatty acid esters was investigated. For this purpose, extraction by pressurized liquid was performed and the product resulting from this step was directly used in the reaction in which different conditions of temperature and pressure were evaluated. High operating temperature (<300 °C) led to a reduction in fatty acid esters. At 10 MPa, a high esters yield and low content of unreacted compounds was obtained, in addition, due to the simultaneous use of dimethyl carbonate and ethanol as acyl acceptors, the presence of glycerol in the reaction product was not verified, with the production of glycerol carbonate as a co-product. The reaction carried out at 250 °C and 10 MPa allowed to preserve the active compounds of the oil used as raw material, thus providing a material with high quality and stability, demonstrating that the proposed process has potential applicability.