Lipase-catalyzed preparation of mono- and diesters of ferulic acid

Lipophilic and stable derivatives of ferulic acid are required to improve its efficacy in fatty foods and to optimize its use in cosmetic and pharmaceutical preparations. We report an improved synthesis of ferulic acid monoesters (ethyl ferulate and lauryl ferulate) using immobilized lipase from Candida antarctica B (CALB) in diisopropyl ether (DIPE). Maximum yields were 89% and 85% in 200 h for ethyl and lauryl ferulate, respectively. Ethyl ferulate was further acylated with vinyl esters to form ferulate diesters. 4-Acetoxy-ethyl ferulate was obtained with the immobilized lipase from Alcaligenes sp. (QLG) with 59% yield in 72 h, whereas 4-dodecanoyloxy-ethyl ferulate (a new compound) was synthesized with 52% yield in 72 h using CALB. DIPE was the best solvent for the transesterifications. Finally, the anti-inflammatory activity of the synthesized derivatives was evaluated in vitro; the compounds bearing a dodecyl chain showed improved anti-inflammatory activity compared with short-chain esters.     

Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max)

The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H₂O₂) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H₂O₂ content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H + G + S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.     

Solvent-free synthesis of glyceryl ferulate using a commercial microbial lipase

A process was optimized for the enzymatic synthesis of glyceryl ferulate with a yield of up to 96% using a vacuum-rotary evaporation strategy under following conditions: 15 mmol glycerol, 1.5 mmol ethyl ferulate, 170 mg Candida antarctica lipase, at 60°C for 10 h and under a vacuum of 10 mm Hg. The immobilized lipase can be used 10 times.     

Redirection of flux through the phenylpropanoid pathway by increased glucosylation of soluble intermediates

The phenylpropanoid pathway is used in biosynthesis of a wide range of soluble secondary metabolites including hydroxycinnamic acid esters, flavonoids and the precursors of lignin and lignans. In Arabidopsis thaliana a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases (GTs) that glucosylate phenylpropanoids in vitro. This study explores the effect of constitutively over-expressing two of these GTs (UGT72E1 and E3) in planta using the CaMV-35S promoter to determine whether phenylpropanoid homeostasis can be altered in a similar manner to that achieved by over-expression of UGT72E2 as previously reported. The data show that impact of over-expressing UGT72E3 in leaves is highly similar to that of UGT72E2 in that the production of massive levels of coniferyl and sinapyl alcohol 4-O-glucosides and a substantial loss in sinapoyl malate. In contrast, the over-expression of UGT72E1 in leaves led only to minimal changes in coniferyl alcohol 4-O-glucoside and no effect was observed on sinapoyl malate levels. In roots, over-expression of both UGTs led to some increase in the accumulation of the two glucosides. The cell specificity expression of the whole UGT72E gene cluster was investigated and interestingly only UGT72E3 was found to be wound and touch responsive.     

Novel screening assay for the detection of phenolic acid esterases

A novel plate assay method, developed for the screening of microorganisms or enzyme preparations for phenolic acid esterases, involves incorporating ethyl cinnamate into an agar medium. After inoculation and incubation, the plate is flooded with a pH-sensitive dye to reveal yellow zones around positive cultures against a blue background. A number of yeasts (Rhodotorula spp. and Candida spp.) and fungi (Penicillium sp. and Aspergillus sp.) gave positive results, while a number of commercial enzymes, particularly pectinases, also exhibited good phenolic acid esterase.J.A. Donaghy and A.M. McKay are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK. A.M. McKay is also with the Department of Food Science (Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, UK.     

Solvent-free enzymatic synthesis of feruloylated diacylglycerols and kinetic study

The enzymatic esterification of glyceryl ferulate (FG) and oleic acid (OA) for feruloylated diacylglycerols (FDAG) synthesis in a solvent-free system was studied in this work. The reactions were catalyzed by different commercially available lipases, among which Novozym 435 was found to be the most active biocatalyst. The effects of glycerol in the reaction mixture and various synthesis parameters on yield of FDAG and the initial reaction rate were studied. The optimum synthesis conditions were as follows: temperature, 65 °C; enzyme load, 7.5%; substrate ratio, 7.5:1 (OA/(FG + glycerol), w/w); and reaction time, 12 h. Under the optimum conditions, the conversion of FG and yield of FDAG reached 98.0 ± 1.0% and 82.6 ± 2.2%, respectively. A linear relationship was established between the initial reaction rate and enzyme load up to 10%, which demonstrated that the influence of external mass transfer limitations on the reaction could be eliminated. The relationship between initial reaction rate and temperature was also established, based on the Arrhenius law. Novozym 435 in the present work can be used 18 times under the optimum conditions without essential losses in activity. The reaction kinetics agrees with the Ping-Pong Bi-Bi mechanism characterized by V_m and K^′_m values of 5.26 × 10⁻⁴ mol/(L min) and 0.26 mol/L, respectively.     

Changes in ligno-suberization of cell walls of tomato hairy roots produced by salt treatment: the relationship with the release of a basic peroxidase

A highly basic peroxidase isoenzyme was shown to be released to the culture medium of tomato (Lycopersicon esculentum) hairy roots grown in Murashige-Skoog (MS) liquid medium when it was supplemented with 100 mM NaCl. In this paper we demonstrate that this enzyme is ionically bound to cell walls and that the release was a consequence of the continuous agitation of the tissue in a high ionic strength medium with salt addition. In order to establish the physiological role of this isoenzyme we partially purified it, and we analysed its kinetic properties as coniferyl alcohol peroxidase. The peroxidase isoenzyme showed a high catalytic efficiency for this substrate, which suggests that it would be associated with the ligno-suberization process. To confirm the involvement of this isoenzyme in that process, we studied the pattern of ligno-suberization of the tissue under different conditions of growth. Our results suggest that this basic peroxidase would be indeed involved in ligno-suberization since its leakage from cell walls, induced by 100 mM NaCl in liquid MS, caused less ligno-suberization of exo and endodermis. On the contrary, more ligno-suberization was seen in cell walls when the hairy roots were grown in a salt-supplemented MS solid medium without contact with it, a condition in which the release of the isoenzyme would be avoided. Thus, through the changes produced by the release of the enzyme from its site of action, we could demonstrate the physiological role of this peroxidase in the processing of root cell walls, being part of control mechanisms of ion and water fluxes through the root.     

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.