Laccase-catalyzed oxidative cross-linking of tyrosine and potato patatin- and lysozyme-derived peptides: Molecular and kinetic study.

Laccase can catalyze the oxidative cross-linking of peptides, which is useful in the production of proteinaceous materials with enhanced functional properties. However, the kinetics and the pathway of this reaction remain unclear. In the present study, laccase-catalyzed oxidative cross-linking reaction was investigated through a combination of computational analysis, kinetic studies and end-product profiling using selected substrate models, including peptide AG-10 (AKKIVSDGNG) (without tyrosine) derived from lysozyme and tyrosine-containing peptide ST-10 (SYMTDYYLST) from potato protein (patatin), and tyrosine. Both laccases from Trametes versicolor (LacTv) and Coriolus hirsutus (LacCh) were used as biocatalysts. Laccase exhibited higher binding affinity and catalytic efficiency (kcat/Km) towards ST-10 and AG-10 than tyrosine. Among the laccases, LacCh showed higher kcat towards the substrate models than LacTv. Through the molecular docking, this result was attributed to the presence of the ASN206 at the cavity of LacCh. The end product profiles reveal the formation of homo-oligomers (> 5 units) of ST-10 in the reaction catalyzed by LacTv, while polymerization was favored by LacCh. These cross-linked products were identified to have a mix of oligo-tyrosine linkages. In contrast, the cross-linking of AG-10 required the presence of ferulic acid as mediator, which resulted in the formation of hetero-oligomers and polymers of AG-10. The knowledge obtained in the present study provide insight into an effective reaction for peptide cross-linking.

Assessment of the antioxidant capacity and oxidative stability of esterified phenolic lipids in selected edible oils.

The research work was aimed at the determination of the antioxidant capacity (AOC) and the oxidative stability of phenolic lipids (PLs), obtained by lipase-catalyzed transesterification of phenolic acids (PAs) with selected edible oils (EOs), including flaxseed (FSO), fish liver (FO), and krill (KO) oils. The statistical analyses (Tukey's test at P < 0.05) revealed that the difference in AOC between that of the esterified FSO (EFSO) and the esterified krill oil (EKO) containing PLs and their control trials of EOs was significant (P < 0.05). To evaluate the storage stability, the EOs and their esterified products were subjected to 2 oxidation treatments. The experimental findings showed that the esterified EOs had higher oxidative stability when they were subjected to light, oxygen, and agitation at 50 °C as compared to that of the EOs; however, only the esterified fish oil (EFO) showed a significant difference in its peroxide value, when the esterified EOs were placed in the dark at 25 °C. Overall, the phenolic mono- and diacyglycerols present in the EOs have shown to be potential antioxidants in improving the oxidative stability of the oil and enhancing its AOC.

Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates.

Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day.

Lipase-catalyzed transesterification of dihydrocaffeic acid with flaxseed oil for the synthesis of phenolic lipids.

Lipase-catalyzed transesterification reaction of dihydrocaffeic acid (DHCA) with flaxseed oil in organic solvent media was investigated. Using equal molar concentration of DHCA and flaxseed oil, only phenolic monoacylglycerols were obtained with a transesterification yield (TY) of 18.9%. A 1:4 DHCA to flaxseed oil ratio resulted in the production of both phenolic mono and diacylglycerols, with TY of 39.6 and 27.8%, respectively. On the other hand, when 1:8 ratio of DHCA to flaxseed oil was used, the TY of phenolic diacylglycerols (46.0%) was higher than that of the phenolic monoacylglycerols (33.3%). The TY of phenolic diacylglycerols increased from 25.1 to 55.8%, when the ratio of the hexane/2-butanone reaction medium was changed from 65:35 to 85:25 (v/v); however, the TY of phenolic monoacylglycerols decreased slightly from 34.0 to 31.8%. The relative proportion of the C(18:3)n-3 was higher in the phenolic mono and diacylglycerols, 64.9 and 59.5%, respectively, as compared to the original flaxseed oil, 53.1%. The radical scavenging ability of phenolic lipids was significant; however, it was about half than that of alpha-tocopherol.

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems.

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.