An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.

Chemical and chemoenzymatic routes to bridged homoarabinofuranosylpyrimidines: Bicyclic AZT analogues.

Conformationally restricted diastereomeric homoarabinofuranosylpyrimidines (AZT analogue), i.e., (5'R)-3'-azido-3'-deoxy-2'-O,5'-C-bridged-ß-ᴅ-homoarabinofuranosylthymine and -uracil had been synthesized starting from diacetone ᴅ-glucofuranose following chemoenzymatic and chemical routes in 34-35% and 24-25% overall yields, respectively. The quantitative and diastereoselective acetylation of primary hydroxy over two secondary hydroxy groups present in the key nucleoside precursor was mediated with Lipozyme® TL IM in 2-methyltetrahydrofuran following a chemoenzymatic pathway. Whereas, the protection of the primary hydroxy over the lone secondary hydroxy group in the key azido sugar precursor was achieved using bulky tert-butyldiphenylsilyl chloride (TBDPS-Cl) in pyridine in 92% yield following a chemical synthetic pathway. The chemoenzymatic method was found to be superior over the chemical method in respect of the number of synthetic steps and overall yield of the final product.

Synthesis and Characterization of N-Methyl Fatty Hydroxamic Acids from Ketapang Seed Oil Catalyzed by Lipase.

N-methyl fatty hydroxamic acid (N-MFHA), which is a derivative of hydroxamic acid (HA), was synthesized from ketapang seed oil (Terminalia catappa L.). In general, HAs have wide applications due to their chelating properties and biological activities. N-MFHAs were synthesized using immobilized lipase (Lipozyme TL IM) in biphasic medium which was the ketapang seed oil dissolved in hexane and N-methylhydroxylamine dissolved in water. The products were characterized through color testing and FT-IR spectroscopy after purification. Various factors affecting the enzyme activity investigated in the study included the effect of incubation time, the amount of lipase used, and the temperature. On the basis of the results, the optimum conditions for the synthesis of N-MFHA obtained are 25 h of incubation time, a temperature of 40 °C, and a ratio of 1:100 for the amount of enzyme (g)/oil (g). At the optimum conditions of the reaction, 59.7% of the oils were converted to N-MFHA.

Effective enzymatic caffeoylation of natural glucopyranosides.

Reaction system was developed for enzymatic caffeoylation of model saccharidic acceptor methyl ß-d-glucopyranoside to obtain exclusively methyl 6-O-caffeoyl-ß-D-glucopyranoside. Reaction with starting concentration of acceptor 0.2 M provided 73% yield of purified product within 17 days. Reactions with low acceptor concentrations (0.04 and 0.08 M) run to the completion within 7 days. Such highly effective and regioselective reaction was promoted by Lipozyme TL IM in tert-butanol, using vinyl caffeate as acylation donor. The optimized reaction conditions were used in preparative caffeoylation of natural substances-arbutin and salidroside, giving 75% of 6-O-caffeoylated arbutin (robustaside B) and 74% of 6-O-caffeoylated salidroside as the only products after 12 and 16 days, respectively.

Lipase-mediated lipid removal from propolis extract and its antiradical and antimicrobial activity.

BACKGROUND: Propolis contains many antioxidants such as polyphenols and flavonoids. However, propolis-derived lipid components interrupt an efficient isolation of antioxidants from propolis extract. We examined the effectiveness of various lipase treatments for the removal of lipids from propolis extract and evaluated the biological features of the extract. RESULTS: Lipase OF and Novozyme 435 treatments did not reduce fatty acid level in propolis extract. However, Lipozyme TL IM-treated propolis extract showed a significant decrease in fatty acid level, suggesting the removal of lipids. Lipozyme RM IM also significantly decreased the fatty acid level of the extract, but was accompanied by the reduction of polyphenols and flavonoids, which are antioxidants. In Lipozyme TL IM treatment, an increase in active flavonoids, such as Artepillin C and kaempferide, was observed, with a slight increase of ferric reducing/antioxidant power (FRAP) radical-scavenging activity. In addition, antimicrobial activity towards skin health-related bacteria such as Staphylococcus epidermidis and Propionibacterium acnes was enhanced by Lipozyme TL IM treatment. CONCLUSION: Lipozyme TL IM treatment effectively removes lipids from propolis extract and enhances antibacterial activity. Therefore, we suggest that Lipozyme TL IM is a useful lipase for lipid removal of propolis extract.

Effect of acyl chain length on selective biocatalytic deacylation on O-aryl glycosides and separation of anomers.

It has been demonstrated that Lipozyme® TL IM (Thermomyces lanuginosus lipase immobilised on silica) can selectively deacylate the ester function involving the C-5' hydroxyl group of α-anomers over the other acyl functions of anomeric mixture of peracylated O-aryl α,ß-D-ribofuranoside. The analysis of results of biocatalytic deacylation reaction revealed that the reaction time decreases with the increase in the acyl chain length from C1 to C4. The unique selectivity of Lipozyme® TL IM has been harnessed for the separation of anomeric mixture of peracylated O-aryl α,ß-D-ribofuranosides, The lipase mediated selective deacylation methodology has been used for the synthesis of O-aryl α-D-ribofuranosides and O-aryl ß-D-ribofuranosides in pure forms, which can be used as chromogenic substrate for the detection of pathogenic microbial parasites containing glycosidases.

Impact of solvent and water activity on lipase selectivity and acyl migration of ARA-rich 2-monoacylglycerols in catalytic systems: Kinetic study by particle swarm optimization.

The 2-arachidonoylglycerol enzymatic alcoholysis reaction model was established and kinetic parameters were calculated to explore the effects of solvent and water activity (aw) on the lipase positional selectivity and 2-monoacylglycerol acyl migration. Six rate constants (k1-k6) with the lowest mean square error were obtained using the particle swarm optimization algorithm, and the calculated molar concentration-time curves were well-fitted to the actual curves. As an indicator to characterize the positional selectivity of lipase, k5/k3 was significantly associated with log P of solvent, which first increased and then decreased with the increase of aw. The highest sn-1,3 selectivity of Lipozyme TL IM was found at the aw of 0.53. The changes of acyl migration with the solvent and aw in the enzymatic and non-catalytic systems showed a consistent law. This study provides theoretical support for the targeted synthesis of structural lipids and enzymatic production of diverse structural lipid products.

Production of Margarine Fat Containing Medium- and Long-Chain Triacylglycerols by Enzymatic Interesterification of Peony Seed Oil, Palm Stearin and Coconut Oil Blends.

This paper reports the preparation of margarine fat using Lipozyme TL IM as a catalyst and peony seed oil (PSO), palm stearin (PS) and coconut oil (CO) as raw materials. The results indicate that there were no significant changes in fatty acid composition before or after interesterification of the oil samples. However, the total amount of medium- and long-chain triglycerides (MLCTs) increased from 2.92% to 11.38% in sample E1 after interesterification, mainly including LaLaO, LaMO, LaPM, LaOO, LaPO and LaPP. Moreover, the slip melting point (SMP) of sample E1 decreased from 45.9 °C (B1) to 33.5 °C. The solid fat content (SFC) of all the samples at 20 °C was greater than 10%, indicating that they could effectively prevent oil exudation. After interesterification, the samples exhibited a ß' crystal form and could be used to prepare functional margarine.

From Hamamelitannin Synthesis to the Study of Enzymatic Acylations of D-Hamamelose.

The bioactive natural substance, hamamelitannin, was effectively synthesized in two ways. The chemical acylation of 2,3-O-isopropylidene-α,ß-D-hamamelofuranose promoted by Bu2SnO using 3,4,5-tri-O-acetylgalloyl chloride, followed by the deprotection provided hamamelitannin in 79%. Pilot enzymatic benzoylation of D-hamamelose using vinyl benzoate (4 equiv.) and Lipozyme TL IM as a biocatalyst in t-butyl methyl ether (t-BuMeO) gave mainly benzoylated furanoses (89%), of which tribenzoates reached (52%). Enzymatic galloylation of 2,3-O-isopropylidene-α,ß-D-hamamelofuranose with vinyl gallate under the catalysis of Lipozyme TL IM in t-butyl alcohol (t-BuOH) or t-BuMeO provided only the 5-O-galloylated product. The reaction in t-BuMeO proceeded in a shorter reaction time (61 h) and higher yield (82%). The more hydrophobic vinyl 3,4,5-tri-O-acetylgallate in the same reactions gave large amounts of acetylated products. Vinyl gallate and triacetylgallate in the enzymatic acylation of D-hamamelose with Lipozyme TL IM in t-BuMeO yielded 2',5-diacylated hamamelofuranoses in a yield below 20%. The use of other vinyl gallates hydrophobized by methylation or benzylation provided 2',5-diacylated hamamelofuranoses in good yields (65-84%). The reaction with silylated vinyl gallate did not proceed. The best results were obtained with vinyl 2,3,5-tri-O-benzyl gallate, and the only product, 2',5-diacylated hamamelofuranoside precipitated from the reaction mixture (84% in 96 h). After debenzylation, hamamelitannin was obtained an 82% yield from hamamelose in two steps. This synthesis is preparatively undemanding and opens the way to multigram preparations of bioactive hamamelitannin and its analogues.

Insight into the medium-long-medium structured lipids made from Camellia oil: Composition-structure relationship.

Medium-long-medium (MLM) structured lipid (SL) as a new SL is a potential functional ingredient in food and nutraceutical products, but its composition-structure-physicochemical properties relationship has not been revealed in food industry. MLM type of medium-long chain triacylglycerol (MLCT) was synthesized from Camellia oil by combi-lipase; its physicochemical properties and composition-structure relationship were investigated in this research. The higher MLCT (67.24% ± 0.09) and MLM (52.71% ± 0.53) productivities were achieved after parameter optimization. The physicochemical characterization of SLs exhibited mild thermal property, intermediate Fourier transform infrared spectroscopy absorption intensity, and better crystal morphology. Joint characterizations identified that MLM and long-medium-long type SL were rich in 1,3-dioctanoyl-2-linoleoyl glyceride (CaLCa), 1,3-dioctanoyl-2-oleoyl glyceride (CaOCa), 1,3-dilinoleoyl-2-octanoyl glyceride (LCaL), and 1,3-dilinoleoyl-2-decanoyl glyceride (LCL) components, respectively. This is ascribed to the higher proportion of caprylic and linoleic acid in 1,3-specific enzyme. The 3D structural analysis further demonstrated that the CaLCa, CaOCa, LCaL, and LCL molecules had lower steric energy to form symmetrical structure at 1,3-position. This research provides a practical method to produce MLM-type SL from edible oils and fats in food industry.

Improved Synthesis of α-Glycerol Monolaurate Using Lipozyme TL IM.

This study aimed to synthesize α-Glycerol Monolaurate from protected glycerol (glycerol 1,2-acetonide) using Lipozym TL IM as a catalyst. In the first step, transesterification of methyl laurate and glycerol 1,2-acetonide with Lipozyme TL IM produced 1,2-acetonide-3-lauryl glycerol. In the second step, deprotection of 1,2-acetonide-3-lauryl glycerol with Amberlyst-15 produced α-Glycerol Monolaurate. Furthermore, the optimum yield (82.1%) of 1,2-acetonide-3-lauryl glycerol (light yellow liquid, purity of 92%) was achieved at a reactant mole ratio of 1, n-hexane (4 mL) with a reaction time of 12 hours, and total Lipozyme TL IM of 5% (w/w of the total weight of reactants) at a temperature of 35°C. Deprotection of 1,2-acetonide-3-lauryl glycerol with Amberlyst-15 was conducted at room temperature for 24 hours. At a melting point of 62.8°C, and purity of 100% α-Glycerol Monolaurate in the form of a white solid was obtained with a yield of 74.6% after the recrystallization of the crude product. This α-glycerol monolaurate synthesis reaction pathway can be referred to as a green α-monoacylglyceride synthesis method.

Enzymatic interesterification effect on the physicochemical and technological properties of cupuassu seed fat and inaja pulp oil blends.

The objective of this work was to evaluate the effect of enzymatic interesterification process in blends with different proportions (w:w) of cupuassu fat and inaja oil (80:20, 70:30, 60:40, 50:50 and 40:60). The interesterification reaction was carried out at 65 °C, agitation at 150 rpm, and enzyme concentration of 5% (w/w), for 6 h. Acidity index, melting point, consistency and solid fat content of the blends were characterized before and after the interesterification process. Fatty acid content was characterized in cupuassu fat and inaja oil and, nutritional quality indexes of atherogenicity (AI) and thrombogenicity (TI) were calculated. Enzymatic interesterification promoted a decrease in acidity (<0.6%) and changes in the blends' properties, making them suitable for food product preparation. All esterified blends (cupuassu seed fat:inaja pulp oil) presented suitable consistency properties, plasticity and spreadability to be used for the preparation of functional, table and soft table types of margarine and used in food preparation such as special fats.

Separation of saturated fatty acids from docosahexaenoic acid-rich algal oil by enzymatic ethanolysis in tandem with molecular distillation.

Algal oil, rich in docosahexaenoic acid (DHA) and an environmentally sustainable source of ω-3 fatty acids, is receiving increasing attention. In the present study, a novel approach combining ethanolysis with a 1,3-specific immobilized lipase (Lipozyme® TL IM) and molecular distillation was investigated to increase the DHA content of algal oil. Algal oil with a 45.94% DHA content was mixed with ethanol, pumped into a column filled with Lipozyme® TL IM, and then circulated for 4 hr at room temperature. The ethanol was then recycled by vacuum distillation. At an evaporator temperature of 150°C, the residue was separated by molecular distillation into a heavy component enriched with DHA glycerides (in the form of triglyceride (TG), diglyceride (DG), and monoglyceride (MG)) and a light component enriched with palmitic acid (PA) and DHA ethyl ester (EE). As a result, 76.55% of the DHA from the algal oil was present in the heavy component, whose DHA content was 70.27%. DHA-MG was collected in the heavy component mostly in the form of 1-MG. Lipozyme® TL IM appeared to specifically target PA rather than DHA at the sn-1(3) position. The Lipozyme® TL IM allowed 90.03% of the initial DHA yield to be retained after seven reaction cycles. Therefore, an eco-friendly and simple method for increasing the DHA content in algal oil has been developed.

Production of stearidonic acid-rich triacylglycerol via a two-step enzymatic esterification.

The aim of this study was to synthesize stearidonic acid (SDA)-rich triacylglycerol (TAG) via a two-step lipase-catalyzed esterification under vacuum. SDA-rich fatty acid, which was prepared from echium oil via Candida rugosa lipase-catalyzed selective esterification, was used as the substrate. Two different immobilized lipases, Novozym 435 from Candida antarctica and Lipozyme TL IM from Thermomyces lanuginosus, were employed for the synthesis of SDA-rich TAG. In the first step, Novozym 435-catalyzed esterification of the SDA-rich fatty acid with glycerol was carried out for 2 h. In the second step, Lipozyme TL IM-catalyzed esterification of the reaction mixture from the first step was performed for an additional 10 h. The optimal reaction conditions for the second step were a temperature of 65 °C, an enzyme loading of 20%, and a vacuum of 0.7 kPa. Consequently, the maximum TAG conversion of ca. 86.4 wt% was obtained after 12 h via a two-step lipase-catalyzed esterification.

Lipase-catalyzed Production and Purification of Palm Esters Using Stirred Tank Reactors (STR).

Lipase-catalyzed production of palm esters was performed via alcoholysis of palm oil and oleyl alcohol in solvent and solvent-free systems using a 2 L stirred tank reactor (STR). Two immobilized lipases were tested and Lipozyme RM IM exhibited superior performance in both reaction systems. Reusability studies of the enzymes in a solvent-free system also demonstrated the high stability of Lipozyme RM IM as shown by its ability to yield more than 70% palm esters with up to 19 cycles of reusing the same enzymes. Modification of the enzyme washing process improved the stability of Lipozyme TL IM in a solvent system as demonstrated by maintaining 65% yield after 5 times of repeated enzyme use. The scale up process for both lipases was conducted in the presence of solvents by using the impeller tip speed approach. Lipozyme RM IM-catalyzed reaction in a 15 L STR produced 85.7% yield and there was a significant drop to 60.7% in the 300 L STR, whereas Lipozyme TL IM had a lower yield (65%) when the reaction volume was increased to 15 L. The low yields could be due to the accumulation of enzymes at the bottom of the vessel. Purification of palm esters via solvent-solvent extraction revealed that more than 90% of oleyl alcohol was extracted after the third extraction cycle at 150 rpm impeller speed with reduced palm esters: ethanol ratio (v/v) from 1:4 to 1:3.

Enzymatic synthesis of lysophosphatidylcholine with n-3 polyunsaturated fatty acid from sn-glycero-3-phosphatidylcholine in a solvent-free system.

The n-3 polyunsaturated fatty acids (PUFA)-rich lysophosphatidylcholine (LPC) was successfully synthesized by Thermomyces lanuginosus lipase (TL IM)-catalyzed esterification of glycerylphosphorylcholine (GPC) and n-3 PUFA-rich fatty acids in a solvent-free system. Effects of reaction temperature, enzyme loading and substrate mole ratio on the yield of LPC and incorporation of n-3 PUFA were evaluated. The acyl-specificities of five enzymes were tested for direct esterification of n-3 PUFA, and Lipozyme TL IM was found to be more effective than others for production of LPC with n-3 PUFA. Substrate mole ratio and reaction temperature, however, had no significant effect on the incorporation. The maximal yield of LPC was obtained under the following conditions: temperature 45°C, enzyme loading 15% by weight and substrate mole ratio (GPC/n-3 PUFA) 1:20. Furthermore, the composition of products were further investigated in the study. The 1-acyl-sn-glycero-3-lysophosphatidylcholine (2-LPC) was predominant in the mixtures at early stages of reaction, whereas less increment of 2-acyl-sn-glycero-3-lysophosphatidylcholine (1-LPC) and PC was observed at later stages.

The combined use of ultrasound and molecular sieves improves the synthesis of ethyl butyrate catalyzed by immobilized Thermomyces lanuginosus lipase.

In this work, the combined use of ultrasound energy and molecular sieves was investigated for the synthesis of ethyl butyrate, ester with mango and banana notes, catalyzed by the immobilized lipase from Thermomyces lanuginosus (Lipozyme TL-IM). Initially, the best concentrations of biocatalysts (35%) and butyric acid (0.7M) were tested using ultrasound as an alternative to mechanical agitation. The amount of acid in the reaction could be increased by 2-fold when compared to previous works where mechanical agitation was used. In the next step, substrate molar ratio and reaction temperature were optimized and the best conditions were at their lowest levels: 1:1 (acid:alcohol), and 30°C, reaching 61% of conversion in 6h. Molecular sieves (3Å) were added to optimized reaction medium in order to remove the formed water and improve the maximum yield. The reaction yield increased 1.5 times, reaching 90% of conversion in 6h, when 60mg of molecular sieves per mmol of butyric acid was used. Finally, the reuse of Lipozyme TL-IM for the ultrasound-assisted synthesis of ethyl butyrate was verified for 10 batches, without any appreciable loss of activity, whereas in systems using mechanical agitation, the biocatalyst was completely inactivated after 5 batches. These results suggest that the combined use of ultrasound and molecular sieves greatly improve esterification reactions by stabilizing the enzyme and increasing yields.

Lipase-catalysed interesterification between canola oil and fully hydrogenated canola oil in contact with supercritical carbon dioxide.

The processing parameters in enzymatic reactions using CO2-expanded (CX) lipids have strong effects on the physical properties of liquid phase, degree of interesterification, and physicochemical properties of the final reaction products. CX-canola oil and fully hydrogenated canola oil (FHCO) were interesterified using Lipozyme TL IM in a high pressure stirred batch reactor. The effects of immobilised enzyme load, pressure, substrate ratio and reaction time on the formation of mixed triacylglycerols (TG) from trisaturated and triunsaturated TG were investigated. The optimal immobilised enzyme load, pressure, substrate ratio and time for the degree of interesterification to reach the highest equilibrium state were 6% (w/v) of initial substrates, 10 MPa, blend with 30% (w/w) of FHCO and 2h, respectively. The physicochemical properties of the initial blend and interesterified products with different FHCO ratios obtained at optimal reaction conditions were determined in terms of TG composition, thermal behaviour and solid fat content (SFC). The amounts of saturated and triunsaturated TG decreased while the amounts of mixed TG increased as a result of interesterification. Thus, the interesterified product had a lower melting point, and broader melting and plasticity ranges compared to the initial blends. These findings are important for better understanding of CX-lipid reactions and for optimal formulation of base-stocks of margarine and confectionary fats to meet industry demands.