Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l^?1 sucrose, 0.3 mol l^?1 vinyl palmitate), a maximum of 45 g l^?1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l^?1 in 120 h using 20% DMSO with 0.2 mol l^?1 sucrose and 0.6 mol l^?1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.     

Synthesis of mixed esters of ascorbic acid using methyl esters of palm and soybean oils

Mixed esters of ascorbic acid were synthesized using methyl esters of palm and soybean oils as acyl donors, in acetone at 50 degrees C, and catalyzed by Novozym 435. A conversion of 62% was obtained with palm oil methyl ester at an ascorbic acid to acyl donor molar ratio of 1:4; the mixed ester contained 45.89% ascorbyl palmitate, 42.59% ascorbyl oleate and 10.1% ascorbyl linoleate. Acylation with soybean oil methyl ester resulted in 17% conversion, yielding a mixed ester containing 10.08% ascorbyl palmitate, 20.68% ascorbyl oleate, and 64.96% of ascorbyl linoleate. The mixed esters of ascorbic acid can find direct use in food and cosmetics.     

Lipase-catalyzed regioselective synthesis of steryl (6′-<Emphasis Type="Italic">O</Emphasis>-acyl)glucosides

The regioselective acylation of cholesteryl β-d-glucoside, at the C-6 of the glucose moiety, was achieved using microbial lipases in organic solvents. With palmitic acid as an acyl donor 81 or 63% conversions of cholesteryl glucoside to its 6′-O-palmitoyl derivative were obtained using Candida antarctica or Rhizomucor miehei enzymes, respectively. High yields (64–92%) were also obtained with fatty acids 6:0–22:0 and 16:1 (n-7). The synthesis of cholesteryl (6′-O-palmitoyl)glucoside was also achieved via transesterification, using mono-, di- and tri-palmitoylglycerols or methyl and ethyl palmitate as acyl sources. With R. miehei lipase transesterification between methyl palmitate (80 mM) and cholesteryl glucoside (1 mM) proceeded after 24 h with a nearly quantitative yield (97%).     

Enzymic synthesis of fructose monooleate in a reduced pressure pilot scale reactor using various acyl donors

Enzymic synthesis of fructose esters was studied under reduced pressure. Different acyl donors were tested, and immobilized Candida antarctica lipase was used as biocatalyst. Influences of pressure, nature of the acyl donor, molar ratio sugar/acyl donor were investigated. Pressure had the greatest influence. At 200 mbar, more than 90% of fructose was acylated compared to 50% under atmospheric pressure. This is explained by the evaporation of reaction by-product (methanol or water) that shifted the equilibrium. C. antarctica lipase catalyzed sugar ester synthesis very efficiently using rapeseed oil as acyl donor. Moreover, synthesis performed with an equimolar mixture of both substrates gave promising results. Although the reaction rate was slower than synthesis performed with an excess of fatty acid, fructose monooleate concentration was still high (44 g l⁻¹ instead of 56 g l⁻¹) and the residual acyl donor concentration was very low. Downstream processes for the recovery of pure fructose monooleate were simplified in this case.     

Production of ascorbic acid glucoside by alginate-entrapped mycelia of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

α-Chymotrypsin-Catalyzed Peptide Synthesis Using <Emphasis Type="Italic">N</Emphasis>-Protected <Emphasis Type="SmallCaps">D</Emphasis>-Amino Acid Carbamoylmethyl Esters as Acyl Donors

α-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml⁻¹; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h.     

Resolution of d,l-menthol by interesterification with triacetin using the free and immobilized lipase of Candida cylindracea

Summary Interesterification in isooctane with triacetin as an acyl donor was found to be a new and effective method of racemic resolution of d,l-menthol, when using the free and immobilized lipase of Candida cylindracea. No water was produced by this highly stereoselective type of reaction in contrast to ester synthesis with acetic acid as an acyl donor. Even with diacetin no possible back reaction occurred and the enzyme was easily separated from the reaction solution as opposed to ester hydrolysis in aqueous systems. Inhibition of interesterification was caused by increasing concentrations of the acyl donor triacetin by more than 10 mmol·l^-1 on the one hand, and especially by diacetin on the other hand. The reaction product menthyl acetate had no influence. By adding water the interesterification activity of the lipase was reduced significantly. An alteration of the acyl donor triacetin to longerchained triglycerides caused changes in higher specific activities but poor enantioselectivities of the products, as in the case of ester synthesis starting from longer-chained organic acids.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 60th birthday     

Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor

The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.     

Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides

Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.     

Fatty Acid Specificity and Selectivity of the Chloroplast sn-Glycerol 3-Phosphate Acyltransferase of the Chilling Sensitive Plant, Amaranthus lividus

Chilling sensitivity of plants is strongly correlated with the presence of high levels of a species of chloroplast phosphatidylglycerol that contains two saturated fatty acids. The most straightforward synthetic pathway for this lipid would require the primary acylation of sn-glycerol 3-phosphate (G3P) with a saturated fatty acid (palmitic acid) rather than with oleic acid, an unsaturated acid. This selective incorporation would differ markedly from the reported properties of the chloroplast G3P acyltransferases of pea and spinach, two chilling resistant plants and thus we have studied the chloroplast G3P acyltransferase of Amaranthus lividus, a chilling sensitive plant. In contrast to our results and those of others (M. Frentzen et al. 1983 Eur J Biochem 129: 629-636 and previous work) with the pea and spinach enzymes, the amaranthus chloroplast G3P acyltranferase did not select oleic acid donors from a mixture of oleic and palmitic acid donors (either coenzyme A or acyl carrier protein thioesters). Instead the fatty acid composition of the synthesized 1-acyl G3P faithfully reflected the composition of the acyl donor mixture. However, the amaranthus enzyme did strongly select against incorporation of stearic acid. The properties of the amaranthus G3P acyltransferase are consistent with this enzyme having the major role in synthesis of the disaturated phosphatidylglycerol species.     

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-¹⁴C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentK _m values andV _max values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentK _m and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.     

Application of several types of substrates to ficin-catalyzed peptide synthesis

Summary. The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72–96% yield using 10 different substrates, within a few minutes of reaction time. Authors’ address: Prof. Haruo Sekizaki, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan     

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.     

A comparison of the metabolic fate of Fatty acids of different chain lengths in developing oilseeds

To determine if medium and long chain fatty acids can be appropriately metabolized by species that normally produce 16 and 18 carbon fatty acids, homogenates of developing Cuphea wrightii, Carthamus tinctorius, and Crambe abyssinica seeds were incubated with radiolabeled lauric, palmitic, oleic, and erucic acids. In all three species, acyl-CoA synthetase showed broad substrate specificity in synthesis of acyl-coenzyme A (CoA) from any of the fatty acids presented. In Carthamus, two- to fivefold less of the foreign FAs, lauric, and erucic acid was incorporated into acyl-CoAs than palmitic and oleic acid. Lauric and erucic acid also supported less glycerolipid synthesis in Carthamus than palmitic and oleic acid, but the rate of acyl-CoA synthesis did not control rate of glycerolipid synthesis. In all species examined, medium and long chain fatty acids were incorporated predominantly into triacylglycerols and were almost excluded from phospholipid synthesis, whereas palmitic and oleic acid were found predominantly in polar lipids. However, the rate of esterification of unusual fatty acids to triacylglycerol is slow in species that do not normally synthesize these acyl substrates.     

Substrate Selectivity of Glycerol-3-phosphate Acyl Transferase in Rice

Substrate selectivity of glycerol-3-phosphate acyltransferase (EC 2. 3. 1. 15) of rice ( Oryza sativa L.) was explored in a comparative study of acyltransferases from seven plant species. In vitro labeling of acyl carrier protein (ACP) with ¹⁴C or ³H showed that acyltransferase from chill-sensitive plants, such as rice that uses either oleic (18:1) or palmitic acid (16:0) as acyl donor at comparable rates, displays lower selectivity than the enzyme from chill-resistant plants, such as spinach, which preferentially uses oleic acid (18:1) rather than palmitic acid (16:0) as an acyl donor. This may be a result of the size and character of the substrate-binding pocket of acyltransferase. Homology modeling and protein structure-based sequence alignment of acyltransferases revealed that proteins from either chill-sensitive or chill-tolerant plants shared a highly conserved domain containing the proposed substrate-binding pocket. However, the aligned residues surrounding the substrate-binding pocket are highly heterogeneous and may have an influence mainly on the size of the substrate binding pockets of acyltransferases. The substrate selectivity of acyltransferase of rice can be improved by enlarging the substrate-binding pocket using molecular biological methods.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.     

Efficient kinetic resolution of dl-menthol by lipase-catalyzed enantioselective esterification with acid anhydride in fed-batch reactor

Acid anhydrides were used as highly reactive and non-water-producing acyl donors for hydrolase-catalyzed enantioselective esterification. Efficient kinetic resolution of dl-menthol has been achieved via lipase-catalyzed enantioselective esterification in cyclohexane when propionic anhydride as an acyl donor was continuously fed into a reactor containing dl-menthol and Candida cylindracea lipase OF 360, while a high concentration of the acid anhydride in a batch reaction system with a dehydrated organic solvent did not facilitate the reaction, because water necessary for the enzyme function was consumed by the competing hydrolysis of the anhydride catalyzed by the same enzyme. The efficiency of this fed-batch reaction system using acid anhydride was higher and the enzyme stability in repeated use was much better than those of conventional batch and fed-batch reaction systems using propionic acid as an acyl donor. The optical purity (more than 98% e.e.) of the l-menthyl ester produced in the fed-batch system using the anhydride was comparable to that in the system using the corresponding acid. *** DIRECT SUPPORT *** AG903062 00002     

生物法合成维生素C棕榈酸酯

研究了不同的脂肪酶在有机溶剂体系中催化合成L-维生素C棕榈酸酯的反应。针对维生素C在有机溶剂中溶解度较低这一问题,对催化合成维生素C棕榈酸酯反应的脂肪酶和反应介质进行比较,同时对影响合成维生素C棕榈酸酯反应的因素（温度、底物浓度、底物摩尔比、反应时间和酶量等）进行探讨,优化了反应条件：在10mL的丙酮中,1.094g棕榈酸与0.107g维生素C在酶量为20％（W/W, 固定化酶/维生素C）的固定化脂肪酶催化下,初始含0.4nm分子筛20%,温度为60℃,转速为200r/min,反应48h转化率可以达到80%,产物维生素C棕榈酸酯的浓度可达20g/L。     

Enzymatic modification of cassava starch by bacterial lipase

Enzymatic modification of starch using long chain fatty acid makes it thermoplastic suitable for a myriad of industrial applications. An industrial lipase preparation produced by Burkholderia cepacia (lipase PS) was used for modification of cassava starch with two acyl donors, lauric acid and palmitic acid. Reactions performed with palmitic acid by liquid-state and microwave esterification gave a degree of substitution (DS) of 62.08% (DS 1.45) and 42.06% (DS 0.98), respectively. Thermogravimetric analysis showed that onset of decomposition is at a higher temperature (above 600°C) for modified starch than the unmodified starch (280°C). Modified starch showed reduction in α-amylase digestibility compared to native starch (76.5–18%). Swelling power lowered for modified starch as esterification renders starch more hydrophobic, making it suitable for biomedical applications as materials for bone fixation and replacements, carriers for controlled release of drugs and bioactive agents. Thus enzymatic esterification is ecofriendly.