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.     

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger

Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL “Amano” produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 °C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by ¹H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.     

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger

Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL "Amano" produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 degrees C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by (1)H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.     

Enzymatic synthesis of geraniol esters in a solvent-free system by lipases

Geraniol esters were synthesised by direct esterification catalysed by esterases and lipases (five enzymes were tested) in a solvent-free system at 37°C. The best conversions yields, about 85%, on geranyl butyrate and valerate obtained with esterase 30 000 from Mucor miehei. The effect of substrate molar ratio alcohol/acid variation was studied. A study of the water production was made in parallel during the esterification reaction.     

Synthesisof fructose laurate esters catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst in a non-aqueous system

Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose, temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4Å molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.

     

Production of mannitol usingLeuconostoc mesenteroides NRRL B-1149

A process for the production of mannitol from fructose (5% to 25%) usingLeuconostoc mesenteroides NRRL B-1149 was investigated. Fermentations were carried out in batch or fed-batch fermentations without aeration at 28°C, pH 5.0. When 5% fructose was used in batch culture fermentation, the yield of mannitol was 78% of that expected theoretically. When the fructose concentration was increased to 10%, the yield dropped to 59.6% of the theoretical value. However, in the fed-batch culture, using 10% fructose, the yield was 81.9% of the theoretical value. In a 15% fructose fed-batch culture, with 5% fructose being added initially and the other 10% fructose being added as a continuous supply, the final yield was 83.7% of the theoretical yield. When 20% fructose was used in the same manner, the yield was 89.5% of theoretical yield.     

The immediate effects of insulin and fructose on the metabolism of the perfused liver. Changes in lipoprotein secretion, fatty acid oxidation and esterification, lipogenesis and carbohydrate metabolism

1. When livers from fed rats were perfused with blood containing elevated concentrations of rat insulin or blood to which fructose was added, the oxidation of free fatty acids was depressed and their esterification was increased. 2. Raised concentrations of insulin or addition of fructose increased secretion of triglyceride in very-low-density lipoproteins, but only insulin caused more of the free fatty acids taken up by the liver to be incorporated into very-low-density lipoproteins. 3. When insulin and fructose were added together the combined effect on oxidation and esterification of free fatty acids and on secretion of very-low-density lipoproteins was equal to the sum of the effects of either alone. No statistically significant interaction between the effects of fructose and insulin was found for any of the parameters investigated. 4. Bovine insulin had similar effects, in most respects, to comparable studies with raised concentrations of rat insulin. 5. Lipogenesis was increased in the livers treated with fructose plus bovine insulin. 6. A significant proportion of the fatty acids in very-low-density lipoproteins were derived either from the liver triglyceride pool or from lipogenesis. This fraction was increased both by treatment with insulin or fructose, and was augmented further when both insulin and fructose were present together. 7. The uptake of fructose by the perfused liver was similar to that found in vivo. It was unaffected by the presence of insulin. 8. Addition of fructose to the perfused liver caused perfusate lactate concentrations to increase, as a result of diminished hepatic uptake of lactate. 9. The uptake of free fatty acids by the perfused liver was unaffected by the addition of either insulin or fructose. 10. The distribution among the various lipid classes in plasma lipoproteins of label arising from the hepatic uptake of [(14)C]oleate was unaltered by the addition of either fructose or insulin. 11. It is suggested that the effects described are due principally to control of the balance between esterification of fatty acids and lipolysis of the ensuing triglyceride, fructose enhancing esterification and insulin inhibiting lipolysis.     

Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-hish speed supernatant system.

The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.     

Application of a substrate inhibition model to estimate the effect of fructose concentration on the growth of diverse <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis> strains

In this study, we performed an analysis of the ability of four Saccharomyces cerevisiae and one S. bayanus var. uvarum strains, isolated from different industrial processes, to ferment increasing amounts of fructose (from 0 to 70%, w/v). Overall yeast growth was estimated by integration of the area under optical density vs. time curves. Subsequently, this parameter was modeled by means of a substrate inhibition model. All strains showed a similar behavior against fructose concentration in spite of their different origins, but with slight differences. The optimum fructose concentrations to stimulate yeast growth were obtained between 4.33 and 6.05%, while the maximum concentrations above which growth was completely inhibited were attained between 59.56 and 63.85%. Statistically, model parameters calculated for wine yeast strains were significantly different than those obtained for yeasts from Agave and table olive fermentations, except for the maximum inhibitory concentration. The methodology used in this work could be useful for the industry in general as a first procedure to select yeast strains with higher fructose preferences or tolerances, and especially for winemakers, where the risk of spoilage increases by the presence of a marked residual fructose concentration in the finished wine.     

Chemical Defense in Millipedes (Myriapoda,Diplopoda): Do Representatives of the Family Blaniulidae Belong to the ‘Quinone’ Clade?

The defensive secretions of two blaniulid millipedes, Nopoiulus kochii and Cibiniulus phlepsii, were characterized by GC‐FID and GC/MS analyses, which showed the presence of a complex mixture of benzoquinones, hydroquinones, and oleates. Altogether, 13 compounds were identified. The major compound in the secretions of both analyzed species was 2‐methyl‐1,4‐benzoquinone (toluquinone). The second major constituent in the N. kochii secretion was 2‐methyl‐3,4‐(methylenedioxy)phenol, while in that of C. phlepsii, it was 2‐methoxy‐3‐methyl‐1,4‐benzoquinone. The defensive secretion of N. kochii also showed a high content of hydroquinones (13.5%) in comparison to that of C. phlepsii (0.8%). Hexyl oleate and octyl oleate were detected for the first time in defensive millipede fluids. The chemical composition of the defensive secretions supports the chemotaxonomic position of the family Blaniulidae in the ‘quinone’ millipede clade.     

Continuous production of fructose syrup and ethanol from hydrolysed Jerusalem artichoke juice

Summary The results from this study showed that Jerusalem artichoke juice can be used for the production of very enriched fructose syrup by selective conversion of glucose to ethanol in a continuous process using immobilized cells ofSaccharomyces cerevisiae ATCC 36859. The product contained up to 99% of the total carbohydrates as fructose compared to 76% in the feed. Using Jerusalem artichoke juice supplemented with some glucose a product was obtained with 7.5% w/v ethanol which made ethanol recovery economically favourable. It was found that some fructose was consumed in these continuous processes; the glucose/fructose conversion rate ratio was regulated by the glucose concentration in the product stream.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

A Grape Seed Procyanidin Extract Ameliorates Fructose-Induced Hypertriglyceridemia in Rats via Enhanced Fecal Bile Acid and Cholesterol Excretion and Inhibition of Hepatic Lipogenesis

The objective of this study was to determine whether a grape seed procyanidin extract (GSPE) exerts a triglyceride-lowering effect in a hyperlipidemic state using the fructose-fed rat model and to elucidate the underlying molecular mechanisms. Rats were fed either a starch control diet or a diet containing 65% fructose for 8 weeks to induce hypertriglyceridemia. During the 9^th week of the study, rats were maintained on their respective diet and administered vehicle or GSPE via oral gavage for 7 days. Fructose increased serum triglyceride levels by 171% after 9 weeks, compared to control, while GSPE administration attenuated this effect, resulting in a 41% decrease. GSPE inhibited hepatic lipogenesis via down-regulation of sterol regulatory element binding protein 1c and stearoyl-CoA desaturase 1 in the fructose-fed animals. GSPE increased fecal bile acid and total lipid excretion, decreased serum bile acid levels and increased the expression of genes involved in cholesterol synthesis. However, bile acid biosynthetic gene expression was not increased in the presence of GSPE and fructose. Serum cholesterol levels remained constant, while hepatic cholesterol levels decreased. GSPE did not modulate expression of genes responsible for esterification or biliary export of the newly synthesized cholesterol, but did increase fecal cholesterol excretion, suggesting that in the presence of GSPE and fructose, the liver may secrete more free cholesterol into the plasma which may then be shunted to the proximal small intestine for direct basolateral to apical secretion and subsequent fecal excretion. Our results demonstrate that GSPE effectively lowers serum triglyceride levels in fructose-fed rats after one week administration. This study provides novel insight into the mechanistic actions of GSPE in treating hypertriglyceridemia and demonstrates that it targets hepatic de novo lipogenesis, bile acid homeostasis and non-biliary cholesterol excretion as important mechanisms for reducing hypertriglyceridemia and hepatic lipid accumulation in the presence of fructose.     

Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size,but only small changes in carbohydrate metabolism

A potato (Solanum tuberosum L. ) cDNA coding for the chloroplastic isoform of fructose 1,6-bisphosphatase (cp-FBPase) was utilized to repress its activity in tomatoes (Lycopersicon esculentum Mill.) using antisense techniques. The patatin B33 promoter was used to ensure fruit specificity of the antisense effect. Transgenic plants were isolated in which fructose 1,6-bisphosphatase activity was reduced by more than 50% of the control in green fruits. Immunoblots indicated that the plastidial isoform was almost completely eliminated in the most strongly inhibited lines. Fruits of the transgenic plants were analyzed for levels of metabolites during fruit development. Glucose and fructose concentrations were increased in green fruits in the transgenic lines, but unchanged at later stages of development. The sucrose concentration was low, and was not significantly altered in the transgenic lines. There was net degradation of starch over the developmental period, but the starch content was not decreased. In green fruit the levels of hexose phosphates were unchanged, whilst the level of 3-phosphoglyceric acid was significantly increased in one line. Most importantly the deduced ratio of hexose phosphate to 3-phosphoglyceric acid decreased, consistent with an in vivo inhibition of fructose 1,6-bisphosphatase activity. One consequence of this reduction of in vivo activity of cp-FBPase was that the average weight of fully ripe fruits was significantly decreased by up to 20% in all transgenic lines in comparison with the control.Abbreviations AGPase ADP-glucose pyrophosphorylase - cp-FBPase Chloroplastidic fructose 1,6-bisphosphatase - cy-FBPase Cytoplasmic fructose 1,6-bisphosphatase - DAF Days after flowering     

A novel lipase/acyltransferase from the yeast Candida albicans: expression and characterisation of the recombinant enzyme

A gene encoding an extracellular lipase (CaLIP4) from Candida albicans was successfully expressed in Saccharomyces cerevisiae after mutagenesis of its unusual CUG serine codon into a universal one. The ability of this lipase, which shares 60% sequence homology with the lipase/acyltransferase from Candida parapsilosis, to synthesise esters was investigated. CaLIP4 behaved as a true lipase, displaying activity towards insoluble triglycerides and having no activity in the presence of short-chain fatty acid (FA) esters and phosphatidylcholine. Methyl, ethyl and propyl esters were efficiently used. The lipase exhibited highest selectivity for unsaturated FA. With saturated FAs, C14–C16 acyl chains were preferred. In a biphasic aqueous/lipid system, CaLIP4 displayed a high alcoholysis activity with a range of alcohols (e.g. methanol, ethanol, propanol and isopropanol) as acyl acceptor. During the course of the alcoholysis reaction, new esters are produced at concentrations above the thermodynamic equilibrium of the esterification reaction, indicating that ester synthesis does not proceed by esterification but mainly by direct acyltransfer. Ester synthesis is under kinetic control due to the high rate of alcoholysis. Unwanted hydrolysis is limited by competition between the acyl acceptor (alcohol) and water for the acyltransfer reaction, favouring the alcohol.     

Enrichment of ω-3 Polyunsaturated Fatty Acids of Sardine Cannery Effluents Using Lipozyme™: Optimization of Reaction Conditions

Enrichment of n-3 polyunsaturated fatty acids from sardine cannery effluents upon enzymatic esterification by Lipozyme® was optimized in batch and in continuous processes. In these processes, the yield of docosahexaenoic acid (DHA) (Y₁) and the yield of eicosapentaenoic acid (EPA) (Y₂), in the residual acid fraction were maximized. In batch, a two-factor Doehlert design was used to study the effects of temperature and ratio of fatty acid to alcohol. Second order polynomial regression models for Y₁ and Y₂ were postulated to generate response surfaces. After esterification the fraction of fatty acid was enriched to 70% with DHA or to 44% with EPA. In a continuous process, a three-factor central composite design was employed to study the effects of temperature, ratio of fatty acid to alcohol and flow rate. Second order polynomial regression models for Y₁ and Y₂ were used to generate response surfaces. After esterification, a quantity of DHA close to 30% and 17% of EPA.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

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.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.