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AIR3-CT94-2291
Production of Sugar Fatty Acid Esters from Renewable Agricultural Resources: An Integrated Optimization of Enzymatic-Purification Processes and of Surfactive Properties |
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Proposal No: | AIR3-CT94-2291 |
| Date Prepared: | October 1997, September 1999 | |
| Source: | Final Report June 1998
Second Annual Progress Report |
Summary
The aim of the project was to acquire an improved knowledge of enzymatic and purification processes for the synthesis of sugar fatty acid esters, with the objective of developing economically feasible enzymatic processes for the preparation of new non-toxic biodegradable surface-active agents derived entirely from renewable resources.
The project developed several conjugates of sugars and fatty acids:
Compared to chemically synthesised sugar derivatives, enzymatically synthesised sugar esters are characterised by greater purities and specificities. They exhibit surfactive properties comparable to those of commercially available alkylpolyglycosides. They also show competitive ecotoxicological and biodegradability characteristics.
A major outcome of the project is novel enzymatic processes for the synthesis of alkyl and acyl sugars using lipases and glycosidases in organic solvents. As a result of an in-depth investigation of the influences of operational parameters, reaction conditions were optimised to reach high reaction performances in terms of final titers and conversion yields. The enzymatic technologies show a great flexibility, as they can be readily adapted to fatty acids of different chain lengths.
The project also developed purification schemes of the produced acyl and alkyl sugars:
From a more fundamental point of view, the project generated numerous quantitative data on the biosynthesis and purification of sugar based surfactants:
In the future the thermodynamic and kinetic database will be most valuable for the further optimisation and scale-up of the novel enzyme technologies for sugaresters synthesis and purification.
Introduction
The aim of the project was to acquire an improved knowledge of enzymatic and purification processes for the synthesis of sugar fatty acid esters, with the objective to develop economically feasible enzymatic processes for the preparation of new non-toxic biodegradable surface-active agents derived entirely from renewable resources. The project was organised in an integrated and multidisciplinary approach. Starting from several sugars, namely glucose, galactose, fructose, sucrose, lactose, and from fatty acids of different chain lengths, it examined the performances of the different reaction and purification processes leading to sugar esters.
Activities
Overall the processes involved the following four steps:
The enzymatic alkylation of sugars Three different enzymatic reactions were developed for the transformation of glucose or lactose into butyl glucose and galactose:
Major process parameters, namely the initial sugar concentration, the water/butanol volume ratio and the temperature, were optimised to achieve 25 g/l final butylgalactoside concentration with a 80% galactose conversion yield and 45g/l butylglucoside concentration with a 50% glucose conversion yield. Purification schemes based on liquid chromatography were developed to recover purified butyl glucose and galactose from the reaction medium.
The production of sugar fatty acid esters by lipase esterification Enzymatically synthesised alkyl sugars were used as substrates for the lipase esterification reaction. The central part of the project was the development of lipase based processes for the synthesis of sugar fatty acid esters. A wide range of data were collected for reaction conditions involving different sugars (glucose, fructose, galactose and sucrose), fatty acids (oleate, stearate, palmitate and laurate), solvents and lipases. The results provided:
The main contributions can be organised according to the sugar molecule used and solvent conditions. They included the synthesis of fructose esters in 2-methyl-2-butanol, using an immobilised lipase from Candida antarctica and 2-methyl-2-butanol as a solvent.
For the production of fructose oleate, 60 g/l with conversion yields of around 85% were obtained when operating the reaction under reduced pressure, at 60 degrees C, with a sugar/fatty acid ratio of 1. Under these conditions, the reaction yielded mainly monoesters of fructose. A lower sugar/fatty acid ratio resulted in a high proportion of fructose diesters. The same process was used for the production of other fructose esters, namely stearate, palmitate and laurate.
The synthesis of glucose and fructose eaters in hexane and supercritical C02 During the screening of solvent conditions appropriate for the synthesis of monosaccharide esters, emphasis was also given to the comparison of the behaviour of lipases in hexane and supercritical CO2. With these solvents a major problem to be solved was sugar solubility. Two techniques were assessed: the adsorption of the sugars onto a solid support (e.g. silica gel or cellulose) and the addition of a polar co-solvent. Highest reaction activities with the adsorption on silica gel. Lipases from Mucor miahei, Candida antarctica and Candida cylindracea catalyzed the esterification of monosaccharides with relatively high yields in these solvents. Lipase from Candida antarctica gave the best conversion yield (85%) for the esterification of fructose by lauric acid in n-hexane.
The synthesis of sucrose esters The synthesis of fatty acids esters of sucrose was another important target of the project. The main difficulty to be overcome was the very low solubility of sucrose in most organic solvents as only a few hydrogen-bonding solvents (DMSO, DMF) can solubilize the sugar to a significant extent. Two different approaches were evaluated leading to the production first of butyryl-sucrose and second of sucrose laurate, palmitate and oleate. The former product was made using a two-step synthesis of di- or tri substituted sucrose using short-chain fatty acids. In a first step, the synthesis of monobutyrylsucrose was performed in DMF using the lipase from Pseudomonas sp. Pure mono-butyrylsucrose was isolated with a yield of more than 90% in two days. In a second step, the acylation of mono-butyrylsucrose was performed in acetone with the immobilized lipase from Candida antarctica. During the first hours of reaction, the acylation took place preferentially at position 6' of the fructose ring yielding the diester 1',6'-o-dibutyrylsucrose. Further acylation occurred to yield the triester 1',6,6'-o-tributyrylsucrose.
The synthesis of monoacylsucrose derivatives using long-chain acyl donors (e.g. lauric, palmitic or oleic acid) As proteases were not effective on acyl chains longer than C8, this was achieved using vinyl esters, chosen as acylating agents in order to shift the equilibrium towards ester formation (the vinyl alcohol forms tautomerizes to the low-boiling-point acetaldehyde). Best results were observed with lipase from Humicola lanuginosa with a 45 % conversion of sucrose was reached, with 65% selectivity to l'-O-laurylsucrose. With the enzyme covalently linked to a hydrophilic support (Eupergit C), the reaction rate was very fast: after 20 hours less than 10% of the initial sucrose remained in the system (56% monoesters and 37% higher esters). Significant enzyme activities were only observed in solvents (DMSO, DMF, DMA, pyridine) that significantly solubilize sucrose. DMSO gave the best results. The molar ratio of acylating-agent to sucrose was shown to be of particular importance; its optimal value was determined at around 1.5:1. Temperature was also found a crucial parameter for the yield of the process. The enzyme activity was notably higher at 60oC than at 40oC and led to a productivity of 90g monoester per litre in 45 minutes.
With respect to the synthesis of sucrose monopalmitate, several lipases were found efficient, but the activities were lower than for sucrose laurate. Best results were observed with the lipase from Humicola lanuginosa immobilised on Celite and with a preparation of lipase from Penicillium sp. obtained from industrial wastes. With the latter a productivity of 95 g/L was obtained in 144 h using dimethylacetamide as solvent. Due to the relative toxicity of the solvents employed (DMSO, DMA), we also developed processes using less toxic non-aqueous media. Trans-esterification was studied in mixtures of solvents containing a high percentage (70-100%) of a less toxic solvents (a tertiary alcohol, acetone, etc.) and a low amount (0-30%) of a solvent where sucrose is soluble (DMSO, DMF, DMA, pyridine). It was found that the mixture 2-methyl-2-butanol: dimethylacetamide 90:10 (VN) was the best for the acylation of sucrose using lauric and palmitic vinyl esters.
Oleate esters of butylglucoside and butylgalactoside Because of the extremely low glucose and galactose solubility in solvents such as methyl-butanol, only limited conversion yields around 20% were obtained for the lipass acylation of glucose and galactose. The feasibility of using the previously synthesised alkyl sugars in the esterification reaction. Using the reaction conditions defined for the production of fructose esters, a high activity and conversion yield of 65% for the esterification of butylglucoside and butylgalactoside with oleic acid were obtained.
The extraction and purification of sugar esters The third part of the project dealt with the development of extraction and purification processes. An advantage of the enzymatic process, as compared to the established chemical synthesis, is generation of products of higher purities under relatively mild reaction conditions that do not generate by-products. When sufficient conversion yields of sugar esters were achieved at the end of the reaction, the downstream processing of the reaction medium was minimised and limited to the filtration of the enzyme particles and the evaporation of the solvent. At lower conversion yields, or where highly purified products were required, additional extraction steps can be utilised (liquid-liquid extraction, supercritical fluid extraction and liquid chromatography).
Characterisation of the sugar esters Preliminary studies of the physico-chemical and environmental properties of the novel synthesised sugar esters were carried out using either a purified fraction of fructose monooleates (99% purity), or a crude mixture of fructose palmitate and fructose stearates (purity around 60%). Studies included investigations of surface tension, oil/water interfacial tension, emulsion stability, biodegradability and ecotoxicological properties.
The preliminary results suggested that enzymatically synthesised sugar esters have excellent physico-chemical and environmental properties and have good potential as biosurfactants for use in detergency, cosmetics and foods.
Conclusions
The project achieved its objectives and resulted in several major technological innovations and scientific contributions: The project generated several novel biosurfactants: - acylsugars: oleate, stearate, palmitate, and laurate of fructose, palmitate and laurate of sucrose alkyl sugars: butyl glucose and butyl galactose acyl alkyl sugars: butyl glucose oleate. These molecules represent a new class of non-toxic biodegradable surface-active agents derived entirely from renewable resources. Compared to chemically synthesised sugar derivatives they have much greater purities and specificities.
A major outcome of the project is several novel enzymatic processes for the synthesis of alkyl and acyl sugars. With the use of lipases and galactosidases in organic solvents, high reaction performances can be achieved in terms of final titers and conversion yields. The enzymatic technologies are also very flexible and can be adapted to various sugars and fatty acids. In addition the project also developed purification schemes of the produced acyl and alkyl sugars.
A data base on enzymatically produced sugar biosurfactants in non aqueous media was built up, to include thermodynamic data on sugars, fatty acids, alkyl and acyl sugars as well as solubilities and activity coefficients in organic media and supercritical CO2. Equilibrium constants for the synthesis reactions were also produced, as was kinetic data for lipases and glucosidases in non aqueous media, specially with respect to the influence of environmental parameters: nature of the solvent, water activity, sugar and fatty acid concentrations, temperature, overhead pressure. This will be a valuable tool for future optimisation and scale-up of the novel enzyme technologies.
SUMMARY
This project is investigating both chemical and enzyme based methods
of producing surface active materials (surfactants and emulsifiers) based
on esterification of any of a number of different sugars with any of a number
of different long chain fatty acids. Some such products, such as sucrose esters,
are available commercially, finding use in cosmetics and foods. However, in general
they are relatively costly due to difficulties in production and purification. These
compounds consist of the combination of a sugar molecule, which is water soluble,
with a long chain fatty acid which is soluble in organic solvents. The problem is to identify
solvents in which both components will dissolve and then react as required. Such reactions
may require the production of complex intermediates and reaction sequences. A number of
the solvents which can be used are potentially harmful and have to be removed from the products.
An alternative is to use enzymes which catalyse esterifications or transesterifications. However,
again there is a need to find conditions under which the reactants will dissolve, the enzymes are
stable and the products formed in sufficient quantities to be separated from the reaction mixture.
Progress in reaching these objectives over the first two years of the project are described in this
item.
INTRODUCTION
The aim of this project is to investigate synthesis, extraction and purification processes for the
preparation of sugar-fatty acid esters. From the results, it is hoped to develop economic routes
for the preparation of new non toxic biodegradable surface active agents derived entirely from
renewable resources.
OBJECTIVE
The aim of the project is to acquire an improved knowledge of
enzymatic and purification processes for the synthesis of sugar
fatty acid esters, with a view to develop economically feasible
processes for the preparation of new non-toxic biodegradable surface-active
agents derived entirely from renewable resources.
The project proposes an integrated and multidisciplinary approach towards the optimization of enzymatic processes for the production of new sugar fatty acid esters surfactants.
Starting from the sugars sucrose and lactose and from fatty acids of different chain length, it examines the performances of the different reaction and purification steps leading to sugar esters.
The study mainly focuses on the following aspects :
The knowledge gained on these processes are expected to contribute to the development of economically competitive technologies and of novel surfactants interesting as detergents or as additives for cosmetics, food and pharmaceuticals.
SYNTHESIS
Studies on synthesis cover both alkylation and acylation reactions, with the objective of
optimising both chemical and enzymatic alkylation of sugar and enzymatic acylation in
organic solvents. In addition the chemical alkylation of lactose was studied with analytical
methods developed to determine the various reaction products. The enzymatic alkylation
of butyl glycoside and butyl galactoside were also investigated. It was found that:
This result was due to an increase in the concentration of glucose in the medium. Synthesis of butyl galactoside was not depended on the molar ratio of butanol/galactose because of the poor solubility of galactose in the medium.
The enzymatic acylation of fructose, developed in the first year, was extrapolated to pilot scale, operated under reduced pressure. Yields of about 90% were obtained and fructose oleates concentrations up to 85 g l l were obtained.
Other work focused on the acylation of sucrose in organic solvents and the purification of the resulting esters. Two processes were considered (the synthesis of sucrose monolaureate and sucrose monopalmitate) in order to obtain sucrose esters with different surface active properties. The study of the optimal conditions for the synthesis of sucrose monolaureate was carried out using the lipase from Humicola lanuginosa immobilised on Eupergit C which was tested under various conditions of humidity (water activity, aw). Following this various solvents were assayed. The reaction only took place in solvents (DMSO, DMF, DMA. pyridine) that solubilise sucrose to a significant extent. DMSO was best in terms of production. The molar ratio acylating agent:sucrose was found to be of particular important on the time course of the reaction, due to the low solubility of vinyl laureate in this solvent. Surprisingly, a molar ratio 1.5:1 gave better results than 4:1. It was found that reaction temperature was crucial for the yield of the process, with the reaction proceeding significantly faster at 60°C than at 40°C. Under optimal conditions, at 60°C, a productivity of 90 g monoester per litre (175mM) was achieved in about 45 minutes. The biocatalyst (immobilised enzyme) did not loose any activity during the process and could be re-used. Investigations of the synthesis of sucrose monopalmitate showed that the reaction took place at a much slower rate. Several lipases were found to catalysed transesterification. The best results were obtained with the lipase from Humicola lanuginosa immobilised on Celite and with a preparation of lipase from Penicillium sp. obtained from industrial wastes. With the latter biocatalyst a productivity of 95 g.l-1 was obtained in 144 hours using dimethylacetamide as solvent.
SEPARATION
Equilibrium constants, required for process optimisation purposes, were estimated with
preliminary results suggesting that the UNIFAC model could be used for the estimation
and possibly for the prediction of equilibrium constants. In addition process were developed
for the recovery of fatty acid sugar esters through supercritical extraction and liquid liquid
extraction and their performance evaluated. Product recovery using supercritical extraction
with carbon dioxide appeared promising on the basis of results achieved for the recovery of
fructose laureate. For product recovery through liquid liquid extraction, application of the
UNIFAC model for the screening of potential solvents gave encouraging results showing
a good fit with experimental results.
A liquid chromatography separation process for the recovery of butyl glycoside was developed which gave a very pure product (>99%) with a yield of 70%. A method for the purification of fructose monooleates was developed, resulting in recovery of small quantities of pure sugar esters. The structure and some surfactant properties of these molecules was determined. In addition two purification processes were developed. These combined a filtration step (removal of the immobilised enzyme) and distillation (removal of the solvent) or liquid liquid extraction (removal of the residual sugar). The two methods differed in the separation of the residual fatty acid. The first (analytical scale) used medium pressure liquid chromatography to separate sugar ester from the acyl donor. This method allowed the recovery of pure sugar ester used for structural analysis. The second one used another liquid liquid extraction for the removal of fatty acid. The purification of sucrose monolaureate and monopalmitate was attempted using liquid liquid extraction steps. However, a problem arose from the surfactant properties of the monopalmitate, since the emulsions were very stable and difficult to break.
CHARACTERISATION
The products recovered as above were used in evaluation of the surface active properties.
The structural analysis of products demonstrated that the enzymatic synthesis of fructose
monooleate led to a mixture of 4 isomers (a and b anomers of 6 fructofuranose and b anomers
of l fructofuranose and l fructopyranose). In addition surface and interfacial tensions as well as
the foaming and emulsifying powers were determined. Fructose monooleates caused a significant
decrease in both surface and interfacial tensions, even at low concentrations. The critical micelle
concentration of fructose monooleates was determined as 2.4x10-4 M. This biosurfactant showed
a high capacity to stabilise emulsions. In practice, 20% separation of phases were obtained with
0.1 % fructose monooleates previously solubilised in xylene phase. In addition, on beating, this
surfactant formed a foam which was very stable with time.
PARTICIPANTS
The project is co-ordinated by INPL (Vandoeuvre, France) in collaboration with CSIC (Madrid, Spain)
and NTUA (Athens, Greece).
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