Final Report Summary - AQUACAT (Tailor made lipases for synthetic catalysis in biphasic media: From poly (lactone) applications towards novel sugar esters)
AquaCat (Grant no. 911723) is a return phase of an IIF project (Grant no. 301723), funded by the European Union FP7 in accordance with the strategic research agenda in the area of “biocatalytic process design”. The project began its activity on June, 2014 and ended May, 2015, with all experiments performed in Thailand (Bangkok) at Department of Biotechnology, Faculty of Science, Mahidol University. AquaCat designs the control substitution on the sucrose molecules by applying lipases (a class of enzymes, i.e. biocatalysts) to a tailored microenvironment of fatty acid esters, resulting in the synthesis of myriad sucrose fatty esters with different end-used properties, i.e. bio-surfactant and fat substitutes.
Sucrose fatty esters are commercially and technologically available, however, production of these molecules are based on only chemical process involving chemical catalysts such as sodium methoxide, or alkaline catalysts. The industrial relevant process has been firstly developed for the synthesis of sucrose esters as bio-surfactants (with the degree of substitution lower than 3). The return phase aims to use the previously studied technology to increase the degree of substitution of the sucrose to higher than 3, leading to the synthesis of sucrose-based fat substitute with nutritional properties similar to triglycerides, but cannot be digested by lipolytic enzymes. The commercial sucrose-based fat substitutes contain six or more fatty acids per sucrose molecule.
At first, the characterization of the biphasic system and the main sucrose monoester products were performed. The reaction system was visualized by light microscope, the dispersion of ester droplets of approximately 25 micron in the continuous phase of sucrose solution was observed. At 20 min, after lipase addition, the conversion of sucrose reached the maximum of 85 % with the major product of monoester. The product was purified by column chromatography and its purity and chemical structure were characterized by High Performance Liquid Chromatography and 13C-Nuclear Magnetic Resonance Spectroscopy, respectively. The substitution was occurred on the secondary hydroxyl groups of glucose moiety of sucrose. The structure of sucrose monoester provides insight of the reaction mechanism and selectivity of lipase, which were crucial for the fat-substituted synthesis. When the molar ratio of esters to sucrose was fixed at 3:1, it is possible to increase the degree of substitution of sucrose fatty esters to 3.
Sucrose has 8 hydroxyl groups, available for the substitution by the fatty acid chain. Consequently, the synthesis of sucrose esters with degree of substitution higher than 3 might be done by increasing the mole ratio of ester to sucrose to higher than 3 to 1. The degree of substitution remained unchanged, whereas the reaction rate decreased gradually. The selectivity of C. rugosa lipase towards only the secondary hydroxyl groups of glucopyranosyl ring might limit further use of esters for sucrose substitution, leading to the inhibition from the high concentration of esters. To circumvent the problem of enzyme selectivity, a cocktail of lipases is needed for such a synthesis as both primary /secondary hydroxyl groups on the sucrose could be substituted.
The research was then moved to the screening of the commercial lipases with different selectivity from C. rugosa lipases. The potential targeted lipases with different selectivity from C. rugosa were chosen from intensive literature review. Only the commercially available lipases, for instance lipase from Pseudomonas sp., Mucor miehei, Candida antarctica and Thermomyces lanuginosus, were tested for their activities using the optimized system setup, as mentioned previously. Surprisingly, none of them were active, sucrose and esters stayed intact, limiting the possibility to achieve AquaCat return phase objective. It is important to note that the biphasic system setup in AquaCat was different from the general organic solvent system reported in the literature, in terms of both substrate load and type of solvent used as reaction medium. This may probably has the consequence on the operational stability of tested lipases. This hypothesis was proven by the visualization of lipase form during the catalysis by Transmission Electron Microscopy. Only the pretreated C. rugosa lipase used in this study can maintain its globular form while the native one lost its tridimensional structure during catalysis. The targeted lipases were then treated as C. rugosa lipase, followed by the evaluation of their activities in the same optimized system. None of those lipases can catalyze the substitution of fatty acid chain onto sucrose in AquaCat biphasic system. The unpredictable molecular properties of lipases towards process and starting materials represent the main hurdle to general application of the AquaCat concept towards the synthesis of fat substitute. However, the high purity of sucrose monoester from this technique cannot be found in commercially available products. The surfactant properties were studied and compared with commercial products of closed chemical structure.
AquaCat relates to a process for production of novel nanoparticles and sugar esters which are mid- to high-value compounds with a high application potential in the European Chemical, Food and Pharmaceutical Industry. In accordance with the strategic research agenda in the area of “Biocatalytic process design”, the novel principle of lipase-catalyzed synthesis in aqueous biphasic systems bring technological solutions to the conventional process involving the conversion of oleochemicals and carbohydrates substrates in general. The publication and application of AquaCat principle is thus beneficial, not only for the scientific society working in the domain of green chemistry, but also for the industries and policy maker involved in the sustainable chemistry.
Sucrose fatty esters are commercially and technologically available, however, production of these molecules are based on only chemical process involving chemical catalysts such as sodium methoxide, or alkaline catalysts. The industrial relevant process has been firstly developed for the synthesis of sucrose esters as bio-surfactants (with the degree of substitution lower than 3). The return phase aims to use the previously studied technology to increase the degree of substitution of the sucrose to higher than 3, leading to the synthesis of sucrose-based fat substitute with nutritional properties similar to triglycerides, but cannot be digested by lipolytic enzymes. The commercial sucrose-based fat substitutes contain six or more fatty acids per sucrose molecule.
At first, the characterization of the biphasic system and the main sucrose monoester products were performed. The reaction system was visualized by light microscope, the dispersion of ester droplets of approximately 25 micron in the continuous phase of sucrose solution was observed. At 20 min, after lipase addition, the conversion of sucrose reached the maximum of 85 % with the major product of monoester. The product was purified by column chromatography and its purity and chemical structure were characterized by High Performance Liquid Chromatography and 13C-Nuclear Magnetic Resonance Spectroscopy, respectively. The substitution was occurred on the secondary hydroxyl groups of glucose moiety of sucrose. The structure of sucrose monoester provides insight of the reaction mechanism and selectivity of lipase, which were crucial for the fat-substituted synthesis. When the molar ratio of esters to sucrose was fixed at 3:1, it is possible to increase the degree of substitution of sucrose fatty esters to 3.
Sucrose has 8 hydroxyl groups, available for the substitution by the fatty acid chain. Consequently, the synthesis of sucrose esters with degree of substitution higher than 3 might be done by increasing the mole ratio of ester to sucrose to higher than 3 to 1. The degree of substitution remained unchanged, whereas the reaction rate decreased gradually. The selectivity of C. rugosa lipase towards only the secondary hydroxyl groups of glucopyranosyl ring might limit further use of esters for sucrose substitution, leading to the inhibition from the high concentration of esters. To circumvent the problem of enzyme selectivity, a cocktail of lipases is needed for such a synthesis as both primary /secondary hydroxyl groups on the sucrose could be substituted.
The research was then moved to the screening of the commercial lipases with different selectivity from C. rugosa lipases. The potential targeted lipases with different selectivity from C. rugosa were chosen from intensive literature review. Only the commercially available lipases, for instance lipase from Pseudomonas sp., Mucor miehei, Candida antarctica and Thermomyces lanuginosus, were tested for their activities using the optimized system setup, as mentioned previously. Surprisingly, none of them were active, sucrose and esters stayed intact, limiting the possibility to achieve AquaCat return phase objective. It is important to note that the biphasic system setup in AquaCat was different from the general organic solvent system reported in the literature, in terms of both substrate load and type of solvent used as reaction medium. This may probably has the consequence on the operational stability of tested lipases. This hypothesis was proven by the visualization of lipase form during the catalysis by Transmission Electron Microscopy. Only the pretreated C. rugosa lipase used in this study can maintain its globular form while the native one lost its tridimensional structure during catalysis. The targeted lipases were then treated as C. rugosa lipase, followed by the evaluation of their activities in the same optimized system. None of those lipases can catalyze the substitution of fatty acid chain onto sucrose in AquaCat biphasic system. The unpredictable molecular properties of lipases towards process and starting materials represent the main hurdle to general application of the AquaCat concept towards the synthesis of fat substitute. However, the high purity of sucrose monoester from this technique cannot be found in commercially available products. The surfactant properties were studied and compared with commercial products of closed chemical structure.
AquaCat relates to a process for production of novel nanoparticles and sugar esters which are mid- to high-value compounds with a high application potential in the European Chemical, Food and Pharmaceutical Industry. In accordance with the strategic research agenda in the area of “Biocatalytic process design”, the novel principle of lipase-catalyzed synthesis in aqueous biphasic systems bring technological solutions to the conventional process involving the conversion of oleochemicals and carbohydrates substrates in general. The publication and application of AquaCat principle is thus beneficial, not only for the scientific society working in the domain of green chemistry, but also for the industries and policy maker involved in the sustainable chemistry.