The integrated enzymatic production and downstream processing of glucosides

<p>Glucosides are of commercial interest for the industry in general and for the pharmaceutical and food industry in particular. Chemical preparation of glycosides is not applicable in the food industry, and therefore an enzyme-catalyzed reaction would be an alternative. However, until now the low yield in the enzymatic reaction prevents the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and a continuous removal of the product. The objectives of the research described in this thesis were to develop a glucosylation reaction mediated by almondβ-glucosidase, together with a reactor that integrates the enzymatic production and downstream processing of glucosides.</p><p>The enzyme appeared to be specific for aliphatic aglycons. It appeared impossible to glucosylate phenolic aglycons. This different reactivity was investigated, explained and described in chapter 2. The successful enzymatic glucosylation of an aglycon appeared to be mainly dependent on the nucleophilicity of the aglycon. Although their chemical glucosylation was facile, phenolic aglycons were not nucleophilic enough to be glucosylated enzymatically. By using PM3 and AM1 semi-empirical methods, the magnitude of this nucleophilicity was calculated and was found to correlate with the charge on the reacting atom of the aglycon. Based on this trend, the aglycons were divided in reacting and non-reacting, which allowed a prediction of their reactivity in the glucosylation reaction.</p><p>Based on this knowledge, the optimum reaction conditions were determined and described in chapter 3.1. The highest yield and enzyme half-life in the glucosylation reaction of cyclohexanol was found at a glucose concentration of 500 g per kg of buffer solution, an organic phase/buffer phase ratio of 9:1 and a temperature of 50°C.</p><p>In chapter 3.2, the production of glyceryl glucoside with almondβ-glucosidase was described. In this case, downstream processing of the glucoside from a mixture of compounds with highly similar (solubility) properties was complex. Therefore, efforts were made to optimize the composition of the mixture at the equilibrium to facilitate downstream processing. Using the molar fraction based equilibrium constant and the mass balances, the glucoside yield was calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. This was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. A fivefold reduction of the equilibrium molar fraction of glucose was possible with only a twofold lower glucoside yield. Optimization to a minimum equilibrium molar fraction of glycerol was found to be impossible without seriously compromising the glucoside yield.</p><p>The development of a bioreactor with an integrated downstream process was described in chapter 4. In chapter 4.1, the glucosylation of hexanol in a two-phase system in a spray column reactor was described. A hexyl glucoside production of 2.5 g.l <sup>-1</sup> , and an initial production rate of 2.24 mg.U <sup>-1</sup> .h <sup>-1</sup> was achieved. The two phases were separated with a flat sheet polypropylene membrane, which was pretreated using block copolymers to prevent breakthrough of water. In-line adsorption was used to semi-continuously remove the produced glucoside. From equilibrium adsorption experiments with ten different adsorbents, alumina was chosen for in-line adsorption. Although the maximum glucoside adsorption in the full process appeared to be much lower than in the equilibrium experiments, an average glucoside adsorption of 11.15 mg.g <sup>-1</sup> was achieved. Alumina was regenerated, yielding a pure glucoside and a reusable column.</p><p>The same bioreactor was used for the production of geranyl glucoside, which is described in chapter 4.2. Geranyl glucoside was produced with an initial production rate of 0.58 mg.U <sup>-1</sup> .h <sup>-1</sup> . Based on examples from the literature, four downstream processes were tested on their viability for this system. Both extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol. Adsorption on alumina and destillation under reduced pressure were successfully applied and tested in-line with the bioreactor. A maximum glucoside adsorption of 7.86 mg.g <sup>-1</sup> was achieved on alumina. After desorption, the pure glucoside was obtained quantitatively. A pure product could not be obtained after destillation due to the fact that a small amount of glucose was present in the permeate as well.</p><p>Finally, in chapter 5, the results from the previous chapters were evaluated and placed into perspective. Furthermore, additional results that were not discussed in the previous chapters were presented. The results from glucoside stability experiments were presented and the implications of these results for an application were discussed. It appeared that glucosides are very stable under extreme conditions with respect to a food application. Therefore, formulating the glucoside together with a glucoside hydrolase might be necessary. Furthermore, alternative enzymes for the glycosylation reaction were reviewed.α-Amylase andβ-galactosidase appeared to be possible attractive alternatives, although in the first case a mixture of glucosides is produced, while in the second case the sugar donor is not used very efficiently. Preliminary results withβ-glucosidase from <em>Pyrococcus furiosus</em> were shown as an example of a potentially interesting alternative glucosidase source. The results were promising, but unfortunately the enzyme is not commercially available yet. In addition, attempts to downstream process hexyl glucoside and glyceryl glucoside that were not shown in the previous chapters were discussed in this chapter. Finally, the industrial viability of the bioreactor system and two successfully applied downstream processes were presented by calculations of the minimum equipment requirements. It was shown that a high space time yield can be achieved with a minimum of requirements and without a high waste production.</p&gt