Albert van der Padt

Perspectives for the industrial enzymatic production of glycosides.

Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus, enzyme‐catalyzed reactions are a good alternative. However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product. Unfortunately, a bioreactor for the commercial scale production of glycosides is not available. The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.

Substrate sorption into the polymer matrix of Novozym 435 and its effect on the enantiomeric ratio determination

In the enantioselective esterification of 4-methyloctanoic acid with ethanol by immobilised Candida antarctica lipase B (Novozym 435®), the enantiomeric excesses determined during the course of the reaction deviated strongly from the theoretical values, leading to unacceptably large confidence intervals for the enantiomeric ratio (E value). This observation was in contrast to our previous findings for transesterification and hydrolysis reactions with this enzyme. Herein, the three reactions are compared; the anomalous results in the esterification reaction appear to be caused by adsorption of 4-methyloctanoic acid inside the enzyme beads. We found that on average 1.19 g of 4-methyloctanoic acid was incorporated per g of Novozym 435®. If the concentration of this substrate was adjusted accordingly in the calculations, the resulting E values showed acceptable confidence intervals. In previous research on transesterification reactions in excess apolar solvent (comparable affinity for the beads), sorption does not play an important role because only small amounts of substrate were lost. For hydrolysis reactions, sorption takes place but the acid is released from the beads upon titration and no effect on the E value is found. However, for esterification reactions, sorption should not be neglected since there is no driving force to release the acid from the beads.

Why are some alcohols easy to glucosylate with β-glucosidases while others are not? A computational approach

A method is presented for predicting the reactivity of alcoholic aglycons in the β-glucosidase mediated glucosylation reaction. The successful enzymatic glucosylation of an aglycon appears to be mainly dependent on the nucleophilicity of the aglycon. Vinylic and phenolic aglycons are not nucleophilic enough to be glucosylated enzymatically, although their chemical glucosylation is facile. By using PM3 and AM1 semi-empirical methods, the magnitude of this nucleophilicity can be calculated and was found to correlate with the charge on the reacting atom of the aglycon. Based on this trend, the aglycons can be classified as reacting or non-reacting. The orbital related parameters seem to have a limited influence on the reaction behaviour. In addition to these calculations, the energy of the transition state of two enzymatic reactions has been calculated using a simplified model of the enzyme active site for both an experimentally reacting and an experimentally non-reacting aglycon (cyclohexanol and phenol, respectively). The activation energy for the cyclohexanol complex was computed to be 1.3 kcal mol−1, while the calculated activation energy for the phenol complex is 15.8 kcal mol−1. This difference can indeed explain the fact that cyclohexanol is easily glucosylated while phenol is not.Finally, it is pointed out that facile and fast calculation methods can be used to make a confident prediction about the reaction behaviour of aglycons without performing the actual laboratory experiments. p

Downstream Processing of Enzymatically Produced Geranyl Glucoside

Geraniol plays an important role in the fragrance and flavor industry. The corresponding glucoside has interesting properties as a “slow release” aroma compound. Therefore, the enzymatic production and downstream processing of geranyl glucoside were investigated. Geranyl glucoside was produced in a spray column reactor with an initial production rate of 0.58 mg U−1 h−1. A pretreated hydrophobic microfiltration membrane was used to prevent migration of the aqueous, enzyme‐containing phase to the downstream process. No retention of the glucoside, which accumulated in the geraniol phase, was found. On the basis of examples from the literature, four downstream processes were tested on their viability for this system. Extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol. In the first case, the glucoside selectivity for the geraniol phase was found to be high, which made extraction with water unsuccessful. In the second case it was possible to obtain a stable foam, but significant enrichment of the foam with glucoside did not occur. Adsorption on alumina and distillation under reduced pressure were applied successfully and tested in‐line with the bioreactor. A maximum glucoside adsorption of 7.86 mg g−1 was achieved on alumina. After desorption and evaporation of the extractant the pure glucoside was obtained quantitatively. A pure product could not be obtained after distillation because a small amount of glucose was present in the permeate as well, which accumulated in the bottom fraction. It was shown that with this reactor system a production of 1 kg of geranyl glucoside in 2 days is possible using an initial amount of 50,000 units of enzyme.

The catalytic potency of β-glucosidase from Pyrococcus furiosus in the direct glucosylation reaction

Abstract Enzymes from extremophiles operate at conditions that are different from their ‘normal’ counterparts, and are therefore a useful extension of the enzyme toolbox. In this paper, the direct glucosylation reaction mediated by a hyperthermophilic β-glucosidase from Pyrocuccus furiosus was investigated. Hexanol was successfully coupled to glucose with this enzyme. A preliminary study was conducted to improve the product yield. A maximum product concentration of 12.9 g.l−1 was attainable by increasing the glucose concentration to the maximum solubility of 2000 g.(kg buffer solution)−1 at the reaction temperature. The highest glucose based yield of 2.64% was achieved with a glucose concentration of 900 g.(kg buffer solution)−1 at a reaction temperature of 65°C and a pH of 6.0. Performing the reaction at higher pH and temperature led to lower product concentrations. This was caused by deactivation of the enzyme accompanied by browning of the reaction mixture. A pH of 4.4 did have a negative effect on both the storage and the operational stability of the enzyme.

Optimization of production and downstream processing of the almond β-glucosidase-mediated glucosylation of glycerol

This article describes the synthesis of glyceryl glucoside from glycerol and glucose with almond beta-glucosidase as the catalyst. A yield of 54% (0.45 mmol/g) was obtained. The influence of the enzyme stability, the water concentration, and the water activity on the glucoside yield were determined. A molar fraction-based equilibrium constant of 2.4 +/- 0.6 was found, with which the glucoside yield could be calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. A model was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. This straightforward model gives a good prediction of the measured glucoside yield, according to a parity plot.