In-situ product removal to enhance the yield of biocatalytic reactions with competing equilibria: alpha-glucosidase catalysed synthesis of disaccharides

Transglycosylations are an important class of enzyme-catalysed reaction tha t occur in most living organisms and which are finding increasing applicati on for the synthesis of therapeutic compounds. Compared with other bioconve rsion processes, however, they generally suffer from low product yields. Th is is due to the fact that in aqueous environments water is able to undergo a nucleophilic attack of the enzyme-substrate complex, increasing the rate of the competing hydrolysis reaction. The equilibrium yield of such reacti ons is consequently only around 10% (w/w). Here, the potential of applying in-situ product removal (ISPR), with the boronate-containing affinity resin Affi-Gel (R) 601, to the alpha -glucosidase mediated conversion of phenyl alpha -D-glucoside to phenyl alpha -maltoside has been examined. ISPR can i ncrease the product yield from such kinetically-controlled reactions by rem oving the product from the bulk aqueous phase as soon as it is formed. In t his way the competing hydrolysis reaction can be prevented and conversions potentially driven to completion. Initial experiments revealed that the opt imum pH of the alpha -glucosidase reaction in water-acetonitrile mixtures w as between 5.5 and 6.5, whereas the optimum pH for binding of the product t o the Affi-Gel (R) 601 resin was between 8.0 and 8.5. Despite having to com promise on both the optimal conditions for glucosidation and for binding, a n increase in product yield of 25% (w/w) was still possible following the i mplementation of ISPR at pH 8 in an aqueous medium containing 50% (v/v) ace tonitrile. Similar results were found with the beta -galactosidase catalyse d synthesis of phenyl alpha -galactobiose, indicating the potentially gener ic nature of the ISPR methodology. While these initial results are promisin g, they indicate the need for more highly selective resins for carbohydrate adsorption (with higher capacities) if further increases in product yield are to be obtained. (C) 2001 Society of Chemical Industry.