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

Abstract

Transglycosylations are an important class of enzyme-catalysed reaction that occur in most living organisms and which are ®nding increasing application for the synthesis of therapeutic compounds. Compared with other bioconversion processes, however, they generally suffer from low product yields. This 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 reactions is consequently only around 10% (w/w). Here, the potential of applying in-situ product removal (ISPR), with the boronate-containing af®nity resin Af®-Gel 1 601, to the a-glucosidase mediated conversion of phenyl a-D-glucoside to phenyl a-maltoside has been examined. ISPR can increase the product yield from such kinetically-controlled reactions by removing the product from the bulk aqueous phase as soon as it is formed. In this way the competing hydrolysis reaction can be prevented and conversions potentially driven to completion. Initial experiments revealed that the optimum pH of the a-glucosidase reaction in water±acetonitrile mixtures was between 5.5 and 6.5, whereas the optimum pH for binding of the product to the Af®-Gel 1 601 resin was between 8.0 and 8.5. Despite having to compromise on both the optimal conditions for glucosidation and for binding, an increase in product yield of 25% (w/w) was still possible following the implementation of ISPR at pH 8 in an aqueous medium containing 50% (v/v) acetonitrile. Similar results were found with the b-galactosidase catalysed synthesis of phenyl a-galactobiose, indicating the potentially generic nature of the ISPR methodology. While these initial results are promising, 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.

Glycosyl Hydrolases and Glycosyltransferases in the Synthesis of Oligosaccharides

“…With this boronate-containing affinity resin the equilibrium is shifted driving the reaction to completion removing the product from the aqueous phase. An increase in product yield of 25% was reported despite having to compromise among the pH of enzymatic reaction (phenyl α-maltoside formation using α-glucosidase from baker's yeast, pH 5.5-6.5) and binding of product into the resin (optimal pH 8) [106].…”

Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspectives

“…General criteria in the bioreactor design and in the selection of the operating conditions could be: use of reactors or reaction regimes that allow a rapid reduction of the glucose concentration; running of the reactions at low to medium substrate concentrations in order to maintain higher conversion rates and hence obtain higher volumetric productivity of the reactor . The integration of the bioreactor with a separation unit (reaction-separation hybrids) has shown promising results with product inhibited or equilibrium limited enzyme-catalyzed conversions, because it is possible to remove the products as they are formed (Ahmed et al, 2001;Gan et al, 2002). In this regard, membrane (bio) reactors could be a viable process configuration.…”

Methods for Improving Enzymatic Trans-glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics

“…In β-galactosidase-catalyzed production of GOS from lactose, this has been achieved by adsorption of oligosaccharides to activated carbon, resulting in a yield improvement of 30% . In a model reaction studying β-galactosidase-catalyzed self-condensation of Gal-β- p NP, the adsorption of p NP-β-galactobioside to a boronate resin gave a yield improvement of 10–15%, the major issue being the discrepancy between the optimal reaction pH and the optimal binding pH . In the sialidase-catalyzed production of 3′-sialyl-lactose from lactose and CGMP, the large size of the limiting substate, that is, CGMP, has been exploited to facilitate continuous product removal in an enzymatic membrane reactor .…”