Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5'-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 A degrees C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.