Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose

Abstract

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 °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.

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

“…The thermodynamic constraints of enzymatic glycosylations of sugar acceptors with nucleotide donors for the synthesis of di-, oligo-, or polysaccharides has been explored to a lesser extent. Sucrose synthase has been employed for the regeneration of nucleotide sugars [25,42,80,176,177]. The equilibrium constant (K eq ) of the reaction of sucrose with UDP to afford the sugar donor UDP-glucose was determined [178].…”

Glycosyltransferase Co‐Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C‐Glucoside Nothofagin with Efficient Reuse of Enzymes

“…Immobilization is an important method from the general applied bio‐catalysis toolbox for reaction development at the interface with process engineering. [14] Besides occasional reports scattered across glycosyltransferase types (e. g., sialyltransferase, [15a] galactosyltransferase, [15b] antibiotic glycosyltrans‐ferase, [15c] sucrose synthase [15d,e] ), enzyme immobili‐zation for (semi)continuous synthesis with reuse of solid catalyst is not well developed for the Leloir glycosyltransferases. In particular, broadly applicable technology for glycosyltransferase co‐immobilization to establish glycosylation cascades on an insoluble support is lacking.…”

Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

“…This immobilization strategy offers important stabilization effects, due to the short spacer arms that lead to the rigidification of the enzyme structure [25]. Thus, more than 100 industrial enzymes have been highly stabilized by this immobilization protocol [8,[26][27][28]. Figure 1 shows the standard protocol for protein immobilization on glyoxyl-activated supports.…”

“…and structures (monomeric or multimeric enzymes, cofactor-dependent or cofactor-independent enzymes, etc.) [9,[28][29][30][31][32]. However, one of the main disadvantages of using this immobilization methodology is the reduction step with sodium borohydride (NaBH4) that may adversely affect the enzyme activity.…”