Glycosylation of polyphenolic compounds: Design of a self-sufficient biocatalyst by co-immobilization of a glycosyltransferase, a sucrose synthase and the cofactor UDP.

Glycosyltransferases catalyze the regioselective glycosylation of polyphenolic compounds, increasing their solubility without altering their antioxidant properties. Leloir-type glycosyltransferases require UDP-glucose as a cofactor to glycosylate a hydroxyl of the polyphenol, which is expensive and unstable. To simplify these processes for industrial implementation, the preparation of self-sufficient heterogeneous biocatalysts is needed. In this study, a glycosyltransferase and a sucrose synthase (as an UDP-regenerating enzyme) were co-immobilized onto porous agarose-based supports coated with polycationic polymers: polyethylenimine and polyallylamine. In addition, the UDP cofactor was strongly ionically adsorbed and co-immobilized with the enzymes, eliminating the need to add it separately. Thus, the optimal self-sufficient heterogeneous biocatalyst was able to catalyze the glycosylation of three polyphenolic compounds (piceid, phloretin and quercetin) with in situ regeneration of the UDP-glucose, allowing multiple consecutive reaction cycles without the addition of exogenous cofactor. A TTN value of 50 (theoretical maximum) was obtained in the reaction of piceid glycosylation, after 5 reaction cycles, using the self-sufficient biocatalyst based on an improved sucrose synthase variant. This result was 5-fold higher than the obtained using soluble cofactor and the co-immobilized enzymes, and much higher than those reported in the literature for similar processes.

Omega-3 production by fish oil hydrolysis using a lipase from Burkholderia gladioli BRM58833 immobilized and stabilized by post-immobilization techniques.

Immobilization of lipase from Burkholderia gladioli BRM58833 on octyl sepharose (OCT) resulted in catalysts with higher activity and stability. Following, strategies were studied to further stabilize and secure the enzyme to the support using functionalized polymers, like polyethylenimine (PEI) and aldehyde-dextran (DEXa), to cover the catalyst with layers at different combinations. Alternatively, the construction of a bifunctional layer was studied using methoxypolyethylene glycol amine (NH 2 -PEG) and glycine. The catalyst OCT-PEI-DEXa was the most thermostable, with a 263.8-fold increase in stability when compared to the control condition. When evaluated under alkaline conditions, OCT-DEXa-PEG 10 /Gly was the most stable, reaching stability 70.1 times greater than the control condition. Proportionally, the stabilization obtained for B. gladioli BRM58833 lipase was superior to that obtained for the commercial B. cepacia lipase. Preliminary results in the hydrolysis of fish oil demonstrated the potential of the coating technique with bifunctional polymers, resulting in a stable catalyst with greater catalytic capacity for the production of omega-3 PUFAs. According to the results obtained, it is possible to modulate B. gladioli BRM58833 lipase properties like stability and catalytic activity for enrichment of omega-3 fatty acids.

Coimmobilization and colocalization of a glycosyltransferase and a sucrose synthase greatly improves the recycling of UDP-glucose: Glycosylation of resveratrol 3-O-ß-D-glucoside.

Glycosylation is one of the most efficient biocompatible methodologies to enhance the water solubility of natural products, and therefore their bioavailability. The excellent regio- and stereoselectivity of nucleotide sugar-dependent glycosyltransferases enables single-step glycosylations at specific positions of a broad variety of acceptor molecules without the requirement of protection/deprotection steps. However, the need for stoichiometric quantities of high-cost substrates, UDP-sugars, is a limiting factor for its use at an industrial scale. To overcome this challenge, here we report tailor-made coimmobilization and colocalization procedures to assemble a bi-enzymatic cascade composed of a glycosyltransferase and a sucrose synthase for the regioselective 5-O-ß-D-glycosylation of piceid with in situ cofactor regeneration. Coimmobilization and colocalization of enzymes was achieved by performing slow immobilization of both enzymes inside the porous support. The colocalization of both enzymes within the porous structure of a solid support promoted an increase in the overall stability of the bi-enzymatic system and improved 50-fold the efficiency of piceid glycosylation compared with the non-colocalized biocatalyst. Finally, piceid conversion to resveratrol 3,5-diglucoside was over 90% after 6 cycles using the optimal biocatalyst and was reused in up to 10 batch reaction cycles accumulating a TTN of 91.7 for the UDP recycling.

Immobilization of Enzymes on Hetero-Functional Supports: Physical Adsorption Plus Additional Covalent Immobilization.

The immobilization of proteins on heterofunctional amino-epoxy and amino-glyoxyl supports is described in this chapter. Immobilization on both supports is performed through a two-step mechanism: in the first step, the enzyme is physically adsorbed to the support, and in the second step, the intramolecular covalent attachment between the adsorbed enzyme and the support is promoted. On the one hand, amino-epoxy supports present a ratio between amino and epoxy groups of 1:1 to allow the rapid adsorption of the enzyme and promote a strong multipoint covalent linkage. On the other hand, amino-glyoxyl supports contain the highest concentration of glyoxyl groups capable of reacting covalently with primary amino groups on the enzyme surface to promote increased rigidification. The intensity of the covalent enzyme-support interaction can be modulated by modifying the ratio between glyoxyl and amino groups of the support. These heterofunctional supports are able to immobilize and rigidify proteins through different orientations, leading to biocatalysts with different enzyme properties (activity, stability, and selectivity).

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

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.

Immobilization-stabilization of a complex multimeric sucrose synthase from Nitrosomonas europaea. Synthesis of UDP-glucose.

Sucrose synthases (SuSys) can be used to synthesize cost-effective uridine 5'-diphosphate glucose (UDP-glc) or can be coupled to glycosyltransferases (GTs) for the continuous recycling of UDP-glc. In this study, we present the first report of the immobilization-stabilization of a SuSy by multipoint covalent attachment. This stabilization strategy is very complex for multimeric enzymes because a very intense multipoint attachment can promote a dramatic loss of activity and/or stability. The homotetrameric SuSy from Nitrosomonas europaea (SuSyNe) was immobilized on a glyoxyl agarose support through two different orientations. The first occurred at pH 8.5 through the surface area containing the greatest number of amino termini from several enzyme subunits. The second orientation occurred at pH 10 through the region of the whole enzyme containing the highest number of Lys residues. The multipoint covalent immobilization of SuSy on glyoxyl agarose at pH 10 provided a very significant stabilization factor under reaction conditions (almost 1000-fold more stable than soluble enzyme). Unfortunately, this important enzyme rigidification led to a dramatic loss of catalytic activity. A less stabilized conjugate, which was 65-fold more stable than the soluble form, preserved 64% of its initial catalytic activity. This derivative could be used for 3 reaction cycles and yielded approximately 210mM of UDP-glc per cycle. This optimal biocatalyst was modified with a polycationic polymer, polyethyleneimine (PEI), increasing its stability in the presence of the organic co-solvents necessary to glycosylate apolar antioxidants by GTs coupled to SuSy.