Improvement of functional properties of a thermostable β‐glycosidase for milk lactose hydrolysis

Abstract

In this work, a new exploitation of the thermostable β-glycosidase from Sulfolobus solfataricus expressed in Saccharomyces cerevisiae to create functional foods for low lactose diets was evaluated. For this purpose, the lactose hydrolysis reaction using immobilized and soluble enzymes was investigated. Activity and stability at different conditions of pH and temperature were tested. The immobilization process had a big impact on the catalysis performance, leading to an enhancement of the enzymatic reaction rate on lactose, as demonstrated by the increasing of 2 and 2.5 folds of K and K /K , respectively. Moreover, the maximal activity for the immobilized form was referred at pH 6.5 instead of 7.0, leading to an improvement of the catalytic performance at milk pHs. Although the soluble enzyme was already weakly inhibited by the reaction products, the immobilization further reduced the inhibitory action of glucose increasing the K from 96.7 to 110.4 mM. Finally, the immobilized enzyme showed high hydrolysis rate in whole milk that yielded 99% of lactose breakdown in 10 and 30 min at 60 and 40°C, respectively. These results support the application of the immobilized β-glycosidase for the development of new functional foods particularly suitable to the alleviation of lactose intolerance.

Forty years of study on the thermostable β‐glycosidase fromS. solfataricus: Production, biochemical characterization and biotechnological applications

“…The recovery of lacS expressed in S. cerevisiae is particularly advantageous since after thermoprecipitation treatments, high degrees of enzyme purification were obtained. As reported by Ionata et al [19], after only two sequential thermoprecipitation steps (at 60 and then 70°C), a 90% recovery of partially pure enzyme with a specific activity of 106 U/mg was obtained; the addition of only one hydrophobic chromatographic step allowed to obtain 2 mg/L of pure enzyme with a specific activity of 166 U/mg [43]. The purification of the enzyme in native form, instead, required three chromatographic steps to separate Sβgly from the archaeal thermostable proteins of S. solfataricus crude extract and yielded only a 28% of enzyme recovery [20].…”

“…The efficiency during a biocatalytic process could decrease for the inhibition of increasing concentrations of the product, for the effect on the balance of the reaction, as reported for several galactosidases in the presence of free glucose [58][59][60]. Studies carried out on the Sβgly catalytic efficiency in the presence of large amount of free glucose, used as inhibitor (K i value between 96.7 and 110.4 mM), demonstrated that the monosaccharide did not substantially affect the enzyme affinity and hydrolytic capability for the synthetic and natural substrates [19].…”

“…However, denaturation and aggregation processes started at temperature higher than 85°C reaching a plateau at about 100°C [27,72]. Small differences in the thermal denaturation process are also dependent on the structural changes caused by PTMs occurring in the native and in some expressed enzymes, as above described [19,27].…”

“…In the Sβgly sequence, only one consensus sequence for N-glycosylation is present; thus, it could be hypothesized that glycosylation may occur Applied Biochemistry principally to serine or threonine residues. Further analyses should be carried on to characterize the glycosylation sites and the bound oligosaccharide, in order to elucidate the peculiar features of the enzyme expressed in S. cerevisiae for milk hydrolysis [19], and also for the possible importance in its biotechnological applications.…”

“…The β-glycosidase isolated by S. solfataricus (Sβgly) [12] represents an interesting example of a well-characterized hydrolytic enzyme, which has been proposed for several biotechnological applications [13][14][15][16][17][18][19]. Based on its ability to hydrolyze the substrate 5-bromo-4-chloro-3-indolyl-βd-galactopyranoside, this enzyme was initially described as β-galactosidase [20].…”