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Biotechnol Adv ; : 107465, 2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31689470


ß-Galactosidases, an important class of glycosidases, naturally catalyze the hydrolysis of ß-galactosidic bonds in oligosaccharides and polysaccharides. Traditionally, these enzymes have been used to degrade lactose in dairy products, which are beneficial for lactose-intolerant people. Attractively, ß-galactosidases exhibit glycosyl transfer activity under certain conditions in vitro. They are capable of synthesizing carbohydrates from cheap starting substrates in a facile, efficient, and environment-friendly way. The condensation of lactose into the well-known prebiotic galacto-oligosaccharides by ß-galactosidases has become a key aspect of the industrial interest in the synthetic activity in recent years. At present, the transglycosylation activity of these enzymes has been greatly extended. It can be used not only in building glycan blocks of crucial glycoconjugates to elucidate their biological functions, but also in glycosylation of vital molecules, which have been applied in food, medicine and cosmetic industries to improve solubility, stability and bioactivity. Further molecular engineering of ß-galactosidases has significantly improved their synthetic activity, expanded the substrate spectrum and made them more powerful in carbohydrate synthesis. This review covers the classification, structure and mechanism of ß-galactosidases, galactosylation reactions catalyzed by these enzymes, and various strategies of enzyme engineering, with an emphasis on recent advances.

J Agric Food Chem ; 67(7): 2012-2019, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30678460


α-Amylases are among the most important and widely used industrial enzymes for starch processing. In this work, an α-amylase from Bacillus subtilis XL8 was purified and found to possess both hydrolysis and transglycosylation activities. The optimal pH and temperature for starch hydrolysis were pH 5.0 and 70 °C, respectively. The enzyme could degrade soluble starch into beneficial malto-oligosaccharides ranging from dimer to hexamer. More importantly, it was able to catalyze α-glycosyl transfer from the soluble starch to salidroside, a medicinal plant-derived component with broad pharmacological properties. The transglycosylation reaction catalyzed by the enzyme generated six derivatives in a total high yield of 73.4% when incubating with 100 mg/mL soluble starch and 50 mM salidroside (pH 7.5) at 50 °C for 2 h. These derivatives were identified as α-1,4-glucosyl, maltosyl, maltotriosyl, maltotetraosyl, maltopentaosyl, and maltohexaosyl salidrosides, respectively. They were novel promising compounds that might integrate the bioactive functions of malto-oligosaccharides and salidroside.

Glucosídeos/metabolismo , Fenóis/metabolismo , Amido/metabolismo , Bacillus subtilis/enzimologia , Glicosilação , Hidrólise , Maltose/metabolismo , Oligossacarídeos/metabolismo , alfa-Amilases/metabolismo
Appl Environ Microbiol ; 84(13)2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29678922


Gut bacteria provide a rich source of glycosidases that can recognize and/or hydrolyze glycans for nutrition. Interestingly, some glycosidases have also been found to catalyze transglycosylation reactions in vitro and thus can be used for oligosaccharide synthesis. In this work, six putative and one known exo-α-sialidase genes-three from Bacteroides fragilis NCTC9343, three from Clostridium perfringens ATCC 13124, and one known from Bifidobacterium bifidum JCM1254-were subjected to gene cloning and heterogeneous expression in Escherichia coli The recombinant enzymes were purified, characterized for substrate specificity, and screened for transglycosylation activity. A sialidase, named BfGH33C, from B. fragilis NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide. The native BfGH33C was a homodimer with a molecular weight of 113.6 kDa. The Km and kcat values for 4-methylumbelliferyl N-acetyl-α-d-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.2 s-1, and 0.75 mM and 329.6 s-1, respectively. The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict α2-6 regioselectivity. The influences of the initial substrate concentration, pH, temperature, and reaction time on transglycosylation were investigated in detail. Using 40 mM sialic acid dimer (or 40 mg/ml oligomer) and 1 M lactose (pH 6.5) at 50°C for 10 min, BfGH33C could specifically produce 6'-sialyllactose, a dominant sialylated human milk oligosaccharide, at a maximal conversion ratio above 20%. It provides a promising alternative to the current chemical and enzymatic methods for obtaining sialylated oligosaccharides.IMPORTANCE Sialylated human milk oligosaccharides are significantly beneficial to the neonate, as they play important roles in supporting resistance to pathogens, gut maturation, immune function, and brain and cognitive development. Therefore, access to the sialylated oligosaccharides has attracted increasing attention both for the study of saccharide functions and for the development of infant formulas that could mimic the nutritional value of human milk. Nevertheless, nine-carbon sialic acids are rather complicated for the traditional chemical modifications, which require multiple protection and deprotection steps to achieve a specific glycosidic bond. Here, the exo-α-sialidase BfGH33C synthesized 6'-sialyllactose in a simple step with high transglycosylation activity and strict regioselectivity. Additionally, it could utilize oligosialic acid, which was newly prepared in an easy, economical way to reduce the substrate cost, as a glycosyl donor. All the studies laid a foundation for the practical use of BfGH33C in large-scale synthesis of sialylated oligosaccharides in the future.