| Summary: | Three novel glycosidases produced from <i>Lactobacillus plantarum</i>, so called Lp_0440, Lp_2777, and Lp_3525, were isolated and overexpressed on <i>Escherichia coli</i> containing a His-tag for specific purification. Their specific activity was evaluated against the hydrolysis of <i>p</i>-nitrophenylglycosides and <i>p</i>-nitrophenyl-6-phosphate glycosides (glucose and galactose) at pH 7. All three were modified with hyaluronic acid (HA) following two strategies: A simple coating by direct incubation at alkaline pH or direct chemical modification at pH 6.8 through preactivation of HA with carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at pH 4.8. The modifications exhibited important effect on enzyme activity and specificity against different glycopyranosides in the three cases. Physical modification showed a radical decrease in specific activity on all glycosidases, without any significant change in enzyme specificity toward monosaccharide (glucose or galactose) or glycoside (C-6 position free or phosphorylated). However, the surface covalent modification of the enzymes showed very interesting results. The glycosidase Lp_0440 showed low glycoside specificity at 25 °C, showing the same activity against <i>p</i>-nitrophenyl-glucopyranoside (<i>p</i>NP-Glu) or <i>p</i>-nitrophenyl-6-phosphate glucopyranoside (<i>p</i>NP-6P-Glu). However, the conjugated cHA-Lp_0440 showed a clear increase in the specificity towards the <i>p</i>NP-Glu and no activity against <i>p</i>NP-6P-Glu. The other two glycosidases (Lp_2777 and Lp_3525) showed high specificity towards <i>p</i>NP-6P-glycosides, especially to the glucose derivative. The HA covalent modification of Lp_3525 (cHA-Lp_3525) generated an enzyme completely specific against the <i>p</i>NP-6P-Glu (phosphoglycosidase) maintaining more than 80% of the activity after chemical modification. When the temperature was increased, an alteration of selectivity was observed. Lp_0440 and cHA-Lp_0440 only showed activity against <i>p</i>-nitrophenyl-galactopyranoside (<i>p</i>NP-Gal) at 40 °C, higher than at 25 °C in the case of the conjugated enzyme.
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