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Chemical Modification of Novel Glycosidases from Lactobacillus plantarum Using Hyaluronic Acid: Effects on High Specificity against 6-Phosphate Glucopyranoside

Department of Biocatalysis, Institute of Catalysis (CSIC), Marie Curie 2, Campus UAM, Cantoblanco, E-28049 Madrid, Spain
Department of Microbial Biotechnology, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), José Antonio Novais 10, 28040 Madrid, Spain
Institute of Medicinal Chemistry (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain
Author to whom correspondence should be addressed.
Coatings 2019, 9(5), 311;
Received: 9 April 2019 / Revised: 30 April 2019 / Accepted: 7 May 2019 / Published: 9 May 2019
(This article belongs to the Special Issue Surface Chemical Modification)
PDF [2349 KB, uploaded 10 May 2019]


Three novel glycosidases produced from Lactobacillus plantarum, so called Lp_0440, Lp_2777, and Lp_3525, were isolated and overexpressed on Escherichia coli containing a His-tag for specific purification. Their specific activity was evaluated against the hydrolysis of p-nitrophenylglycosides and p-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 p-nitrophenyl-glucopyranoside (pNP-Glu) or p-nitrophenyl-6-phosphate glucopyranoside (pNP-6P-Glu). However, the conjugated cHA-Lp_0440 showed a clear increase in the specificity towards the pNP-Glu and no activity against pNP-6P-Glu. The other two glycosidases (Lp_2777 and Lp_3525) showed high specificity towards pNP-6P-glycosides, especially to the glucose derivative. The HA covalent modification of Lp_3525 (cHA-Lp_3525) generated an enzyme completely specific against the pNP-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 p-nitrophenyl-galactopyranoside (pNP-Gal) at 40 °C, higher than at 25 °C in the case of the conjugated enzyme. View Full-Text
Keywords: glycosidades; phosphate-glycopyranosides; phosphoglucosidase; chemical modification; hyaluronic acid glycosidades; phosphate-glycopyranosides; phosphoglucosidase; chemical modification; hyaluronic acid

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Perez-Rizquez, C.; Lopez-Tejedor, D.; Plaza-Vinuesa, L.; de las Rivas, B.; Muñoz, R.; Cumella, J.; Palomo, J.M. Chemical Modification of Novel Glycosidases from Lactobacillus plantarum Using Hyaluronic Acid: Effects on High Specificity against 6-Phosphate Glucopyranoside. Coatings 2019, 9, 311.

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