Characterization and improvement of a new glycosidase from Lactobacillus plantarum

Póster presentado a la VII International Conference on Environmental Industrial and Applied Microbiology, celebrada en Madrid (España) del 18 al 20 de octubre de 2017.

In recent years, the high stereoselectivity and efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavour enhancement in foods. In recent years, the recent advances in carbohydrate synthesis by glycosidases are based on two complementary approaches: the use of wild-type enzymes with engineered substrates, and mutant glycosidases. In particular, much attention has been focused on the use of β-glucosidases for the enzymatic hydrolysis of flavourless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to found a novel glycosidase with potential applications in food industry it has been produced and biochemically characterized Lp_3629 glycosidase, a member of GH1 glycosil hydrolase family, from Lactobacillus plantarum. For that purpose, we have clone, using a LIC strategy described previously, and heterologously expressed lp_3629 gene in Escherichia coli. The recombinant Lp_3629 protein containing an amino terminal His6 tag has been produced in a soluble form. Purified recombinant enzyme showed galactosidase but not glucosidase activity. However when 6-phospho-β-D-glucopyranoside was synthetized, it was demonstrated that the enzyme mainly exhibited 6- phospho-β-glucosidase activity. Lp_3629 showed solubility problems since it is a metastable protein. In order to avoid the protein instability, a double mutant Cys211Ser /Cys 292Ser was constructed by site-directed mutagenesis PCR technique. The Lp_3629mutant showed high solubility although it maintain its kinetic parameters.

Financial support from the MINEICO (AGL2014-52911-R).

Peer Reviewed