Glucansucrase Gtf180-?N of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.

ABSTRACT: Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of ?-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing ?-glucan synthesis by mutational engineering of the Gtf180-?N enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-?N mutants (L938F, L981A, and N1029M) with an impaired ?-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-?-D-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-?N protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired ?-glucan synthesis, thus yielding mutants with an improved glycosylation potential.

SUBMITTER: Devlamynck T 

PROVIDER: S-EPMC4980424 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature