Mutational Analysis of the Role of the Glucansucrase Gtf180-Delta N Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate

Hien Pham, Tjaard Pijning, Lubbert Dijkhuizen*, Sander Sebastiaan van Leeuwen

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Glucansucrase Gtf180-Delta N from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (alpha 1 -> 2)/(alpha 1 -> 3)/(alpha 1 -> 4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (alpha 1 -> 3) linkage and increased F4 4',2-glc-lac with (alpha 1 -> 4)/(alpha 1 -> 2) linkages. Formation of F2 2-glc-lac with an (alpha 1 -> 2) linkage and F4 was negatively affected in most W1065 and N1029 mutants, respectively. Mutant N1029G synthesized four new products with additional (alpha 1 -> 3)-linked glucosyl moieties (2xDP4 and 2xDP5). Sucrose/lactose strongly reduced Gtf180-Delta N hydrolytic activity and increased transferase activity of Gtf180-Delta N and mutant N1029G, in comparison to activity with sucrose alone. N1029/W1065/Q1140 thus are key determinants of Gtf180-Delta N linkage and product specificity in the acceptor reaction with lactose. Mutagenesis of key residues in Gtf180-Delta N may allow synthesis of tailor-made mixtures of novel lactose-derived oligosaccharides with potential applications as prebiotic compounds in food/feed and in pharmacy/medicine.

Original languageEnglish
Article numberacs.jafc.8b04486
Pages (from-to)12544-12554
Number of pages11
JournalJournal of Agricultural and Food Chemistry
Volume66
Issue number47
Early online date6-Nov-2018
DOIs
Publication statusPublished - 28-Nov-2018

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