Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118

Abstract

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K(M) 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0-6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-D-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked D-glucopyranosyl units and a low degree of branching.

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

“…Glucansucrase assays and polysaccharide structure determinations were carried out as before (Côté and Skory 2012). Enzyme assays were based on the incorporation of 14 C-labeled glucose from 14 C-(U)-labeled sucrose (PerkinElmer-NEN, Waltham, MA) into alcoholinsoluble polysaccharide (i.e., includes water-soluble and insoluble fractions), according to a modification of the method described by Germaine et al (1974).…”

“…In addition, the enzymes that form this type of glucan react more readily with the water-soluble co-product, fructose, than with the nascent water-insoluble glucan resulting in significantly higher yields of oligosaccharides Lee et al 1997). We previously cloned a glucansucrase, DsrI, from the type strain of L. mesenteroides NRRL B-1118 that catalyzes the synthesis of a waterinsoluble α-D-glucan (Côté and Skory 2012). In an effort to alter production of the desired glucan, we further modified this enzyme with a series of site-directed amino acid substitutions within the catalytic domain (Côté and Skory 2014).…”

Production of isomelezitose from sucrose by engineered glucansucrases

“…We previously cloned a glucansucrase (DsrI, EC 2.4.1.125) from Leuconostoc mesenteroides strain NRRL B-1118 [9] that synthesizes a water-insoluble glucan, and demonstrated that amino acid substitutions within the active site of the enzyme at threonine residue 654 exhibit altered linkage specificity with respect to the ratios of α(1→3) and α(1→6) d-glucopyranosyl linkages [27]. Several of those strains produced higher proportions of α(1→3) linkages, but also gave lower yields of glucan.…”

“…Glucan formation was measured directly by monitoring the incorporation of 14 C-glucose into methanol-insoluble glucan using a modified filter-paper technique [9,18]. Alternatively, glucansucrase activity could be determined indirectly by measuring the accumulation of fructose released under the same reaction conditions using the Megazyme D-Glucose/ D-Fructose Assay Kit with a modified microplate protocol from those previously described [19].…”

“…The glucansucrase, DsrI (EC 2.4.1.125), from L. mesenteroides NRRL B-1118 was previously cloned and expressed in E. coli using a small ubiquinone-like modifier (SUMO) fusion tag to improve solubility [9]. After removal of the SUMO tag with SUMO protease 1, the purified enzyme is expected to have the same amino acid sequence as the mature full-length protein without the native DsrI signal peptide, which is normally removed during secretion in the wild-type L. mesenteroides host.…”