Biochemical characterization of a GH70 protein from Lactobacillus kunkeei DSM 12361 with two catalytic domains involving branching sucrase activity

Abstract

The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ). Sequence analysis revealed that GtfZ harbors two separate catalytic cores (CD1 and CD2), predicted to have glucansucrase and branching sucrase specificity, respectively. GtfZ-CD1 was not characterized biochemically due to its unsuccessful expression. With only sucrose as substrate, GtfZ-CD2 was found to mainly catalyze sucrose hydrolysis and leucrose synthesis. When dextran was available as acceptor substrate, GtfZ-CD2 displayed an efficient transglycosidase activity with sucrose as donor substrate. Kinetic analysis showed that the GtfZ-CD2-catalyzed transglycosylation reaction follows a Ping Pong Bi Bi mechanism, indicating the in-turn binding of donor and acceptor substrates in the active site. Structural characterization of the products revealed that GtfZ-CD2 catalyzes the synthesis of single glucosyl (α1 → 3) linked branches onto dextran, resulting in the production of highly branched comb-like α-glucan products. These (α1 → 3) branches can be formed on adjacent positions, as shown when isomaltotriose was used as acceptor substrate. Homology modeling of the GtfZ-CD1 and GtfZ-CD2 protein structure strongly suggests that amino acid differences in conserved motifs II, III, and IV in the catalytic domain contribute to product specificity. Our present study highlights the ability of beneficial lactic acid bacteria to produce structurally complex α-glucans and provides novel insights into the molecular mechanism of an (α1 → 3) branching sucrase.

Detection of fructophilic lactic acid bacteria (FLAB) in bee bread and bee pollen samples and determination of their functional roles

“…Importantly, two L. kunkeei strains, AP15 and AP37, produced 2.79 and 2.01 g/L EPS, respectively, suggesting that in some L. kunkeei strains, EPS production can be an important characteristic. In accordance with this finding, recently, glucansucrases were shown to be presented in some L. kunkeei strains responsible for the glucan type EPS production (Meng et al, 2018;Vasileva et al, 2017). The highest EPS production level in this study was detected for L. mesenteroides AP-40 and was found to be 5.37 g/L EPS.…”

“…In accordance with this finding, recently, glucansucrases were shown to be presented in some L . kunkeei strains responsible for the glucan type EPS production (Meng et al., 2018; Vasileva et al., 2017). The highest EPS production level in this study was detected for L. mesenteroides AP‐40 and was found to be 5.37 g/L EPS.…”

Antagonistic Activity against Ascosphaera apis and Functional Properties of Lactobacillus kunkeei Strains

“…Bacterial biofilm production is a highly complex process depending on the expression of specific genes, and this process is a means by which to adapt to new nutritional and environmental conditions [70]. Several studies have reported that in this phase of adaptation, bacteria produce extracellular polymeric substances (EPS), especially exopolysaccharides and proteinaceous compounds, that may be constituents of the biofilm and could exert an antimicrobial action [72][73][74][75][76].…”

“…apis DSM 3116 and L. kunkeei DSM 12361, belonging to the DSMZ collection (German Collection of Microorganism and Cell Cultures GmbH), were used as reference cultures. We used the DSM 12361 strain as a reference because it has demonstrated a capacity in previous research to produce EPS [76][77][78].…”

Structures, physico-chemical properties, production and (potential) applications of sucrose-derived α-d-glucans synthesized by glucansucrases

“…35 g/L (Miao, Bai et al, 2014;Miao, Jia, Hamaker, et al, 2016) L. NR (Brison et al, 2010;Passerini et al, 2015) L. citreum NRRL B-742 /Brs-B L. fallax KCTC3537 /Brs-C L. kunkeei DSM 12361/GtfZ-CD2 α-(1→3) branched dextran 0−50% α-(1→3) and 50−100% α-(1→6) Resistant to hydrolysis by digestive enzymes Potential prebiotics (stimulating the production of short chain fatty acids) NR (Meng et al, 2018;Vuillemin et al, 2016) NR: not reported.…”