Dataset Information

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-?-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

ABSTRACT: 4,6-?-Glucanotransferase (4,6-?-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some ?1?4 linkages but mostly (relatively long) linear chains with ?1?6 linkages and are soluble dietary starch fibers. 4,6-?-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-?N (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-?-GTase enzymes. The data show that GTFB-?N is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-?N were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.

SUBMITTER: Bai Y

PROVIDER: S-EPMC4579422 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

Bai Yuxiang Y van der Kaaij Rachel Maria RM Leemhuis Hans H Pijning Tjaard T van Leeuwen Sander Sebastiaan SS Jin Zhengyu Z Dijkhuizen Lubbert L

Applied and environmental microbiology 20150807 20

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have sig ...[more]

PMID: 26253678

Similar Datasets

Project description:BackgroundThe GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme.MethodsDenaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC.ResultsExpression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.ConclusionIn view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Project description:Previously, we have identified and characterized 4,6-?-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (?1?4)-linkages in amylose and introduce (?1?6)-linkages in linear chains. The 4,6-?-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (?1?6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1?2,6)-?-d-Glcp-(1?6)- (Leuconostoc citreum enzyme) or -(1?3,6)-?-d-Glcp-(1?6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1?3,6)-?-d-Glcp-(1?6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (?1?3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various ?-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (?1?3)-linkages present. The treatment of amylose with 4,6-?-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived ?-glucans with markedly reduced digestibility. These starch derived ?-glucans may find applications in the food industry.

Dataset Information

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-?-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

Publications

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets