Biochemical characterization of Lactobacillus reuteri Glycoside Hydrolase family 70 GTFB type of 4,6-α-Glucanotransferase enzymes that synthesize soluble dietary starch fibersBai, Y., van der Kaaij, R. M., Leemhuis, H., Pijning, T., van Leeuwen, S. S., Jin, Z. & Dijkhuizen, L., 7-Aug-2015, In : Applied and environmental microbiology. 81, 20, p. 7223-7232 10 p.
Research output: Contribution to journal › Article › Academic › peer-review
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/malto-polysaccharides (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 significant potential for industrial applications. Here we report that an N-terminal truncation (1-733 amino acids) strongly enhances the soluble expression level of fully active GTFB-ΔN (approx. 75 fold compared to full length wild type GTFB) in Escherichi coli. In addition, quantitative assays based on amylose V as substrate are described, allowing 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-α-GTase enzymes. The data shows that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by NMR analysis of their final products. Using these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB-ΔN enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.
|Number of pages||10|
|Journal||Applied and environmental microbiology|
|Publication status||Published - 7-Aug-2015|