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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; van der Kaaij, Rachel Maria; Leemhuis, Hans; Pijning, Tjaard; van Leeuwen, Sander Sebastiaan; Jin, Zhengyu; Dijkhuizen, Lubbert.
Afiliación
  • Bai Y; Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, China.
  • van der Kaaij RM; Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands.
  • Leemhuis H; Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands.
  • Pijning T; Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands.
  • van Leeuwen SS; Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands.
  • Jin Z; The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, China.
  • Dijkhuizen L; Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands L.Dijkhuizen@rug.nl.
Appl Environ Microbiol ; 81(20): 7223-32, 2015 Oct.
Article en En | MEDLINE | ID: mdl-26253678
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 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-ΔN (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-α-GTase enzymes. The data show that GTFB-ΔN 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-ΔN 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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Almidón / Proteínas Bacterianas / Limosilactobacillus reuteri / Glucosiltransferasas Idioma: En Revista: Appl Environ Microbiol Año: 2015 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Almidón / Proteínas Bacterianas / Limosilactobacillus reuteri / Glucosiltransferasas Idioma: En Revista: Appl Environ Microbiol Año: 2015 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos