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

Abstract

c 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. Starch is the second-most-abundant carbohydrate on earth and a major dietary carbohydrate for humans; as a storage carbohydrate it is present in seeds, roots, and tubers of plants (1). It consists of ␣-glucan polymers with ␣1¡4 linkages and a low percentage of ␣1¡6 linkages, in the form of amylose and branched amylopectin (2). Starches are applied in various industrial products such as food, paper, and textiles, often after processing by physical, chemical, or enzymatic treatment (3-6).Dietary fibers and low-glycemic-index (low-GI) food are considered healthy food contributing to our long-term well-being (7,8). Of all the nutritional types of starch, slowly digestible starch with low GI has drawn the strongest interest. Annealing/heatmoisture treatment, recrystallization, and enzymatic treatment are recognized approaches to obtain slowly digestible starch (9-11). Slowly digestible starch materials prepared by physical processing suffer losses upon boiling; therefore, structural modifications through enzymatic treatment of starch are more desirable. In the human digestive system, the ␣1¡6 linkages in starch are hydrolyzed at a lower rate than ␣1¡4 linkages (12, 13). Branching enzymes, alone or in combination with ␤-amylase, are used to increase the percentage of ␣1¡4,6 branches in starches (12)(13)(14).The 4,6-␣-glucanotransferase (4,6-␣-GTase) enzymes, such as GTFB, GTFW, and GTFML4, of Lactobacillus reuteri strains constitute a subfamily of glycoside hydrolase family 70 (GH70); GH70 ...

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

“…Determination of the total activity of GtfC on amylose, in the absence or presence of 1 mM CaCl 2 , revealed that the Ca 2ϩ ions slightly activated GtfC enzyme activity, as is the case for L. reuteri 121 GtfB (20). The effects of pH and temperature on the activity of the full-length GtfC enzyme were examined with amylose V as a substrate.…”

“…Apart from the well-recognized I-to-IV homology regions, the three additional conserved regions (VI, V, and VII) proposed to be related to the maintenance of the structure and to a given enzyme specificity in GH13 family proteins (6,31,32) were identified in the GH70 and GH13 sequences. Interestingly, GtfC-and GtfB-like proteins had some Left, predicted GtfB domain organization plotted along the primary structure (20). Right, primary structure of the E. sibiricum GtfC protein showing functional regions identified by the Pfam server, annotated with information about the pairwise alignment of the different regions, the predicted domain order, and the locations of conserved regions I to IV in GtfC.…”

“…The initial total GtfC enzyme activity was determined by the amylose-iodine assay described by Bai et al (20), with minor modifications. This method is based on monitoring of the decrease in the absorbance at 660 nm of the ␣-glucan-iodine complex resulting from transglycosylation and/or hydrolytic activity (20). The reaction mixture contained 0.125% (wt/vol) amylose V (AVEBE, Foxhol, The Netherlands) in 25 mM sodium acetate buffer (pH 6.0) in a total volume of 325 l. After incubation at 40°C for 10 min, the reaction was started by the addition of 8 g of the purified enzyme.…”

“…Subsequently, the reaction mixture was neutralized by the addition of 12.5 l of 0.4 M HCl. These samples were used to quantify the amylose content by the iodine staining method (20). One unit of activity corresponds to the amount of enzyme converting 1 mg of amylose V/min.…”

Starch meets biotechnology : in planta modification of starch composition and functionalities

“…linkages, generating a linear chain with α-1,6 linkages (Bai et al, 2015;Leemhuis et al, 2014). This specific activity makes GTFB an interesting target enzyme for producing novel starches in planta.…”