Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase:  Assessment of Directed Evolution Strategies

Kelly, Ronan M.; Leemhuis, Hans; Dijkhuizen, Lubbert

doi:10.1021/bi701160h

Cited by 54 publications

(38 citation statements)

References 43 publications

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“…The introduction of a bulky hydrophobic Trp in the active site often enhances the transglycosylation capacity (Fujita and Takegawa, 2002;Tang et al, 2006). The finding that only a few amino acids are essential to discriminate between transferases and hydrolases was also reported by Schroeven et al (2008), by Chambert and PetitGlatron (1991), and by Kelly et al (2007).…”

Section: Understanding the Complex Role Of W343mentioning

confidence: 71%

Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

et al. 2008

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Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase , sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase , fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.Fructans are Fru polymers that can be considered as an extension of Suc, accumulating above a certain threshold Suc concentration (Cairns and Pollock, 1988;Guerrand et al., 1996;Maleux and Van den Ende, 2007). Fructans occur in many dicot and monocot plant species mainly belonging to Asteraceae, Liliaceae, and Poaceae (Hendry, 1993;Prud'homme et al., 2007;Shiomi et al., 2007;Van den Ende and Van Laere, 2007;Yoshida et al., 2007). Nowadays, research is mainly focused on economically important cereals (wheat [Triticum aestivum] and barley [Hordeum vulgare]) and forage grasses (mainly Lolium species). Depending on the linkage type between the fructosyl residues and the position of the Glc residue (Lewis, 1993), several fructan types can be distinguished. Fructans with a terminal Glc residue include the b(2-1)-type fructans (inulin, principally occurring in dicots) and the linear b(2-6) (levan)-or branched-type fructans (graminan) with both b(2-6) and b(2-1) linkages (as occurring in bacteria and cereals, respectively). Fructans with an internal Glc residue include the neoinulin and neolevan types (occurring in monocots such as Allium, Asparagus, and Lolium).Apart from their function as a vacuolar storage carbohydrate, fructans may help to protect plants ag...

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Section: Understanding the Complex Role Of W343mentioning

confidence: 71%

Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

et al. 2008

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“…[107] Mutants displaying switched selectivity at high activity were obtained by using a series of small saturation mutagenesis libraries originating from sites aligning the binding pocket, screening between 320 and 720 trasnsformants, and applying ISM. In contrast, much larger epPCR libraries showed hits having only moderately improved catalytic profiles.…”

Section: Modulating Lignin Biosynthesis For Better Utilization Of Plamentioning

confidence: 99%

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

2010

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Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long-standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non-ending source of robust and selective catalysts for a variety of useful applications.

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“…3). Reducing power and glucose measurements are widely accepted methods to determine H of starchconverting enzymes with either endo-or exohydrolyzing activity, such as ␣-amylase, ␣-glucosidase, and cyclodextrin glucanotransferase (30)(31)(32). In the case of 4,6-␣-GTases, the measurement of free glucose specifically may lead to an underestimation of H. The reducing power assay representing both free glucose and products of the transferase reaction with glucose as the acceptor substrate may give a more precise estimate of H (Fig.…”

Section: Figmentioning

confidence: 99%

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

Kaaij

Leemhuis

et al. 2015

Appl Environ Microbiol

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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 ...

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Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

Cited by 54 publications

References 43 publications

Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

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

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