Exploring the glucosylation potential of glucansucrases : from enzyme to product

De introductie van een glycosylgroep kan zowel de fysicochemische als biologische eigenschappen van organische moleculen, zoals antioxidanten, antibiotica en zoetstoffen, beïnvloeden. Een industrieel relevant voorbeeld is de glycosylatie van steviolglycosiden, zoete verbindingen die geëxtraheerd worden uit Stevia, om zo hun ongewenste bitterheid te elimineren. De chemische synthese van glycosiden is een meerstapsproces dat gekenmerkt wordt door de productie van een grote hoeveelheid afvalproducten. Enzymatische glycosylatie, in het bijzonder met glucansucrases, geniet de voorkeur; de hoge specificiteit van enzymen leidt immers tot de productie van 5 keer minder afval. Het eerste deel van deze thesis spitste zich toe op het geschikter maken van glucansucrases als glycosylatiebiokatalysatoren door middel van reaction – en enzymengineeringstrategieën. De resulterende kennis werd vervolgens toegepast op de glycosylatie van steviolglycosiden, met als ultiem doel het verbeteren van hun smaakeigenschappen. Na het selecteren van de meest geschikte enzymmutant werden de reactiecondities geoptimaliseerd en werd een efficiënte downstream processing ontwikkeld. De beoordeling van de smaakeigenschappen van de geglycosyleerde producten door een getraind smaakpanel onthulde dat de typische Stevia-bitterheid geëlimineerd was. Gezien de ontwikkelde technologie commercieel potentieel vertoonde werd een kostenanalyse van het proces uitgevoerd. Hierdoor konden verschillende opportuniteiten aangeduid worden voor procesverbetering

Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri

Previously, we have shown that the glucansucrase GtfA-ΔN enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937-946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-ΔN used both α-D-Glcp-catechol and α-D-Glcp-(1→4)-α-D-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-ΔN used α-D-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-D-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates

Trans-α-glucosylation of stevioside by the mutant glucansucrase enzyme Gtf180-ΔN-Q1140E improves its taste profile

The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient alpha-glucosylation of stevioside using the mutant glucansucrase Gtf180-Delta N-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the alpha-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties

Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties

Glucansucrase Gtf180-Delta N of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules

Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of alpha-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing alpha-glucan synthesis by mutational engineering of the Gtf180-Delta N enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-Delta N mutants (L938F, L981A, and N1029M) with an impaired alpha-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-alpha-d-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-Delta N protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired alpha-glucan synthesis, thus yielding mutants with an improved glycosylation potential

Biphasic catalysis with disaccharide phosphorylases: chemoenzymatic synthesis of α-D-glucosides using sucrose phosphorylase

Thanks to its broad acceptor specificity, sucrose phosphorylase (SP) has been exploited for the transfer of glucose to a wide variety of acceptor molecules. Unfortunately, the low affinity (K-m > 1 M) of SP towards these acceptors typically urges the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of alpha-D-glucosides, such as cinnamyl alpha-D-glucopyranoside, geranyl alpha-D-glucopyranoside, 2-O-alpha-D-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-alpha-D-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial pyrogallol was converted when using biphasic catalysis

Pilot scale recovery of lignin from black liquor and advanced characterization of the final product

Recently, the academic and industrial interest in lignin as a renewable resource for many valuable applications has been on the rise. However, the current biomass separation technologies are focused on obtaining high quality cellulose which can be further processed, e.g., in the paper industry, resulting in a lignin of rather low quality. Moreover, lignin recovery from black liquor is often accompanied with filter clogging and a severe flux decline, limiting the cost-efficiency of its valorization. In this work, the pilot scale recovery of lignin from a black liquor derived from a mild soda pulping process of Miscanthus x giganteus chips is studied with the aim to develop a straightforward procedure that yields a high quality final product. A first pilot scale experiment demonstrated the pH to be crucial for optimal precipitation. Moreover, adding an enzyme mixture containing cellulases, hemicellulases and beta-glucosidases, clearly enhanced the flocculation and filterability. Thorough characterization of the obtained lignin showed a native-like structure which can be related to the mild pulping conditions and revealed that the p-coumarates and ferulates were converted to the free acids as a result of the base catalyzed hydrolysis as well as the enzymatic cleavage of the ester linkages leading to the complete removal of the hydrophilic (poly)saccharides. Moreover, this resulted in a slightly more hydrophobic lignin material that was more amenable to flocculation. Building on lab scale experiments aimed at optimization of the process conditions, a second pilot scale experiment was performed resulting in improved precipitation and flocculation by means of acidification, an enzymatic treatment as well as the addition of a flocculant. This allowed for smooth filtration and resulted in a high purity of the isolated lignin

Pilot scale recovery of lignin from black liquor and advanced characterization of the final product

Recently, the academic and industrial interest in lignin as a renewable resource for many valuable applications has been on the rise. However, the current biomass separation technologies are focused on obtaining high quality cellulose which can be further processed, e.g., in the paper industry, resulting in a lignin of rather low quality. Moreover, lignin recovery from black liquor is often accompanied with filter clogging and a severe flux decline, limiting the cost-efficiency of its valorization. In this work, the pilot scale recovery of lignin from a black liquor derived from a mild soda pulping process of Miscanthus x giganteus chips is studied with the aim to develop a straightforward procedure that yields a high quality final product. A first pilot scale experiment demonstrated the pH to be crucial for optimal precipitation. Moreover, adding an enzyme mixture containing cellulases, hemicellulases and β-glucosidases, clearly enhanced the flocculation and filterability. Thorough characterization of the obtained lignin showed a native-like structure which can be related to the mild pulping conditions and revealed that the p-coumarates and ferulates were converted to the free acids as a result of the base catalyzed hydrolysis as well as the enzymatic cleavage of the ester linkages leading to the complete removal of the hydrophilic (poly)saccharides. Moreover, this resulted in a slightly more hydrophobic lignin material that was more amenable to flocculation. Building on lab scale experiments aimed at optimization of the process conditions, a second pilot scale experiment was performed resulting in improved precipitation and flocculation by means of acidification, an enzymatic treatment as well as the addition of a flocculant. This allowed for smooth filtration and resulted in a high purity of the isolated lignin