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

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

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

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