Enzyme Engineering for Oligosaccharide Biosynthesis

Talens-Perales, David; Polaina, Julio; Marín-Navarro, Julia

doi:10.1007/978-81-322-2610-9_2

Cited by 5 publications

(4 citation statements)

References 149 publications

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“…The crystal structure of ArthβDG, as well as establishing purification and crystallization protocols, gives a good starting point for further crystallographic analysis of this enzyme aiming at its activity engineering [48]. The comparison of its catalytic center, with other βDGs indicates putative residues involved in the transglycosylation reaction.…”

Section: Discussionmentioning

confidence: 99%

In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

et al. 2018

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Abstract:There is an increasing demand for cold-adapted enzymes in a wide range of industrial branches. Nevertheless, structural information about them is still scarce. The knowledge of crystal structures is important to understand their mode of action and to design genetically engineered enzymes with enhanced activity. The most difficult task and the limiting step in structural studies of cold-adapted enzymes is their crystallization, which should provide well-diffracting monocrystals. Herein, we present a combination of well-established crystallization methods with new protocols based on crystal seeding that allowed us to obtain well-diffracting crystals of two cold-adapted β-D-galactosidases (βDGs) from Paracoccus sp. 32d (ParβDG) and from Arthrobacter sp. 32cB (ArthβDG). Structural studies of both βDGs are important for designing efficient and inexpensive enzymatic tools for lactose removal and synthesis of galacto-oligosaccharides (GOS) and hetero-oligosaccharides (HOS), food additives proved to have a beneficial effect on the human immune system and intestinal flora. We also present the first crystal structure of ArthβDG (PDB ID: 6ETZ) determined at 1.9 Å resolution, and compare it to the ParβDG structure (PDB ID: 5EUV). In contrast to tetrameric lacZ βDG and hexameric βDG from Arthrobacter C2-2, both of these βDGs are dimers, unusual for the GH2 family. Additionally, we discuss the various crystallization seeding protocols, which allowed us to obtain ParβDG and ArthβDG monocrystals suitable for diffraction experiments.

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Section: Discussionmentioning

confidence: 99%

In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

et al. 2018

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“…Their use in biocatalysis has been extensively researched, but is still limited for a large scale by the high cost of the nucleotide-phosphate donors and difficulty in expression and handling of these mostly membrane bound enzymes [ 7 ]. Alternative carbohydrate active enzymes are glycohydrolases or glycosidases, of which a high variety are readily available due to their occurrence in the metabolic pathways of all organisms [ 8 ]. This group of enzymes naturally degrade glycosidic structures and are defined into two groups depending on their catalytic mechanism ( Scheme 1 A,B) [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of Glycosides by Glycosynthases

Hayes

Pietruszka

2017

Molecules

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The many advances in glycoscience have more and more brought to light the crucial role of glycosides and glycoconjugates in biological processes. Their major influence on the functionality and stability of peptides, cell recognition, health and immunity and many other processes throughout biology has increased the demand for simple synthetic methods allowing the defined syntheses of target glycosides. Additional interest in glycoside synthesis has arisen with the prospect of producing sustainable materials from these abundant polymers. Enzymatic synthesis has proven itself to be a promising alternative to the laborious chemical synthesis of glycosides by avoiding the necessity of numerous protecting group strategies. Among the biocatalytic strategies, glycosynthases, genetically engineered glycosidases void of hydrolytic activity, have gained much interest in recent years, enabling not only the selective synthesis of small glycosides and glycoconjugates, but also the production of highly functionalized polysaccharides. This review provides a detailed overview over the glycosylation possibilities of the variety of glycosynthases produced until now, focusing on the transfer of the most common glucosyl-, galactosyl-, xylosyl-, mannosyl-, fucosyl-residues and of whole glycan blocks by the different glycosynthase enzyme variants.

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“…Oligosaccharides are important compounds for the food and pharmaceutical industries because of their growing use as prebiotics and antioxidants and for drug delivery. 1 A relevant type of oligosaccharides are isomaltooligosaccharides (IMOs), roughly defined from a chemical point of view as short glucose oligomers (between two and nine units) containing α-(1→6), α-(1→3), or α-(1→2) linkages. Examples are isomaltose, panose, isomaltotriose, nigerosylglucose, and kojibiosylglucose, among others.…”

Section: Introductionmentioning

confidence: 99%

“…Enzymatic synthesis has become a preferred procedure for oligosaccharide production over alternative chemical synthesis or polysaccharide hydrolysis. Transferases and retaining glycoside hydrolases which are naturally endowed with transferase activity and do not require activated substrates are generally used . In the specific case of IMO synthesis, the enzymes used are dextransucrases or alternansucrases from lactic acid bacteria, or fungal transferases and α-glucosidases. − The use of glycoside hydrolases, belonging to family GH31, for IMO synthesis is possible because these are retaining enzymes, whose catalytic mechanism allows them to act as transferases.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α-Glucosidase

2017

Self Cite

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The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.

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Enzyme Engineering for Oligosaccharide Biosynthesis

Cited by 5 publications

References 149 publications

In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

Synthesis of Glycosides by Glycosynthases

Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α-Glucosidase

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