Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM‐1 towards Donor Substrates Glucose/Mannose 1‐Phosphate by Using X‐ray Crystallography and Saturation Transfer Difference NMR Spectroscopy

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1‐phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM‐1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (β‐d‐mannopyranosyl‐(1→3)‐d‐glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α‐mannose 1‐phosphate (Man1P). To investigate how the enzyme recognises different sugar 1‐phosphates, the X‐ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1‐phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1‐phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these -glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in -glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4- -glucan 4- -glucosyltransferase from GH31. Distinct from 1,4- -glucan 6- -glucosyltransferases (EC 2.4.1.24) and 4- -glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from (134)- glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro--glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme

Rational design of an improved transglucosylase for production of the rare sugar nigerose

The sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) can be used as a transglucosylase for the production of rare sugars. We designed variants of BaSP for the efficient synthesis of nigerose from sucrose and glucose, thereby adding to the inventory of rare sugars that can conveniently be produced from bulk sugars

Structural Insights into the Epimerization of β-1,4-Linked Oligosaccharides Catalyzed by Cellobiose 2-Epimerase, the Sole Enzyme Epimerizing Non-anomeric Hydroxyl Groups of Unmodified Sugars

Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of β1,4-mannobiose to 4-O-β-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the β5-β6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand

Trehalose analogues: latest insights in properties and biocatalytic production

Trehalose (alpha-d-glucopyranosyl alpha-d-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (alpha-d-glucopyranosyl alpha-d-galactopyranoside) or galactotrehalose (alpha-d-galactopyranosyl alpha-d-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. "Greener" alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis

Sucrose phosphorylase and related enzymes in glycoside hydrolase family 13 : discovery, application and engineering

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield alpha-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6(F)-phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years

β-Glucan phosphorylases in carbohydrate synthesis

beta-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of beta-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of beta-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 (www.cazy.org). Since the discovery of beta-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of beta-glucan and associated beta-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules

Efficient Enzymatic Synthesis of α-(1→4)-glucosidic Disaccharides by Maltose Phosphorylase from Lactobacillus acidophilus NCFM

A gene cluster involved in maltose/maltodextrin metabolism was identified in the genome of Lactobacillus acidophilusNCFM. Enzymatic properties was described for MalP belonging to glycoside hydrolase family 65 (GH65), which is encodedby malP (GenBank: AAV43670.1) located in the gene cluster. MalP catalyses phosphorolysis of maltose with inversion ofthe anomeric configuration releasing β-glucose 1-phosphate and glucose. Broad acceptor specificity was demonstrated byreverse phosphorolysis using various carbohydrate acceptors and β-glucose 1-phosphate as donor. MalP showed strongpreference for monosaccharide acceptors with equatorial 3-OH and 4-OH, and reacted also with the 2-deoxy amino sugars.By contrast none of the tested di- and trisaccharides served as acceptor. Disaccharide yields obtained from 50 mM β-glucose 1-phosphate and 50 mM glucose, glucosamine, N-acetyl glucosamine, mannose, xylose, or L-fucose were 99, 80, 53,93, 81, and 13%, respectively. Product structures were determined by NMR and ESI-MS to be α-D-glucopyranosyl-(1→4)-Dglucopyranose,α -D-glucopyranosyl-(1→4)-D-glucosaminopyranose,α -D-glucopyranosyl-(1→4)-N-acetyl-D-glucosaminopyranose,α -D-glucopyranosyl-(1→4)-D-mannopyranose, α-D-glucopyranosyl-(1→4)-D-xylopyranose, and α-D-glucopyranosyl-(1→4)-L-fucopyranose. Additionally MalP catalysed synthesis of the α-(1 → 4)-glucosidic disaccharides from maltose in a coupledphosphorolysis/reverse phosphorolysis one-pot reaction. Thus phosphorolysis of maltose to β-glucose 1-phosphatecircumvented addition of costly β-glucose 1-phosphate for reverse phosphorolysis with different monosacchaide acceptors.This strategy can be applied to large-scale production of valuable oligosaccharides from low-cost carbohydrates as catalysedby phosphorylases with different substrate specificity.Lactobacillus acidophilus NCFM のゲノム内に、マルトース/マルトデキストリン代謝に関与する遺伝子クラスターを見出した。本クラスター中には、グルコシドハイドロレースファミリー65に属する機能未知な糖質関連酵素（MalP）をコードする遺伝子が含まれている。そこで大腸菌を宿主として組み換え酵素を生産し、その酵素化学的諸性質を調査した結果、MalP はマルトースに特異的に作用する糖質加リン酸分解酵素であることが明らかになった。加えてMalP が触媒する糖質合成反応を用いて、６種のα（- 1→4）- グルコ二糖を高収率生産に成功した。departmental bulletin pape

Uporaba celulaz v procesu plemenitenja

Cellulases are enzymes that are used for the surface modifi cations of cellulosic materials primarily during fi nishing. It is a multi-component enzymatic system which hydrolyzes cellulose chains, on the surface of the fi bres, to glucose. During their applications in the fi nishing of textiles, surface fi bres are removed and the surfaces of the treated textiles become smooth. The most important application is in the processing of denim for providing special eff ects without signifi cant fabric loss of strength. Enzymes are eff ective over mild conditions of pH and temperatures and are easily biodegradable.Celulaze so encimi, namenjeni površinski modifi kaciji celuloznih tekstilij, predvsem pri plemenitenju tekstilij. Večkomponentni sistem encimov hidrolizira celulozne makromolekule na površju vlaken do glukoze. Z uporabo celulaz pri plemenitenju tekstilij se odstranijo štrleča vlakna na površju tekstilije, s čimer postane obdelano površje gladko. Med najpomembnejše vrste uporabe celulaz spada plemenitenje denim jeansa, kjer dosežejo posebne učinke brez bistvenega znižanja trdnosti tkanine. Encimi so učinkoviti v blagih pogojih vrednosti pH in temperature in so enostavno biorazgradljivi

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose-6-P phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-a-glucosidase, which in B. subtilis hydrolyses maltose 6-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phosphosubstrates revealed that MapP preferably dephosphorylates disaccharides containing an O-aglycosyl linkageFil: Mokhtari, Abdelhamid. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; Francia. University Mentouri. Faculty of Natural Science and Life. Department of Biochemistry-Microbiology. Laboratory of Environmental Biology; ArgeliaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Henry, Céline. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; FranciaFil: Pikis, Andreas. Center for Drug Evaluation and Research. Food and Drug Administration; Estados UnidosFil: Bourand, Alexa. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; FranciaFil: Alvarez, Maria de Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Immel, Stefan. Technische Universität Darmstad. Institut für Organische Chemie; AlemaniaFil: Mechakra Maza, Aicha. University Mentouri. Faculty of Natural Science and Life. Department of Biochemistry-Microbiology. Laboratory of Environmental Biology; ArgeliaFil: Hartke, Axel. Universite de Caen Basse Normandie; FranciaFil: Thompson, John. National Institutes of Health. Laboratory of Cell and Developmental Biology. Microbial Biochemistry and Genetics Section; Estados UnidosFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Deutscher, Josef. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; Franci