512-66-3Relevant articles and documents
A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile transfructosylation reactions
Lu, Lili,Fu, Feng,Zhao, Renfei,Jin, Lan,He, Chunjuan,Xu, Li,Xiao, Min
, p. 1503 - 1510 (2015/01/06)
This work disclosed the broad transglycosylation capability of the levansucrase from Bacillus licheniformis 8-37-0-1 for the first time. The levansucrase was firstly purified from the strain 8-37-0-1 and found to be a monomer of ～51 kDa with KETQDYKKSY as the N-terminus. Then, the gene encoding the enzyme was cloned and it contained an ORF of 1449 nucleotides, encoding a 482 amino-acid protein with a predicted 29 amino-acid signal peptide. The deduced mature protein without the signal showed the same N-terminus to the purified enzyme. The mature enzyme was subsequently expressed in Escherichia coli. The recombinant enzyme showed similar biochemical properties to the native one. It produced maximal yield of 7.1 mg/mL levan (Mr 9.6 × 106) from 0.8 M sucrose (pH 6.5) at 40 °C for 24 h in vitro. When using sucrose as the donor, the enzyme displayed a wide range of acceptor specificity and was able to transfer fructosyl to a series of sugar acceptors including hexose, pentose, β- or α-disaccharides, along with the difficult branched alcohols that have not been investigated before. Chemical structures of the resultant products were analyzed by MS and NMR spectra.
Probing substrate promiscuity of amylosucrase from neisseria polysaccharea
Daude, David,Champion, Elise,Morel, Sandrine,Guieysse, David,Remaud-Simeon, Magali,Andre, Isabelle
supporting information, p. 2288 - 2295 (2013/08/23)
The amylosucrase from Neisseria polysaccharea (NpAS) naturally catalyzes the synthesis of a variety of products from sucrose and shows signs of plasticity of its active site. To explore further this promiscuity, the tolerance of amylosucrase towards different donor and acceptor substrates was investigated. The selection of alternate donor substrates was first made on the basis of preliminary molecular modeling studies. From 11 potential donors harboring selective derivatizations that were experimentally evaluated, only p-nitrophenyl-α-D-glucopyranoside was used by the wild-type enzyme, and this underlines the high specificity of the -1 subsite of NpAS for glucosyl donor substrates. The acceptor substrate promiscuity was further explored by screening 20 hydroxylated molecules, including D- and L-monosaccharides as well as polyols. With the exception of one compound, all were successfully glucosylated, and this showcases the tremendous plasticity of the +1 subsite of NpAS, which is responsible for acceptor recognition. The products obtained from the transglucosylation reactions of three selected acceptors were characterized, and they revealed original structures and enzyme enantiopreference, which were more particularly analyzed by insilico docking analyses.
Method for synthesizing oligosaccharides and glycosylation
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Page/Page column 6, (2009/04/24)
The invention relates to an enzymatic method for synthesizing oligosaccharides, whereby one saccharide group of a sucrose analogue each is transferred onto an acceptor molecule, for example for glycosylating a hydroxyl compound, a saccharide, peptide, or a drug. According to the inventive method, an enzymatic synthesis of β-D-fructofuranosyl-a-D-aldopyranoside is carried out in a first step, and in a second step one of the saccharide groups is enzymatically transferred onto the acceptor molecule.
Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase)
Seibel, Juergen,Moraru, Roxana,Goetze, Sven,Buchholz, Klaus,Na'amnieh, Shukrallah,Pawlowski, Alice,Hecht, Hans-Juergen
, p. 2335 - 2349 (2007/10/03)
In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in 4C1 conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the β-(1→2)-fructosyl transfer to 2-OH of l-sugars (l-glucose, l-rhamnose, l-galactose, l-fucose, l-xylose) presumably in a 1C4 conformation. In those conformations, the l-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.
Biocatalytic and chemical investigations in the synthesis of sucrose analogues
Seibel, Jürgen,Moraru, Roxana,G?tze, Sven
, p. 7081 - 7086 (2007/10/03)
Herein, we report about the synthesis of sucrose analogues, obtained by two different approaches: a chemical and an enzymatic. The one step synthesis of the sucrose analogues with the exo-fructosyltransferase (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, which transfers the fructosyl residue of the substrate sucrose to the monosaccharide acceptors galactose, mannose, xylose and fucose, has been developed. Effects in the fructosylation by variation of the positions of the hydroxyl-groups in glycopyranoside acceptors have been studied in respect to their acceptor properties. In contrast, the chemical equivalent nonenzymatic organic synthesis of galacto-sucrose and manno-sucrose has been achieved including six synthetic steps.
The Donor Substrate Spectrum of Recombinant Sucrose Synthase 1 from Potato for the Synthesis of Sucrose Analogues
R?mer, Ulrike,Rupprath, Carsten,Elling, Lothar
, p. 684 - 686 (2007/10/03)
The donor substrate spectrum of recombinant sucrose synthase 1 (SuSy1) from potato was studied in order to synthesise novel sucrose analogues. With D-fructose as acceptor substrate SuSy1 accepts a variety of UDP-activated sugars, e.g., UDP-N-acetyl-α-D-glucosamine (UDP-GlcNAc, 2) (100%), UDP-α-D-glucuronic acid (UDP-GlcA, 3) (32%), UDP-α-D-xylose (UDP-Xyl, 4) (39%), UDP-α-D-galactose (UDP-Gal, 5) (23%), and UDP-N-acetyl-α-D-galactosamine (UDP-GalNAc, 6) (23%). The kinetic analyses revealed that the non-natural donors 2 (kcat/Km 1.2 s-1 mM-1) and 5 (kcat/Km 0.01 s-1 mM-1) were relative poor substrates compared to UDP-Glc 1 (kcat/Km 310.4 s-1 mM-1). UDP-GlcNAc was used in a preparative synthesis to produce 188 mg (0.5 mmol) 2-acetamido-2-deoxy-D-glucopyranosyl-β-D-fructofuranoside (9). The sucrose analogue 9 was not hydrolysed by invertase.
Characterization of Levansucrase from Rahnella aquatilis JCM-1683
Ohtsuka, Koutaro,Hino, Shiro,Fukushima, Takayuki,Ozawa, Osamu,Kanematsu, Tadashi,Uchida, Takatsugu
, p. 1373 - 1377 (2007/10/02)
Levansucrase (EC 2.4.1.10) was purified to homogeneity from cell-free extracts of Rahnella aquatilis JCM-1683 by streptomycin treatment, ammonium sulfate fractionation, and HPLC with a DEAE-Toyopearl pak 650M, TSKgel G-3000SW, and TSKgel DEAE-5WP.The enzyme had optimum activity around pH 6.0 and 55 - 60 deg C.The molecular weight of the enzyme was 120,000 on gel filtration and it was composed of two identical subunits (64,000).The amount of levan synthesized by the purified enzyme was 2.95 g from 10.0 g sucrose.The enzyme had a broad acceptor specificity and gavetransfer products.D-Xylose, D-arabinose, L-arabinose, lactose, maltose, maltotriose, cellobiose, and melibiose were effective acceptors in the transfructosylation reaction of the enzyme.
Difference in mode of inhibition between alpha-D-xylosyl beta-D-fructoside and alpha-isomaltosyl beta-D-fructoside in synthesis of glucan by Streptococcus mutans D-glucosyltransferase.
Nisizawa,Takeuchi,Imai,Kitahata,Okada
, p. 135 - 144 (2007/10/02)
Both alpha-isomaltosyl beta-D-fructoside and alpha-D-xylosyl beta-D-fructoside show strong inhibition of the synthesis of water-insoluble and water-soluble D-glucans from sucrose by a partially purified preparation of a D-glucosyltransferase (GTase) from Streptococcus mutans 6715; however, the inhibitory modes differ substantially. In the presence of alpha-isomaltosyl beta-D-fructoside, the production of reducing sugars and the consumption of sucrose are remarkably enhanced, compared with a control of sucrose alone. Under these conditions, a large proportion of low-molecular-weight glycan (lmwg) and a series of nonreducing oligosaccharides (both containing D-fructosyl groups or residues) are produced. In contrast, in the presence of alpha-D-xylosyl beta-D-fructoside, the production of reducing sugars and the sucrose consumption are strikingly suppressed, and no lmwg or oligosaccharides are produced. Thus, it may be concluded that alpha-isomaltosyl beta-D-fructoside acts as an alternative acceptor for the D-glucosyl and/or D-glucanosyl transfer reactions of the enzyme, and serves to lessen the formation of insoluble and soluble D-glucan, although it stimulates the transferring activity of the enzyme. On the other hand, alpha-D-xylosyl beta-D-fructoside competitively inhibits the sucrose-splitting activity of the enzyme as an analog to sucrose, and thereby diminishes the synthesis of D-glucan.
