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Gellan gum (GG) is used in many industries. Here, we obtained a low molecular weight GG (L-GG) directly produced by M155, the high-yield mutant strain of Sphingomonas paucimobilis ATCC 31461, which was selected using UV-ARTP combined mutagenesis. The molecular weight of L-GG was 44.6 % lesser than that of the initial GG (I-GG), and the GG yield increased by 24 %. The monosaccharide composition and Fourier transform-infrared spectroscopic patterns of L-GG were similar to those of I-GG, which indicated that the decrease in the molecular weight of L-GG was probably because of reduction in the degree of polymerization. In addition, microstructural analysis revealed that the surface of L-GG was rougher, with smaller pores and tighter network, than that of I-GG. L-GG showed low hardness, gumminess, and chewiness, which are indicative of better taste. The results of rheological analysis revealed that the L-GG solution is a typical non-Newtonian fluid with low viscoelasticity, which exhibited stable dynamic viscoelasticity within 20-65 °C. To the best of our knowledge, this is the first report of direct biosynthesis of low molecular weight GG during fermentation, which will reduce the manufacturing costs. Our observations provide a reference for precise and expanded applications of GG., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr 177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr 177 . In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr 177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this work, the influence of the concentration on the flow behaviour, dynamic viscoelastic and creep properties of diutan gum in aqueous solution was investigated. Diutan gum is a biopolymer which belongs to the sphingans group. To know its rheological properties and its microstructure as a function of the concentration is directly related to the current and future applications of this biological polymer. Mechanical spectra showed a crossover point between G' and G″ which changed as a function of diutan gum concentration. A master curve for the frequency dependence on the η* was obtained. The creep compliance results made it possible to deduce the yield stress value and they were fitted to Burgers model. A shear-thinning behaviour was exhibited by diutan gum aqueous solutions, which was fitted to the Carreau-Yasuda model. Higher G', G″, τ 0 and η 0 values and lower ω c , J e 0 , γ̇ c and n values were obtained by increasing the gum concentration, it is being possible to modulate the viscoelasticity, viscosity and shear resistance as a function of concentration. A more complex structure with stronger entanglements between macromolecules of diutan was obtained when the concentration of diutan increases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Diutan gum is a biological polymer produced by Sphingomonas sp. In aqueous solution it shows gel-like structure under quiescent conditions. However, the flow-induced evolution of its viscoelastic properties and its microstructure are not known. In this work, the viscoelastic moduli were obtained under a flow field, applied in parallel, as a function of the temperature for 0.5 wt% diutan gum aqueous solutions. As both stress and temperature increase a decrease in the viscoelastic properties occurred, due to the fact that the molecular interactions decreased. Nevertheless, at stresses within the zero-shear viscosity region of the flow curves, no changes were observed. In addition, high stress values dampened the effect of the temperature. The results obtained are very interesting from an industrial application perspective., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The tripartite EfeUOB system functions as a low pH iron importer in Gram-negative bacteria. In the alginate-assimilating bacterium Sphingomonas sp. strain A1, an additional EfeO-like protein (Algp7) is encoded downstream of the efeUOB operon. Here we show the metal binding mode of Algp7, which carries a M_75 metallopeptidase motif. The Algp7 protein was purified from recombinant E. coli cells and was subsequently characterized using differential scanning fluorimetry, fluorescence spectrometry, atomic absorption spectroscopy, and X-ray crystallography. The fluorescence of a dye, SYPRO Orange, bound to denatured Algp7 in the absence and presence of metal ions was measured during heat treatment. The fluorescence profile of Algp7 in the presence of metals such as ferric, ferrous, and zinc ions, shifted to a higher temperature, suggesting that Algp7 binds these metal ions and that metal ion-bound Algp7 is more thermally stable than the ligand-free form. Algp7 was directly demonstrated to show an ability to bind copper ion by atomic absorption spectroscopy. Crystal structure of metal ion-bound Algp7 revealed that the metal ion is bound to the cleft surrounded by several acidic residues. Four residues, Glu79, Glu82, Asp96, and Glu178, distinct from the M_75 motif (His115xxGlu118), are coordinated to the metal ion. This is the first report to provide structural insights into metal binding by the bacterial EfeO element., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This in vitro study was conducted in order to determine the effects of hydrocarbons and growth phase on the phospholipid ester-linked fatty acid composition of two marine sedimentary hydrocarbon-degrading bacteria. These two strains, namely Corynebacterium sp. and Sphingomonas sp. 2MPII, were cultivated on either a simple soluble substrate (ammonium acetate) or a hydrocarbon (respectively n-eicosane and phenanthrene). The incubations were stopped at different times corresponding to point of lag (2 days), exponential (7 days) and stationary phases (21 and 56 days). The effects of growth phase and hydrophobic substrates were successfully demonstrated by a simple index, given as the sum of saturated fatty acids divided by the sum of unsaturated fatty acids ( summation operatorSFA/ summation operatorMUFA), ranging from 1.4 to 3, 0.3 to 0.6, and 0.5 to 1.0 for Corynebacterium sp., Sphingomonas sp. 2MPII, and mixed cultures, respectively. This result was validated by a principal component analysis. In pure cultures, the phospholipid fatty acid (PLFA) composition was strongly influenced by both the carbon source and the growth phase. Nevertheless, the two strains showed different "behaviors". For 2MPII, the main PLFA composition changes were observed at 2 days while they were progressive as a function of time for Corynebacterium sp. These differences could explain the evolution of PLFAs of mixed cultures.

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.

Sphingomonas sp. A1 possesses a high molecular mass (average 25,700 Da) alginate uptake system mediated by a novel pit-dependent ABC transporter. The X-ray crystallographic structure of AlgQ2 (57,200 Da), an alginate-binding protein in the system, was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 18.3% for 15 to 2.0 A resolution data. The refined structure of AlgQ2 was comprised of 492 amino acid residues, 172 water molecules, and one calcium ion. AlgQ2 was composed of two globular domains with a deep cleft between them, which is expected to be the alginate-binding site. The overall structure is basically similar to that of maltose/maltodextrin-binding protein, except for the presence of an N2-subdomain. The entire calcium ion-binding site is similar to the site in the EF-hand motif, but comprises a ten residue loop. This calcium ion-binding site is about 40 A away from the alginate-binding site., (Copyright 2002 Elsevier Science Ltd.)

X-Ray diffraction analysis of the sodium salt of the polysaccharide RMDP17, a 2-deoxy rhamsan analog, reveals that it adopts a gellan-like, half-staggered, threefold, left handed, double helix of pitch 57.4 A. The side chain of the branched polymer is hydrogen bonded to the main chain. Sodium ions, linked to the carboxylate groups, promote the association of helices via water molecules. Two helices of opposite polarity occupy a trigonal unit cell of dimensions a=17.6 and c=28.7 A. The packing arrangement displays a series of hydrogen bonds involving main chain and side chain atoms, as well as some water bridges, between the helices.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Zähringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer

A two-step procedure is described in this paper to identify the position of O-acetyl groups in S-657 polysaccharide. Reductive-cleavage experiments performed on the fully methylated (base-catalyzed) polysaccharide, followed by acetylation of anhydroalditols, identified individual sugar residues and their position of linkage. In a second experiment, the polysaccharide was methylated under neutral conditions leaving native acetate groups intact. Reductive cleavage of the neutral methylated polysaccharide using CF3SO3SiMe3 as a catalyst, followed by acetylation in situ, identified sugar residues containing native acetate groups and established their position of substitution. Using this two-step procedure of analysis, S-657 polysaccharide is shown to contain O-acetyl groups on the 2-position and the 2,6-positions of 3-linked glucopyranosyl residues.

The structure of A1-III from a Sphingomonas species A1 complexed with a trisaccharide product (4-deoxy-l-erythro-hex-4-enepyranosyluronate-mannuronate-mannuronic acid) was determined by X-ray crystallography at 2.0 A with an R-factor of 0.16. The final model of the complex form comprising 351 amino acid residues, 245 water molecules, one sulfate ion and one trisaccharide product exhibited a C(alpha) r.m.s.d. value of 0.154 A with the reported apo form of the enzyme. The trisaccharide was bound in the active cleft at subsites -3 approximately -1 from the non-reducing end by forming several hydrogen bonds and van der Waals interactions with protein atoms. The catalytic residue was estimated to be Tyr246, which existed between subsites -1 and +1 based on a mannuronic acid model oriented at subsite +1., (Copyright 2001 Academic Press.)