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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.)

Multiple microbial exopolysaccharides have been reported in recent decade with their structural and functional features. Gellan gum (GG) is among these emerging biopolymers with versatile properties. Low production yield, high downstream cost, and abundant market demand have made GG a high cost material. Hence, an understanding on the various possibilities to develop cost-effective gellan gum bioprocess is desirable. This review focuses on details of upstream and downstream process of GG from an industrial perspective. It emphasizes on GG producing Sphingomonas spp., updates on biosynthesis, strain and media engineering, kinetic modeling, bioreactor design and scale-up considerations. Details of the downstream operations with possible modifications to make it cost-effective and environmentally sustainable have been discussed. The updated regulatory criteria for GG as a food ingredient and analytical tools required to validate the same have been briefly discussed. Derivatives of GG and their applications in various industrial segments have also been highlighted., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central β-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the β2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures., Competing Interests: The authors have declared that no competing interests exist.

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.)

A Gram-stain-negative, aerobic, motile strain, HHU CXW T , was isolated from hair of a healthy 21-year-old female student of Hohai University, Nanjing, China. The 16S rRNA gene sequence analysis indicated that HHU CXW T represents a member of the genus Sphingomonas with the highest sequence similarity (97.6%) to the type strain S. aquatilis JSS7 T . HHU CXW T grew at 4-35 °C and pH 6-8, with optimum growth at 28 °C and pH 7. Tolerance to NaCl was up to 2% (w/v) with optimum growth in 0.5-1.0% NaCl. The major fatty acids were C 16:0 , C 17:1 ω6c, C 18:1 ω7c11-methyl, summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c), and summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c). The predominant isoprenoid quinone was ubiquinone-10. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), sphingoglycolipid (SGL), phosphatidylinositol mannosides (PIM), and an unidentified glycolipid (GL). The DNA G + C content was 67.1%. The average nucleotide identity (ANI) values and digital DNA-DNA hybridization (dDDH) between HHU CXW T and closely related members of the genus Sphingomonas were all below the cut-off level (95-96% and 70%, respectively) for species delineation. On the basis of the phenotypic, phylogenetic and chemotaxonomic characterizations, HHU CXW T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas hominis sp. nov. is proposed. The type strain is HHU CXW T (= KCTC 72946 T  = CGMCC 1.17504 T  = MCCC 1K04223 T ).

This study focuses on isolation of pigment producing bacteria from fresh water sediment. The isolated bacteria were grown in nutrient broth and the maximum absorbance of 2.512 was obtained for the extracted pigment at 500 nm. The effective strains were optimized, pH 11 and temperature 30 °C was found to be more favorable for its maximum growth. The isolates were identified based on their molecular characterestics as Microbacterium arborescens and Sphingomonas paucimobilis, molecular size of the amplified 16S rRNA gene sequence was found to be approximately 1270 and 765 bp respectively. The antioxidant property of the pigment was analyzed using DPPH and ABTS assay. The IC 50 value of Microbacterium arborescens was higher in all the three assays in comparison with Sphingomonas paucimobilis . The extracted pigment was characterized for the presence of compounds using GC-MS and FTIR analysis to determine the functional groups. As the pigment obtained from M. arborescens had shown better antioxidant activity it may be used as colorant in food industrial applications.

Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13 C, 15 N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433-434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13 C and 15 N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.

The species belonging to the Sphingomonas genus possess multifaceted functions ranging from remediation of environmental contaminations to producing highly beneficial phytohormones, such as sphingan and gellan gum. Recent studies have shown an intriguing role of Sphingomonas species in the degradation of organometallic compounds. However, the actual biotechnological potential of this genus requires further assessment. Some of the species from the genus have also been noted to improve plant-growth during stress conditions such as drought, salinity, and heavy metals in agricultural soil. This role has been attributed to their potential to produce plant growth hormones e.g. gibberellins and indole acetic acid. However, the current literature is scattered, and some of the important areas, such as taxonomy, phylogenetics, genome mapping, and cellular transport systems, are still being overlooked in terms of elucidation of the mechanisms behind stress-tolerance and bioremediation. In this review, we elucidated the prospective role and function of this genus for improved utilization during environmental biotechnology.

Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the β-O-4 bonds, the most prevalent intermonomer linkage. In particular, stereospecific cleavage of lignin oligomers by glutathione S -transferases (GSTs) has been reported in several sphingomonads. Here, we apply quantum mechanics/molecular mechanics simulations to study the mechanism of two glutathione-dependent enzymes in the β-aryl ether catabolic pathway of Sphingomonas sp. SYK-6, namely, LigF, a β-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a S N 2 reaction mechanism, with the monoaromatic leaving group being promptly neutralized upon release. Specific interactions with conserved residues are responsible for stereoselectivity and for activation of the cofactor as a nucleophile. A glutathione conjugate is also released by LigF and serves the substrate of LigG, undergoing a S N 2-like reaction, in which Cys15 acts as the nucleophile, to yield the second monoaromatic product. The simulations suggest that the electron-donating substituent at the para-position found in lignin-derived aromatics and the interaction with Tyr217 are essential for reactivity in LigG. Overall, this work deepens the understanding of the stereospecific enzymatic mechanisms in the β-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs.

Gellan gum is a microbial exopolysaccharide, produced after aerobic fermentation using the Gram-negative bacterium strain Sphingomonas elodea ATCC 31461. Due to its unique structure and excellent physical characteristics, gellan gum has a broad range of applications in food, pharmaceutical, and other industries where it is used for stabilizing, emulsifying, thickening, and suspending. During the fermentative production of gellan, strain ATCC 31461 also accumulates large amounts of the metabolic by-products yellow carotenoid pigments and poly-β-hydroxybutyrate (PHB), which is decreasing the gellan production and increasing processing costs. A pigment PHB-free mutant was obtained by knocking out the phytoene desaturase gene ( crtI ) in the carotenoid biosynthetic pathway and the phaC gene, encoding a PHB synthase for the polymerization of PHB. Unfortunately, the double gene knockout mutant produced only 0.56 g liter -1 gellan. Furthermore, blocking PHB and carotenoid synthesis resulted in the accumulation of pyruvate, which reduced gellan production. To elevate gellan production, combined UV irradiation and ethyl methanesulfonate (EMS) mutagenesis treatment were used. A mutant strain with the same level of pyruvate as that of the wild-type strain and higher gellan production was isolated (1.35 g liter -1 , 132.8% higher than the double gene knockout mutant and 14.4% higher than the wild-type strain ATCC 31461). In addition, a new gellan gum recovery method based on the new mutant strain was investigated, in which only 30% isopropanol was required, which is twice for the wild-type strains, and the performance of the final product was improved. Thus, the mutant strain could be an ideal strain for the commercial production of gellan. IMPORTANCE A carotenoid- and PHB-free double gene knockout strain mutant was constructed to simplify the purification steps normally involved in gellan production. However, the production of gellan gum was unexpectedly reduced. A mutant with 14.4% higher gellan production than that of the wild-type strain was obtained and isolated after employing UV and EMS combined mutagenesis. Based on this high-yield and low-impurity-producing mutant, a new recovery method requiring less organic solvent and fewer operating steps was developed. This method will effectively reduce the production costs and improve the economic benefits of large-scale gellan production., (Copyright © 2019 Li et al.)

Objective: Intrinsic skin ageing is mainly caused by cellular senescence. p16 and p21 are two important tumour suppressor proteins that play essential roles during cell proliferation and ageing through regulating the expression of several genes. Moreover, physical changes between the ages of 55 and 60 years affect one's physical and disrupt self-esteem. The cosmetics industry has focused on bioactive substances derived from natural products such as plants, mushrooms and marine algae to counteract the deleterious effect on skin senescence. Besides these products, compounds produced by bacteria may decelerate individual senescence., Methods: In order to evaluate the potential benefits of bacteria extract over skin ageing, we investigated whether a Sphingomonas hydrophobicum (SH) extract is able to protect our skin against senescence mechanisms. We used an ageing full-thickness skin equivalent model, which was treated or not with the bacteria extract in a systemic way for 42 days. p21 and p16 and senescence-associated galactosidase activity were used to detect cellular senescence with immunohistology. Using a psychobiological approach, we evaluated in vivo the effect of SH extract on self-esteem, isotropy and suppleness., Results: Sphingomonas extract significantly suppressed senescence associated with β-galactosidase activation. It also significantly inhibited the expression of cell cycle inhibitors (p21 and p16). At the same time, the bacteria extract has a significant positive impact on the issue by increasing the expression of versican and fibrillin-1. Significant improvements of self-esteem were reported after 56 days of SH extract application. These psychological benefits were accompanied by a significant improvement in skin suppleness and isotropy., Conclusion: Sphingomonas extract delays intrinsic skin ageing process by inhibiting cellular senescence, and has also the capability to restructure the skin. These beneficial physiological effects induced by SH extract have a positive influence on self-esteem. Because skin ageing causes emotional distress, SH extract can serve as an anti-ageing cosmeceutical agent and help to build a better psychological health, and help individuals to assume ageing., (© 2019 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Tributyl phosphate (TBP) is recognised as a global environmental contaminant because of its wide use in floatation reagents, nuclear fuel reprocessing and plasticisers. This contaminant is hardly degraded by hydrolysis in the environment due to its special physicochemical properties. In this study, one TBP-degrading strain was isolated from TBP-contaminated abandoned mine tailings, and 16S rRNA identification revealed that the strain belonged to the genus Sphingomonas. Results validated that the strain could utilise TBP as the sole carbon source, and vitamin was not the essential factor for its growth. Liquid chromatography time-of-flight mass spectrometry analysis identified di-n-butyl phosphate (DnBP) and mono-n-butyl phosphate (MnBP) as the intermediate metabolites for TBP biodegradation. No obvious change in carbon and hydrogen isotope composition was observed in biodegradation processes (cell suspension and crude extract degradation), which indicated that the first irreversible bond cleavage did not involve carbon or hydrogen. Hence, the TBP degradation scheme by Sphingomonas sp. proposed that the first irreversible step of TBP transferred to DnBP would lead to PO bond cleavage. This study combined the identification of products and isotope fractionation in substrates to investigate the transformation mechanism, thereby providing an eco-friendly and cost-effective way for the in situ bioremediation of TBP-contaminated sites by the isolated TBP degradation strain., (Copyright © 2019. Published by Elsevier Ltd.)

Three strains L3B27 T , 3CNBAF, L1A4 isolated from a brackish cultivated pokkali rice rhizosphere were characterised using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA and recA gene sequences revealed that these strains were highly similar among each other and formed a separate monophyletic cluster within the genus Sphingomonas with Sphingomonas pituitosa DSM 13101 T , Sphingomonas azotifigens DSM 18530 T and Sphingomonas trueperi DSM 7225 T as their closest relatives sharing 97.9-98.3% 16S rRNA similarity and 91.3-94.0% recA similarity values, respectively. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridisation (dDDH) values between L3B27 T (representative of the novel strains) and its phylogenetically closest Sphingomonas species were well below the established cut-off <94% (ANI/AAI) and <70% (dDDH) for species delineation. Further, the novel strains can be distinguished from its closest relatives based on several phenotypic traits. Thus, based on the polyphasic approach, we describe a novel Sphingomonas species for which the name Sphingomonas pokkalii sp. nov (type strain L3B27 T =KCTC 42098 T =MCC 3001 T ) is proposed. In addition, the novel strains were characterised for their plant associated properties and found to possess several phenotypic traits which probably explain its plant associated lifestyle. This was further confirmed by the presence of several plant associated gene features in the genome of L3B27 T . Also, we could identify gene features which may likely involve in brackish water adaptation. Thus, this study provides first insights into the plant associated lifestyle, genome and taxonomy of a novel brackish adapted plant associated Sphingomonas., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

β-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized β-xylosidases of glycoside hydrolase family (GH) 39 were classified into the same subgroup with conserved amino acid residue positions participating in substrate recognition. Protein-ligand docking revealed that seven of these positions were probably essential to bind xylose-glucose, which is linked by a β-1,2-glycosidic bond. Amino acid residues in five of the seven positions are invariant, while those in two of the seven positions are variable with low frequency. Both the wild-type β-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited β-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-β-d-xylopyranoside and transformed notoginsenosides R 1 and R 2 to ginsenosides Rg 1 and Rh 1 , respectively. The results suggest that all of these characterized GH 39 β-xylosidases probably show β-1,2-xylosidase activity, which should be assigned an EC number with these β-xylosidases as representatives.

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 present study aimed to develop a plate-screening method, based on the specific color development of complexes formed between chlorogenic acid, a valuable plant-derived compound, and aluminum (III), to detect chlorogenic acid-producing microbial strains. Modified media with 0.75 mM aluminum chloride were developed to identify CGA-producing bacteria (based on beef extract agar medium) or fungi (based on the potato dextrose agar medium). Compared with conventional screening, the modified media let to 3.3 times more CGA producers from plants, at 90.9% selective accuracy. Novel chlorogenic acid-biosynthesizing strains included Brevibacillus borstelensis B14, Bacillus amyloliquefaciens B17, Bacillus badius B19, Sphingomonas yabuuchiae N21, Enterobacter tabaci N22, and Lodderomyces elongisporus S216 and P212. Strain S216 produced the highest chlorogenic acid yield (23.39 mg L -1 ). This study provides a highly efficient and low-cost tool for quick detection and subsequent identification of several newly isolated strains with chlorogenic acid-producing potential.

Strain DRJ-4 T was isolated from forest soil of a low-altitude mountain and was taxonomically characterized by a polyphasic approach. This strain was yellow-colored, Gram-stain-negative, strictly aerobic, motile, non-sporulating, catalase-positive, oxidase-negative, and rod-shaped. Strain DRJ-4 T was able to grow at 20-42 °C, pH 6.5-10.0, and 0-1.0% (w/v) NaCl concentration. Based on the 16S rRNA gene sequence analysis, strain DRJ-4 T formed a distinct lineage within the members of the family Sphingomonadaceae of the phylum Proteobacteria and moderately related to Sphingomonas faucium E62-3 T (96.67% sequence similarity), 'Sphingosinicella ginsenosidimutans' BS11 (96.45% sequence similarity), Sphingosinicella vermicomposti YC7378 T (96.27% sequence similarity), and Sphingobium boeckii 469 T (95.82% sequence similarity). The sole respiratory quinone was ubiquinone Q-10 and the major polyamine was homospermidine. The polar lipid profile revealed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, and sphingoglycolipid. The predominant fatty acids of strain DRJ-4 T were summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c), summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c), C 16:0 , and C 17:1 ω6c. The genomic DNA G + C was 64.0 mol %. On the basis of phenotypic, genotypic, and phylogenetic analysis, strain DRJ-4 T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas montis sp. nov. is proposed. The type strain of Sphingomonas montis is DRJ-4 T (= KEMB 9005-708 T  = NBRC 113142 T ).

Mining for novel enzymes from new microorganisms is a way to obtain β-xylosidases with promising applications. A Sphingomonas β-xylosidase was expressed in Escherichia coli. The purified recombinant enzyme (rJB13GH39) was most active at pH 4.5 and 50 °C, retaining 10%-50% of its maximum activity at 0-20 °C. Most salts and chemical reagents including 3.0%-20.0% (w/v) NaCl showed little or no effect on the enzymatic activity. rJB13GH39 exhibited 71.9% and 55.2% activity in 10.0% and 15.0% (v/v) ethanol, respectively. rJB13GH39 was stable below 60 °C in 3.0%-30.0% (w/v) NaCl, 3.0%-20.0% (v/v) ethanol, and 2.2-87.0 mg/mL trypsin. The enzyme transferred one xylosyl moiety to certain sugars and alcohols. The salt/ethanol tolerance and low-temperature activity of the enzyme may be attributed to its high structural flexibility caused by high proportions of small amino acids ACDGNSTV and random coils.

Rare sugars and sugar derivatives that can be obtained from abundant sugars are of great interest to biochemical and pharmaceutical research. Here, we describe the substrate scope of a short-chain dehydrogenase/reductase from Sphingomonas species A1 (SpsADH) in the oxidation of aldonates and polyols. The resulting products are rare uronic acids and rare sugars respectively. We provide insight into the substrate recognition of SpsADH using kinetic analyses, which show that the configuration of the hydroxyl groups adjacent to the oxidized carbon is crucial for substrate recognition. Furthermore, the specificity is demonstrated by the oxidation of d-sorbitol leading to l-gulose as sole product instead of a mixture of d-glucose and l-gulose. Finally, we applied the enzyme to the synthesis of l-gulose from d-sorbitol in an in vitro system using a NADH oxidase for cofactor recycling. This study shows the usefulness of exploring the substrate scope of enzymes to find new enzymatic reaction pathways from renewable resources to value-added compounds., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Polymer sanxan is a novel microbial polysaccharide produced by Sphingomonas sanxanigenens NX02, which can produce poly-3-hydroxybutyrate (PHB) simultaneously. A strategy of combining rational and random gene manipulation was investigated to improve the yield of sanxan. Several crucial PHB biosynthesis genes were deleted through homologous recombination, then the PHB-deficient mutant was treated with plasma mutagenesis to obtain NXdP, an engineering strain. Ultimately, the yield of purified sanxan produced by strain NXdP increased from 14.88 ± 0.83 g/L to 21.20 ± 0.38 g/L in a 5 L bioreactor, while the cell dry weight (CDW) was decreased from 9.61 ± 0.14 g/L to 3.12 ± 0.15 g/L. The total precipitable material (crude sanxan) produced from NXdP showed higher zero-shear viscosity, light transmittance, and greater gel strength than that from NX02 due to the enhancement of its purity. The engineering strategies explored here are useful for engineering cell factories to produce other valuable metabolites., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The red diketocarotenoid, astaxanthin, exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster, which may potentially protect against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue. These numerous health benefits and consumer interest in natural products have therefore increased the market demand of astaxanthin as a nutraceutical and medicinal ingredient in food, aquaculture feed, and pharmaceutical industries. Consequently, many research efforts have been made to discover novel microbial sources with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diode array-MS methods for carotenoids analysis, we isolated a novel astaxanthin-producing bacterium (strain TDMA-17 T ) that belongs to the family Sphingomonadaceae (Asker et al., FEMS Microbiol Lett 273:140-148, 2007).In this chapter, we provide a comprehensive description of the methods used for the analysis and identification of carotenoids produced by strain TDMA-17 T . We will also describe the methods of isolation and identification for a novel bacterial carotenoid (an astaxanthin derivative), a major carotenoid that is produced by the novel strain. Finally, the identification methods of the novel strain will be summarized.

An orange pigmented, Gram-staining negative, aerobic, motile, rod-shaped bacterium isolated from a soil from the Tanggula Mountain, China was studied using a polyphasic approach. Based on 16S rRNA gene sequence similarity, strain W16RD T was found to be closely related to Sphingomonas prati DSM 103336 T (99%), Sphingomonas fennica DSM 13665 T (97.21%), followed by Sphingomonas laterariae DSM 25432 T (96.44%), Sphingomonas haloaromaticamans CGMCC 1.10206 T (96.36%) and Sphingomonas formosensis DSM 24164 T (96.06%). The strain was found to be catalase and oxidase positive and was found to grow optimally at temperatures of 20-25 °C, pH 8 and tolerated NaCl concentration up to 1% (w/v). The major fatty acids identified were summed feature eight comprising C 18:1 ω 7c and/or C 18:1 ω 6c (39.2%), summed feature three comprising of C 16:1 ω7c and/or C 16:1 ω6c (36.7%) and C 16:0 (7.0%). The polar lipids detected were phosphatidylcholine, sphingoglycolipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, and three unidentified lipids. The strain possessed ubiquinone-10 (Q-10) as the predominant respiratory quinone. Along with other distinguishing characteristics, we also describe the draft genome of strain W16RD T . The final assembled draft genome sequence is 3,722,743 bp with 3390 coding and 48 RNA (45 tRNA and 3 rRNA) genes. The DNA G+C content of the genomic DNA was determined to be 67%. The DNA-DNA relatedness value between the strain W16RD T and its closest phylogenetic relatives S. prati DSM 103336 T , S. fennica DSM 13665 T , S. laterariae DSM 25432 T , and S. haloaromaticamans CGMCC 1.10206 T were 52.17, 47.60, 20.93 and 17.09% respectively. The strain W16RD T could be distinguished genotypically and phenotypically from the recognized species belonging to the genus Sphingomonas and thus represents a novel species, for which the name Sphingomonas montana sp. nov. is proposed. The type strain is W16RD T (=CGMCC 1.15646 T  = DSM 103337 T ).

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&#95;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&#95;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.)

The micro-network structure and functional properties of sanxan, a novel polysaccharide produced by Sphingomonas sanxanigenens NX02, were investigated. Transparent hydrogel sanxan was a high acyl polymer containing 8.96% acetyl and 4.75% glyceroyl. The micro-network structure of sanxan was mainly cyclic configurations composed of side-by-side intermolecular associations, with many rounded nodes found. Sanxan exhibited predominant gelation behavior at concentrations above 0.1%, which was enhanced by adding cations, especially Ca 2+ . The gel strength of sanxan was much higher than that of low acyl gellan, but slightly lower than that of high acyl gellan. Furthermore, the conformation transition temperature was increased in the presence of added cations. Moreover, sanxan showed excellent emulsifying and emulsion stabilizing properties. Consequently, such excellent functional properties make sanxan a good candidate as a gelling, stabilizing, emulsifying, or suspending agent in food and cosmetics industries, and in medical and pharmaceutical usage., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The previously developed Sphingomonas sp. based optical microplate biosensor for methyl parathion (MP) was good as it detected multiple samples but had poor stability and low sensitivity. The present study aims to overcome these limitations. Silica nanoparticles (Si NP) were thus functionalized with polyethyleneimine (PEI) and the functionalized silica nanoparticles ( f Si NP) were then integrated with Sphingomonas sp. cells. The process was optimized for hydrolysis of MP into p-nitrophenol (PNP). Integration of f Si NP with cells was confirmed by FT-IR analysis. Biohybrid of Sphingomonas sp.- f Si NP was immobilized on the wells of microplate and associated directly with the optical transducer of microplate reader. Immobilized biohybrid of Sphingomonas sp.- f Si NP was characterized using SEM. A detection range of 0.1-1ppm MP was achieved from the linear range of calibration plot. After integration with f Si NP the storage stability of biohybrid was enhanced ten times from 18 to 180 days. This study proves that after interaction of cells with f Si NP, improved the sensitivity and stability of the biosensor. Spiked samples were also analyzed and correlated using this biohybrid based biosensor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Two-component systems are major signal transduction pathways, which consist of histidine kinases and response regulators that communicate through phosphorylation. Here, we highlight a distinct class of single-domain response regulators containing the PFXFATG[G/Y] motif that are activated by a mechanism distinct from the Y-T coupling described for prototypical receiver domains. We first solved the structures of inactive and active SdrG, a representative of the FAT GUY family, and then biochemically and genetically characterized variants in which residues in this motif were mutated. Our results support a model of activation mainly driven by a conserved lysine and reveal that the rotation of the threonine induces the reorganization of several aromatic residues in and around the PFXFATG[G/Y] motif to generate intermediates resembling those occurring during classical Y-T coupling. Overall, this helps define a new subfamily of response regulators that emerge as important players in physiological adaptation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Endophytic bacteria have been recently known for their potential to bioaccumulate metal from contaminated mediums. However, little is known about the physiological responses of phytohormone producing (gibberellins and auxins) endophytes during metal stressed environment. Endophytic bacteria Sphingomonas sp. LK11 was assessed for metals bioaccumulation and its physiological responses towards metal stress. The endophyte was grown in cadmium (Cd), zinc (Zn), aluminum (Al), manganese (Mn), and copper (Cu) contaminated mediums. The results revealed significantly higher endophytic growth potentials in Cd, Cu and Zn contaminations; however, the bio-accumulation rate of Cd was more prolific as compared to Zn and Cu. Interestingly, the SDS-PAGE profile showed increased expressions of proteins in Zn and Cu than in Cd. A similar attenuate response of amino acids was also observed for Cd than in case of Zn and Cu. Only asparagine, glutamate and proline showed significant impact in Cd while Cu and Zn had significantly higher responses of almost all amino acids. Detailed protein profile showed the activation of chaperone, antioxidative and detoxification proteins. Increased regulations of oxidoreductases, superoxide dismutase, thioredoxin, malate dehydrogenase, 2-oxoisovalerate dehydrogenase, 2-oxoisovalerate dehydrogenase, and dihydrolipoyl dehydrogenase were observed. The cellular defense-related protein responses were potent against Cd stress. The results conclude that Sphingomonas sp. LK11 reprogram its amino acids and proteomic expressions and maintain a steady growth during Cd stress. Using such phytohromones producing endophytic bacterium can be ideal approach to increase the phytoextraction potential of metal remediating plants. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 887-896, 2016., (© 2014 Wiley Periodicals, Inc.)

Sphingomonas sanxanigenens, a new species of the genus Sphingomonas, synthesizes extracellular biopolymer termed sanxan. Sanxan polysaccharide was purified from the fermentation broth by Sephacryl S-400 column chromatography. The molecular weight of sanxan polysaccharide was 408kDa by the method of size-exclusion chromatography combined with laser light scattering. Based on FT-IR, periodate oxidation, Smith degradation, composition analysis and nuclear magnetic resonance experiments, the structure of sanxan polysaccharide was elucidated as follows: The solution of sanxan polysaccharide showed properties of high viscosity and shear-thinning. By cooling hot solutions, sanxan polysaccharide could form elastic thermoreversible gel., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, designated X1(T), was isolated from the permafrost region of Qilian Mountains in northwest of China. Phylogenetic analyses of 16S rRNA gene sequence revealed that strain X1(T) was a member of the genus Sphingomonas and shared the highest 16S rRNA gene sequence similarity with Sphingomonas oligophenolica JCM 12082(T) (96.9%), followed by Sphingomonas glacialis CGMCC 1.8957(T) (96.7%) and Sphingomonas alpina DSM 22537(T) (96.4%). Strain X1(T) was able to grow at 15-30 °C, pH 6.0-10.0 and with 0-0.3% NaCl (w/v). The DNA G+C content of the isolate was 64.8 mol%. Strain X1(T)-contained Q-10 as the dominant ubiquinone and C(18:1)ω7c, C(16:1)ω7c, C(16:0) and C(14:0) 2-OH as the dominant fatty acids. The polar lipid profile of strain XI(T)-contained sphingoglycolipid, phosphatidylglycerol, phosphatidylethanolamine, one unidentified glycolipid and two unidentified phospholipid. Due to the phenotypic and genetic distinctiveness and other characteristic studied in this article, we consider X1(T) as a novel species of the genus Sphingomonas and propose to name it Sphingomonas qilianensis sp. nov. The type strain is X1(T) (=CGMCC 1.15349(T) = KCTC 42862(T)).

Electrokinetic-microbial remediation (EMR) has emerged as a promising option for the removal of polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. The aim of this study was to enhance degradation of phenanthrene (Phe)-contaminated soils using EMR combined with biosurfactants. The electrokinetic (EK) remediation, combined with Phe-degrading Sphingomonas sp. GY2B, and biosurfactant obtained by fermentation of Pseudomonas sp. MZ01, degraded Phe in the soil with an efficiency of up to 65.1 % at the anode, 49.9 % at the cathode after 5 days of the treatment. The presence of biosurfactants, electricity, and a neutral electrolyte stimulated the growth of the degrading bacteria as shown by a rapid increase in microbial biomass with time. The electrical conductivity and pH changed little during the course of the treatment, which benefitted the growth of microorganisms and the remediation of Phe-contaminated soil. The EMR system with the addition of biosurfactant had the highest Phe removal, demonstrating the biosurfactant may enhance the bioavailability of Phe and the interaction with the microorganism. This study suggests that the EMR combined with biosurfactants can be used to enhance in situ bioremediation of PAH-contaminated soils.

Strain GP20-2(T) was isolated from a soil cultivated with ginseng in Korea. The 16S rRNA gene sequence of this strain showed the highest sequence similarity with Sphingomonas daechungensis CH15-11(T) (96.7%) and Sphingomonas sediminicola Dae 20(T) (96.2%) among the type strains. The strain GP20-2(T) was a strictly aerobic, Gram-negative, non-motile, rod-shaped bacterium that formed very tiny colonies, less than 0.3 mm in diameter after 10 days on R2A agar. The strain grew at 10-35-C (optimum, 35-C), at a pH of 5.0-8.0 (optimum, pH 6.0), and in the absence of NaCl. The DNA G+C content of strain GP20-2(T) was 67.2 mol%. It contained ubiquinone Q-10 as the major isoprenoid quinone, and summed feature 8 (C18:1ω6c and/or C18:1ω7c, 49.8%) and C16:0 (17.0%) as the major fatty acids. On the basis of evidence from our polyphasic taxonomic study, we concluded that strain GP20-2(T) should be classified as a novel species of the genus Sphingomonas, for which the name Sphingomonas parvus sp. nov. is proposed. The type strain is GP20-2(T) (=KACC 12865(T) =DSM 100456(T)).

2-Pyrone-4,6-dicarboxylate lactonase (LigI) is the first identified enzyme from amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. It catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. In this paper, a combined quantum mechanics and molecule mechanics (QM/MM) approach was employed to study the reaction mechanism of LigI from Sphingomonas paucimobilis. According to the results of our calculations, the whole catalytic reaction contains three elementary steps, including the nucleophilic attack, the cleavage of CO of lactone (substrate) and the intramolecular proton transfer. The intermediate has two intramolecular proton transfer pathways, due to which, two final hydrolysis products can be obtained. The energy profile indicates that 4-carboxy-2-hydroxymuconate (CHM) is the main hydrolysis product, therefore, the isomerization between 4-carboxy-2-hydroxymuconate (CHM) and 4-oxalomesaconate (OMA) is suggested to occur in solvent. During the catalytic reaction, residue Asp248 acts as a general base to activate the hydrolytic water molecule. Although His31, His33 and His180 do not directly participate in the chemical process, they play assistant roles by forming electrostatic interactions with the substrate and its involved species in activating the carbonyl group of the substrate and stabilizing the intermediates and transition states., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A yellow-colored, Gram-negative, strictly aerobic, non-motile, rod-shaped bacterium, designated THG-MM5(T), was isolated from road soil in Yongin-si, Gyeonggi-do, Republic of Korea. Based on 16S rRNA gene sequence, strain THG-MM5(T) was moderately related to Sphingomonas sediminicola KACC 15039(T) (96.1%), Sphingomonas ginsengisoli KACC 16858(T) (96.1%) and Sphingomonas jaspsi KACC 13230(T) (96.0%). Chemotaxonomic data revealed that strain THG-MM5(T) possesses ubiquinone-10 as the only respiratory quinone, sym-homospermidine as the major polyamine and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C18:1 ω7c and C16:0 as the major fatty acids. The polar lipid profile included sphingoglycolipid. The DNA G + C content was 60.7 mol%. These data, together with phenotypic characterization, corroborated the affiliation of strain THG-MM5(T) to the genus Sphingomonas. Thus, the isolate represents a novel species, for which the name Sphingomonas flavus sp. nov. is proposed, with THG-MM5(T) as the type strain (=KACC 18277(T) = CCTCC AB 2014320(T)).

The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Degradation of chrysene, a four ringed highly carcinogenic polycyclic aromatic hydrocarbon (PAH) has been demonstrated by bacterial mixed culture Biorem-CGBD comprising Achromobacter xylosoxidans, Pseudomonas sp. and Sphingomonas sp., isolated from crude oil polluted saline sites at Bhavnagar coast, Gujarat, India. A full factorial Central Composite Design (CCD) using Response Surface Methodology (RSM) was applied to construct response surfaces, predicting 41.93% of maximum chrysene degradation with an experimental validation of 66.45% chrysene degradation on 15th day, using a combination of 0.175, 0.175 and 0.385 mL of OD600 = 1 inoculum of A. xylosoxidans, Pseudomonas sp. and Sphingomonas sp., respectively and a regression coefficient (R2) of 0.9485 indicating reproducibility of the experiment. It was observed that chrysene degradation can be successfully enhanced using RSM, making mixed culture Biorem-CGBD a potential bioremediation target for PAH contaminated saline sites.

Sphingomonas is a novel and abundant microbial resource for biodegradation of aromatic compounds. It has great potential in environment protection and industrial production. The use of microorganisms for the synthesis of nanoparticles is in the limelight of modern nanotechnology, since it is cost effective, non-toxic and friendly to the ever-overwhelmed environment. In this paper, the biosynthesis of silver nanoparticles (AgNPs) using Sphingomonas paucimobilis sp. BDS1 under ambient conditions was investigated for the first time. Biosynthesised AgNPs were characterised with powder ultraviolet-visible spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy and energy dispersive X-ray spectroscopy. The overall results revealed that well-dispersed face centred cubic spherical AgNPs in the range of 50-80 nm were produced on the surface of Sphingomonas paucimobilis sp. BDS1, after challenging pure wet biomass with silver nitrate solution. This suggests that the capture of silver ions may be a complex process of physical and chemical adsorption and the proteins on the surface of the bacteria may play the role of reduction and stabilising agent with regard to the result of FTIR.

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.)

A universal method to enhance productivity and viscosity of bacterial exopolysaccharides was developed. The technique was based on the principle that ampicillin can inhibit the biosynthesis of peptidoglycan, which shares a common synthetic pathway with that of bacterial exopolysaccharides. Serial passages of three typical representatives of bacterial EPS-producing strains, namely Sphingomonas elodea, Xanthomonas campestris, and Paenibacillus elgii, were subjected to ampicillin, which was used as a stressor and a mutagen. These mutant strains are advantageous over other strains because of two major factors. First, all of the resulting strains were almost mutants with increase in EPS productivity and viscosity. Second, isolated serial strains showed different levels of increase in EPS production and viscosity to satisfy the different requirements of practical applications. No differences were observed in the monosaccharide composition produced by the mutant and parent strains; however, high-viscosity mutant strains exhibited higher molecular weights. The results confirmed that the developed method is a controlled universal one that can improve exopolysaccharides productivity and viscosity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Plant growth promoting endophytic bacteria have been identified as potential growth regulators of crops. Endophytic bacterium, Sphingomonas sp. LK11, was isolated from the leaves of Tephrosia apollinea. The pure culture of Sphingomonas sp. LK11 was subjected to advance chromatographic and spectroscopic techniques to extract and isolate gibberellins (GAs). Deuterated standards of [17, 17-(2)H2]-GA4, [17, 17-(2)H2]-GA9 and [17, 17-(2)H2]-GA20 were used to quantify the bacterial GAs. The analysis of the culture broth of Sphingomonas sp. LK11 revealed the existence of physiologically active gibberellins (GA4: 2.97 ± 0.11 ng/ml) and inactive GA9 (0.98 ± 0.15 ng/ml) and GA20 (2.41 ± 0.23). The endophyte also produced indole acetic acid (11.23 ± 0.93 μM/ml). Tomato plants inoculated with endophytic Sphingomonas sp. LK11 showed significantly increased growth attributes (shoot length, chlorophyll contents, shoot, and root dry weights) compared to the control. This indicated that such phyto-hormones-producing strains could help in increasing crop growth.

Sphingomonas sp. HXN-200 bacteria were immobilized onto gelatin and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres in an aqueous system for industrial scale biohydroxylation. Scanning electron microscopy and confocal laser scanning microscopy were used to confirm the bacterial immobilization. The gelatin microsphere-immobilized cells successfully transformed N-benzyloxycarbonyl-pyrrolidine and N-tert-butoxycarbonyl-pyrrolidine into R-N-benzyloxycarbonyl-3-hydroxypyrrolidine and R-N-tert-butoxycarbonyl-3-pyrrolidinol at various substrate concentrations showing an improved conversion yield and product efficiency with respect to the freely suspended cells system at each reaction interval time. Additionally, in the immobilized cells system, it showed a faster reaction rate as compared to the freely suspended cells system for reaching the same product concentration. The inhibition effect due to substrate and product concentrations was significantly lower after immobilizing the bacteria onto the microspheres, which is beneficial for continuous reaction. The efficient whole-cell biocatalytic process was feasibly conducted in a 500mL preparative scale bioreactor and the conversion yield of N-benzyloxycarbonyl-pyrrolidine did not decrease after consecutive repeated use, suggesting that the microsphere-cells immobilization system was stable and reusable., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct α/β hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.

The microbial exopolysaccharides are water-soluble polymers secreted by microorganisms during fermentation. The biopolymer gellan gum is a relatively recent addition to the family of microbial polysaccharides that is gaining much importance in food, pharmaceutical and chemical industries due to its novel properties. It is commercially produced by C.P. Kelco in Japan and the USA. This article presents a critical review of the available information on the gum synthesized by Sphingomonas paucimobilis with special emphasis on its fermentative production. Factors affecting the fermentative production of gellan gum and problems associated with mass transfer have been addressed. Classification and trade names of gellan gum has been specified. Characteristics of gellan gum with respect to its structure, physicochemical properties are discussed. An attempt has also been made to review the current and potential applications of gellan gum in food, pharmaceutical and other industries., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The α-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of < or =97.2% with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 α-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas alpha-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas α-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-α-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and 60 degrees C and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant alpha-galactosidases showing thermolability at 50 degrees C or 60°C and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at 60°C) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas α-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

Mild hydrolysis of Sphingomonas sp. ATCC 31555 polysaccharide WL-26 afforded a new oligosaccharide, F21. Structural resolution based on sugar and methylation analyses as well as NMR data revealed the oligosaccharide to have the following structure: [Formula, see text:]., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Alginate is a heteropolysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.

Sphingomonas sp. A1 directly incorporates alginate polysaccharides through a 'superchannel' comprising a pit on the cell surface, alginate-binding proteins in the periplasm and an ABC transporter (alginate importer) in the inner membrane. Alginate importer, consisting of four subunits, AlgM1, AlgM2 and two molecules of AlgS, was crystallized in the presence of the binding protein AlgQ2. Preliminary X-ray analysis showed that the crystal diffracted to 3.3 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 72.5, b = 136.8, c = 273.3 Å, suggesting the presence of one complex in the asymmetric unit., (© 2012 International Union of Crystallography)

A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis (FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T (iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin(ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1α, IL-1β, IL-17, TNF-α, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-β analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-β could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-β, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-β through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management.

A new diketopiperazine, glionitrin B (1), was produced using a microbial coculture of the fungus Aspergillus fumigatus KMC-901 and the bacterium Sphingomonas sp. KMK-001 that were isolated from acidic coal mine drainage. The structure of 1 was determined to be (3S,10aS)-dithiomethylglionitrin A. This structure was determined by the analyses of extensive NMR data and the circular dichroism spectra of the natural product and a semisynthetic compound derived from glionitrin A. In contrast to glionitrin A (2), glionitrin B (1) is not cytotoxic against the human prostate cancer cell line DU145. However, compound 1 caused suppression of DU145 cell invasion, producing 46% inhibition at 60 μM.

Natural killer T (NKT) cells recognize glycolipids produced by Sphingomonas bacteria, and these glycolipids contain C6-oxidized sugars, either glucuronic acid or galacturonic acid, linked to ceramides. Glycolipids with gluco stereochemistry are the most prevalent. Multiple studies have demonstrated that galactosylceramides are more potent stimulators of NKT cells than their glucose isomers. To determine if this stereoselectivity is retained in the context of the C6-oxidized sugars found in bacterial glycolipids, we prepared two sets of gluco and galacto-glycolipids oxidized at their C6 positions and compared their NKT stimulatory properties. In the context of carboxylic acid groups at C6, gluco stereochemistry gave the more potent responses. We also prepared bacterial glycolipids containing more complex ceramide groups to determine if these chains impact NKT cell responses.