Biosynthesis and physicochemical properties of low molecular weight gellan produced by a high-yield mutant of Sphingomonas paucimobilis ATCC 31461.

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.

Advances in fermentative production, purification, characterization and applications of gellan gum.

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.

Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

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 ß-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 ß2 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.

Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

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 Tyr177 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 Tyr177. 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 Tyr177 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.

Sphingomonas hominis sp. nov., isolated from hair of a 21-year-old girl.

A Gram-stain-negative, aerobic, motile strain, HHU CXWT, 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 CXWT represents a member of the genus Sphingomonas with the highest sequence similarity (97.6%) to the type strain S. aquatilis JSS7T. HHU CXWT 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 C16:0, C17:1ω6c, C18:1ω7c11-methyl, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). 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 CXWT 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 CXWT represents a novel species of the genus Sphingomonas, for which the name Sphingomonas hominis sp. nov. is proposed. The type strain is HHU CXWT (= KCTC 72946T = CGMCC 1.17504T = MCCC 1K04223T).

Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins.

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 13C, 15N 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 13C and 15N 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.

Sphingomonas: from diversity and genomics to functional role in environmental remediation and plant growth.

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.

Molecular characterization and antioxidant assay of pigment producing bacteria, Sphingomonas paucimobilis and Microbacterium arborescens isolated from fresh water sediments.

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 IC50 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.

Catalytic Mechanism of Aryl-Ether Bond Cleavage in Lignin by LigF and LigG.

Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the ß-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 ß-aryl ether catabolic pathway of Sphingomonas sp. SYK-6, namely, LigF, a ß-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a SN2 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 SN2-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 ß-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs.

A Carotenoid- and Poly-ß-Hydroxybutyrate-Free Mutant Strain of Sphingomonas elodea ATCC 31461 for the Commercial Production of Gellan.

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-ß-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.

Attenuation of negative effects of senescence in human skin using an extract from Sphingomonas hydrophobicum: development of new skin care solution.

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 ß-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.

OBJECTIF: Le vieillissement intrinsèque de la peau est principalement causé par la sénescence cellulaire. p16 et p21 sont deux importantes protéines suppressives de tumeurs qui jouent un rôle essentiel dans la prolifération et le vieillissement cellulaire en régulant l'expression de plusieurs gènes. De plus, les changements physiques survenant entre 55 et 60 ans affectent le physique et perturbent l'estime de soi. L'industrie cosmétique s'est concentrée sur les substances bioactives dérivées de produits naturels tels que les plantes, les champignons et les algues marines pour contrer les effets délétères sur la sénescence de la peau. En plus de ces produits, les composés produits par les bactéries peuvent ralentir la sénescence individuelle. MÉTHODES: Afin d'évaluer les bénéfices potentiels de l'extrait de bactérie sur le vieillissement cutané, nous avons étudié si un extrait de Sphingomonas hydrophobicum (SH) est capable de protéger notre peau des mécanismes de sénescence. Nous avons utilisé un modèle équivalent de peau vieillissante de pleine épaisseur, qui a été traitée ou non avec l'extrait de bactérie de façon systémique pendant 42 jours. p21 et p16, et l'activité galactosidase associée à la sénescence ont été utilisés pour détecter la sénescence cellulaire par immunohistologie. En utilisant une approche psychobiologique, nous avons évalué in vivo l'effet de l'extrait de SH sur l'estime de soi, l'isotropie et la souplesse. RÉSULTATS: L'extrait de Sphingomonas a considérablement supprimé la sénescence associée à l'activation de ß-galactosidase. Il a également inhibé de manière significative l'expression des inhibiteurs du cycle cellulaire (p21 et p16). En même temps, l'extrait de bactérie a un impact positif significatif sur le problème en augmentant l'expression du versican et de la fibrilline-1. Des améliorations significatives de l'estime de soi ont été rapportées après 56 jours d'application de l'extrait de SH. Ces bienfaits psychologiques s'accompagnaient d'une amélioration significative de la souplesse et de l'isotropie de la peau. CONCLUSION: L'extrait de Sphingomonas retarde le processus de vieillissement intrinsèque de la peau en inhibant la sénescence cellulaire et a également la capacité de restructurer la peau. Ces effets physiologiques bénéfiques induits par l'extrait de SH ont une influence positive sur l'estime de soi. Parce que le vieillissement de la peau provoque une détresse émotionnelle, l'extrait de SH peut servir d'agent cosméceutique anti-âge et aider à construire une meilleure santé psychologique, ainsi qu'aider les individus à assumer le vieillissement.

Elucidating the biodegradation mechanism of tributyl phosphate (TBP) by Sphingomonas sp. isolated from TBP-contaminated mine tailings.

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.

Glycoside Hydrolase Family 39 ß-Xylosidases Exhibit ß-1,2-Xylosidase Activity for Transformation of Notoginsenosides: A New EC Subsubclass.

ß-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized ß-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 ß-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 ß-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited ß-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-ß-d-xylopyranoside and transformed notoginsenosides R1 and R2 to ginsenosides Rg1 and Rh1, respectively. The results suggest that all of these characterized GH 39 ß-xylosidases probably show ß-1,2-xylosidase activity, which should be assigned an EC number with these ß-xylosidases as representatives.

Sphingomonas pokkalii sp. nov., a novel plant associated rhizobacterium isolated from a saline tolerant pokkali rice and its draft genome analysis.

Three strains L3B27T, 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 13101T, Sphingomonas azotifigens DSM 18530T and Sphingomonas trueperi DSM 7225T 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 L3B27T (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 L3B27T=KCTC 42098T=MCC 3001T) 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 L3B27T. 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.

Flow, dynamic viscoelastic and creep properties of a biological polymer produced by Sphingomonas sp. as affected by concentration.

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″, τ0 and η0 values and lower ωc, Je0, γ̇c 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.

A novel design to screen chlorogenic acid-producing microbial strains from the environment.

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.

Purification and Identification of Astaxanthin and Its Novel Derivative Produced by Radio-tolerant Sphingomonas astaxanthinifaciens.

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-17T) 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-17T. 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.

Glycoside Hydrolase Family 39 ß-Xylosidase of Sphingomonas Showing Salt/Ethanol/Trypsin Tolerance, Low-pH/Low-Temperature Activity, and Transxylosylation Activity.

Mining for novel enzymes from new microorganisms is a way to obtain ß-xylosidases with promising applications. A Sphingomonas ß-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.

Effect of temperature and shear on the microstructure of a microbial polysaccharide secreted by Sphingomonas species in aqueous solution.

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.

Sphingomonas montis sp. nov., Isolated from Forest Soil of Low-Altitude Mountain.

Strain DRJ-4T 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-4T 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-4T formed a distinct lineage within the members of the family Sphingomonadaceae of the phylum Proteobacteria and moderately related to Sphingomonas faucium E62-3T (96.67% sequence similarity), 'Sphingosinicella ginsenosidimutans' BS11 (96.45% sequence similarity), Sphingosinicella vermicomposti YC7378T (96.27% sequence similarity), and Sphingobium boeckii 469T (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-4T were summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0, and C17:1ω6c. The genomic DNA G + C was 64.0 mol %. On the basis of phenotypic, genotypic, and phylogenetic analysis, strain DRJ-4T 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-4T (= KEMB 9005-708T = NBRC 113142T).