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A New Source of Industrially Important Enzyme Systems JSM Central Biotechnology & Biomedical Engineering Special Issue on Industrial Biotechnology-Made in Germany: The path from policies to sustainable energy, commodity and specialty products Edited by: Dr. Thomas Brück Professor of Industrial Biocatalysis, Dept. of Chemistry, Technische Universität München (TUM), Germany Research Article *Corresponding authors Prof. Dr. Garabed Antranikian, Institute of Technical Microbiology, Hamburg University of technology, The Psychrophile Shewanella Kasernenstr, 12, 21073 Hamburg, Germany, Tel: 4904042878311; Fax: 49040428782582; Email: arctica sp. Nov: A New Source Prof. Dr. Thomas Brück, Department of Chemistry, Industrial Biocatalysis, Technical University of Munich, Lichtenbergstr, 4, 85748 Garching, Germany, Tel: of Industrially Important 4908928913253; Fax: 4908928913255; Email: Submitted: 19 March 2014 Enzyme Systems Accepted: 12 May 2014 Qoura, F.1, Brück, T.2* and Antranikian, G.3* Published: 14 May 2014 1Department of Chemistry, Industrial Biocatalysis, Technical University of Munich, Germany ISSN: 2333-7117 2Department of Chemistry, Industrial Biocatalysis, Technical University of Munich, Germany Copyright 3 Institute of Technical Microbiology, Hamburg University of technology, Germany © 2014 Qoura et al. Keywords OPEN ACCESS Spitsbergen; Psychrophile; Shewanella arctica; Industrial enzymes Abstract A new psychrophilic, strictly aerobic bacterium, strain 40-3, was isolated from seawater samples collected at Spitsbergen in the Arctic. The cells are gram negative, straight or curved rod shaped and non-spore forming (2-3 µm long and 0.4-0.6 µm wide). Colonies on agar medium are slightly orange, circular, smooth and convex. 40-3 strain grows optimally over the temperature range of 10-15 °C and a pH range of 7-8 in media containing 8 to 9 % NaCl (w/v). Growth occurs with α cyclo-dextrin, dextrin, tween 80, N-acetyl-D-glucosamine, α-D-glucose, maltose, sucrose, methyl pyruvate, D,L-lactitate, succiniate , bromo succinic acid, inosine, esculin ferric citrate, L-arabinose, potassium gluconate, malic acid and trisodium citrate. In the presence of glucose H2S was produced and nitrates are reduced to nitrites. The fatty acid methyl ester (FAME) are composed of 17.89 % straight chain saturated FAMEs, 14,85 % terminally branched saturated FAMEs and 17.73 % monounsaturated FAMEs. The DNA base ratio is 48 mol % G + C. Phylogenetic analysis reveals as close relationship to Shewanella putrefaciens with 99 % 16S rDNA composition identity and 50 % DNA- DNA similarity. The phylogenetic evidence, together with phenotypic characteristics, show that this psychrophilic strain constitute a novel species of the genus Shewanella. The name Shewanella arctica is proposed. Interestingly, when grown on glucose as a carbon source Shewanella arctica produced numerous industrially important enzyme systems including amylase, pullulanase, protease, ornithine decarboxylase, alkaline phospatase, esterase (C4), lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β– glucosaminidase. Due to the functional new physochrophilic Shewanella strainresource for the isolation of new enzyme system, which may operate at low reaction thereby increasing the energy efficiency of industrial processes. ABBREVIATION Recently, a comprehensive study of the phylogenetic FAME: Fatty Acid Methyl Ester INTRODUCTION thisrelationships study a noveland taxonomy psychrophilic of the bacterium genus provided was isolated an improved from seawaterapproach samplesfor identification obtained offrom newly the isolatedarea of Spitsbergen,wild strains [6].in the In Recently, increasing attention has been directed to cold- Arctic. The novel isolate was found to be similar to, but distinct adapted microorganisms able to grow at/or close to the from in a number of characteristics, a previously described genus, freezing point of water, namely psychrophiles [1]. Cold adapted Shewanella microorganisms are found in both permanently and temporarily isolated strain demonstrated that the represent is a novel specie cold habitats, which comprise more than 80 % of the Earth’s of the genus. Taxonomic Shewanella and, and physiological we propose analysisthe name of Shewanella the newly biosphere. The genus MacDonell and Colwell 1985 Shewanella arctica sp. nov. For the species represented by the strain 40- comprises a ubiquitous group of gram negative, aerobic and 3. Further, the isolated Shewanella strain secreted numerous . This genus comprises -Proteobacteria industrially relevant enzyme systems including hydrolyases more than 25 species inhabiting a wide range of environments facultatively anaerobic δ such, as amylase, protease and, esterases and lipases. Enzymes derived from physchiphilic organism have the potential to and freshwater, cold water and sediments of the deep sea [2-6]. operate at low temperatures thereby improving the energy Duringincluding the spoiled last decade, food, oil the field bacteria wastes, of thisredox genus interfaces have received in marine a physochophilic enzymes have shown the propensity to operate metabolic bioremediation of halogenated organic pollutants [7], efficiency of biotechnological process [10-12]. Further, many destructivesignificant amount souring of of attention crude petroleum due to their [8] importantand the dissimilatory roles in co- mesophilic organism [13,14]. More recently, various Shewanella over a wider temperature profile than those derived from reductionCite this article: of magnesium Qoura F, Brueck and iron T, Antranikian oxides [9]. G (2014) The Psychrophile Shewanella arctica sp. Nov: A New Source of Industrially Important Enzyme Systems. JSM Biotechnol Bioeng 2(1): 1026. Qoura et al. (2014) Email: (and) Central sp. were inoculated with the new strain cells suspension (cells were relevant enzyme systems [15-17]. Therefore, the enzymes systems have beenthat can flagged be produced as excellent using sources the current for novel bacterial industrially strain nm) and incubated at 15 °C overnight. have potential use in the biofuels and food (amylase, lipase) as resuspended in NaCl 0.85 % (w/v) medium to a final OD of 0.5 well as the chemical industry (pullulanase, alkaline phospatase, leucine arylamidase, valine arylamidase). A large number of carbon source were tested: α cyclo- MATERIALS AND METHODS acid,dextrin, succinic dextrin, acid, Tween bromo 80, succinic Tween 40,acid, N-acetyl-D-glucosamine, inosine, esculin ferric citrate,α-D-glucose, L-arabinose, maltose, potassium sucrose, gluconate, methylpyruvate, malic acid, trisodiumD,L-lactic Sample collection, media and culturing conditions Seawater samples were collected in 1998 during an acid, D-arabitol, cellobiose, L-erythol, D-fructose, L-fructose, citrate, glycogen, N-acetyl-D-galacto-samine, adonitol, capric to the laboratory at temperature between 2 to 10 °C. A 1 ml expedition at Spitsbergen, Norway. Samples were transported D-galactose, gentiobiose, m-inositol, α-D-lactose, lactulose, turanose,D-mannitol, Xylitol, D-mannose, mono-methyl D-mellobiose, succinate, β-methyl-D-glucose, acetic acid, cis- basalvolume medium of liquid supplemented sample was with incubated a solution in ofa complexdifferent marinecarbon aconiticD-psicose, acid, D-raffinose, citric acid,L-rhamnose, formic acid,D-sorbitol, D-galactonic D-trehalose, acid sources.liquid medium. The basal The medium complex contained marine (volume medium g lconsisted-1 of a lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic g; KCl 0.77 g; CaCl2 2O 0.02 g; MgSO4 2 4Cl 1.0 ): NaClg; 10-fold 28.13 concentration trace x2Helement solution (DSM x7H 141)O 0.5 1 g;ml; NH 10-fold acid, D-glucuronic acid, α-hydroxy-butyric acid, β-hydroxy- g; iron-ammonium-citrate 0.02 g; yeast extract 0.5 butyric acid, γ-hydroxy-butyric acid, p-hydroxyphenylacetic 2PO4 2.3 acid, itaconic acid, α-keto butyric acid, α-keto glutaric acid, 2 4 2 D-saccharic acid, seabacic acid, succinamic acid, glucuronamide, concentration-1 vitamin solution (DSM 141) 1 ml; KH α-keto valeric acid, malonic acid, propionic acid, quinic acid, (volume g l 2 alaninamide, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, g; Na HPO x2H O 2.9 g. The carbon source mixture solution 0.5 g; DL-malate 0.5 g; D-mannitol 0.5 g and glucose 2 g. The L-aspartic acid, L-glutamic acid, glycyl-L-aspartic acid, glycyl- ): NA-acetate 0.5 g; Na -succinate 0.5 g; Na-pyruvate out at 4 °C for about 4-7 days, before growth and colonies on L-ornithine, L-phenyl-alanine, L-proline, L-pyroglutamic acid, agarfinal platespH of thewere complex observed. medium Colonies was were 7. Incubation selected onwas the carried basis L-glutamic acid, L-histidine, hydroxy L-proline, L-leucine, of morphological differences. For the isolation of a pure culture, acid, urocanic acid, uridine, thymidine, phenylthylamine, serial dilution and plating techniques were applied. The pure putrescine,D-serine, L-serine, 2-aminoethanol, L-threonine, 2,3-butanediol,D,L-carnitine, γ-aminoglycerol, butyric D,L agar plates at 15 °C for 4 days. phenylacetic acid. isolates were routinely cultivated on complex marine medium α-glycerol phosphate, glucose 1-phosphate, glucose 6-phosphate, Cellular characterization Screening for enzyme production Gram staining test was performed by staining cells using the The ability of the new strain to produce: alkaline phospatase, arginine dihydrolase, esterase (C4), esterase lipase (C8), for up to 6 days in medium containing no carbon source other lipase, leucine arylamidase, valine arylamidase, cystine
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