Abstract
Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.
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References
Ammar YB, Matsubara T, Ito K, Iizuka M, Limpaseni T, Pongsawasdi P, Minamiura N (2002) Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature. J Biotechnol 99:111–119. https://doi.org/10.1016/S0168-1656(02)00160-8
Andersone I, Auzina L, Vigants A, Mutere O, Zikmanis P (2004) Formation of levan from raffinose by levansucrase of Zymomonas mobilis. Eng Life Sci 4:56–59. https://doi.org/10.1002/elsc.200400006
Ates O, Arga KY, Toksoy Öner E (2013) The stimulatory effect of mannitol on levan biosynthesis: lessons from metabolic systems analysis of Halomonas smyrnensis AAD6T. Biotechnol Prog 29:1386–1397. https://doi.org/10.1002/btpr.1823
Avigad G (1957) Enzymatic synthesis and characterization of a new trisaccharide, α-lactosyl-β-fructofuranoside. J Biol Chem 229:121–129
Avsar G, Agirbasli D, Agirbasli MA, Gunduz O, Toksoy Öner E (2018) Levan based fibrous scaffolds electrospun via co-axial and single-needle techniques for tissue engineering applications. Carbohydr Polym 193:316–325. https://doi.org/10.1016/j.carbpol.2018.03.075
Axente E, Sima F, Sima LE, Erginer M, Eroglu MS, Serban N, Ristoscu C, Petrescu SM, Toksoy Öner E, Mihailescu IN (2014) Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts. Biofabrication 6:035010. https://doi.org/10.1088/1758-5082/6/3/035010
Aydin B, Ozer T, Toksoy Öner E, Arga KY (2018) The genome-based metabolic systems engineering to boost levan production in a halophilic bacterial model. OMICS 22:198–209. https://doi.org/10.1089/omi.2017.0216
Baciu IE, Jördening HJ, Seibel J, Buchholz K (2005) Investigations of the transfructosylation reaction by fructosyltransferase from B. subtilis NCIMB 11871 for the synthesis of the sucrose analogue galactosyl-fructoside. J Biotechnol 116:347–357. https://doi.org/10.1016/j.jbiotec.2004.10.019
Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258. https://doi.org/10.1093/nar/gku340
Biton J, Michel J, Beller DL, Pelenc V, Paul F, Monsan PF, Gellf G (1995) Enzymatic synthesis of low-calorie sugar substitutes cellobiofructose and gentiobiofructose. Ann N Y Acad Sci 750:321–324. https://doi.org/10.1111/j.1749-6632.1995.tb19973.x
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Caputi L, Nepogodiev SA, Malnoy M, Rejzek M, Field RA, Benini S (2013) Biomolecular characterization of the levansucrase of Erwinia amylovora, a promising biocatalyst for the synthesis of fructooligosaccharides. J Agric Food Chem 61:12265–12273. https://doi.org/10.1021/jf4023178
Chambert R, Petit-Glatron MF (1991) Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis. Biochem J 279:35–41. https://doi.org/10.1042/bj2790035
Chen GQ, Jiang XR (2018) Next generation industrial biotechnology based on extremophilic bacteria. Curr Opin Biotechnol 50:94–100. https://doi.org/10.1016/j.copbio.2017.11.016
Choi HJ, Kim CS, Kim P, Jung HC, Oh DK (2004) Lactosucrose bioconversion from lactose and sucrose by whole cells of Paenibacillus polymyxa harboring levansucrase activity. Biotechnol Prog 20:1876–1879. https://doi.org/10.1021/bp049770v
DasSarma S, DasSarma P (2012) Halophiles. eLS. https://doi.org/10.1038/npg.els.0004356
Diken E, Özer T, Arikan M, Emrence Z, Toksoy Öner E, Ustek D, Arga KY (2015) Genomic analysis reveals the biotechnological and industrial potential of levan producing halophilic extremophile, Halomonas smyrnensis AAD6T. SpringerPlus 4:393. https://doi.org/10.1186/s40064-015-1184-3
Elevi Bardavid R, Oren A (2012) Acid-shifted isoelectric point profiles of the proteins in a hypersaline microbial mat: an adaptation to life at high salt concentrations? Extremophiles 16:787–792. https://doi.org/10.1007/s00792-012-0476-6
El-Refai HA, Abdel-Fattah AF, Mostafa FA (2009) Enzymic synthesis of levan and fructo-oligosaccharides by Bacillus circulans and improvement of levansucrase stability by carbohydrate coupling. World J Microbiol Biotechnol 25:821–827. https://doi.org/10.1007/s11274-009-9957-x
Erkorkmaz BA, Kirtel O, Ateş Duru Ö, Toksoy Öner E (2018) Development of a cost-effective production process for Halomonas levan. Bioprocess Biosyst Eng:1–13. https://doi.org/10.1007/s00449-018-1952-x
Gao S, Qi X, Hart DJ, Gao H, An Y (2017) Expression and characterization of levansucrase from Clostridium acetobutylicum. J Agric Food Chem 65:867–871. https://doi.org/10.1021/acs.jafc.6b05165
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy Server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, pp 571–607
Goldman D, Lavid N, Schwartz A, Shoham G, Danino D, Shoham Y (2008) Two active forms of Zymomonas mobilis levansucrase an ordered microfibril structure of the enzyme promotes levan polymerization. J Biol Chem 283:32209–32217. https://doi.org/10.1074/jbc.M805985200
Gomes TD, Caridade SG, Sousa MP, Azevedo S, Kandur MY, Toksoy Öner E, Alves NM, Mano JF (2018) Adhesive free-standing multilayer films containing sulfated levan for biomedical applications. Acta Biomater 69:183–195. https://doi.org/10.1016/j.actbio.2018.01.027
Graziano G, Merlino A (2014) Molecular bases of protein halotolerance. Biochim Biophys Acta Proteins Proteomics 1844:850–858. https://doi.org/10.1016/j.bbapap.2014.02.018
Hernández L, Arrieta J, Menendez C, Vazquez R, Coego A, Suarez V, Selman G, Petit-Glatron MF, Chambert R (1995) Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochem J 309:113–118. https://doi.org/10.1042/bj3090113
Hettwer U, Gross M, Rudolph K (1995) Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola. J Bacteriol 177:2834–2839. https://doi.org/10.1128/jb.177.10.2834-2839.1995
Homann A, Biedendieck R, Götze S, Jahn D, Seibel J (2007) Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochem J 407:189–198. https://doi.org/10.1042/BJ20070600
Ishida R, Sakaguchi K, Matsuzaki C, Katoh T, Ishida N, Yamamoto K, Hisa K (2016) Levansucrase from Leuconostoc mesenteroides NTM048 produces a levan exopolysaccharide with immunomodulating activity. Biotechnol Lett 38:681–687. https://doi.org/10.1007/s10529-015-2024-9
Jang KH, Song KB, Kim CH, Chung BH, Kang SA, Chun UH, Choue RW, Rhee SK (2001) Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis. Biotechnol Lett 23:339–344. https://doi.org/10.1023/A:1005641220946
Kazak Sarilmiser H, Ates O, Ozdemir G, Arga KY, Toksoy Oner E (2015) Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T. J Biosci Bioeng 119(4):455–463
Khmelenina VN, Kalyuzhnaya MG, Starostina NG, Suzina NE, Trotsenko YA (1997) Isolation and characterization of halotolerant alkaliphilic methanotrophic bacteria from Tuva soda lakes. Curr Microbiol 35:257–261. https://doi.org/10.1007/s002849900249
Kim MG, Seo JW, Song KB, Kim CH, Chung BH, Rhee SK (1998) Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis. Biotechnol Lett 20:333–336. https://doi.org/10.1023/A:1005310926399
Kırtel O, Versluys M, Van den Ende W, Toksoy Öner E (2018) Fructans of the saline world. Biotechnol Adv 36:1524–1539. https://doi.org/10.1016/j.biotechadv.2018.06.009
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Lammens W, Le Roy K, Schroeven L, Van Laere A, Rabijns A, Van den Ende W (2009) Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications. J Exp Bot 60:727–740. https://doi.org/10.1093/jxb/ern333
Li M, Seo S, Karboune S (2015a) Bacillus amyloliquefaciens levansucrase-catalyzed the synthesis of fructooligosaccharides, oligolevan and levan in maple syrup-based reaction systems. Carbohydr Polym 133:203–212. https://doi.org/10.1016/j.carbpol.2015.07.010
Li W, Yu S, Zhang T, Jiang B, Mu W (2015b) Recent novel applications of levansucrases. Appl Microbiol Biotechnol 99:6959–6969. https://doi.org/10.1007/s00253-015-6797-5
Li W, Yu S, Zhang T, Jiang B, Mu W (2017) Synthesis of raffinose by transfructosylation using recombinant levansucrase from Clostridium arbusti SL206. J Sci Food Agric 97:43–49. https://doi.org/10.1002/jsfa.7903
Liu Q, Yu S, Zhang T, Jiang B, Mu W (2017) Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii. Carbohydr Polym 157:1732–1740. https://doi.org/10.1016/j.carbpol.2016.11.057
Loukas A, Kappas I, Abatzopoulos TJ (2018) HaloDom: a new database of halophiles across all life domains. J Biol Res (Thessaloniki) 25:2. https://doi.org/10.1186/s40709-017-0072-0
Lu L, Fu F, Zhao R, Jin L, He C, Xu L, Xiao M (2014) A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile transfructosylation reactions. Process Biochem 49:1503–1510. https://doi.org/10.1016/j.procbio.2014.05.012
MacRae E, Lunn JE (2012) Photosynthetic sucrose biosynthesis: an evolutionary perspective. In: Eaton-Rye J, Tripathy B, Sharkey T (eds) Photosynthesis. Advances in photosynthesis and respiration. Springer, Dordrecht, pp 675–702
Martínez-Fleites C, Ortíz-Lombardía M, Pons T, Tarbouriech N, Taylor EJ, Arrieta JG, Hernández L, Davies GJ (2005) Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus. Biochem J 390:19–27. https://doi.org/10.1042/BJ20050324
Méndez-Lorenzo L, Porras-Domínguez JR, Raga-Carbajal E, Olvera C, Rodríguez-Alegría ME, Carrillo-Nava E, Costas M, Munguía AL (2015) Intrinsic levanase activity of Bacillus subtilis 168 levansucrase (SacB). PloS One 10:e0143394. https://doi.org/10.1371/journal.Pone.0143394
Meng G, Fütterer K (2003) Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Mol Biol 10:935–941. https://doi.org/10.1038/nsb974
Miller GL (1959) Modified DNS method for reducing sugars. Anal Chem 31:426–428
Morales-Arrieta S, Rodríguez ME, Segovia L, López-Munguía A, Olvera-Carranza C (2006) Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F. Gene 376:59–67. https://doi.org/10.1016/j.gene.2006.02.007
Nakapong S, Pichyangkura R, Ito K, Iizuka M, Pongsawasdi P (2013) High expression level of levansucrase from Bacillus licheniformis RN-01 and synthesis of levan nanoparticles. Int J Biol Macromol 54:30–36. https://doi.org/10.1016/j.ijbiomac.2012.11.017
Oren A (2013) Life at high salt concentrations, intracellular KCl concentrations, and acidic proteomes. Front Microbiol 4:315. https://doi.org/10.3389/fmicb.2013.00315
Oren A (2015) Halophilic microbial communities and their environments. Curr Opin Biotechnol 33:119–124. https://doi.org/10.1016/j.copbio.2015.02.005
Osman A, Toksoy Öner E, Eroglu MS (2017) Novel levan and pNIPA temperature sensitive hydrogels for 5-ASA controlled release. Carbohydr Polym 165:61–70. https://doi.org/10.1016/j.carbpol.2017.01.097
Ozimek LK, Euverink GJW, Van Der Maarel MJEC, Dijkhuizen L (2005) Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes. FEBS Lett 579:124–1128. https://doi.org/10.1016/j.febslet.2004.11.113
Ozimek LK, Kralj S, Van der Maarel MJ, Dijkhuizen L (2006) The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions. Microbiol 152:1187–1196. https://doi.org/10.1099/mic.0.28484-0
Papadopoulos JS, Agarwala R (2007) COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics 23:1073–1079. https://doi.org/10.1093/bioinformatics/btm076
Park HE, Park NH, Kim MJ, Lee TH, Lee HG, Yang JY, Cha J (2003) Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from Microbacterium laevaniformans ATCC 15953. Enzym Microb Technol 32:820–827. https://doi.org/10.1016/S0141-0229(03)00062-0
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. https://doi.org/10.1038/nmeth.1701
Poli A, Kazak H, Gürleyendag B, Tommonaro G, Pieretti G, Toksoy Öner E, Nicolaus B (2009) High level synthesis of levan by a novel Halomonas species growing on defined media. Carbohydr Polym 78:651–657. https://doi.org/10.1016/j.carbpol.2009.05.031
Porras-Domínguez JR, Ávila-Fernández Á, Miranda-Molina A, Rodríguez-Alegría ME, Munguía AL (2015) Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight. Carbohydr Polym 132:338–344. https://doi.org/10.1016/j.carbpol.2015.06.056
Rairakhwada D, Seo JW, Seo MY, Kwon O, Rhee SK, Kim CH (2010) Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens. J Ind Microbiol Biotechnol 37:195–204. https://doi.org/10.1007/s10295-009-0664-2
Santos-Moriano P, Fernandez-Arrojo L, Poveda A, Jimenez-Barbero J, Ballesteros AO, Plou FJ (2015) Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions. J Mol Catal B Enzym 119:18–25. https://doi.org/10.1016/j.molcatb.2015.05.011
Seibel J, Moraru R, Götze S, Buchholz K, Na’amnieh S, Pawlowski A, Hecht HJ (2006) Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase). Carbohydr Res 341:2335–2349. https://doi.org/10.1016/j.carres.2006.07.001
Sezer AD, Kazak Sarilmiser H, Rayaman E, Çevikbaş A, Toksoy Öner E, Akbuğa J (2015) Development and characterization of vancomycin-loaded levan-based microparticular system for drug delivery. Pharm Dev Technol 22:627–634. https://doi.org/10.3109/10837450.2015.1116564
Shaheen S, Aman A, Siddiqui NN (2017) Influence of metal ions, surfactants and organic solvents on the catalytic performance of levansucrase from Zymomonas mobilis KIBGE-IB14. J Basic Appl Sci 13:41–46. https://doi.org/10.6000/1927-5129.2017.13.07
Silvério SC, Macedo EA, Teixeira JA, Rodrigues LR (2015) Perspectives on the biotechnological production and potential applications of lactosucrose: a review. J Funct Foods 19:74–90. https://doi.org/10.1016/j.jff.2015.09.014
Strube CP, Homann A, Gamer M, Jahn D, Seibel J, Heinz DW (2011) Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs. J Biol Chem 286:17593–17600. https://doi.org/10.1074/jbc.M110.203166
Szwengiel A, Czarnecka M, Czarnecki Z (2007) Levan synthesis during associated action of levansucrase and Candida cacaoi DSM 2226 yeast. Pol J Food Nutr Sci 57:433–440
Toksoy Öner E, Hernández L, Combie J (2016) Review of levan polysaccharide: from a century of past experiences to future prospects. Biotechnol Adv 34:827–844. https://doi.org/10.1016/j.biotechadv.2016.05.002
Ua-Arak T, Jakob F, Vogel RF (2017) Fermentation pH modulates the size distributions and functional properties of Gluconobacter albidus TMW 2.1191 levan. Front Microbiol 8:807. https://doi.org/10.3389/fmicb.2017.00807
Versluys M, Kirtel O, Toksoy Öner E, Van den Ende W (2018) The fructan syndrome: evolutionary aspects and common themes among plants and microbes. Plant Cell Environ 41:16–38. https://doi.org/10.1111/pce.13070
Visnapuu T (2012) Levansucrases encoded in the genome of Pseudomonas syringae pv. tomato DC3000: heterologous expression, biochemical characterization, mutational analysis and spectrum of polymerization products. Doctoral dissertation, University of Tartu
Visnapuu T, Mardo K, Mosoarca C, Zamfir AD, Vigants A, Alamäe T (2011) Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation. J Biotechnol 155:338–349. https://doi.org/10.1016/j.jbiotec.2011.07.026
Visnapuu T, Mardo K, Alamaee T (2015) Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo-and polysaccharides. New Biotechnol 32:597–605. https://doi.org/10.1016/j.nbt.2015.01.009
Wuerges J, Caputi L, Cianci M, Boivin S, Meijers R, Benini S (2015) The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site. J Struct Biol 191:290–298. https://doi.org/10.1016/j.jsb.2015.07.010
Xu W, Yu S, Liu Q, Zhang T, Jiang B, Mu W (2017) Enzymatic production of melibiose from raffinose by the levansucrase from Leuconostoc mesenteroides B-512 FMC. J Agric Food Chem 65:3910–3918. https://doi.org/10.1021/acs.jafc.7b01265
Yin J, Chen JC, Wu Q, Chen GQ (2015) Halophiles, coming stars for industrial biotechnology. Biotechnol Adv 33:1433–1442. https://doi.org/10.1016/j.biotechadv.2014.10.008
Yun JW, Lee MG, Song SK (1994) Batch production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase. J Ferment Bioeng 77:159–163. https://doi.org/10.1016/0922-338X(94)90316-6
Zhang T, Li R, Qian H, Mu W, Miao M, Jiang B (2014) Biosynthesis of levan by levansucrase from Bacillus methylotrophicus SK 21.002. Carbohydr Polym 101:975–981. https://doi.org/10.1016/j.carbpol.2013.10.045
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WVdE and MV are supported by funds from FWO Vlaanderen. The financial support of The Scientific and Technological Research Council of Turkey (TUBITAK) (grant number: 115O495) and Marmara University Scientific Research Fund (grant number: FEN-A-130515-0178) are gratefully acknowledged.
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Kirtel, O., Menéndez, C., Versluys, M. et al. Levansucrase from Halomonas smyrnensis AAD6T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized. Appl Microbiol Biotechnol 102, 9207–9220 (2018). https://doi.org/10.1007/s00253-018-9311-z
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DOI: https://doi.org/10.1007/s00253-018-9311-z