This article was downloaded by: [Eindhoven Technical University] On: 13 February 2015, At: 07:32 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
PRODUCTION AND CHARACTERIZATION OF L-FUCOSE DEHYDROGENASE FROM NEWLY ISOLATED Acinetobacter sp. STRAIN SA-134 a
Takashi Ohshiro & Noriyuki Morita
a
a
Department of Biotechnology , Tottori University , Tottori , Japan Accepted author version posted online: 16 Aug 2013.Published online: 09 Dec 2013.
Click for updates To cite this article: Takashi Ohshiro & Noriyuki Morita (2014) PRODUCTION AND CHARACTERIZATION OF L-FUCOSE DEHYDROGENASE FROM NEWLY ISOLATED Acinetobacter sp. STRAIN SA-134, Preparative Biochemistry and Biotechnology, 44:4, 382-391, DOI: 10.1080/10826068.2013.833107 To link to this article: http://dx.doi.org/10.1080/10826068.2013.833107
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:382–391, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.833107
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
PRODUCTION AND CHARACTERIZATION OF L-FUCOSE DEHYDROGENASE FROM NEWLY ISOLATED Acinetobacter sp. STRAIN SA-134
Takashi Ohshiro and Noriyuki Morita Department of Biotechnology, Tottori University, Tottori, Japan
& Microorganisms producing L-fucose dehydrogenase were screened from soil samples, and one of the isolated bacterial strains SA-134 was identified as Acinetobacter sp. by 16S rDNA gene analysis. The strain grew well utilizing L-fucose as a sole source of carbon, but all other monosaccharides tested such as D-glucose and D-arabinose did not support the growth of the strain in the absence of L-fucose. D-Arabinose inhibited the growth even in the culture medium containing L-fucose. Although the strain grew on some organic acids and amino acids such as citric acid and L-alanine as sole sources of carbon, the enzyme was produced only in the presence of L-fucose. The fucose dehydrogenase was purified to apparently homogeneity from the strain, and the native enzyme was a monomer of 25 kD. L-Fucose and D-arabinose were good substrates for the enzyme, but L-galactose was a poor substrate. The enzyme acted on both NADþ and NADPþ in the similar manner. Keywords Acinetobacter, enzyme purification, enzyme properties, fucose dehydrogenase
INTRODUCTION L-Fucose is widely distributed in microorganisms, plants, and animals. Especially, it is found at the nonreducing termini of biological active carbohydrates in mammalian systems, such as immunoglobulins and mucin glycoproteins.[1] Since the fucosylated glycans are known to act as adherence recognition targets for intestinal bacteria, some pathogenic bacteria release fucose through the hydrolytic activity toward the mucin and facilitate the dispersion of the pathogens.[2] In addition, free fucose has been reported to play a role as a mediator in the modulation of immune response in human intestinal epithelium.[3] Therefore, free fucose has been recognized as a marker of several diseases, and the accurate measurement of L-fucose is significantly important in the field of clinical Address correspondence to Takashi Ohshiro, Department of Biotechnology, Tottori University, Koyamachominami 4-101, Tottori 680-8552, Japan. E-mail: [email protected]
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
383
inspection.[4] One of the promising methods to determine L-fucose is an enzymatic analysis using L-fucose dehydrogenase. L-Fucose is also known to be a main component of fucoidan, which is the sulfated macromolecule substance contained in brown algae. Fucoidan has attracted much attention in terms of healthy food because it exhibits a variety of biological activities, including antitumor,[5,6] antioxidant,[7] anticoagulant,[8] and anti-inflammatory activities.[9] In spite of the significance of sulfate content in fucoidan being proposed before now, the reason why fucoidan shows these physiological activities has not been fully elucidated. Moreover, it is unclear whether high-molecular-weight fucoidan is incorporated through the intestinal wall or digested before the incorporation. Recently, it has been demonstrated that the biological activities of fucoidan change due to the size of its molecular weight.[10] Thus, the molecular size appears to be a significant factor, as well as the sulfate content. Low-molecular-weight fucoidans were prepared from native fucoidan with high molecular weight, by partial acid hydrolysis, radical depolymerization, or enzymatic treatment. We found some microorganisms utilizing fucoidan from Cladosiphon okamuranus for the purpose of the enzymatic treatment,[11] and identified one bacterial strain as Luteolibacter algae, which had been classified as an unculturable bacterium.[12] Fucoidan from C. okamuranus consists of fucose moieties attached to sulfate and acetate as the main chain, and glucuronic acid as the branching part. Accordingly, it is assumed that L-fucose is released during lowering of the molecular weight of the native fucoidan. In this case, L-fucose dehydrogenase has been thought to be an important tool to measure the content of nonbound free fucose. Up to now, three microbial L-fucose dehydrogenases have been investigated from Pseudomonas sp. no. 1143,[13] Agrobacterium radiobacter,[14] and A. tumefaciens,[15] and the gene of no. 1143 strain was cloned.[16] Although L-fucose is converted to L-fucono-1,5-lactone by L-fucose dehydrogenase, more metabolites have not been detected in these microorganisms. Among these, the enzyme from A. tumefaciens was constitutively produced, but the detailed production conditions have not been investigated about these three strains. In this study, we found a new bacterial strain producing L-fucose dehydrogenase, examined the medium composition for the enzyme production, and characterized the purified enzyme.
EXPERIMENTAL Strain Isolation, Identification, and Culture Conditions Enrichment culture was performed at 30 C with reciprocal shaking (300 strokes=min) in test tubes containing 5 mL of the synthetic medium
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
384
T. Ohshiro and N. Morita
(pH 7.0) previously used[11] except using fucose as a sole source of carbon. The composition of the medium was 0.25% fucose, 0.4% K2HPO4, 0.05% KH2PO4, 0.2% (NH4)2SO4, 0.041% MgSO47H2O, metal solution, and vitamin mixture. To prepare the purified enzyme, the isolated strain SA-134 was cultivated at 30 C for 1 day with reciprocal shaking (120 strokes= min) in 2-L flasks containing 500 mL of LB medium (1% peptone, 0.5% yeast extract, and 0.5% NaCl) with 0.1% fucose. Whole DNA extraction was performed from cells of the strain SA134 as previously reported,[11] and the 16S rDNA gene was amplified using universal bacterial 16S rDNA primers 27f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1525r (50 -AAAGGAGGTGATCCAGCC-30 ). The amplified DNA fragment of approximately 1500 bp was purified, sequenced, and compared against samples deposited with the database. Enzyme Assay Fucose dehydrogenase activity was determined at 37 C using the increase in absorbance at 340 nm due to reduction of NADPþ. The reaction mixture contained 35 mM Tris-HCl buffer (pH 8.0), 50 mM L-fucose, 10 mM NADPþ, and the enzyme in a total volume of 1 mL. One unit of activity was defined as the amount of fucose dehydrogenase necessary to release 1 mmol of NADPH per min. Enzyme Purification All operations were performed at 4 C and 20 mM Tris-HCl buffer (pH 8.0) was used as the basal buffer. Approximately 170 g (wet weight) of frozen cells was suspended in 500 mL of the basal buffer and disrupted with an ultraoscillator (Sonifier 450; Branson Instruments, Danbury, CT) at 20 kHz. The cell debris was removed by centrifugation at 15,000 g for 60 min, and the resultant supernatant was used as the cell-free extract. After the cell-free extract was dialyzed against the basal buffer, the enzyme solution was applied to a DEAE-Sepharose column (5.6 36 cm) that had been equilibrated with the same buffer. The column was washed with the same buffer and bound proteins were eluted with the buffers containing 0.1, 0.25, or 0.5 M NaCl. The active fractions eluted with the buffer containing 0.25 M NaCl were collected and concentrated by ultrafiltration. After the enzyme solution was dialyzed against the basal buffer containing 1 M (NH4)2SO4, the enzyme solution was applied to a Phenyl-Toyopearl column (5.6 11 cm) that had been equilibrated with the same buffer. The column was washed with the same buffer and bound proteins were eluted with the buffer containing 0.8 M (NH4)2SO4. The active fractions were collected, concentrated by
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
385
ultrafiltration, and dialyzed against 10 mM Tris-HCl buffer (pH 8.0). The enzyme solution was applied to a Q-Sepharose column (2.8 38 cm) that had been equilibrated with the same buffer. The column was washed with the same buffer and bound proteins were eluted with 25, 100, 250, and 500 mM Tris-HCl buffers (pH 8.0). The active fractions eluted with 500 mM Tris-HCl buffer (pH 8.0) were collected and concentrated by ultrafiltration. After the buffer solution dissolving the enzyme was replaced by ultrafiltration with the basal buffer, the enzyme solution was applied to a Superdex 200HR 10=30 (1 30 cm) equilibrated with the same buffer. The chromatography was performed at a flow rate of 0.3 mL=min controlled by an AKTA system. Finally, the collected active fraction was applied to a Mono-Q HR 10=10 (1 10 cm) equilibrated with the same buffer. The chromatography was performed with a linear gradient of 0 to 1 M NaCl in the basal buffer at a flow rate of 0.3 mL=min controlled by an AKTA system.
Other Analytical Methods Protein concentration was determined by the method of Bradford[17] using a Bio-Rad protein assay reagent with bovine serum albumin as a standard. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done using 12.5% polyacrylamide gel by the method of Laemmli.[18] The gels were stained with Coomassie brilliant blue dissolved in 50% methanol–10% acetic acid and destained in 30% methanol–10% acetic acid. The N-terminal amino acid sequence was determined by a PPSQ-31A (Shimadzu, Kyoto, Japan).
RESULTS AND DISCUSSION Isolation and Identification of Fucose Dehydrogenase-Producing Microorganism Approximately 200 soil samples were employed for the screening, and we have found some bacterial strains utilizing fucose as a sole source of carbon. Cell-free extract of each strain was prepared, and the enzyme activities were estimated. As a result, one strain, SA-134, showing the maximum activity among the isolated strains was chosen for the following studies. The sequence of the 16S rDNA (AB720732) of strain SA-134 showed the highest similarity (99%) to the sequences of Acinetobacter sp. YNA109 (JQ039977) and Acinetobacter sp. AW 1-18(JQ316540). Hence, strain SA-134 was identified as Acinetobacter sp.
386
T. Ohshiro and N. Morita
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Enzyme Production by Acinetobacter sp. SA-134 Acinetobacter sp. SA-134 was isolated using a synthetic medium containing fucose as a sole source of carbon. However, the growth of this strain in the synthetic medium with 0.25% fucose was not good (OD660 ¼ 2), and fucose is an expensive compound as a carbon source for the large-scale cultivation to purify the enzyme. To enhance enzyme production by strain SA-134, the bacterium was cultured in a variety of media and the enzyme production was estimated. As shown in Table 1, the growth of Acinetobacter sp. SA-134 was enhanced when D-lactate, succinate, L-alanine, and L-glutamate were added to the media containing fucose, but D-glucose, D-fructose, and D-galactose among monosaccharides tested did not influence the growth of the strain. On the contrary, D-mannose and D-xylose had inhibitory effects, and D-arabinose completely inhibited the growth. However, in any case, the enzyme production was not enhanced compared to the medium containing fucose only. Acinetobacter sp. SA-134 could grow in a synthetic media with D-lactate, succinate, L-alanine, and L-glutamate as sole sources of carbon. However, no enzyme activities were detected in TABLE 1 Effects of Organic Compounds on Growth and Enzyme Production of Acinetobacter sp. SA-134 in Media With or Without L-Fucose Growth (OD660) Additive (0.5%) None D-Arabinose D-Glucose D-Fructose D-Galactose D-Mannose D-Xylose Maltose Sucrose Lactose Glycerol Sorbitol Mannitol D-Lactate Succinate L-Alanine L-Glutamate Fucoidan
Specific Activity (units=mg)
With Fucose
Without Fucose
With Fucose
Without Fucose
2.01 0.07 1.87 1.61 1.63 0.99 0.51 1.62 1.73 1.86 1.90 1.73 1.91 3.40 2.57 4.64 3.37 2.16
0 0.06 0.06 0.11 0.07 0.05 0.16 0.05 0.03 0.05 0.02 0.02 0.02 1.89 0.85 1.31 1.10 0.13
2.81 N.D. 2.31 2.46 2.95 1.99 1.92 2.37 2.28 2.56 2.22 1.96 1.72 1.46 2.52 1.15 1.61 2.36
N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0 0 0 0 N.D.
The strain SA-134 was cultivated in the synthetic medium with or without fucose adding indicated compounds at 0.5%. The medium with fucose in this Table corresponds to the synthetic medium containing 0.25% L-fucose described in the text. The cultivation was done at 30 C for 1 d. N.D. means not determined.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
387
these cases. Moreover, all sugars tested except for L-fucose did not support the growth of Acinetobacter sp. SA-134 (Table 1). Thus, only fucose appeared to induce enzyme synthesis. Although there are few previous reports describing enzyme production about fucose dehydrogenase, Itoh et al. demonstrated that Agrobacterium tumefaciens K-28 constitutively produced the enzyme.[15] In the case of Acinetobacter sp. SA-134, we demonstrated that fucose induces the production of fucose dehydrogenase and other sugars do not repress the enzyme production. Next, we tried to investigate enzyme production in nutrient broth with various concentrations of fucose. Both growth (OD660 ¼ 3.8) and enzyme production (2.3 units=mg) were the highest when Acinetobacter sp. SA-134 was cultured in LB medium with 0.1% fucose. Needless to say, the enzyme production was not observed in the cells grown in only LB medium. Hence, the strain was cultivated in LB medium containing 0.1% fucose to obtain the purified enzyme. Enzyme Purification and N-Terminal Amino Acid Sequence Fucose dehydrogenase was purified to apparent homogeneity from the cell-free extracts of Acinetobacter sp. A-134 and the molecular mass of the subunit was 25 kD (Figure 1). The purification steps are summarized in Table 2. However, the enzyme inactivation occurred significantly during the purification steps, so it is necessary to examine factors causing the inactivation in the future. The native molecular mass of the enzyme was estimated to be approximately 23 kD by the analysis of Superdex column chromatography (data not shown), indicating that the enzyme is a monomer. Although the molecular weight of the enzyme from Acinetobacter sp.
FIGURE 1 SDS-PAGE of fucose dehydrogenase from Acinetobacter sp. SA-134. Lane 1 and 8, marker proteins; lane 2, cell-free extract (28 mg); lane 3, pooled fraction after DEAE-Sepharose (28 mg); lane 4, pooled fraction after Phenyl-Toyopearl (18 mg); lane 5, pooled fraction after Q-Sepharose (3.5 mg); lane 6, pooled fraction after Superdex (2.5 mg); lane 7, purified enzyme after Mono Q (0.8 mg) (color figure available online).
388
T. Ohshiro and N. Morita
TABLE 2
Summary of Purification of Fucose Dehydrogenase From Acinetobacter sp. SA-134
Step
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Cell-free extracts DEAE-Sepharose Phenyl-Toyopearl Q-Sepharose Superdex MonoQ
Total Protein (mg)
Total Activity (units)
Specific Activity (units=mg)
Purification (fold)
Yield (%)
20100 5240 788 16.4 1.01 0.332
46200 18000 12900 1520 141 72.0
2.30 3.44 16.4 92.7 140 217
1 1.50 7.13 40.3 60.9 94.3
100 39.0 27.9 3.29 0.305 0.156
A-134 was similar to that from Pseudomonas sp. no. 1143, the substrate specificities of two enzymes differed from each other as described in the following. The N-terminal amino acid sequence of the enzyme was determined to be MDYHEKNKVFIVTGGGAGIGGAIS, but the BLAST analysis found no enzymes having a significant similarity to this sequence. Although the gene of L-fucose dehydrogenase was reported from only Pseudomonas sp. no. 1143,[16] any parts of amino acid sequences from the strain no. 1143 show no homology to the N-terminal sequence of the enzyme from Acinetobacter sp. A-134.
Substrate Specificity and Kinetic Parameters The reactivity of the enzyme from Acinetobacter sp. SA-134 was investigated for various sugars (Table 3). The activities for D-arabinose and TABLE 3 Substrate Specificity of Fucose Dehydrogenase From Acinetobacter sp. SA-134 Compound
Relative Activity (%)
L-Fucose D-Glucose D-Fructose D-Galactose L-Galactose L-Arabinose D-Arabinose D-Mannose L-Rhamnose D-Xylose Maltose Sucrose Lactose
100 1 0 0 11 0 65 0 0 0 0 0 0
Note. The enzyme reaction mixture contained 35 mM Tri-HCl buffer (pH 8.0), 10 mM NADPþ, and 50 mM indicated compounds.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
389
L-galactose were 65 and 11% of that for L-fucose, respectively, and the latter activity was different from those of other enzymes. In the cases of three microbial L-fucose dehydrogenases from Agrobacterium tumefaciens K-28, A. radiobacter, and Pseudomonas sp. no. 1143, the activities for L-galactose were 60% or more. On the contrary, the enzyme of strain SA-134 showed a little activity for L-galactose. The activity for D-arabinose of strain SA-134 was similar to those of two Agrobacterium strains (75 and 71%). All other sugars were inert. Although the enzyme of strain SA-134 acted on D-arabinose as a substrate, it is apparently curious that the growth of the strain was completely inhibited by D-arabinose. Any metabolites derived from D-arabinose might inhibit the growth of the strain. The Km values for L-fucose, NADþ, and NADPþ were 2.6, 2.7, and 1.7 mM, respectively. Since the affinity for L-fucose of the enzyme was similar to those of other enzymes, it is believed that the enzyme from Acinetobacter sp. SA-134 will be applicable to the determination of L-fucose. The enzyme of strain SA-134 acted on both NADþ and NADPþ; however, the activity for NADPþ was lower compared to previous reported enzymes.
Other Enzymatic Properties When the enzyme activity was measured at various pH values and temperatures, the optimum pH was found to be around 8 and the optimum temperature was about 50 C. Stability of the enzyme was examined at different pH at room temperature for 60 min, and at various temperatures for 30 min in 20 mM Tris-HCl buffer (pH 8.0). The enzyme was stable between pH 6 and 10, and below 35 C (data not shown). The effects of metal ions and inhibitors on the enzyme activity were investigated. It was found that heavy metals such as Hg2þ, Agþ, Cu2þ, and Co2þ completely inhibited the enzyme activity at 1 mM; in addition, ethylenediamine tetraacetic acid (EDTA) and N-ethylmaleimide also had significant inhibitory effects at 1 mM (data not shown). The enzymatic properties described in this paragraph were almost similar to previous reported enzymes. In this study, we isolated a new bacterial strain, Acinetobacter sp. SA-134, producing fucose dehydrogenase. We have demonstrated that the strain utilizes no monosaccharides other than L-fucose as a sole source of carbon, and the enzyme production is induced only in the presence of L-fucose. Since the enzyme showed some different properties compared to other previous reported enzymes, the gene cloning and expression of the enzyme from the strain SA-134 are now in progress in our laboratory. Recently, it has been reported that a gastrointestinal pathogenic bacterium, Campylobacter jejuni, utilizes fucose as a sole source of carbon but not common carbohydrates on the glycolytic pathway, the Entner–Doudoroff pathway, and the pentose
390
T. Ohshiro and N. Morita
phosphate pathway.[19,20] These two reports indicate that the pathogenic strain C. jejuni has a novel metabolic pathway for L-fucose. Furthermore, Yew et al. also proposed a novel matabolic pathway for the catabolism of L-fucose by Xanthomonas campestris on the basis of the expected functions of homologues of the proteins predicted from the genome sequence.[21] It will be interesting to examine whether Acinetobacter sp. SA-134 has a similar pathway.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
CONCLUSION In this study, we isolated one L-fucose dehydrogenase-producing microorganism from soil samples, and identified as Acinetobacter sp. by 16S rDNA gene analysis. The strain grew well utilizing L-fucose as a sole source of carbon, but not all other monosaccharides tested such as D-glucose and D-arabinose. Although the strain grew on some organic acids and amino acids such as citric acid and L-alanine as sole sources of carbon, the enzyme was produced only in the presence of L-fucose. The fucose dehydrogenase was purified to apparent homogeneity from the strain, and the native enzyme was a monomer of 25 kD. L-Fucose and D-arabinose were good substrates for the enzyme, but L-galactose was a poor substrate. The enzyme acted on both NADþ and NADPþ in a similar manner. REFERENCES 1. Robbe, C.; Capon, C.; Coddeville, B.; Michalski, J.C. Structural Diversity and Specific Distribution of O-Glycans in Normal Human Mucins Along the Intestinal Tract. Biochem. J. 2004, 384, 307–316. 2. Cone, R.A. Barrier Properties of Mucin. Adv. Drug Deliv. Rev. 2009, 61, 75–85. 3. Chow, W.L.; Lee, Y.K. Free Fucose Is a Danger Signal to Human Intestinal Epithelial Cells. Br. J. Nutr. 2008, 99, 449–454. 4. Mrochek, J.E.; Dinsmore, S.R.; Tormey, D.C.; Waalkes, T.P. Protein-Bound Carbohydrates in Breast Cancer. Liquid-Chromatographic Analysis for Mannose, Galactose, Fucose, and Sialic Acid in Serum. Clin. Chem. 1976, 22, 1516–1521. 5. Fukahori, S.; Yano, H.; Akiba, J.; Ogasawara, S.; Momosaki, S.; Sanada, S.; Kuratomi, K.; Ishizaki, Y.; Moriya, F.; Yagi, M.; Kojiro, M. Fucoidan, a Major Component of Brown Seaweed, Prohibits the Growth of Human Cancer Cell Lines In Vitro. Mol. Med. Rep. 2008, 1, 537–542. 6. Cho, M.L.; Lee, B.-Y.; You, S. Relationship Between Oversulfation and Conformation of Low and High Molecular Weight Fucoidans and Evaluation of Their In Vitro Anticancer Activity. Molecules 2011, 16, 291–297. 7. Wang, J.; Zhang, Q.; Zhang, Z.; Song, H.; Li, P. Potential Antioxidant and Anticoagulant Capacity of Low Molecular Weight Fucoidan Fractions Extracted From Laminaria japonica. Int. J. Biol. Macromol. 2010, 46, 6–12. 8. Zoysa, M.D.; Nikapitiya, C.; Jeon, Y.-J.; Jee, Y.; Lee, J. Anticoagulant Activity of Sulfated Polysaccharide Isolated From Fermented Brown Seaweed Sargassum fulvellum. J. Appl. Phycol. 2008, 20, 67–74. 9. Park, H.Y.; Han, M.H.; Park, C.; Jin, C.-Y.; Kim, G.-Y.; Choi, I.-W.; Kim, N.D.; Nam, T.-J.; Kwon, T.K.; Choi, Y.H. Anti-Inflammatory Effects of Fucoidan Through Inhibition of NF-jB, MAPK and Akt Activation in Lipopolysaccharide-Induced BV2 Microglia Cells. Food Chem. Toxicol. 2011, 49, 1745–1752.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
391
10. Morya, V.K.; Kim, J.; Kim, E.-K. Algal Fucoidan: Structural and Size-Dependent Bioactivities and Their Perspectives. Appl. Microbiol. Biotechnol. 2012, 93, 71–82. 11. Ohshiro, T.; Ohmoto, Y.; Ono, Y.; Ohkita, R.; Miki, Y.; Kawamoto, H.; Izumi, Y. Isolation and Characterization of a Novel Fucoidan-Degrading Microorganism. Biosci. Biotechnol. Biochem. 2010, 74, 1729–1732. 12. Ohshiro, T.; Harada, N.; Kobayashi, Y.; Miki, Y.; Kawamoto, H. Microbial Fucoidan Degradation by Luteolibacter algae H18 With Deacetylation. Biosci. Biotechnol. Biochem. 2012, 76, 620–623. 13. Horiuchi, T.; Suzuki, T.; Himura, M.; Saito, N. Purification and Characterization of L-Fucose (L-galactose) Dehydrogenase From Pseudomonas sp. No. 1143. Agric. Biol. Chem. 1989, 53, 1493–1501. 14. Tsuji, Y.; Koike, A.; Yamamoto, K.; Tochikura, T. Purification and Some Properties of L-Fucose Dehydrogenase From Agrobacterium radiobacter and Its Application to the Assay of Bound-Fucose in Glycoconjugate. Biochim. Biophys. Acta 1992, 1117, 167–173. 15. Itoh, H.; Miyoshi, T.; Yoshino, A.; Shiragami, K.; Izumori, K. Purification and Characterization of L-Fucose Dehydrogenase From Agrobacter tumefaciens K-28. J. Ferment. Bioeng. 1994, 77, 100–102. 16. Yamamoto-Ohtake, H.; Nakano, E.; Koyama, Y. Cloning and Sequencing of the L-Fucose Dehydrogenase Gene From Pseudomonas sp. No. 1143. Biosci. Biotech. Biochem. 1994, 58, 2281–2282. 17. Bradford, M.M. A Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein–Dye Binding. Anal. Biochem. 1976, 72, 248–254. 18. Laemmli, U.K. Cleavage of Structural Proteins During the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. 19. Muraoka, W.T.; Zhang, Q. Phenotype and Genotype Evidence for L-Fucose Utilization by Campylobacter jejuni. J. Bacteriol. 2011, 193, 1065–1075. 20. Stahl, M.; Friis, L.M.; Nothaft, H.; Liu, X.; Li, J.; Szymanski, C.M.; Stintzi, A. L-Fucose Utilization Provides Campylobacter jejuni With a Competitive Advantage. Proc. Natl. Acad. Sci. USA 2011, 108, 7194–7199. 21. Yew, W.S.; Fedorov, A.A.; Fedorov, E.V.; Rakus, J.F.; Pierce, R.W.; Almo, S.C.; Gerlt, J.A. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase From Xanthomonas campestris. Biochemistry 2006, 45, 14582–14597.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
PRODUCTION AND CHARACTERIZATION OF L-FUCOSE DEHYDROGENASE FROM NEWLY ISOLATED Acinetobacter sp. STRAIN SA-134 a
Takashi Ohshiro & Noriyuki Morita
a
a
Department of Biotechnology , Tottori University , Tottori , Japan Accepted author version posted online: 16 Aug 2013.Published online: 09 Dec 2013.
Click for updates To cite this article: Takashi Ohshiro & Noriyuki Morita (2014) PRODUCTION AND CHARACTERIZATION OF L-FUCOSE DEHYDROGENASE FROM NEWLY ISOLATED Acinetobacter sp. STRAIN SA-134, Preparative Biochemistry and Biotechnology, 44:4, 382-391, DOI: 10.1080/10826068.2013.833107 To link to this article: http://dx.doi.org/10.1080/10826068.2013.833107
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:382–391, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.833107
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
PRODUCTION AND CHARACTERIZATION OF L-FUCOSE DEHYDROGENASE FROM NEWLY ISOLATED Acinetobacter sp. STRAIN SA-134
Takashi Ohshiro and Noriyuki Morita Department of Biotechnology, Tottori University, Tottori, Japan
& Microorganisms producing L-fucose dehydrogenase were screened from soil samples, and one of the isolated bacterial strains SA-134 was identified as Acinetobacter sp. by 16S rDNA gene analysis. The strain grew well utilizing L-fucose as a sole source of carbon, but all other monosaccharides tested such as D-glucose and D-arabinose did not support the growth of the strain in the absence of L-fucose. D-Arabinose inhibited the growth even in the culture medium containing L-fucose. Although the strain grew on some organic acids and amino acids such as citric acid and L-alanine as sole sources of carbon, the enzyme was produced only in the presence of L-fucose. The fucose dehydrogenase was purified to apparently homogeneity from the strain, and the native enzyme was a monomer of 25 kD. L-Fucose and D-arabinose were good substrates for the enzyme, but L-galactose was a poor substrate. The enzyme acted on both NADþ and NADPþ in the similar manner. Keywords Acinetobacter, enzyme purification, enzyme properties, fucose dehydrogenase
INTRODUCTION L-Fucose is widely distributed in microorganisms, plants, and animals. Especially, it is found at the nonreducing termini of biological active carbohydrates in mammalian systems, such as immunoglobulins and mucin glycoproteins.[1] Since the fucosylated glycans are known to act as adherence recognition targets for intestinal bacteria, some pathogenic bacteria release fucose through the hydrolytic activity toward the mucin and facilitate the dispersion of the pathogens.[2] In addition, free fucose has been reported to play a role as a mediator in the modulation of immune response in human intestinal epithelium.[3] Therefore, free fucose has been recognized as a marker of several diseases, and the accurate measurement of L-fucose is significantly important in the field of clinical Address correspondence to Takashi Ohshiro, Department of Biotechnology, Tottori University, Koyamachominami 4-101, Tottori 680-8552, Japan. E-mail: [email protected]
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
383
inspection.[4] One of the promising methods to determine L-fucose is an enzymatic analysis using L-fucose dehydrogenase. L-Fucose is also known to be a main component of fucoidan, which is the sulfated macromolecule substance contained in brown algae. Fucoidan has attracted much attention in terms of healthy food because it exhibits a variety of biological activities, including antitumor,[5,6] antioxidant,[7] anticoagulant,[8] and anti-inflammatory activities.[9] In spite of the significance of sulfate content in fucoidan being proposed before now, the reason why fucoidan shows these physiological activities has not been fully elucidated. Moreover, it is unclear whether high-molecular-weight fucoidan is incorporated through the intestinal wall or digested before the incorporation. Recently, it has been demonstrated that the biological activities of fucoidan change due to the size of its molecular weight.[10] Thus, the molecular size appears to be a significant factor, as well as the sulfate content. Low-molecular-weight fucoidans were prepared from native fucoidan with high molecular weight, by partial acid hydrolysis, radical depolymerization, or enzymatic treatment. We found some microorganisms utilizing fucoidan from Cladosiphon okamuranus for the purpose of the enzymatic treatment,[11] and identified one bacterial strain as Luteolibacter algae, which had been classified as an unculturable bacterium.[12] Fucoidan from C. okamuranus consists of fucose moieties attached to sulfate and acetate as the main chain, and glucuronic acid as the branching part. Accordingly, it is assumed that L-fucose is released during lowering of the molecular weight of the native fucoidan. In this case, L-fucose dehydrogenase has been thought to be an important tool to measure the content of nonbound free fucose. Up to now, three microbial L-fucose dehydrogenases have been investigated from Pseudomonas sp. no. 1143,[13] Agrobacterium radiobacter,[14] and A. tumefaciens,[15] and the gene of no. 1143 strain was cloned.[16] Although L-fucose is converted to L-fucono-1,5-lactone by L-fucose dehydrogenase, more metabolites have not been detected in these microorganisms. Among these, the enzyme from A. tumefaciens was constitutively produced, but the detailed production conditions have not been investigated about these three strains. In this study, we found a new bacterial strain producing L-fucose dehydrogenase, examined the medium composition for the enzyme production, and characterized the purified enzyme.
EXPERIMENTAL Strain Isolation, Identification, and Culture Conditions Enrichment culture was performed at 30 C with reciprocal shaking (300 strokes=min) in test tubes containing 5 mL of the synthetic medium
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
384
T. Ohshiro and N. Morita
(pH 7.0) previously used[11] except using fucose as a sole source of carbon. The composition of the medium was 0.25% fucose, 0.4% K2HPO4, 0.05% KH2PO4, 0.2% (NH4)2SO4, 0.041% MgSO47H2O, metal solution, and vitamin mixture. To prepare the purified enzyme, the isolated strain SA-134 was cultivated at 30 C for 1 day with reciprocal shaking (120 strokes= min) in 2-L flasks containing 500 mL of LB medium (1% peptone, 0.5% yeast extract, and 0.5% NaCl) with 0.1% fucose. Whole DNA extraction was performed from cells of the strain SA134 as previously reported,[11] and the 16S rDNA gene was amplified using universal bacterial 16S rDNA primers 27f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1525r (50 -AAAGGAGGTGATCCAGCC-30 ). The amplified DNA fragment of approximately 1500 bp was purified, sequenced, and compared against samples deposited with the database. Enzyme Assay Fucose dehydrogenase activity was determined at 37 C using the increase in absorbance at 340 nm due to reduction of NADPþ. The reaction mixture contained 35 mM Tris-HCl buffer (pH 8.0), 50 mM L-fucose, 10 mM NADPþ, and the enzyme in a total volume of 1 mL. One unit of activity was defined as the amount of fucose dehydrogenase necessary to release 1 mmol of NADPH per min. Enzyme Purification All operations were performed at 4 C and 20 mM Tris-HCl buffer (pH 8.0) was used as the basal buffer. Approximately 170 g (wet weight) of frozen cells was suspended in 500 mL of the basal buffer and disrupted with an ultraoscillator (Sonifier 450; Branson Instruments, Danbury, CT) at 20 kHz. The cell debris was removed by centrifugation at 15,000 g for 60 min, and the resultant supernatant was used as the cell-free extract. After the cell-free extract was dialyzed against the basal buffer, the enzyme solution was applied to a DEAE-Sepharose column (5.6 36 cm) that had been equilibrated with the same buffer. The column was washed with the same buffer and bound proteins were eluted with the buffers containing 0.1, 0.25, or 0.5 M NaCl. The active fractions eluted with the buffer containing 0.25 M NaCl were collected and concentrated by ultrafiltration. After the enzyme solution was dialyzed against the basal buffer containing 1 M (NH4)2SO4, the enzyme solution was applied to a Phenyl-Toyopearl column (5.6 11 cm) that had been equilibrated with the same buffer. The column was washed with the same buffer and bound proteins were eluted with the buffer containing 0.8 M (NH4)2SO4. The active fractions were collected, concentrated by
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
385
ultrafiltration, and dialyzed against 10 mM Tris-HCl buffer (pH 8.0). The enzyme solution was applied to a Q-Sepharose column (2.8 38 cm) that had been equilibrated with the same buffer. The column was washed with the same buffer and bound proteins were eluted with 25, 100, 250, and 500 mM Tris-HCl buffers (pH 8.0). The active fractions eluted with 500 mM Tris-HCl buffer (pH 8.0) were collected and concentrated by ultrafiltration. After the buffer solution dissolving the enzyme was replaced by ultrafiltration with the basal buffer, the enzyme solution was applied to a Superdex 200HR 10=30 (1 30 cm) equilibrated with the same buffer. The chromatography was performed at a flow rate of 0.3 mL=min controlled by an AKTA system. Finally, the collected active fraction was applied to a Mono-Q HR 10=10 (1 10 cm) equilibrated with the same buffer. The chromatography was performed with a linear gradient of 0 to 1 M NaCl in the basal buffer at a flow rate of 0.3 mL=min controlled by an AKTA system.
Other Analytical Methods Protein concentration was determined by the method of Bradford[17] using a Bio-Rad protein assay reagent with bovine serum albumin as a standard. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done using 12.5% polyacrylamide gel by the method of Laemmli.[18] The gels were stained with Coomassie brilliant blue dissolved in 50% methanol–10% acetic acid and destained in 30% methanol–10% acetic acid. The N-terminal amino acid sequence was determined by a PPSQ-31A (Shimadzu, Kyoto, Japan).
RESULTS AND DISCUSSION Isolation and Identification of Fucose Dehydrogenase-Producing Microorganism Approximately 200 soil samples were employed for the screening, and we have found some bacterial strains utilizing fucose as a sole source of carbon. Cell-free extract of each strain was prepared, and the enzyme activities were estimated. As a result, one strain, SA-134, showing the maximum activity among the isolated strains was chosen for the following studies. The sequence of the 16S rDNA (AB720732) of strain SA-134 showed the highest similarity (99%) to the sequences of Acinetobacter sp. YNA109 (JQ039977) and Acinetobacter sp. AW 1-18(JQ316540). Hence, strain SA-134 was identified as Acinetobacter sp.
386
T. Ohshiro and N. Morita
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Enzyme Production by Acinetobacter sp. SA-134 Acinetobacter sp. SA-134 was isolated using a synthetic medium containing fucose as a sole source of carbon. However, the growth of this strain in the synthetic medium with 0.25% fucose was not good (OD660 ¼ 2), and fucose is an expensive compound as a carbon source for the large-scale cultivation to purify the enzyme. To enhance enzyme production by strain SA-134, the bacterium was cultured in a variety of media and the enzyme production was estimated. As shown in Table 1, the growth of Acinetobacter sp. SA-134 was enhanced when D-lactate, succinate, L-alanine, and L-glutamate were added to the media containing fucose, but D-glucose, D-fructose, and D-galactose among monosaccharides tested did not influence the growth of the strain. On the contrary, D-mannose and D-xylose had inhibitory effects, and D-arabinose completely inhibited the growth. However, in any case, the enzyme production was not enhanced compared to the medium containing fucose only. Acinetobacter sp. SA-134 could grow in a synthetic media with D-lactate, succinate, L-alanine, and L-glutamate as sole sources of carbon. However, no enzyme activities were detected in TABLE 1 Effects of Organic Compounds on Growth and Enzyme Production of Acinetobacter sp. SA-134 in Media With or Without L-Fucose Growth (OD660) Additive (0.5%) None D-Arabinose D-Glucose D-Fructose D-Galactose D-Mannose D-Xylose Maltose Sucrose Lactose Glycerol Sorbitol Mannitol D-Lactate Succinate L-Alanine L-Glutamate Fucoidan
Specific Activity (units=mg)
With Fucose
Without Fucose
With Fucose
Without Fucose
2.01 0.07 1.87 1.61 1.63 0.99 0.51 1.62 1.73 1.86 1.90 1.73 1.91 3.40 2.57 4.64 3.37 2.16
0 0.06 0.06 0.11 0.07 0.05 0.16 0.05 0.03 0.05 0.02 0.02 0.02 1.89 0.85 1.31 1.10 0.13
2.81 N.D. 2.31 2.46 2.95 1.99 1.92 2.37 2.28 2.56 2.22 1.96 1.72 1.46 2.52 1.15 1.61 2.36
N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0 0 0 0 N.D.
The strain SA-134 was cultivated in the synthetic medium with or without fucose adding indicated compounds at 0.5%. The medium with fucose in this Table corresponds to the synthetic medium containing 0.25% L-fucose described in the text. The cultivation was done at 30 C for 1 d. N.D. means not determined.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
387
these cases. Moreover, all sugars tested except for L-fucose did not support the growth of Acinetobacter sp. SA-134 (Table 1). Thus, only fucose appeared to induce enzyme synthesis. Although there are few previous reports describing enzyme production about fucose dehydrogenase, Itoh et al. demonstrated that Agrobacterium tumefaciens K-28 constitutively produced the enzyme.[15] In the case of Acinetobacter sp. SA-134, we demonstrated that fucose induces the production of fucose dehydrogenase and other sugars do not repress the enzyme production. Next, we tried to investigate enzyme production in nutrient broth with various concentrations of fucose. Both growth (OD660 ¼ 3.8) and enzyme production (2.3 units=mg) were the highest when Acinetobacter sp. SA-134 was cultured in LB medium with 0.1% fucose. Needless to say, the enzyme production was not observed in the cells grown in only LB medium. Hence, the strain was cultivated in LB medium containing 0.1% fucose to obtain the purified enzyme. Enzyme Purification and N-Terminal Amino Acid Sequence Fucose dehydrogenase was purified to apparent homogeneity from the cell-free extracts of Acinetobacter sp. A-134 and the molecular mass of the subunit was 25 kD (Figure 1). The purification steps are summarized in Table 2. However, the enzyme inactivation occurred significantly during the purification steps, so it is necessary to examine factors causing the inactivation in the future. The native molecular mass of the enzyme was estimated to be approximately 23 kD by the analysis of Superdex column chromatography (data not shown), indicating that the enzyme is a monomer. Although the molecular weight of the enzyme from Acinetobacter sp.
FIGURE 1 SDS-PAGE of fucose dehydrogenase from Acinetobacter sp. SA-134. Lane 1 and 8, marker proteins; lane 2, cell-free extract (28 mg); lane 3, pooled fraction after DEAE-Sepharose (28 mg); lane 4, pooled fraction after Phenyl-Toyopearl (18 mg); lane 5, pooled fraction after Q-Sepharose (3.5 mg); lane 6, pooled fraction after Superdex (2.5 mg); lane 7, purified enzyme after Mono Q (0.8 mg) (color figure available online).
388
T. Ohshiro and N. Morita
TABLE 2
Summary of Purification of Fucose Dehydrogenase From Acinetobacter sp. SA-134
Step
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Cell-free extracts DEAE-Sepharose Phenyl-Toyopearl Q-Sepharose Superdex MonoQ
Total Protein (mg)
Total Activity (units)
Specific Activity (units=mg)
Purification (fold)
Yield (%)
20100 5240 788 16.4 1.01 0.332
46200 18000 12900 1520 141 72.0
2.30 3.44 16.4 92.7 140 217
1 1.50 7.13 40.3 60.9 94.3
100 39.0 27.9 3.29 0.305 0.156
A-134 was similar to that from Pseudomonas sp. no. 1143, the substrate specificities of two enzymes differed from each other as described in the following. The N-terminal amino acid sequence of the enzyme was determined to be MDYHEKNKVFIVTGGGAGIGGAIS, but the BLAST analysis found no enzymes having a significant similarity to this sequence. Although the gene of L-fucose dehydrogenase was reported from only Pseudomonas sp. no. 1143,[16] any parts of amino acid sequences from the strain no. 1143 show no homology to the N-terminal sequence of the enzyme from Acinetobacter sp. A-134.
Substrate Specificity and Kinetic Parameters The reactivity of the enzyme from Acinetobacter sp. SA-134 was investigated for various sugars (Table 3). The activities for D-arabinose and TABLE 3 Substrate Specificity of Fucose Dehydrogenase From Acinetobacter sp. SA-134 Compound
Relative Activity (%)
L-Fucose D-Glucose D-Fructose D-Galactose L-Galactose L-Arabinose D-Arabinose D-Mannose L-Rhamnose D-Xylose Maltose Sucrose Lactose
100 1 0 0 11 0 65 0 0 0 0 0 0
Note. The enzyme reaction mixture contained 35 mM Tri-HCl buffer (pH 8.0), 10 mM NADPþ, and 50 mM indicated compounds.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
389
L-galactose were 65 and 11% of that for L-fucose, respectively, and the latter activity was different from those of other enzymes. In the cases of three microbial L-fucose dehydrogenases from Agrobacterium tumefaciens K-28, A. radiobacter, and Pseudomonas sp. no. 1143, the activities for L-galactose were 60% or more. On the contrary, the enzyme of strain SA-134 showed a little activity for L-galactose. The activity for D-arabinose of strain SA-134 was similar to those of two Agrobacterium strains (75 and 71%). All other sugars were inert. Although the enzyme of strain SA-134 acted on D-arabinose as a substrate, it is apparently curious that the growth of the strain was completely inhibited by D-arabinose. Any metabolites derived from D-arabinose might inhibit the growth of the strain. The Km values for L-fucose, NADþ, and NADPþ were 2.6, 2.7, and 1.7 mM, respectively. Since the affinity for L-fucose of the enzyme was similar to those of other enzymes, it is believed that the enzyme from Acinetobacter sp. SA-134 will be applicable to the determination of L-fucose. The enzyme of strain SA-134 acted on both NADþ and NADPþ; however, the activity for NADPþ was lower compared to previous reported enzymes.
Other Enzymatic Properties When the enzyme activity was measured at various pH values and temperatures, the optimum pH was found to be around 8 and the optimum temperature was about 50 C. Stability of the enzyme was examined at different pH at room temperature for 60 min, and at various temperatures for 30 min in 20 mM Tris-HCl buffer (pH 8.0). The enzyme was stable between pH 6 and 10, and below 35 C (data not shown). The effects of metal ions and inhibitors on the enzyme activity were investigated. It was found that heavy metals such as Hg2þ, Agþ, Cu2þ, and Co2þ completely inhibited the enzyme activity at 1 mM; in addition, ethylenediamine tetraacetic acid (EDTA) and N-ethylmaleimide also had significant inhibitory effects at 1 mM (data not shown). The enzymatic properties described in this paragraph were almost similar to previous reported enzymes. In this study, we isolated a new bacterial strain, Acinetobacter sp. SA-134, producing fucose dehydrogenase. We have demonstrated that the strain utilizes no monosaccharides other than L-fucose as a sole source of carbon, and the enzyme production is induced only in the presence of L-fucose. Since the enzyme showed some different properties compared to other previous reported enzymes, the gene cloning and expression of the enzyme from the strain SA-134 are now in progress in our laboratory. Recently, it has been reported that a gastrointestinal pathogenic bacterium, Campylobacter jejuni, utilizes fucose as a sole source of carbon but not common carbohydrates on the glycolytic pathway, the Entner–Doudoroff pathway, and the pentose
390
T. Ohshiro and N. Morita
phosphate pathway.[19,20] These two reports indicate that the pathogenic strain C. jejuni has a novel metabolic pathway for L-fucose. Furthermore, Yew et al. also proposed a novel matabolic pathway for the catabolism of L-fucose by Xanthomonas campestris on the basis of the expected functions of homologues of the proteins predicted from the genome sequence.[21] It will be interesting to examine whether Acinetobacter sp. SA-134 has a similar pathway.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
CONCLUSION In this study, we isolated one L-fucose dehydrogenase-producing microorganism from soil samples, and identified as Acinetobacter sp. by 16S rDNA gene analysis. The strain grew well utilizing L-fucose as a sole source of carbon, but not all other monosaccharides tested such as D-glucose and D-arabinose. Although the strain grew on some organic acids and amino acids such as citric acid and L-alanine as sole sources of carbon, the enzyme was produced only in the presence of L-fucose. The fucose dehydrogenase was purified to apparent homogeneity from the strain, and the native enzyme was a monomer of 25 kD. L-Fucose and D-arabinose were good substrates for the enzyme, but L-galactose was a poor substrate. The enzyme acted on both NADþ and NADPþ in a similar manner. REFERENCES 1. Robbe, C.; Capon, C.; Coddeville, B.; Michalski, J.C. Structural Diversity and Specific Distribution of O-Glycans in Normal Human Mucins Along the Intestinal Tract. Biochem. J. 2004, 384, 307–316. 2. Cone, R.A. Barrier Properties of Mucin. Adv. Drug Deliv. Rev. 2009, 61, 75–85. 3. Chow, W.L.; Lee, Y.K. Free Fucose Is a Danger Signal to Human Intestinal Epithelial Cells. Br. J. Nutr. 2008, 99, 449–454. 4. Mrochek, J.E.; Dinsmore, S.R.; Tormey, D.C.; Waalkes, T.P. Protein-Bound Carbohydrates in Breast Cancer. Liquid-Chromatographic Analysis for Mannose, Galactose, Fucose, and Sialic Acid in Serum. Clin. Chem. 1976, 22, 1516–1521. 5. Fukahori, S.; Yano, H.; Akiba, J.; Ogasawara, S.; Momosaki, S.; Sanada, S.; Kuratomi, K.; Ishizaki, Y.; Moriya, F.; Yagi, M.; Kojiro, M. Fucoidan, a Major Component of Brown Seaweed, Prohibits the Growth of Human Cancer Cell Lines In Vitro. Mol. Med. Rep. 2008, 1, 537–542. 6. Cho, M.L.; Lee, B.-Y.; You, S. Relationship Between Oversulfation and Conformation of Low and High Molecular Weight Fucoidans and Evaluation of Their In Vitro Anticancer Activity. Molecules 2011, 16, 291–297. 7. Wang, J.; Zhang, Q.; Zhang, Z.; Song, H.; Li, P. Potential Antioxidant and Anticoagulant Capacity of Low Molecular Weight Fucoidan Fractions Extracted From Laminaria japonica. Int. J. Biol. Macromol. 2010, 46, 6–12. 8. Zoysa, M.D.; Nikapitiya, C.; Jeon, Y.-J.; Jee, Y.; Lee, J. Anticoagulant Activity of Sulfated Polysaccharide Isolated From Fermented Brown Seaweed Sargassum fulvellum. J. Appl. Phycol. 2008, 20, 67–74. 9. Park, H.Y.; Han, M.H.; Park, C.; Jin, C.-Y.; Kim, G.-Y.; Choi, I.-W.; Kim, N.D.; Nam, T.-J.; Kwon, T.K.; Choi, Y.H. Anti-Inflammatory Effects of Fucoidan Through Inhibition of NF-jB, MAPK and Akt Activation in Lipopolysaccharide-Induced BV2 Microglia Cells. Food Chem. Toxicol. 2011, 49, 1745–1752.
Downloaded by [Eindhoven Technical University] at 07:32 13 February 2015
Fucose Dehydrogenase From Newly Isolated Bacterium
391
10. Morya, V.K.; Kim, J.; Kim, E.-K. Algal Fucoidan: Structural and Size-Dependent Bioactivities and Their Perspectives. Appl. Microbiol. Biotechnol. 2012, 93, 71–82. 11. Ohshiro, T.; Ohmoto, Y.; Ono, Y.; Ohkita, R.; Miki, Y.; Kawamoto, H.; Izumi, Y. Isolation and Characterization of a Novel Fucoidan-Degrading Microorganism. Biosci. Biotechnol. Biochem. 2010, 74, 1729–1732. 12. Ohshiro, T.; Harada, N.; Kobayashi, Y.; Miki, Y.; Kawamoto, H. Microbial Fucoidan Degradation by Luteolibacter algae H18 With Deacetylation. Biosci. Biotechnol. Biochem. 2012, 76, 620–623. 13. Horiuchi, T.; Suzuki, T.; Himura, M.; Saito, N. Purification and Characterization of L-Fucose (L-galactose) Dehydrogenase From Pseudomonas sp. No. 1143. Agric. Biol. Chem. 1989, 53, 1493–1501. 14. Tsuji, Y.; Koike, A.; Yamamoto, K.; Tochikura, T. Purification and Some Properties of L-Fucose Dehydrogenase From Agrobacterium radiobacter and Its Application to the Assay of Bound-Fucose in Glycoconjugate. Biochim. Biophys. Acta 1992, 1117, 167–173. 15. Itoh, H.; Miyoshi, T.; Yoshino, A.; Shiragami, K.; Izumori, K. Purification and Characterization of L-Fucose Dehydrogenase From Agrobacter tumefaciens K-28. J. Ferment. Bioeng. 1994, 77, 100–102. 16. Yamamoto-Ohtake, H.; Nakano, E.; Koyama, Y. Cloning and Sequencing of the L-Fucose Dehydrogenase Gene From Pseudomonas sp. No. 1143. Biosci. Biotech. Biochem. 1994, 58, 2281–2282. 17. Bradford, M.M. A Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein–Dye Binding. Anal. Biochem. 1976, 72, 248–254. 18. Laemmli, U.K. Cleavage of Structural Proteins During the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. 19. Muraoka, W.T.; Zhang, Q. Phenotype and Genotype Evidence for L-Fucose Utilization by Campylobacter jejuni. J. Bacteriol. 2011, 193, 1065–1075. 20. Stahl, M.; Friis, L.M.; Nothaft, H.; Liu, X.; Li, J.; Szymanski, C.M.; Stintzi, A. L-Fucose Utilization Provides Campylobacter jejuni With a Competitive Advantage. Proc. Natl. Acad. Sci. USA 2011, 108, 7194–7199. 21. Yew, W.S.; Fedorov, A.A.; Fedorov, E.V.; Rakus, J.F.; Pierce, R.W.; Almo, S.C.; Gerlt, J.A. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase From Xanthomonas campestris. Biochemistry 2006, 45, 14582–14597.
