A Polysaccharide-Degrading Marine Bacterium Flammeovirga Sp. MY04 and Its Extracellular Agarase System

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J. Ocean Univ. China (Oceanic and Coastal Sea Research) DOI 10.1007/s11802-012-1929-3 ISSN 1672-5182, 2012 11 (3): 375-382 http://www.ouc.edu.cn/xbywb/ E-mail:[email protected]

A Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. MY04 and Its Extracellular Agarase System

HAN Wenjun1), 2), 3), GU Jingyan1), 3), YAN Qiujie2), LI Jungang2), WU Zhihong3), *, GU Qianqun1), *, and LI Yuezhong3)

1) Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, P. R . C hi n a 2) School of Life Science and Biotechnology, Mianyang Normal University, Mianyang 621000, P. R. China 3) State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, P. R. China

(Received February 2, 2012; revised April 8, 2012; accepted May 29, 2012) © Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2012

Abstract Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported re- garding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and -1 -1 grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L ± 18.2 mg L liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These pro- -1 -1 teins have an activity of 310 U mg ± 9.6 U mg proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30–70 kDa. The EAS is stable at a -1 wide range of pH values (6.0–11.0), temperatures (0–50℃), and sodium chloride (NaCl) concentrations (0– 0.9 mol L ). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysaccharide-degradation system hold great promise in industrial applications.

Key words Flammeovirga; polysaccharide degradation; extracellular agarase system; neoagaro-oligosaccharide

while only two α-agarases have been reported, one from 1 Introduction Thalassomonas sp. JAMB-A33 and one from Alteromo- nas agarlyticus GJ1B (Ohta et al., 2005b; Potin et al., Complex polysaccharides (CPs), which are composed 1993). of repeated units of complex sugars, are widely present in Agarases are useful in the preparation of algal proto- plants, animals, and microorganisms, in which they serve plasts (Gupta et al., 2010; Yeong et al., 2008) and the as essential structural and functional components. Agar, recovery of DNA from agarose gels (Cole and Åkerman, composed of agarose and agaropectin, is the main com- 2000) , and have potential in the production of functional ponent of the cell wall of red algae (Rhodophyta) (Rochas oligosaccharides (Hatada et al., 2006; Hu et al., 2006; et al., 1986). Agarose is a polymer composed of 3,6-an- Wang et al., 2004; Wu et al., 2005). Although the agarose hydro-L-galactopyranose-α-1,3-D-galactopyranose units degradation abilities of many microorganisms isolated that are joined by β1-4 bonds (Rees, 1969). Agarose can from marine environment, fresh water, or soil have been be cleaved by either α-agarase (E.C. 3.2.1.158) at the investigated and the corresponding enzymes have been α1-3 linkage (Rochas et al., 1994) or β-agarase (E.C. characterised, only β-agarase I from Pseudoalteromonas 3.2.1.81) at the β1-4 linkage (Morrice et al., 1983a and atlantica has been industrially applied for the gel recov- 1983b), producing a series of oligosaccharides that have ery of DNA (Fu and Kim, 2010). There are many limita- reducing ends. The oligomers yielded from the degrada- tions in the applications of agarases. For example, the tion of agarose by α-agarase and β-agarase are termed heterologous expression levels of agarases are usually agaro- and neoagaro-oligosaccharides, respectively. There low and the use of these enzymes for some purposes, such have been many studies of agarases and their degradation as the enzymatic preparation of functional oligosaccha- of agarose. Most of the reported agarases are β-agarases, rides, is expensive. The use of native agarase-producers or their extracellular agarase systems (EASs) may provide * Corresponding authors. Tel: 0086-532-82032065 an alternative. E-mail: [email protected]; [email protected] Flammeovirga is a newly defined bacterial genus be-

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376 HAN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 375-382

longing to the family Flammeovirgaceae of the class glucose. After incubation at 30℃ for 16 h with agitation, α-Proteobacteria. Five species have been reported in this the turbidity of the broth was measured at 600 nm to genus: F. aprica (Nakagawa et al., 1997), F. arenaria, F. evaluate the cellular growth. The initial pH of the medium yaeyamensis (Takahashi et al., 2006), F. kamogawensis was adjusted to 5.0–11.0 before autoclaving. Culture tem- (Hosoya and Yokota, 2007), and F. pacifica (Xu et al., peratures were between 4℃ and 45℃. The cellular toler- 2011). All of the type strains have a potent ability to de- ance to sodium chloride was assayed over a concentration grade marine CPs, including agarose. Yang et al. have range of 0–5.0%. The requirement of metal ions for growth described the gene cloning and biochemical characteristic was analysed by growing a series of cultures in which a of an agarase, AgaYT, from the F. yaeyamensis strain YT single metal salt had been removed from the medium. (Yang et al., 2011). Although the heterologously expressed enzyme exhibited a high degradation activity on 2.3 Polysaccharide-Utilizing Abilities of the MY04 agarose, when the protein was expressed in E. coli, it was Strain mostly present in the form of inclusion bodies. To date, To evaluate the ability of the bacterial isolate to utilize we know few of the CP-degradation characteristics of different polysaccharides, the sole carbon source in the Flammeovirga strains or their extracellular agarase sys- BM broth was supplemented with various CPs, including tems, although these strains/systems have been suggested algae-derived polysaccharides (alginate, agar, agarose, to be relevant for biotechnological applications. carrageenan, and ι-carrageenan), plant-derived polysac- Herein, we report the polysaccharide-degradation char- charides (cellulose, carboxymethylcellulose (CMC), crude acteristics of a marine isolate, Flammeovirga sp. MY04, mannan, superfine mannan, microcrystalline cellulose and its extracellular agarase system. (MC), pectate, starch, and xylan), and crustacean polysaccharides (chitin and chitosan). Polysaccharides were 2 Materials and Methods added to the media at a final concentration of 0.10%. Af- ter the initial culture in the BM broth supplemented with 2.1 Isolation of Agarose-Degrading Bacteria 0.2% glucose, the MY04 bacteria were collected by cen- Coastal sediments were collected from a laver farm trifugation when the culture reached an OD600 of 0.8 and near Ganyu City in Jiangsu Province, China. A basal me- were washed twice using sterilised seawater. The washed dium (BM) composed of 3.0% NaCl, 0.75% KCl, 0.11% bacterial sludge was resuspended in BM broth, and an

CaCl2, 0.72% Mg2SO4, 0.15% NH4Cl, and 1.5% agar (pH aliquot was transferred into each restricted broth at a 7.0) was used to isolate agarolytic bacteria. After incuba- starting OD600 of 0.08. The cellular growth of MY04 was

tion at 30℃ for 72 h, the colonies that formed deep cra- evaluated as described above. ters in the agar were transferred to a fresh BM plate for further purification. To evaluate the agar-digestion ability 2.4 Preparation of the Crude Extracellular of the isolates, the BM plates were stained with an iodine Enzymes solution (Hodgson and Chater, 1981) following a 16-h To prepare the crude extracellular enzymes, the MY04 ℃ incubation at 30 . strain was cultured at 30℃ for 72 h in 1 L of BM broth supplemented with 0.4% tryptone and 0.25% yeast extract. 2.2 Identification of the MY04 Isolate The following preparation was performed at 4℃. The

Genomic DNA was prepared using a genomic DNA cultures were centrifuged at 12 000\× g for 15 min, and extraction kit (TianGen Inc., Beijing, China). For PCR ammonium sulphate was added into the supernatant to amplification of the 16S ribosomal RNA (rRNA) gene reach 80% saturation. After a 4-h incubation to precipitate sequence, the bacterial universal primer pair 27f the proteins, the mixture was centrifuged at 15 000 × g for -1 (5’-GAGTTTGATCCTGGCTCAG-3’) and 1492r (5’-AA- 30 min. The pellet was dissolved in 50 mL of 50 mmol L -1 GGAGGTGATCCAGC C-3’) (Weisburg et al., 1991) were HEPES buffer (pH 7.5) containing 0.5 mmol L EDTA used. The PCR products were gel-purified with a DNA and 5% glycerol, which was dialysed three times against extraction kit (TAKARA Inc., Dalian, China) according to the same buffer for 6 h. The dialysed enzyme solution was the manufacturer’s instructions and cloned into the stored in aliquots at −20℃ before use. pMD18-T vector (TAKARA Inc., Dalian, China) for se- quencing. The sequence was analysed against the GenBank 2.5 Polysaccharide-Degradation Activities of the database using the on-line BLAST program (Altschul Crude Extracellular Enzymes et al., 1990) to search for the most similar sequences. The polysaccharides described above for cellular utili- For transmission electron microscopy (TEM), MY04 zation were further tested as substrates for the degrada- cells that were cultured for 16 h at 30℃ were mounted on tion-activity assay of the crude enzyme extract. The 3, a Formvar-coated copper grid and negatively stained with 5-dinitrosalicylic acid method (Miller, 1959), with minor 1.0% aqueous uranyl acetate. The cell-mounted grid was modifications, was employed to assay the yielded reduc- observed using a JEOL1010 transmission electron micro- ing sugar. A 75-µL aliquot of the crude enzyme extract -1 scope (TEM, JEOL) operated at 100 kV. was mixed with 425 μL of 50 mmol L HEPES buffer (pH To determine the growth characteristics, strain MY04 7.5) that contained an individual polysaccharide at a final was cultivated in BM broth supplemented with 0.20% concentration of 0.20%. Following a 30-min incubation at

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40℃, 500 μL of 3, 5-dinitrosalicylic acid was added to the were optimised if necessary. The relative activity was

reaction mixture and boiled for 10 min until red colour defined as the percentage of the residual activity relative

developed, which was measured at 540 nm. The red color to that under the standard conditions described previ- was derived from the reaction of reducing sugars and 3, ously. 5-dinitrosalicylic acids. A control was performed by boiling the enzyme solution for 5 min before adding the solu- 2.9 Identification of the Agarose-Derived tion to the reaction mixture. All of the assays were per- Oligosaccharides formed in triplicate. D-Galactose was used as the standard Oligosaccharides derived from agarose were prepared reducing sugar. The enzyme activity (U) was defined as using the method described previously by Li et al. (Li et al., the amount of enzyme that produced 1 μmol of D-galac- 2007) with some modifications. Briefly, 100 mg of aga- tose per minute. The concentration of protein was deter- -1 rose was dissolved in 100 mL of buffer (50 mmol L mined using the Bradford assay (Bradford, 1976) with -1 -1 Tris-Cl, 150 mmol L NaCl, 10 mmol L CaCl , pH 7.5). bovine serum albumin as a standard. 2 After sterilisation, the substrate was placed in a 45℃ water bath to prevent gelling. The solution was mixed with 2.6 Zymogrammatic Analysis of the EAS 10 mL of the crude extracellular enzyme extracts. After a The extracellular agarase system (EAS) in the crude 48-h incubation at 45℃, the reaction was stopped by

enzyme extracts was analysed to identify the agarase heating the mixture in a boiling water bath for 10 min. components. An aliquot of 10 µL of the crude extracellu- The mixture was subsequently cooled to 4℃ and centri-

lar enzyme extract was mixed with an equal volume of fuged at 12 000 × g for 30 min. The supernatant was con- loading buffer and separated using 13.2% non-denaturing centrated by rotary evaporation at 45℃. A 2-mL aliquot polyacrylamide gel electrophoresis (PAGE). After elec- of the solution of the enzymatic products was loaded onto trophoresis, the gel was washed three times using 50 a Sephadex G25 column (superfine, GE-Healthcare, USA) -1 mmolL HEPES buffer (pH 7.5) for a total of 30 min. The or a Bio-gel P6 column (ﬁne, Bio-Rad Laboratories, USA) gel was then overlaid onto a 1.5% agarose sheet prepared -1 for separation using 0.1 mol L NH4HCO3 as the elution -1 with 50 mmol L HEPES (pH 7.5) and incubated over- solution. The classic phenol-H2SO4 method was performed night at 37℃. The zymogram was developed using iodine to analyse the oligosaccharide fractions. The NH4HCO3 staining. was removed by repeated evaporation under reduced pres- sure at 45℃ for further component identification. 2.7 Effects of pH and Temperature on the EAS The crude enzymatic products of agarose and the main Activity and Stability products from the above preparation, two oligosaccharide The optimal temperature of the EAS was determined fractions (F2 and F3), were applied to silica gel 60 (F254) by carrying out the enzyme activity assay at different plates (Merck KGaA Darmstadt, Germany) for TLC analy- -1 temperatures (15–70℃) in 50 mmol L HEPES buffer sis. The plates were developed with the solvent n-butanol/

(pH 7.0) for 30 min. The optimal pH of the EAS was as- ethanol/water (3:1:1), sprayed with a staining solution sayed at 45℃ in preheated buffers with different pH val- (1% diphenylamine and aniline in acetone) (Zhang et al., -1 ues, including 50 mmol L acetate buffer (pH 4.0–6.0), 50 2010), and heated at 110℃ for 10 min. For MALDI-TOF -1 -1 mmol L HEPES buffer (pH 7.0–8.0), and 50 mmolL mass spectrometry, the oligosaccharide fractions F2 and F3 glycine-NaOH buffer (pH 8.0–12.0). In addition, the ther- were individually observed as pseudo-molecular ions mostability of the EAS was evaluated by measuring the [M+Na]+ and [M+H]+. The mass spectrometry was per- residual activity of the crude enzyme extract after a pre- formed on a Biﬂex III instrument (Bruker Daltonics, Bil- incubation at different temperatures for 1 h. The pH sta- lerica, USA) that was set to the positive-ion mode, with 13 bility of the EAS was determined by pre-incubating the formic acid as the matrix. For C spectroscopy, 10 mg of

crude enzyme extract in each solution (1:20 dilution) of each oligosaccharide fraction was dissolved in 0.5 mL of

varying pH (4.0–12.0) at 45℃ for 1 h and then assaying D2O in 5-mm NMR tubes. The spectra were recorded on a the residual activity. All of the assays were performed in JNM-ECP600 (JEOL, Japan) apparatus set at 150 MHz, triplicate. The relative activity was defined as the per- with acetone-d6 as the internal standard. centage of activity with respect to the maximum agarase activity. 3 Results 2.8 Effects of Different Compounds on the 3.1 Isolation and Identification of the Agarolytic EAS Activity MY04 Strain Various metal ions, chelators, denaturants, reducing A basal medium (BM), described in the Materials and reagents, and a thiol modifier were added to the reaction Methods, was used for the screening and isolation of bac- mixtures to separately assay the effects of these sub- terial strains that are able to utilize agar as their sole car- stances on the EAS activity. The reaction was performed bon source. The agarolytic activity of the colonies formed -1 in 50 mmolL HEPES buffer (pH 7.5) at 45℃ for 30 min. on BM plates was detected based on etched agar and io- The final concentration employed for each reagent was 1, dine staining. The agarolytic strain MY04 was isolated -1 10 or 100 mmol L . The concentrations of the metal ions from coastal sediments collected from a laver farm near

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378 HAN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 375-382

Ganyu City in Jiangsu Province, China, in 2004. On a BM plate, strain MY04 spread and formed large and 3.2 Growth Characterisation of MY04 deeply etched colonies. After the plate was stained with MY04 grew well in the presence of 1.0%−5.0% NaCl, the iodine solution, a clear agar-degradation zone devel- with an optimal concentration of 3.0%, suggesting that oped around individual colonies (Fig.1a). The solid me- this strain is a halophilic bacterium. Among the metal ion dium on which MY04 was cultured turned to liquid after + 2+ 2+ components in the BM, NH4 , Mg , and Ca were es- 5 d of incubation at 30℃. These culture characteristics sential for the growth of MY04, whereas K+ was not es- suggested that MY04 has potent agarolytic activities. sential. MY04 grew optimally at 30℃. The tolerable pH range for the growth of MY04 was between 6.0 and 10.0, with optimal growth at pH 7.0. MY04 was able to grow with most of the tested CPs as the sole carbon source (Fig.2a). Agarose was the best carbon source for the growth of this strain. Some of the other CPs, such as mannan and xylan, were also good carbon sources for MY04 growth. It is interesting to note that, in contrast to the type strains of Flammeovirga, the MY04 strain grew weakly on carboxylmethyl cellulose (CMC). Moreover, the MY04 strain was unable to utilize chitosan.

Fig.1 Phenotypic morphologies of the marine isolate MY04. (A) Colonies of MY04 on a BM plate stained using the iodine method. The regions with yellow or red color denote the presence of reducing sugars produced from the degradation of agar by the extracellular agarase secreted from MY04 colonies, which are in the middle of the yellow regions (the dark dots or lines). (B) Transmission electron micrograph of negatively stained MY04 cells. Scale bar = 1 μm.

The 16S rRNA gene of MY04 was sequenced, and the sequence was deposited in the GenBank database under accession number AY849869. According to the BLASTn results using the GenBank databases, MY04 is highly Fig.2 Utilization and degradation abilities of Flammeo- similar (99.6% similarity with respect to the 16S rRNA T virga sp. MY04 and its extracellular enzyme extracts. (A) gene sequence) to F. yaeyamensis NBRC 100898 (Taka- Growth ability of MY04 cells in BM containing various hashi et al., 2006). However, the MY04 strain had two CPs as the sole carbon source. (B) Degradation activities phenotypic characteristics that were different from the of the crude extracellular enzyme extracts from strain type strain of F. yaeyamensis. One characteristic is that MY04 on CPs. * OD540 = 18.4. The results are means ± the MY04 culture is reddish-orange during exponential S.D. of three different experiments. growth but changes to a white color during the late sta- tionary phase, which is similar to the colors reported for F. 3.3 Polysaccharide-Degradation Abilities of the kamogawensis (Hosoya and Yokota, 2007). Additionally, Crude Extracellular Enzymes × MY04 cells are curved with a size of (0.4– 0.7) μm (4 – 6) To investigate the CP-degradation abilities of the ex- μm (Fig.1b), whereas the reported Flammeovirga mem- tracellular enzymes, enzyme extract was prepared from a bers are longer rods. 72-h culture of MY04. The crude enzyme extracts exhib-

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ited varying levels of activity on different polysaccharides We were interested in whether the agarose degradation (Fig.2b). However, the degradation ability of the ex- products generated by the MY04 EAS are similar. The tracellular enzymes was different from the growth utiliza- TLC detection of the EAS degradation products of aga- tion ability of the cells. For example, whereas agar and rose demonstrated two main products, F2 and F3 (Fig.4a). agarose were still among the best substrates, the single F2 and F3 were individually purified from the crude en- best substrate for the prepared extracellular enzymes was zymatic products through gel chromatography and char- starch. Furthermore, the crude enzymes could not degrade acterized using MALDI-TOF MS. The molecular weights many of the tested polysaccharides, including mannan, of F2 and F3 were 630 and 936, respectively. The results chitin, chitosan, ι-carrageenan, CMC, and cellulose. Some indicated that the main digestion products of agarose of these non-degradable polysaccharides supported the generated by the MY04 EAS are tetraose and hexaose, growth of MY04, such as cellulose and mannan. The re- which are different from the products produced by sults suggest that the prepared extracellular enzymes did r-AgaYT. not include the whole degradation enzyme suite of MY04 for CPs, or the mechanisms by which MY04 cells degrade CPs are different.

3.4 Zymogram of the EAS After a 72-h culture in media broth at 30℃, the MY04 strain produced high concentrations of extracellular pro- -1 -1 teins, reaching approximately 490 mg L ± 18.2 mg L -1 liquid culture, with an agarase activity of 310 U mg ± -1 9.6 U mg proteins. Accordingly, the calculated production of agarases in the supernatant of the MY04 culture -1 was approximately 150 000 U L . The results show that the MY04 strain secretes a powerful extracellular agarase system. The extracellular enzyme extracts were run using native-PAGE to estimate the agarase components in the EAS. The iodine staining of the agarose gel showed that there were four transparent bands on the developed sheet (Fig.3). The molecular masses of these four agarolytic

enzyme bands were between 30 kDa and 70 kDa. The results suggested that there were at least four agarose depolymerases in the MY04 EAS.

Fig.4 Characterization of the neoagaro-oligosaccharides prepared using the MY04 EAS. (A) TLC detection of the crude enzymatic products of agarose. (–), the control of agarose; (+) the crude enzymatic products of agarose by the MY04 EAS; F2 and F3 are the major oligosaccharide products purified from the crude enzymatic products by gel chromatography. (B) 13C-NMR spectrum of the major oligosaccharide product F2 (neoagarotetraose). G represents the 3-O-linked β-D-galactopyranose; A represents the 4-O-linked 3, 6-anhydro- α-L-galactopyranose; r and nr denote the residues at the reducing and non-reducing ends, respectively; and α/β indicates the respective anomer. (C) Structure of neoa- Fig.3 Zymogram analysis of the crude extracellular en- garotetraose (F2). zyme extract from Flammeovirga sp. MY04. (A) Native PAGE. (B) Agarase activity staining. Moreover, in the 13C-NMR spectrum of the oligosac-

charide F2 (Fig.4b), the resonances at 93 ppm and 97 ppm 3.5 Oligomer Products from the Agarose Digestion showed patterns typical of neoagaro-oligosaccharides by the EAS produced by β-agarase (Rochas et al., 1986; Lahaye et al.,

Yang et al. reported that the main degradation products 1989), whereas the signal at 90.72 ppm, a specific of of agarose by a recombinant agarase (r-AgaYT) were agaro-oligosaccharides produced by α-agarase (Rochas neoagarobiose and neoagarotetraose (Yang et al., 2011). et al., 1994), was not present. Therefore, the tetraose was

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380 HAN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 375-382

a neoagarotetraose (Fig.4c). Similarly, the spectra for was decreased by: the ions K+, Mg2+, Cu2+, and Fe3+; the hexaose indicated that the compound was a neoagaro- chelator ethylenediaminetetraacetic acid (EDTA); the hexaose (data not shown). The main components of the detergent sodium dodecyl sulphate (SDS); and the thiol MY04 EAS are thus β-agarases, which degrade agarose modifier 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). into neoagaro-oligosaccharides. Interestingly, the MY04 EAS was stable over a wide range of NaCl concentrations. The presence of NaCl at -1 3.6 Enzymological Characteristics of the EAS different concentrations up to 0.9 mol L increased its The EAS exhibited maximal agarase activity at 45℃ agarolytic activity, and two peaks (148% and 157%) were and retained more than 80% activity when pre-incubated obtained in the presence of 1.0% and 3.0% NaCl, respectively (Fig.6). at a temperature between 0 and 50℃ for 1 h (Fig.5a). The EAS exhibited a maximum agarase activity at pH 7.5 and Table 1 Effects of various compounds on the agarase retained more than 90% activity after pre-treatment at activity of the EAS

45℃ for 1 h over a pH range of 6.0–11.0 (Fig.5b). The -1 Compound Concentration (mmol L ) Relative activity (%) results suggested that the EAS was relatively stable in None 100 environments with variable temperatures and pH values. K+ 100 95 Na+ 100 123 Ca2+ 100 106 Mg2+ 100 72 Cu2+ 1 72 Mn2+ 1 102 Fe3+ 1 99 DTNB 10 55 DTT 10 128 EDTA 10 65 β-Me 10 117 SDS 10 76 Urea 10 91

Fig.6 Effects of the NaCl concentration on the agarase Fig.5 Effects of temperature (A) and pH (B) on the EAS activity of the MY04 EAS. activity and stability. A: The EAS enzymatic activity was measured at various temperatures (15–70℃) in 50 mmolL-1 HEPES buffer (pH 7.5). B: The enzymatic ac- 4 Discussion tivity at various pH values was measured at 45℃ in 50 -1 -1 Five species have been identified within the Flammeo- mmol L acetate buffer (pH 4.0–6.0), 50 mmol L -1 HEPES buffer (pH 6.0–8.0), and 50 mmol L glycine- virga genus, and all the type strains of these species have NaOH buffer (pH 8.0–12.0). been reported to be agarolytic (Nakagawa et al., 1997; Takahashi et al., 2006; Hosoya and Yokota, 2007; Xu et al., The agarolytic activities of the EAS in the presence of 2011). However, except for the characterisation of the various metal ions, chelators, denaturants, reducing re- agarase AgaYT from the F. yaeyamensis strain YT (Yang agents, and a thiol modifier are shown in Table 1. The et al., 2011), less is known of the polysaccharide depoly- enzymatic activity was increased by the presence of the merases in these microorganisms. In the present study, the metal ions Na+ and Ca2+ and the reducing reagents 16S rRNA gene sequence revealed that the MY04 strain β-mercaptoethanol (β-Me) and dithiothreitol (DTT), but belongs to the Flammeovirga genus. Flammeovirga sp.

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MY04 is able to efficiently utilize multiple polysaccha- Fund of the Sichuan Provincial Education Department rides as a sole carbon source and grows best on agarose, (09ZA181) and by grants from the State Key Laboratory mannan, or xylan. The crude extracellular enzymes ex- of Microbial Technology (M2010-12) and the National hibit extensive degradation activities on diverse polysac- Science Foundation of China (30870001). charides, especially agarose. The extracellular proteins secreted by MY04 can reach a concentration of approxi- -1 References mately 500 mg L liquid culture, with an agarase activity -1 of more than 300 U mg proteins. The degradation of Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, agarose by crude extracellular enzymes predominantly D. J., 1990. Basic local alignment search tool. Journal of produces tetraose and hexaose, which were determined to Molecular Biology, 215: 403-410. Bannikova, G. E., Lopatin, S. A., Varlamov, V. P., Kuznetsov, B. be neoagaro-oligosaccharides instead of agaro-oligosac- B., Kozina, I. V., Miroshnichenko, M. L., Chernykh, N. A., charides. Thus, we suggest that Flammeovirga sp. MY04 Turova, T. P., and Bonch-Osmolovskaya, E. A., 2008. The uses the β-agarase-based mechanism for agarose degrada- thermophilic bacteria hydrolyzing agar: characterization of tion, as reported for P. atlantica (Morrice et al., 1983a) thermostable agarase. Applied Bichemistry and Microbiology, and Vibrio sp. strain JT0107 (Sugano et al., 1993). Fur- 44: 366-371. ther zymogram analysis shows that at least four different Bradford, M. M., 1976. A rapid and sensitive method for the agarose-degrading proteins are present in the EAS, with quantitation of microgram quantities of protein utilizing the

molecular weights ranging from 30 to 70 kD, which are principle of protein-dye binding. Analytical Biochemistry, 72: different from those reported for F. yaeyamensis YT 248-254. (Yang et al., 2011). Cole, K. D., and Åkerman, B., 2000. Enhanced capacity for Recent studies have identified a number of agarases electrophoretic capture of plasmid DNA by agarase treatment of agarose gels. Biomacromolecules, 1: 771-781. from various microorganisms. For example, the Dong, J. H., Tamaru, Y., and Araki, T., 2007. Molecular cloning, ℃ β-agarases from Agarivorans sp. LQ48 (AgaA, 0–50 ) expression, and characterization of a β-agarase gene, agaD, (Long et al., 2010), Agarivorans albus YKW-34 (AgaB34, from a marine bacterium, Vibrio sp. strain PO-303. Biosci- 0–50℃) (Fu et al., 2009), Microbulbifer sp. JAMB-A7 ence, Biotechnology, and Biochemistry, 71 (1): 38-46. (AgaA7, 0–50℃) (Ohta et al., 2004a), Microbulbifer-like Dong. J. H., Hashikawa, S., Konishi, T., Tamaru, Y., and Araki, JAMB-A94 (AgaA, 0–60℃) (Ohta et al., 2004b), and a T., 2006. Cloning of the novel gene encoding β-agarase C hot spring bacterium Thermoanaerobacter wiegelii B5 from a marine bacterium, Vibrio sp. strain PO-303, and char- (0–70℃) (Bannikova et al., 2008) are suggested to be acterization of the gene product. Applied and Environmental thermostable enzymes. The β-agarases in Agarivorans sp. Microbiology, 72: 6399-6401. JAMB-A11 (AgaA11, stable in pH 6.0–11.0) (Ohta et al., Fu, X. T., Pan, C. H., Lin, H., and Kim, S. M., 2009. Gene cloning, expression, and characterization of a β-Agarase, 2005a), Agarivorans sp. LQ48 (AgaA, pH 3.0–11.0) AgaB34, from Agarivorans albus YKW-34. Journal of Mi- (Long et al., 2010), Pseudoalteromonas sp. CY24 (AgaB, crobiology and Biotechnology, 19: 257-264. pH 5.7–10.6) (Ma et al., 2007) and Vibrio sp. PO-303 Fu, X. T., and Kim, S. M., 2010. Agarase: review of major (AgaC, pH 4.0–8.0, AgaD, pH 4.0–9.0) (Dong et al., sources, categories, purification method, enzyme characteris- 2006; Dong et al., 2007) are stable over a wide pH range. tics and applications. Marine Drugs, 8: 200-218. The majority of the above microorganisms belong to the Gupta, V., Kumar, M., Kumari, P., Reddy, C. R. K., and Jha, B., γ-Proteobacteria class, except the Firmicutes bacterium T. 2010. Optimization of protoplast yields from the red algae wiegelii B5. In this study, the EAS from the α-Proteo- Gracilaria dura (C. Agardh), J. Agardh and G. verrucosa bacteria Flammeovirga sp. MY04 was found to be stable (Huds.) Papenfuss. Journal of Applied Phycology, DOI: over a wide range of pH (6.0–11.0) and temperature 10.1007/s10811-010-9579-6. Hatada, Y., Ohta, Y., and Horikoshi, K., 2006. Hyperproduction (0–50℃) and have a higher agarase activity at high NaCl -1 and application of α-agarase to enzymatic enhancement of concentrations (up to 0.9 mol L ). The EAS in the super- antioxidant activity of porphyran. Journal of Agricultural and natant of the MY04 culture has a calculated production Food Chemistry, 54: 9895-9900. -1 rate of approximately 150 000 U L . More interestingly, Hodgson, D. A., and Chater, K. F., 1981. A chromosomal locus the major products of the degradation by MY04 EAS controlling extracellular agarase production by Streptomyces were neoagarotetraose and neoagarohexaose, even after a coelicolor A3 (2), and its inactivation by chromosomal inte- long period. These results indicate that the MY04 strain gration of plasmid SCP 1. Journal of General Microbiology, and its extracellular agarases have great potential for a 124: 339-348. range of applications, such as agar fermentation, func- Hosoya, S., and Yokota, A., 2007. Flammeovirga kamogawensis tional oligosaccharide production, medical research, and sp. nov., isolated from coastal seawater in Japan. Interna- tional Journal of Systematic and Evolutionary Microbiology, general industrial processes. Further studies focusing on 57: 1327-1330. agarase characterisation and gene cloning are required Hu, B., Gong, Q. H., Wang, Y., Ma, Y. M., Li, J. B., and Yu, W. and are in progress in our laboratory. G., 2006. Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe, 12: 260- 266. Acknowledgements Lahaye, M., Yaphe, W., Phan, Viet, M. T., and Rochas, C., 1989. This work was supported by the Scientific Research 13C-NMR spectroscopic investigation of metylated and

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