Aquaculture 372–375 (2013) 74–79

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Purification and characterization of agarase from Rhodococcus sp. Q5, a novelagarolytic bacterium isolated from printing and dyeing wastewater

Zehua Feng, Mengying Li ⁎College of Basic Medical and Biological Science, Soochow University, Suzhou 215123, PR China

⁎ Corresponding author. Tel.: +86 13861313106.E-mail address: lmysz@126.com (M. Li).

0044-8486/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.aquaculture.2012.10.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 May 2012Received in revised form 23 October 2012Accepted 25 October 2012Available online 2 November 2012

Keywords:AgaraseRhodococcusPurificationLinear ion trap mass spectrometry

An extracellular agarase was purified from Rhodococcus sp. Q5, a novel agar-degrading bacterium isolatedfrom printing and dyeing wastewater. Agarase staining of the purified agarase on an SDS-polyacrylamidegel revealed a single band with an apparent molecular weight of 54 kDa. The optimum pH and temperatureof agarase were 6.0 and 40 °C, respectively. The activity of the agarase was stable at low temperatures, andmore than 90% activity was retained until 40 °C. It was stable in the pH range from pH 5.0 to 8.0, andmore than 87% of the residual activity was retained. No significant activation or inhibition of the agarasewas observed in the presence of Na+, K+ or Ca2+; whereas, Ag+, Ba2+, Pb2+, Sn2+, Zn2+, Fe3+, Mg2+, Fe2+,SDS, and EDTA inhibited the enzyme activity. This agarase gave Km and Vmax values of 1.47 mg ml−1 and0.98 μM min−1 ml−1. The components of the hydrolytic product analyzed by the linear ion trapmass spectrom-etry showed that agarase mainly produced trisaccharide, as well as a small amount of disaccharides, tetrose,pentasaccharide, and hexose.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Agar, which is a common gelling substance used in scientificresearch and an ingredient in the food industry, is found in the cellwall of marine red algae. It is a complex molecule consisting of aga-rose and agaropectin, and it contains sulfate, pyruvate, and methoxylin both residues. Agarose is the gelling component and comprises alinear chain of alternating 3-O-linked-(alpha)-D-galactopyranoseand 4-O-linked 3, 6-anhydro-(beta)-L-galactopyranose (Duckworthand Yaphe, 1971).

Agarases are the hydrolytic enzymes that degrade agar into oligo-saccharides, which have various chemical properties and biologicalactivities. Based on the sites of hydrolysis, agarases are characterizedas α-agarases and β-agarases. The α-agarases cleave α-L-(1,3) link-ages of agarose to produce oligosaccharides of the agarobiose serieswith 3,6-anhydro-L-galactopyranose at the reducing end, whereasthe β-agarases cleave β-D-(1,4) linkages of agarose to produceneoagarooligosaccharides with D-galactopyranoside residues at thereducing end (Van der Meulen and Harder, 1975). Moreover, agarasescan be used to prepare protoplasts and extract biological substancessuch as unsaturated fatty acid, vitamins, carotenoids, and betaine,among others, from algae (Araki et al., 1998).

Agarases are produced mainly by bacteria, including Pseudomonas,Pseudoalteromonas, Vibrio, Streptomyces, Agarivorans, Alteromonas,Cytophaga, Bacillus, and Halomonas aquamarina (Feng et al., 2012).

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Because agar is a polysaccharide produced by marine seaweeds, it isnatural that most agar-degrading bacteria are inhabitants of marinehabitats. However, there have been few reports of agarases producedby non-marine bacteria isolated from rivers, hot springs, soils, andsewage.

Agarolytic bacteria can be divided into two groups according totheir effect on solid agar (Agbo and Moss, 1979; Lakshmikanth etal., 2006b). Bacteria in group 1 soften the agar, forming depressionsaround colonies, while those in group 2 cause extensive liquefactionof the agar.

In our previous study, Rhodococcus sp. Q5, a novel agar-degradingbacteriumwas isolated from printing and dyeing wastewater (Feng etal., 2012). In this study, the characteristics of agarase from Q5 and itsenzymatic products were investigated.

2. Materials and methods

2.1. Microorganism and growth medium

The strain Q5 used in this study was originally isolated from thewastewater treatment system of a printing and dyeing factory. Itwas identified as Rhodococcus based on its 16S rDNA gene sequence(GenBank accession number JN644309). The seed culture wasmaintained at−80 °C in a brothmedium containing 20% (v/v) glycer-ol. The inoculum was prepared by incubating the seed culture at 30 °Cwith a shaking speed of 140 rpm for 12 h. For agarase production, theprepared inoculum was transferred to a fermentation medium at aratio of 1:100 and incubated at 30 °C with a shaking speed of 140 rpm

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for 36 h. The fermentation medium was prepared by supplementing amineral-salts medium (MM) with 0.1% agar, 0.1% lactose, and 0.05%peptone. The composition of MM is as follows (g l−1): NH4NO3,0.407; KH2PO4, 0.098; K2HPO4, 0.033; NaCl, 0.03; and MgSO4, 0.20.The initial pH of the medium was adjusted to 6.0. Flasks (250 ml)containing 50 ml of medium were used for both the seed culture andagarase production.

To detect the agarolytic activity on agar plates, the pure strain wastransferred onto a new mineral-salts agar plate and grown at 30 °Cfor several days. Then, Lugol's iodine solution (25 g of iodine and50 g of potassium iodine in 1000 ml of distilled water) was overlaidto detect reducing sugars and degraded products of agar resultingfrom agarase treatment.

2.2. Analytical methods

The cell growth was monitored by measuring the absorbance ofculture fluid at 600 nm. The culture fluid was centrifuged at 6000 gfor 10 min at 4 °C, and the supernatant was used as crude extracellu-lar agarase.

The activity of the enzyme on agar was measured by the release ofthe reducing sugar equivalent using the 3,5-dinitrosalicylic acid(DNS) method (Miller, 1959). A volume of 100 μl of the supernatantobtained as described above was added to 900 μl of 20 mM phos-phate buffered saline (PBS) solution at pH 7.0 with 0.1% agar sub-strate and was incubated at 40 °C for 15 min. The 1 ml reactionsolution was subsequently mixed with 2 ml of DNS reagent. Afterbeing heated at 100 °C for 5 min and cooled, the mixture was dilutedto 10 ml with distilled water. The optical density was read at 520 nm,and values for reducing sugars were expressed as D-galactose equiva-lents. One unit of enzyme activity was defined as the amount of en-zyme that liberated 1 μM galactose equivalents per minute underthe assay conditions. Specific activity of agarase was calculated asunits of enzyme produced per mg of enzyme protein. Protein concen-tration in the culture fluid was determined by Bradford (1976) meth-od using bovine serum albumin as the calibration standard.

2.3. Effect of the medium composition on Q5 growth and agaraseproduction

One agarolytic strain was isolated from compost using platescontaining medium made only from agar and deionized water (Liuet al., 2009). After the test, the authors found that Q5 could alsogrow on plates made only from agar and distilled water. It seemedthat Q5 could fix nitrogen and grow without added mineral elements.

To study the effect of medium components on the growth andagarase production of Q5, three different media were prepared: dis-tilled water supplemented with agar (0.1%, w/v); MM supplementedwith agar (0.1%, w/v); and NFMM (nitrogen-free mineral-salts medi-um) supplemented with agar (0.1%, w/v). To prepare the NFMM,NH4NO3 was eliminated from the MM recipe. Agar was prepared bysoaking and washing the agar gel with distilled water to remove sol-uble contaminants. Exponential growth-phase cultures (0.5 ml) wereinoculated into fresh 250 ml Erlenmeyer flasks containing 50 ml ofthe three different media. The flasks were incubated on a rotary shak-er at 140 rpm and 30 °C. The growth and agarase activity of Q5 wereassayed as described above.

2.4. Preparation of partially purified agarase solution

The culture grown for 36 h was chilled at 4 °C for 30 min, and thecells were separated by centrifugation at 6000 g for 10 min at 4 °C.The enzyme in the supernatant was perturbed by the addition ofsolid ammonium sulfate at 75% saturation with slow stirring for 1 h.The precipitate formed was recovered by centrifugation at 6000 gfor 30 min at 4 °C. The precipitate was dissolved in PBS buffer

(20 mM, pH 7.0) and dialyzed against 120 volumes of the same bufferovernight with four changes. The dialyzed sample was concentratedwith polyethylene glycol 20,000 solution. Then, the concentratedsample was centrifuged again at 6000 g for 10 min. This time, the su-pernatant was used as partially purified agarase solution for the na-tive polyacrylamide gel electrophoresis (native PAGE) (Hu et al.,2009; Lakshmikanth et al., 2006a, 2006b).

2.5. Detection, separation of agarase and molecular weight determination

Native PAGE of the partially purified agarase solution wasperformed on 10% (w⁄v) acrylamide slab gels at 4 °C. After the nativePAGE procedure, the gel was soaked in PBS buffer (20 mM, pH 7.0)for 5 min. The gel was overlaid onto a plate containing 1.5% (w⁄v)agar gel in PBS buffer (20 mM, pH 7.0) and incubated for 6 h at40 °C. The gel was removed from the agar plate and then stained forproteins with Coomassie Brilliant Blue R-250. The agar plate wasflooded with Lugol's iodine solution to visualize agarase activity.

The fractions in the native PAGE gel corresponding to the activitybands in which the agarases were located were then excised. Then,the gel fractions containing agarase were homogenized using a glassstick and soaked in PBS buffer (20 mM, pH 7.0) overnight. The mix-ture was centrifuged at 6000 g for 10 min at 4 °C, and the superna-tant was used as a purified agarase solution (Hu et al., 2009). Next,native PAGE and activity staining were performed to confirmwhetherthe agarase was successfully purified to homogeneity. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed to estimate protein molecular mass with a stacking gel(4% polyacrylamide) and a separating gel (10% polyacrylamide).After SDS-PAGE, the gel was soaked in PBS buffer (20 mM, pH 7.0)for a total period of 30 min to remove the SDS, and activity stainingwas performed as described earlier.

2.6. Effects of temperature and pH on agarase activity and stability

The effect of temperature on agarase activity was determined byperforming the enzyme activity assay at temperatures between 10and 60 °C at pH 7.0. The effect of pH on agarase activity was assayedat 40 °C and various pH levels (3.0–10.0). The pH profiles were mea-sured at 40 °C in different 20 mM buffers: glycine–HCl buffer(pH 3.0), acetic acid buffer (pH 4.0–6.0), sodium phosphate buffer(pH 7.0 and pH 8.0), and glycine–NaOH buffer (pH 9.0 and pH 10.0)(Long et al., 2010). The thermal stability of the agarase was deter-mined by pre-incubating the enzyme solution at temperatures be-tween 10 and 60 °C at pH 7.0 for 30 min and then measuring theresidual enzyme activity. The pH stability of the agarase was deter-mined by pre-incubating the enzyme solution at each pH (3.0–10.0)at 4 °C for 30 min and then measuring the residual enzyme activityafter adjusting the pH with phosphate buffer (20 mM, pH 7.0). Therelative activity was defined as the percentage of activity determinedwith respect to the maximum agarase activity.

2.7. Effects of various reagents on agarase activity

To investigate the effects of various metal ions and reagents on theactivity of the purified agarase, standard enzyme assayswere performedat 40 °C in 20 mM sodium phosphate buffer (pH 7.0) supplementedwith the tested metal ions and reagents at 1 mM concentrations in thereaction mixture.

2.8. Estimation of the kinetic parameters

Km and Vmax values for the agarase acting on agar (final concen-tration of 0.125–2 mg ml−1) were calculated by linear regressionanalysis of Lineweaver–Burk, double-reciprocal plots of initial veloci-ty data obtained under standard assay conditions.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 12 24 36 48 60 72 84

Time (h)

Cel

l den

sity

A60

0

Fig. 2. The growth of Q5 grown in 0.1% agar supplemented with distilled water ( ),MM ( ), or NFMM ( ) was measured after different incubation periods.

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2.9. Identification of the enzyme hydrolysis products

Five grams of agar powder was scattered in 500 ml of PBS buffer(20 mM, pH 7.0). Agarase solution was added to the agar mixture.The reaction was conducted at 40 °C for 12 h and was then stoppedby heating the mixture in boiling water for 10 min. After centrifuga-tion and concentration, the depolymerized end products were isolat-ed by ethanol gradient fractionation. The molecular mass distributionof the agar hydrolysis products was determined by LIT-MS (linear iontrap mass spectrometry). The instrument was operated at the follow-ing parameters: source voltage 3.50 kV, capillary voltage 39.97 V,tube lens voltage 119.94 V, capillary temp 349.96 °C, RF detectortemp 45.76 °C, RF generator temp 25.82 °C, syringe pump (N2) flowrate 5.00 μl min−1.

A B

3. Results and discussion

3.1. Effect of the medium composition on Q5 growth and agaraseproduction

The pure strain was transferred onto a new mineral-salts agarplate and grown at 30 °C for several days. Fig. 1 shows the agar hy-drolyzing ability of the colony after the plate was stained by overlay-ing Lugol's solution.

Q5 grew slowly in distilled water supplemented with agar (Fig. 2).In this medium, maximum growth and agarase production were ob-served after 60 h of incubation; these maxima occurred at 48 h inNFMM supplemented with agar and at 36 h in MM supplementedwith agar. Among the different media tested, MM supplementedwith agar displayed the highest agarase activity and maximumgrowth. This result indicated that Q5 could fix nitrogen and grow inthe absence of mineral elements. The addition of mineral elementsor nitrogen sources facilitated growth and agarase production.

Very fewagar-degrading bacteria capable offixing nitrogenhave beenreported. H. aquamarina, A. macleodii (Sorkhoh et al., 2010), and severalstrains isolated by Shieh et al. (1988) could grow in a nitrogen-freemedi-um. There are also few reports of nitrogen fixation by Rhodococcus spe-cies. Two Rhodococcus species, R. fascians and R. erythropolis, whichwere isolated from spruce humus and from red fescue roots inoculatedwith the humus (Elo et al., 2000), could grow in a nitrogen-free medi-um, as could Rhodococcus sp. R6 (Luan et al., 2009). In this study,Rhodococcus sp. Q5 could both degrade agar and fix nitrogen.

Fig. 1. Detection of agarolytic activity on agar plate. The strain was cultured on amineral-salts agar plate and Lugol's iodine solution was overlaid to detect reducingsugars and degraded products of agar resulting from agarase treatment.

3.2. Separation and identification of agarase

The activity staining by in situ detection after native PAGE showeda single distinct band for agarase activity (Fig. 3), which revealed thepresence of only one extracellular agarase, designated as agarase Q5.

Activity staining after SDS-PAGE also showed a single band foragarase activity, and the apparent molecular mass was estimated tobe 54 kDa (Fig. 4). The reported molecular weight of agarase variesfrom values as low as 12 kDa (Khambhaty et al., 2008), in the case ofB. megaterium, to as high as 210 kDa, in the case of Pseudomonas-likebacteria (Malmqvist, 1978). The agarases from Alteromonas sp. StrainC-1 (52 kDa) (Leon et al., 1992) and Agarivorans sp. HZ105 (57 kDa)(Hu et al., 2009) are closer to that from strain Q5 with respect to theirmolecular weights.

3.3. Effects of temperature and pH on agarase activity and stability

Temperature and pH are considered to be decisive parameters forenzyme activity. Temperature optima of various agarases are higherthan the gelling temperature of agar because compact bundles ofgelled agar hinder enzyme action (Jonnadula and Ghadi, 2011; Ohtaet al., 2005; Suzuki et al., 2003; Van der Meulen and Harder, 1975).It was observed that the activity of agarase Q5 consistently increasedfrom 10–40 °C, with optimum activity at 40 °C (Fig. 5A). However, a

Fig. 3. Activity staining andnative-PAGEof partially purified agarase preparation. (A)Native-PAGE of partially purified agarase preparation on agar gel. (B) Activity staining of partiallypurified agarase preparation on agar gel.

A B

M 1

Fig. 4. Activity staining and SDS-PAGE of purified agarase. (A) Lane M prestained pro-tein mass markers; lane 1 SDS-PAGE of purified agarase. (B) Activity staining of puri-fied agarase.

Table 1Effects of various reagents on the activity of agarase.

Reagent Relative activity (%)

None 100Na+ 100K+ 93Ca2+ 107Mg2+ 46Fe2+ 53Ag+ 15Ba2+ 16Pb2+ 7Sn2+ 30Zn2+ 23Fe3+ 15SDS 54EDTA 77

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drastic decrease was observed when the agarase was incubated attemperatures above 40 °C. The activity of agarase was stable at alow temperature and retained more than 90% of its activity up to atemperature of 40 °C, which is similar to many other agarases(Kirimura et al., 1999; Ohta et al., 2005; Van der Meulen and

A

0

20

40

60

80

100

10 20 30 40 50 60 70

Temperature (°C)

Rel

ativ

e ac

tivity

(%

)

B

0

20

40

60

80

100

3 4 5 6 7 8 9 10 11

pH

Rel

ativ

e ac

tivity

(%

)

Fig. 5. Effects of temperature and pH on activity ( ) and stability ( ) of the agarase.(A) Temperature profiles were checked at different temperatures (10–60 °C) in20 mM PBS buffer (pH 7). (B) pH profiles were checked at 40 °C in different buffers(pH 3–10).

Harder, 1975). However, the agarase AgaA34 (Fu et al., 2008) pos-sessed 95% activity after incubation at 50 °C for 1 h, and the agarasefrom Microbulbifer sp. strain CMC-5 (Jonnadula and Ghadi, 2011)was thermally stable up to 50 °C, with 62% of its residual activityretained. In contrast, agarase Q5 exhibited a narrow temperature sta-bility range, indicating that it is susceptible to temperature.

The enzyme exhibited the maximum agarase activity at pH 6.0(Fig. 5B), whereas the optimum pH for many other agarases is 7.0(Jonnadula and Ghadi, 2011; Long et al., 2010; Shi et al., 2008).Agarases active in alkaline pH have also been reported (Fu et al.,2008; Lee et al., 2006). Agarase Q5 was stable in a wide pH range of5.0–8.0, retaining more than 87% of its residual activity, which wassuperior to some other agarases such as AgaY (6.0–8.0) (Shi et al.,2008), and that from Bacillus sp. MK03 (7.1–8.2) (Suzuki et al.,2003), and Alteromonas sp. E-1 (7.0–9.0) (Kirimura et al., 1999).

3.4. Effects of various reagents on agarase activity

Effects of metal ions and other reagents on the agarase activitywere investigated (Table 1). Metal ions found in seawater, such asNa+, K+, and Ca2+, activated some agarases (Lee et al., 2006; Wanget al., 2006), but these metal ions did not affect the activity of agaraseQ5. However, the agarase activity was inhibited by other metal ions,such as Ag+, Ba2+, Pb2+, Sn2+, Zn2+, Mg2+, Fe2+or Fe3+, whichwas similar to AgaY. These results indicated that agarase Q5 was nota metal-ion-dependent enzyme and that the divalent metal cationswere not essential for its activity. SDS and EDTA exhibited a negativeeffect on the activity of agarase Q5, whereas EDTA is a poor inhibitorfor AgaA34 and AgaA34 is resistant to SDS.

3.5. Kinetic parameters

The Km and Vmax values were obtained by Lineweaver–Burk plotsof agarase activity at 40 °C using various concentrations of agar sub-strate. The Km of agarase was 1.47 mg ml−1, and the Vmax was

y = 1500x + 1.0212

R2 = 0.9906

-2

0

2

4

6

8

10

12

14

-0.002 0 0.002 0.004 0.006 0.008 0.01

1/[S] µg ml -1

1/v

(µ m

ol /

mL

/min

) -1

Fig. 6. Lineweaver–Burk plot to determine the kinetic parameters of the agarase en-zyme acting on agar.

Fig. 7. Linear ion trap mass spectrometry of agar oligosaccharides. The main peaks showed oligosaccharides with different polymerization degrees.

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0.98 μM min−1 ml−1 (Fig. 6). The Michaelis constants of agarasefrom strain N-1 and β-agarase I from P. atlantica X82134 were 0.077and 0.044 mg ml−1, respectively (Vera et al., 1998). The recombinantAgaA had a Km of 3.9 mg ml−1, and a Vmax of 909.1 μM min−1 mg−1

for agarose (Long et al., 2010). And the agarase from B. megateriumgave a Km and Vmax value of 4 mg ml−1 and 2.75 μM min−1 mg−1

(Khambhaty et al., 2008).

3.6. LIT-MS of the agar oligosaccharides

After sufficient hydrolysis and repeated fractionation by ethanol pre-cipitation, the main depolymerized end products were collected. The LITmass spectrum of the sample was shown in Fig. 7. The results of calcula-tion according to the data in the mass spectrum showed the sample wasan oligosaccharide mixture with polymerization degree from 2 to 6. Theagarase mainly produced trisaccharide, as well as a small amount of di-saccharides, tetrose, pentasaccharide, and hexose. Some other agar hy-drolysis products have also been reported. The main oligosaccharidescomprising the agar produced by strain SY37-12 are tetrasaccharide,hexasaccharide, and octosaccharide (Wang et al., 2006). Agar oligosac-charides produced by strain QJH-12 were applied to an anion-exchange chromatography column, and three main peaks were shown.AOS-1 was an oligosaccharide mixture with a polymerization degreeranging from 3 to 14. The main component was trisaccharide,pentasaccharide, hexasaccharide and octosaccharide. AOS-2 wasmainlycomposed of oligosaccharides with polymerization degrees rangingfrom 6 to 22, and AOS-3 was composed of oligosaccharides with aneven polymerization degree ranging from 16 to 24 (Wang et al., 2004).

Acknowledgments

This work was supported by the National Natural Science Founda-tion of China (no. 51079094) and the Natural Science Foundation ofJiangsu Province (no. BK2010215).

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