Purification and the Secondary Structure of Fucoidanase from Fusarium sp. LD8

The fucoidanase from Fusarium sp. (LD8) was obtained by solid-state fermentation. The fermented solid medium was extracted by citric acid buffer, and the extracts were precipitated by acetone and purified by Sephadex G-100 successively. The results showed that the specific fucoidanase activity of purified enzyme was 22.7-fold than that of the crude enzyme. The recovery of the enzyme was 23.9%. The purified enzyme gave a single band on SDS-PAGE gel, and the molecular weight of fucoidanase was about 64 kDa. The isoelectric point of the enzyme was 4.5. The enzyme properties were also studied. The results showed that the optimum temperature and pH were 60°C and 6.0, respectively; the temperature of half inactivation was 50°C, and the most stable pH for the enzyme was 6.0. K M, and the V max of the enzyme was 8.9 mg·L−1 and 2.02 mg·min−1 ·mL−1 by using fucoidan from Fucus vesiculosus as substrate. The compositions of the secondary structure of fucoidanase were estimated by FTIR, the second derivative spectra, and the curve-fitting analysis of the amide I bands in their spectra. The results showed that β-sheet was the dominant component (58.6%) and α-helix was the least (12%); the content of β-turn and random coil were 15.39% and 14.5%, respectively.

There were some papers focusing on fermentation conditions of fucoidanase from marine fungus [26][27][28] and marine bacteria [23,29], a few papers concerning purifications of fucoidanase from hepatopancreas [14] and marine bacteria reported [17,[20][21][22]. In order to elucidate the structure of fungal fucoidanase, we investigate fucoidanases isolated from different species of fungi. The present work is devoted to purification and the structure analysis of a fucoidanase from marine fungus Fusarium sp. LD8.  [31] to estimate the release of reducing sugars at 540 nm as follows: a mixture consisting of 1 mL substrate solution (1% fucoidan (w/v) from Fucus vesiculosus dissolved with 0.02 mol·L −1 citric acid-sodium citric buffer, pH 6.0) and 0.1 mL enzyme solution (crude extract or pure enzyme) was incubated at 50 • C for 10 min, using inactivated enzyme solution as blank CK. Using fucose as standard, the calibration curve function was y = 1.611x (x: the quantity of fucose, mg; y: the absorbance at 540 nm, R 2 = 0.9983).

Materials and Methods
One unit (IU) of fucoidanase activity is defined as the amount of enzyme that releases 1 μmol of fucose per minute under the assay conditions.

Fucosidase Activity.
Fucosidase activity was measured under the following conditions: the reaction mixture contained 1 mL substrate solution (1% ρ-nitrophenyl-α-L-fucoside (w/v) dissolved with 0.02 mol·L −1 citric acidsodium citric buffer, pH 6.0) and 0.1 mL enzyme solution (purified enzyme) was incubated at 40 • C for 2 h. One unit (IU) of fucosidase activity is defined as the amount of enzyme that releases 1 μmol of ρ-nitrophenyl per minute under the assay conditions.

Amylase Assay.
The reaction mixture containing 1 mL of 2% soluble starch (w/v) in acetate buffer (pH 5.5) and 1 mL of enzyme solution was incubated with shaking at 40 • C for 30 min. The reaction was stopped by boiling water for 5 min, and after centrifugation the released reducing sugar was measured by the dinitrosalicylic acid method [31]. One unit (IU) of amylase activity is defined as the amount of enzyme that liberated 1 μmol reducing sugar (as glucose) per min under the assay conditions.

Extraction, Purification, and Purity
Identification of Fucoidanase. The LD8 was cultivated at 28 • C for 48 h on the solid-state medium in 250 mL flask.
All the following steps were accomplished at 4 • C except being indicated specifically. 50 g fermented culture medium was extracted with citric acid-citric sodium buffer (pH 6.0) for 0.5 h. After being filtrated through six-layer carbasus, the filtrate was centrifuged at 15,000 g for 0.5 h, and then ice-cold acetone was added to the supernatant to a final concentration of 66.7% (v/v) with gentle stirring. Insoluble material was obtained by centrifugation at 15,000 g for 0.5 h. The precipitate was dissolved in pH 6.0, 0.02 mol·L −1 citric acid-citric sodium buffer and was centrifuged at 15,000 g for 0.5 h, and the clear solution was collected for use. The enzyme solution was concentrated to about 10 mL by low-temperature vacuum concentration and then loaded to Sephadex G-100 column (2.5 × 100 cm), which has been balanced well with 0.1 mol·L −1 citric acidcitric sodium buffer. The enzyme was eluted at room temperature at the flow rate of 0.33 mL·min −1 ; fractions were collected at 20 min intervals. The fractions with the highest enzymatic activity were pooled, concentrated by lowtemperature vacuum concentration to 10 mL, dialyzed in deionized water, lyophilized, and stored at −18 • C.

Characteristics of the Purified Enzyme.
Isoelectrofocusing was performed on gel rods. The electrode solutions were 2% (w/v) sodium hydroxide solution at the cathode and 5% (v/v) phosphoric acid solution at the anode. Each gel was focused in 100 V, 2 mA for 2 h, followed by 150-160 V, 4 mA for 5 h, which allowed for a sharp focalisation of fucoidanase. Following isoelectric focusing, the gels were cut into 0.5 cm for measuring pH and enzyme activity. The protein was stained by 0.5% amino black (w/v).
The pH relative activity of the fucoidanase was determined by detecting the fucoidanase activity over a pH range of 3-8 with three kinds of buffer solutions (50 mmol·L −1 sodium citrate buffer, 50 mmol·L −1 sodium phosphate buffer, 50 mmol·L −1 sodium carbonate buffer) at 50 • C. The pH stability of the enzyme was studied by detecting the residual activity after the enzyme being incubated for 3 h at room temperature under different pH value. To obtain optimal reactive temperature, a mixture consisting of 1 mL substrate solution (1% fucoidan (w/v) from Fucus vesiculosus dissolved with 0.02 mol·L −1 citric acid-sodium citric buffer, pH 6.0) and 0.1 mL enzyme solution was incubated at different temperatures for 10 min. For thermal stability study, 0.1 mL of enzyme solution was incubated at temperature range from 30 to 80 • C for 1 h, then cooled rapidly in ice bath for 5 min, and then removed to 25 • C. The residual enzyme activity was detected at 50 • C for 10 min.
The substrate specificity of the enzyme was examined by using different fucoidan from Fucus vesiculosus and Laminaria sp. as substrates. Michaelis constants (K M ) and maximum reaction velocities (V max ) were calculated by the double-reciprocal plot method of Lineweaver and Burk [32]. INSTRVMENTW, USA). For the spectrum range from 1500∼2200 cm −1 , 32 scans were collected at a spectral resolution of 4 cm −1 . Pure fucoidanase (10 mg) was mixed with 100 mg of dried potassium bromide (KBr). Water vapor was purged from sample room. The spectrum of the amide I band of fucoidanase was obtained, and self-deconvolution and curve-fitting methods were used to analyze the secondary structure of the fucoidanase.

Purification of Fucoidanase.
Two protein peaks (E1 and E2) were observed. E2 peak showed fucoidanase activities ( Figure 1) whose purity had been detected by SDS-PAGE (see below). The crude enzyme extraction was purified by 66.7% acetone precipitation and Sephadex G-100 gel chromatography ( Table 1). The purification fold of fucoidanase activity of 1 mg protein was enhanced from 1-fold to 23.9-folds while recovery rate was decreased from 100% to 22.7%. The fractions with higher fucoidanase activity (tube no. 65 to 87) were pooled and concentrated to 10 mL. To exclude the reducing carbohydrates from carbon sources used for cultivation (starch, kelp polysaccharides), fucoidanase-related enzymes such as fucosidase and amylase activity of purified protein were determined, but there is no fucosidase or amylase activity (Table 2).

Determination of Isoelectric Point and Molecular
Mass of Fucoidanase. The result exhibited that the purified fucoidanase gave a single band on isoelectric electrophoresis, and the isoelectric point of the enzyme was pH 4.5 ( Figure 2). The purified fucoidanase gave a single band on SDS-PAGE gel, which suggested that relatively pure fucoidanase had been obtained. The molecular mass of the fucoidanase was about 64 kDa by SDS-PAGE ( Figure 3) which was different from that of Dendryphiella arenaria TM94 (180 kDa). Molecular markers used were myosin (220 kDa), α-2 macroglobulin (170 kDa), β-galactosidase (116 kDa), transferrin (76 kDa), and glutamic dehydrogenase (53 kDa), respectively.

Effect of pH on Fucoidanase Activity and Stability.
Effect of pH on the activity of fucoidanase obtained from Fusarium sp. LD8 was shown in Figure 4. The maximum enzyme activity was at pH 6. The optimal pH of this enzyme was very close to that from marine fungus Dendryphiella arenaria TM94 and Vibrio sp. N-5, while the optimal pH of fucoidanase from Hepatopancreas of Patinopecten yessoensis was 5.5.
The effect of pH on stability was also determined. The results showed that the enzyme displayed stability at pH 6.0, whereas at pH 5.0 and 8.0, an activity loss of about 50% occurred after 6 h incubation at room temperature (25 • C), respectively.

Effect of Temperature on Fucoidanase Activity and Stability.
The optimum temperature for maximal activity of the fucoidanase was 60 • C at pH 6.0 ( Figure 5(a)). At 30 • C the activity of fucoidanase decreased to 12.5%, while at 80 • C to 18.75%. The result showed that the optimal temperature of fucoidanase from TM94 was higher than that of the fucoidanase in Vibrio sp. N-5, whose optimum temperature was 40 • C [33].
The residual activity of fucoidanase was examined after preincubating it at different temperatures for 1 hr, and the temperature at which enzyme lost half activity was 50 • C ( Figure 5(b)). It was completely inactivated at above 80 • C. The temperature of lost half activity is different from those of Vibrio sp. N-5 and Dendryphiella arenaria TM94. The enzyme of Dendryphiella arenaria TM94 and Vibrio sp. N-5 showed that their optimal temperature of half lost inactivation was at 56 • C and 40 • C, respectively [33].

Determination of K M , V max and Affinity for Fucoidanase.
The kinetic parameters of fucoidanase were examined using Fucus vesiculosus fucoidan and Laminaria sp. fucoidan as substrates. The K M values of the enzyme determined by Lineweaver-Burk method were 8.9 mg·L −1 and 10.9 mg·mL −1 , respectively. At the same time, the V max values for both substrates were 2.02 mg·min −1 ·mL −1 and 2.06 mg·min −1 ·mL −1 , respectively. The enzyme showed higher affinity to fucoidan from Fucus vesiculosus.
3.6. The Second Structure of Fucoidanase. The original IR spectrum for the fucoidanase was showed in Figure 6. The   bands at about 1620∼1700 cm −1 can be mainly attributed to the (ν(C=O)) and is called amide I. The shape of the amide I band is representative of fucoidanase secondary structure. The bands of the 1650∼1658 cm −1 , 1600∼1640 cm −1 , 1640∼1650 cm −1 , 1660∼1695 cm −1 regions were, respectively, assigned to α-helix, β-sheet, random coil, and β-turn structure.
The second derivative and a curve-fitting treatment can be carried out to estimate quantitatively the relative proportion of each component representing a type of secondary structure. The fourth derivative function was calculated by the PeakFit 4.12 software to determine the number of components in the amide I region for the second derivative spectra and the curve-fitting process (Table 3). According to this band composition, the amide I profile of fucoidanase contains four major components that can be linked with αhelix, β-sheet, random coil, and β-turn structure where βsheet is evidently the most intense component. The results showed that β-sheet was the dominant component (58.6%), α-helix was the least (12%), and the content of β-turn and random coil were 15.39% and 14.5%.
Fucoidanase from Fusarium sp. LD8 was more sensitive to pH and temperature. The catalytic activity of the fucoidanase of LD8 reached maximum at pH 6.0, which is very close to that of the fucoidanases from bacteria Vibrio sp. N-5 and Dendryphiella arenaria TM94. The enzyme activity decreased rapidly in LD8 when pH is below or above 6.0. At pH 5.0 and 7.0, the enzyme retained 68.2% and 86.4% of the enzyme activity at pH 6, respectively. The fucoidanase from LD8 had a relatively higher optimal temperature (60 • C) compared with that of bacteria  Figure 4: Effects of pH on fucoidanase activity (a) and stability. (b) Effects of pH on fucoidanase activity was determined under the following method: 1.0 mL substrate solution (pH 3-8) in buffer solutions (50 mmol·L −1 sodium citrate buffer, 50 mmol·L −1 sodium phosphate buffer, 50 mmol·L −1 sodium carbonate buffer) was added to 0.1 mL enzyme solution and then was incubated at 50 • C for 10 min. The pH stability of the enzyme was determined by assaying the residual activity after incubating enzyme for 3 h at the room temperature at different pH levels ranging from 3 to 9. Residual fucoidanase activity was determined by adding 0.1 mL enzyme solution to 1.0 mL of substrate solution (citric acid-citric sodium buffer, pH 6.0) and then was incubated at 50 • C for 10 min. and marine fungi Dendryphiella arenaria TM94 (50 • C). The temperature of half inactivation of LD8 fucoidanase was 50 • C, while that of bacteria Vibrio sp. N-5 was 65 • C [33].
Effects of temperature on the secondary structure of LD8 fucoidanase were studied by Gaussian fitting to the deconvoluted spectra of fucoidanase at amide I kregion [36,37].
A decrease of the β-turn structure and an increase of αhelix of amide I region had been observed when treated temperature was below 60 • C. While treated temperature was above 60 • C, the contents of α-helix, β-sheet, random coil, and β-turn had no changes. The above result was consistent with our conclusion that the optimal enzyme reaction tem-  Figure 5: Effects of temperature on fucoidanase activity (a) and stability. (b) Effects of temperature on fucoidanase activity was obtained by the following method: adding 0.1 mL of enzyme solution to 1.0 mL of substrate solution (citric acid-citric sodium buffer, pH 6.0), then incubating for 10 min at different temperatures from 30 • C to 80 • C, respectively. Temperature on stability of the fucoidanase was determined by assaying the residual activity under the following method: 0.1 mL of enzyme solution was incubated at 30 to 80 • C for 1 h, rapidly cooled in an ice bath for 5 min, and then removed to 25 • C. When the sample reached 25 • C, 1.0 mL of substrate solution was added, and the residual enzyme activity was determined and expressed relative to the maximum activity. perature was 60 • C; The enzyme activity decreased rapidly in LD8 below or above pH 6.0. It was suggested that the enzyme activity of fucoidanase was closely related to the proportion of α-helix and β-turn structure with no direct relation to βsheet and random coil structure.

Acknowledgments
This work was supported by funds from Anhui Provincial Nature Science Foundation (no. 03043104), Anhui Provin-