Appl Microbiol Biotechnol (2009) 85:85–94 DOI 10.1007/s00253-009-2061-1

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Purification and molecular characterization of exo-β-1, 3-glucanases from the marine yeast Williopsis saturnus WC91-2 Ying Peng & Zhen-Ming Chi & Xiang-Hong Wang & Jing Li

Received: 20 April 2009 / Revised: 22 May 2009 / Accepted: 25 May 2009 / Published online: 10 June 2009 # Springer-Verlag 2009

Abstract The extracellular β-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific β-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40°C, respectively. The enzyme was significantly stimulated by Li+, Ni2+, and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1,10phenanthroline. The Km and Vmax values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLD AFEKR which is the conserved motif of the β-1, 3-glucanases from other yeasts. Keywords Molecular characterization . Marine yeast . β-1,3-Glucanases . Williopsis saturnus . Laminarin

Y. Peng : Z.-M. Chi (*) : X.-H. Wang : J. Li Key Laboratory of Marine Genetics and Gene Resource Exploitation of Ministry of Education, Ocean University of China, Yushan Road, No.5, Qingdao, China e-mail: [email protected]

Introduction β-Glucans are widely found in nature, with the β-1, 4-glucan cellulose being the most abundant. Noncellulolytic β-glucan hydrolyzing enzymes have been reported in many different organisms (Stone and Clarke 1992), including fungi, bacteria, archaea, algae, mollusks, and higher plants (Stone and Clarke 1992; Pitson et al. 1993; Vijayendra and Kashiwagi 2009). β-1,3-Glucanases are the enzymes, which can cleave the beta glycosidic linkages of glucans. They can be divided into two groups: exo- or endo-β-1,3-glucanases. The fungal β-1,3-glucanases play key roles in both metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, and autolysis during development and differentiation (Adams 2004) and in mobilization of βglucans in response to conditions of carbon and energy source exhaustion (Pitson et al. 1993). They also have important nutritional roles in both saprobic and mycoparasitic fungi. As β-glucans are major components of fungal cell walls, it seems likely that β-1,3-glucanases play a crucial role in hydrolyzing localized areas and enabling insertion of new cell wall material without disrupting its overall integrity (Adams 2004). These enzymes are having potential applications in food, feed, pharmaceutical, and fermentation industries. The fungal β-1,3-glucanases gene has been cloned and sequenced from Trichoderma harzianum, Cochliobolus carbonum (EXG1), Trichoderma atroviride (gluc78), Acremonium blochii strain C59 (BGN3.2), Aspergillus saitoi (exgS), Ampelomyces quisqualis, Coniothyrium minitans (cmg1), Trichoderma virens (Martin et al. 2007). Eight yeast genes encoding exo-β-1,3-glucanases: Candida oleophila EXG1 (AAM21469); C. albicans EXG (A47702);

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Saccharomyces kluyveri EXG1 (AAO32563); S. cerevisiae EXG1 (AAA34599), Pichia anomala EXG2 (CAA11018); P. angusta EXG (CAA86948), P. pastoris EXG and P. anomala YF07b have been cloned and characterized (Martin et al. 2007; Wang et al. 2007). However, little has been known about β-1,3-glucanases from marine yeasts. In our previous studies (Wang et al. 2007), we found that exo-β-1,3-glucanases produced by the marine yeast P. anomala YF07b have killer toxin activity against the pathogenic yeast Metschnikowia bicuspidate WCY isolated from diseased crab. In another study (Wang et al. 2008), the marine yeast Williopsis saturnus WC91-2 was found to produce both β-1, 3-glucanases and killer toxin. Therefore, in the present study, β-1,3-glucanases was purified and characterized from the supernatant of the marine yeast W. saturnus WC91-2, and the partial gene encoding the β-1, 3-glucanases was also cloned.

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System (Millipore). These concentrated supernatants were used as the crude β-1,3-glucanases preparation (Wang et al. 2008). Determination of β-1,3-glucanase activity

Materials and methods

The reaction mixture containing 250 μl of 0.25% laminarin (purchased from Sigma-Aldrich Co., St. Louis, USA) in 50.0 mM acetate buffer (pH 4.0) and 20 μl of the crude β-1,3-glucanases preparation obtained above or the purified β-1,3-glucanases described below was incubated at 40°C for 1 h. The reaction was stopped immediately by heating at 100°C for 10 min. The reducing sugar in the mixture was determined by the method of Nelson-Somogyi (Spiro 1966). One unit of β1,3-glucanase activity was defined as the amount of enzyme causing release of reducing sugars equivalent to 1.0 mg glucose from laminarin 30 min under the assay conditions. Protein concentration was measured by the method of Bradford, and bovine serum albumin served as standard (Bradford 1976).

Yeast strains

Purification of β-1,3-glucanases

The yeast strain employed here was the marine yeast W. saturnus WC91-2 (collection number 2E00219 at the Marine Microorganisms Culture Collection of China) which was found to able to produce both β-1,3-glucanase and killer toxin (Wang et al. 2008). M. bicuspidate-sensitive strain WCY (collection number 2E00088 at the Marine Microorganisms Culture Collection of China) is the pathogenic yeast isolated from the diseased crab.

The crude β-1,3-glucanases preparation obtained from the previous step was applied to Sephadex™ G-75 column (Pharmacia 2.5×100 cm), and the column was eluted with 50.0 mM Na2HPO4–citric acid buffer (pH 4.5) by using ÄKTA™ prime with Hitrap™ (Amersham, Biosciences, Sweden). At a flow rate of 0.5 ml/min, 2.0-ml fractions were collected. The β-1,3-glucanase-positive fractions were combined and applied to DEAE Sepharose Fast Flow anion-exchange column (2.5 × 30 cm) that had been equilibrated with 20.0 mM Na2HPO4–citric acid buffer (pH 4.5). The bound proteins were then eluted with a linear gradient of NaCl solution in the range of 0–1.0 M in the equilibrating buffer. The active fractions were concentrated by filtration through an AmiconYM3 (MW cutoff 10,000) membrane.

Media Growth medium was YPD medium containing 2.0% glucose, 2.0% peptone, and 1.0% yeast extract. The medium for β-1,3-glucanase production consisted of 1.0% yeast extract, 2.0% peptone, 2.0% glucose, 2.0% NaCl, and 15% glycerol adjusted to pH 4.5 with 50.0 mM Na2HPO4– citric acid buffer. Production of β-1,3-glucanases by W. saturnus WC91-2 The yeast strain W. saturnus WC91-2 was cultivated for 3 days at 22°C in 500 ml Erlenmeyer flasks with 150 ml of the production medium. The cultures were incubated in a rotary bed shaker (130 rpm). After centrifugation (5,000×g, 10 min, 4°C), the supernatant of the yeast culture was thoroughly mixed with glycerol (the final glycerol concentration was 15 g/100 ml) and the mixture was concentrated to a volume of 15 ml by ultrafiltration with a 5-kDa cutoff™ membrane with a Labscale TFF

Sodium dodecyl sulfate polyacrylamide gel electrophoresis The purity and molecular mass of the β-1,3-glucanases in the concentrated fractions showing the activity was analyzed in noncontinuous denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli 1970) according to the instructions offered by the manufacturer with a two-dimensional electrophoresis system (Amersham, Biosciences, Sweden) and the gels was stained by Coomassie Brilliant Blue R-250 (George and Diwan 1983). The molecular mass standards for SDS-PAGE comprised β-galactosidase (116.2 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydro-

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genase (35 kDa), restriction endonuclease Bsp981 (25 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa). Measurement of killer toxin activity Killer toxin activity was assayed in a diffusion test, using 6-mm-diameter sterile Oxford cups (6 mm×10 mm), which were put on the assay medium seeded with the sensitive yeast strain WCY. Two hundred microliters of the purified β-1,3-glucanases described above was added to each cup and incubated at 24°C for 72 h and the diameter of the inhibition zone was measured for yeast killer activity (Wang et al. 2008). Laminarin hydrolysis The reaction mixture containing 20 μl of 100 U/ml of the purified β-1,3-glucanases and 100 μl of 0.25% laminarin in 50.0 mM acetate buffer (pH 4.0) was incubated at 40°C for 1 h. After that, the mixture was inactivated by heating at 100°C for 10 min immediately. The end products of hydrolysis were determined by thin layer chromatography (Gong et al. 2007). The reaction mixtures in which the purified β-1,3-glucanases was inactivated prior to addition by heating at 100°C for 10 min were used as controls. Mass spectrometry for protein identification Mass spectrometry of the purified β-1,3-glucanases was performed using a Bruker Ultraflex matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDITOF/TOF) mass spectrometer (Bremen, Germany). The tryptic digests were cocrystallized with a matrix of CHCA and spotted on the target wells. The signals of peptide mass fingerprinting (PMF) were acquired in the reflectron mode in the m/z range from 600 to 4,000. Calibration was performed externally using standard peptide mixtures and internally using the peptide fragments of trypsin autolysis products. On the basis of the PMF signals, the one strongest peptide with higher accuracy and higher abundance was further analyzed in the tandem mass spectroscopy (MS/MS) mode. The MS/MS data were exported in a suitable format and submitted to the database for searching for proteins with MASCOT software (Matrix Science, London, UK). The latest version of the fungi database in the NCBInr databases was used in the protein search. Effects of pH and temperature on β-1,3-glucanase activity The optimal temperature for activity of the enzyme was determined at temperatures of 20°C, 30°C, 40°C, 50°C,

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60°C, 70°C, and 80°C. Temperature stability of the purified enzyme was tested by pre-incubating the enzyme at different temperatures ranging from 20°C to 80°C for 1 h, and residual activity was measured as described above immediately. Here, the relative β-1, 3-glucanase activity of the pre-incubated sample at 4°C was regarded as 100%. The effect of pH on the enzyme activity was determined by incubating the purified enzyme in 100 mM Na2HPO4–citric acid buffer between pH 2.0 and 8.0. pH stability was tested via 24 h of pre-incubation of the purified enzyme in 100 mM Na2HPO4–citric acid buffer at different pH values ranging from 2.0 to 8.0 and at temperature of 4°C. The remaining activities of β-1,3-glucanases were measured immediately after this treatment with the standard method as mentioned above. Effects of different metal ions and protein inhibitors on β-1,3-glucanase activity To examine effects of different metal ions on β-1, 3-glucanase activity, enzyme assay was performed in the reaction mixture as described above with various metal ions at a final concentration of 5.0 and 10.0 mM. The activity assayed in the absence of metal ions was defined as control. The metal ions tested include Zn2+, Mg2+, Ca2+, Na+, Hg2+, Cu2+, Mn2+, Fe3+, Fe2+, Ba2+, K+, Co2+, Ag+, Ni2+, and Li+. The effects of protein inhibitors (1, 10-phenanthroline, ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′tetraacetic acid (EGTA), phenylmethylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA), SDS, and iodoacetic acid at a final concentration of 5.0 mM, respectively) on β-1,3-glucanase activity were measured in the reaction mixture as described above. The purified enzyme was pre-incubated with the respective compound for 1 h at 4°C, followed by the standard enzyme assay as described above. The relative activity assayed in the absence of the protein inhibitors was regarded as 100%. Determination of kinetic parameters To obtain Km and Vmax for the purified β-1,3-glucanases, 250 μl of 0.16, 0.31, 0.63, 1.25, and 2.5 mg/ml of laminarin (from Laminaria digitata, Sigma: 73676-1G) in 50.0 mM Na2HPO4–citric acid buffer (pH 4.0) was mixed with 20 μl of the purified β-1,3-glucanase, respectively, and the mixture was incubated at 40°C for 1 h and the reaction was stopped immediately by heating at 100°C for 10 min. Km and Vmax values were obtained from Lineweaver–Burk plot.

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Cloning of the partial gene encoding β-1,3-glucanases

the software offered in NCBI. Multiple alignments of the amino acid sequence were produced using DNAMAN 6.0.

peak3

160

peak2

500

180

140 120

400

100 300

80

200

60

peak1

40 100 20 0 0

20

40

60

Fraction number Absorbance at 280nm

Specific activity

0 80

The extracellular exo-β-1,3-glucanases produced by W. saturnus WC91-2 were purified from the supernatant prepared from the cell culture by ultrafiltration, gel filtration chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The elution profile of gel filtration chromatography indicates that peak 4 with the specific exo-β-1,3-glucanase activity from fractions 20 to 33 showed a single peak (Fig. 1a), while the elution profile of DEAE Sepharose Fast Flow anion exchange chromatography shows that peak 3 with the specific activity from fractions 5 to 15 displayed one single sharp peak (Fig. 1b). Therefore, the fractions were collected and concentrated by ultrafiltration. The results in Table 1 show that the enzyme was purified to homogeneity with a 115-fold increase in specific exo-β-1,3-glucanase activity with a yield of about 27.0% as compared to that in the supernatant. Gel electrophoresis SDS-PAGE was used to determine protein purity and estimate molecular mass of the final concentrated elute as described by Laemmli (1970). The results in Fig. 2 indicate that there was one single protein band from the final concentrated elute and the relative molecular mass of the purified exo-β-1,3-glucanases was estimated to be 47.5 kDa by SDS-PAGE.

b

450

peak3

peak2

800

400

700

350

600

300

500

250

400

200 300

150 100

200

peak1

100

50

0 60

0

0

20

40

Specefic β-1,3-glucanase activity (U/mg)

peak4

600

Purification of exo-β-1,3-glucanases

Absorbance of 280nm (mA)

Absorbsence at 280nm (mA)

a

Results

Specefic β-1,3-glucanase activity (U/mg)

Genomic DNA from W. saturnus WC91-2 was isolated according to the method described by Adams et al (1998). To isolate the consensus sequence of the partial gene encoding β-1,3-glucanases from W. saturnus WC91-2, one set of degenerated primers (PP: 5′-TCNCARAAYGGNTT YGAYAAYTCNGG-3′ and PR: 5′-CKYTTYTCRAAN GCATCYARYTGNGC-3′) were designed based on the information of the amino acid sequence (identified by mass spectrometry described above) of the partial fragment of the β-1,3-glucanases and of the amino acid sequences deduced from β-1,3-glucanases genes of P. anomala (ABK40520), Candida beverwijkiae (O93983), C. albicans SC5314 (XP_721216), C. albicans (CAA21969), P. stipitis CBS 6054 (XP_001385760), Debaryomyces hansenii CBS767 (XP_458827), D. hansenii (CAG86973), Lodderomyces elongisporus NRRL YB-4239 (XP_001526673), Kluyveromyces lactis (XP_452437), and P. pastoris (AAY28969) and were used for polymerase chain reaction (PCR) with the genomic DNA of W. saturnus WC91-2 as template. The reaction system (50 µl) was composed of 5.0 µl of 10× buffer, 4.0 µl (2.5 mM) of dNTP, 1.0 µl (100 µM) of PP, 1.0 µl (100 mM) of PR, 0.5 µl of Taq DNA polymerase (5.0 U/µl), 1.0 µl (25.0 ng/µl) of the genomic DNA, and 37.5 µl of ddH2O. The conditions for the PCR amplification were as follows: initial denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing temperature at 57°C for 30 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min. PCR was run for 30 cycles and the PCR cycler was a GeneAmp PCR System 2400 (PerkinElmer, Waltham, MA, USA). The PCR products were cloned into pMD19-T Vecter (TaKaRa) and sequenced. The amino acid sequence of the cloned DNA sequence was deduced using

Fraction number Absorbance at 280nm

Specific activity

Fig. 1 Elution profiles of the exo-β-1,3-glucanase from W. saturnus WC91-2 on Sephadex G-75 (a) and DEAE Sepharose Fast Flow anion exchange (b)

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Table 1 Summary of the purification procedures Purification steps

Total protein (mg)

Total activity (U)

Yield (%)

Specific activity (U/mg)

Purification fold

Crude enzyme Ultrafiltration Sephadex G-75 DEAE Sepharose Fast Flow

83.6 7.1 2.1 0.17

212 175 120 58

100 82 57 27

17 24 396 1,966

1 1.4 23.3 115

Laminarin hydrolysis

Effects of pH and temperature on β-1,3-glucanase activity

Laminarin is a polymer of D-glucose mainly in a β-1,3 configuration, arranged as helical coils. Laminarin is hydrolyzed mainly by β-1,3-glucanases. These enzymes are further classified as exo- and endo-β-1,3-glucanases. Exo-β-1,3-glucanases hydrolyze laminarin by sequentially cleaving glucose residues from the nonreducing end of polymers or oligomers. Consequently, the sole hydrolysis products are glucose monomers. Endo-β-1,3-glucanases cleave β-1,3 linkages at random sites along the polysaccharide chain, releasing smaller oligosaccharides (CohenKupiec et al. 1999). After laminarin hydrolysis using the purified β-1,3glucanase obtained above, the results in Fig. 3 show that only monosaccharides and disaccharides were released from laminarin. This demonstrates that the purified β-1,3glucanase was exo-β-1,3-glucanases. However, the results in Fig. 4 indicate that the exo-β-1,3-glucanases produced by W. saturnus WC91-2 had no killer toxin activity against the pathogenic yeast M. bicuspidate WCY isolated from diseased crab.

β-1,3-glucanase activity was measured at various pH values in buffers with the same ionic concentrations. Our results (Fig. 5a) show that the maximum activity was observed at pH 4.0. pH stability was tested via 24 h of preincubation of the purified enzyme in appropriate buffers that had the same ionic concentrations at different pH values ranging from 2.0 to 8.0 at 4°C. The remaining activities of β-1,3-glucanases were measured immediately after this treatment with the standard method as mentioned earlier. It can be seen from the results in Fig. 5a that the activity profile of the enzyme was stable from pH 3.0 to 7.5. For example, greater than 74.18% of the residual activity was maintained after treatment at pH from 3.0 to 7.5 and 4°C for 24 h. These results suggest that the enzyme was very stable in the pH range of 3.0 to 7.5. Especially, 100% of the residual activity was maintained after treatment at pH 6.0 and 4°C for 24 h. The exo-β-1,3-glucanase activity measured as a function of temperature from 20°C to 80°C shows that the activity was highest at 40°C (Fig. 5b). Thermostability is considered an important and useful criterion for industrial application of exo-β-1,3-glucanases. Therefore, the thermostability of the purified enzyme was investigat-

1 Fig. 2 SDS-PAGE (12%) of the fractions showing exo-β-1,3-glucanase activity obtained during the purification. Lane M, marker proteins with relative molecular masses indicated on the right. Lane 1, eluate from DEAE Sepharose Fast Flow anion exchange chromatography

2

3

4

Fig. 3 Hydrolysis of laminarin by the purified exo-β-1,3-glucanase. Lane 1, control (laminarin + inactivated β-1,3-glucanase by heating at 100°C for 10 min); lane 2, laminarin + β-1,3-glucanase, incubated at 40°C for 1 h; lane 3, glucose; lane 4, maltobiose

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Fig. 4 Killer activity of the purified β-1,3-glucanase (a), the killer toxin from P. anomala YF07b (b; Wang et al. 2007), and sterile water (c). Two hundred microliters of the purified β-1,3-glucanase was

added to the Oxford cup, which was put on the assay medium seeded with the pathogenic yeast strain WCY and incubated at 24°C for 72 h, and the diameter of the inhibition zone was measured

ed by pre-incubating the enzyme in the same buffer as described in “Materials and methods” for 1 h and its remaining activity was determined. As shown in Fig. 5b, the purified exo-β-1,3-glucanases were stable in the range of 20°C to 50°C and the residual exo-β-1,3-glucanase activity still maintained 92.7% of the control after treatment at 50°C for 1 h, indicating that the enzyme was stable up to 50°C. Figure 5b also reveals that the enzyme was inactivated rapidly at temperatures higher than 50°C and was almost inactivated at 80°C within 1 h. From these results, the exo-β-1,3-glucanases seemed to have considerable thermostability.

Mn2+ (at a concentrations of 10.0 mM) showing the lowest level (0.5%; Table 2), suggesting that they were able to alter the enzyme conformation (Sharon et al. 1998). The inhibition by mercuric ions may indicate the importance of thiol-containing amino acid residues in the β-1, 3-glucanases function (Barth and Gaillardin 1997). Table 3 depicts the effects observed in the presence of protein inhibitors of the purified β-1,3-glucanases. The presence of the chelating agents EDTA, EGTA, and 1,10phenanthroline negatively affected enzyme activity, demonstrating that the purified enzyme was a metalloenzyme (Ramirez-Zavala et al. 2004). The enzyme activity was also strongly inhibited by PMSF and iodoacetic acid, indicating that Ser and Cys residues were important for active sites of the enzyme, respectively. The results in Table 3 also show that SDS inhibited activity of the β-1,3-glucanases obtained in this study.

Effects of different cations and protein inhibitors on activity of the purified β-1,3-glucanases Li+, Ni2+, and Ba2+ (at concentrations of 5.0 and 10.0 mM) significantly stimulated the activity of the purified β-1,3glucanases from W. saturnus WC91-2. However, Mn2+, Ca2+, K+, Na+, Mg2+, Co2+, Zn2+, Hg2+, Ag+, Cu2+, Fe3+, and Fe2+ (at the same concentrations) acted as inhibitors in decreasing activity of the purified β-1,3-glucanases, with

a

Kinetic parameters Lineweaver–Burk plots in this study show that apparent Km and Vmax values of the purified enzyme for laminarin

b

120

100

Relative activity (%)

Relative activity (%)

100 80 60 40 20 0 -20

120

80 60 40 20 0

0

2

4

6

8

pH Activity

10

0

20

40

60

80

100

-20

Temperature (°C) Stability

Activity

Stability

Fig. 5 Effects of different pH (a) and temperatures (b) on β-1,3-glucanase activity and stability. Data are given as means ± SD, n=3

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Table 2 Effect of different cations on the purified β-1,3-glucanase activity Concentrations (mM)

Relative activity (%)

Control Zn2+ Zn2+ Mg2+ Mg2+ Ca2+ Ca2+ Na+ Na+ Hg2+ Hg2+ Cu2+ Cu2+

0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0

100±2.1 84.1±4.1 68.6±3.0 92±2.0 88.8±2.5 81.4±3.5 74.1±2.1 85.8±1.3 84.1±4.4 30.4±2.1 21.6±2.5 32.2±2.1 34.3±3.1

Mn2+ Mn2+ Fe3+ Fe3+ Fe2+ Fe2+ Ba2+ Ba2+ K+ K+ Co2+ Co2+ Ag+ Ag+ Ni2+ Ni2+ Li+ Li+

5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0

3.4±0.1 0.5±0.1 85.3±2.4 82.9±3.4 78.4±4.0 70.2±3.2 156.5±5.2 260.1±4.3 84.7±2.5 81.3±3.5 92.9±3.0 83.2±1.5 19.3±3.3 9.4±3.4 131.2±4.5 163.8±5.0 111.8±2.5 181.6±4.1

Data are given as means ± SD, n=3

Table 3 Effect of protein inhibitors on the purified β-1,3-glucanase activity Inhibitors

Concentrations (mM)

Control 1,10-phenanthroline EGTA PMSF EDTA SDS Iodoacetic acid

0 5.0 5.0 5.0 5.0 5.0 5.0

Data are given as means ± SD, n=3

Relative activity (%) 100±3.0 47.14±4.4 37.72±3.1 71.11±4.1 87.26±2.6 6.31±2.0 5.35±2.0

R2 = 0.9996

5

1/[V] (min*m /mg

Cations

y = 0.7632x + 0.2489

6

4 3 2 1 0 -2

-1

0

1

2

3

4

5

6

7

8

-1

1/[S] ml/mg Fig. 6 Lineweaver–Burk plot for Km and Vmax values of the purified β-1,3-glucanase in the presence of different concentrations of laminarin. Data are given as means ± SD, n=3

were 3.07 mg/ml and 4.02 mg/min ml, respectively (Fig. 6). Protein identification by mass spectrometry Figure 7 shows the mass spectra of the purified β-1, 3-glucanases produced by W. saturnus WC91-2. One peptide fragment of the protein was measured by MALDITOF/TOF MS and the amino acid sequence was YIEAQ LDAFEKR. The measured peptide was matched against one fragment of the exo-β-1,3-glucanases from Candida glabrata CBS138 and P. anomala and the scores were 92 and 90, respectively. These results proved that the purified protein indeed was exo-β-1,3-glucanases. Cloning of the partial gene encoding exo-β-1,3-glucanases After several degenerate primers were tried, only one band of PCR products was obtained by the primers as described in “Materials and methods” and the size of the PCR products was about 650 bp (Fig. 8). After sequencing of the cloned partial DNA fragment (accession number FJ875997) as described in “Materials and methods”, the amino acid sequence was deduced from the partial gene sequence by using the software offered in NCBI. Multiple alignments of the amino acid sequence were produced via DNAMAN6.0 based on amino acid sequences of the exo-β-1,3-glucanases from C. albicans At 1.85 A, exo-β-1,3-glucanases from P. anomala, hypothetical protein CaO19.2990 from C. albicans SC5314, hypothetical protein CaO19.10507 from C. albicans SC5314, RecName Full=Glucan 1, 3-βglucosidases and hypothetical protein CAGL0I00484g from C. glabrata CBS138, and the amino acid sequence deduced from the partial gene sequence cloned from W. saturnus WC91-2 used in this study (Fig. 9). The results in Fig. 9 show that amino acid sequence deduced from the partial

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Fig. 7 MALDI-TOF/TOF mass spectra of the purified β-1,3-glucanase. Inset: A parent ion of 1,482.7692 was selected for MS/MS analysis, and the amino acid sequence, YIEAQLDAFEKR, was confirmed with the mass signals of b-ions and y-ions

gene also contained YIEAQLDAFE of all the exo-β-1, 3-glucanases examined in this study.

Discussion As discussed above, exo-β-1,3-glucanases has many potential applications in food, feed, agricultural, pharmaceutical, and fermentation industries (Zhu et al. 2008; Castoria et al. 1997; Onderci et al. 2008). Therefore, exo-β-1,3-glucanases produced by the marine yeast W. saturnus WC91-2 was purified

M

I

Fig. 8 The PCR products (lane 1) obtained using the degenerate primers as described in “Materials and methods”. M DNA markers

and characterized. The purified exo-β-1,3-glucanases had the relative molecular mass of 47.5 kDa (Fig. 2). It has been reported that the majority of yeast extracellular exo-β-1, 3-glucanases from different sources had a molecular mass in the range of 31.5–83.1 kDa. For example, the molecular mass of the purified exo-β-1,3-glucanases from the marine killer yeast P. anomala YF07b was estimated to be 47.0 kDa (Wang et al. 2007). Therefore, the molecular mass of the β-1,3-glucanases from W. saturnus WC91-2 used in this study was consistent with that of the exo-β-1,3-glucanases from other yeasts. Laminarin could be transformed into monosaccharides and disaccharides by the purified exo-β-1,3-glucanases (Fig. 3). However, the exo-β-1,3-glucanases had no killer activity against the pathogenic yeast strain WCY isolated from diseased crab (Fig. 4). In contrast, we found that the purified exo-β-1,3-glucanases produced by another marine yeast P. anomala YF07b have killer toxin activity against the same pathogenic yeast strain WCY isolated from diseased crab (Wang et al. 2007). Similarly, K5-type yeast killer toxin of P. anomala NCYC 434 cells has exo-β-1, 3-glucanase activity (Izgu et al. 2006). This means that the exo-β-1,3-glucanases produced by W. saturnus WC91-2 were different from that produced by P. anomala YF07b and P. anomala NCYC 434. Vijayendra and Kashiwagi (2009) have also noticed liberation of almost glucose during the hydrolysis of laminarin by the exoglucanase of Rhizoctonia solani. The optimal pH of the purified exo-β-1,3-glucanases was 4.0 and the enzyme was stable in the pH range of 3.0 to

Appl Microbiol Biotechnol (2009) 85:85–94

93

Fig. 9 Alignment of the deduced amino acid sequence (7) of the partial gene (FJ875997) clone from W. saturnus WC91-2 with that of the gene from other eukaryotic cells which included: 1 Chain A, E27q mutant of exo-β-1,3glucanase from C. albicans At 1.85 A, 2 exo-β-1,3-glucanase from P. anomala, 3 hypothetical protein CaO19.2990 from C. albicans SC5314, 4 hypothetical protein CaO19.10507 from C. albicans SC5314, 5 RecName Full=glucan 1,3-β-glucosidase, 6 hypothetical protein CAGL0I00484g from C. glabrata CBS138

......................................GGGHNVAWDYDNNVIRGVNLGGWFVLQP 1.TXT ........MLISTFIISSLLSIALANPIPSRGGTQFYKRGDY...WDYQNDKIRGVNLGGWFVLEP 2.TXT MQLSFILTSSVFILLLEFVKASVISNPFKPNGNLKFKRGGGHNVAWDYDNNVIRGVNLGGWFVLEP 3.TXT MQLSFILTSSVFILLLEFVKASVISNPFKPNGNLKFKRGGGHNVAWDYDNNVIRGVNLGGWFVLEP 4.TXT ........MLISTFIISSLLSIALANPIPSRGGTQFYKRGDY...WDYQNDKIRGVNLGGWFVLEP 5.TXT .MVSFSFATIVAALSVSALAKPVIAPKNKDTSLHFVNEKRYYDYDSKAIGEPIRGVNIGGWFVLEP 6.TXT .................................................................. 7.TXT Consensus

28 55 66 66 55 65 0

1.TXT YMTPSLFEPFQNGNDQS.GVPVDEYHWTQTLGKEAALRILQKHWSTWITEQDFKQISNLGLNFVRI 2.TXT FITPSLFEAFENQGQD...VPVDEYHYTKALGKDLAIERLDQHWSSWIVEADFQSIAGAGLNFVRI YMTPSLFEPFQNGNDQS.GVPVDEYHWTQTLGKEAASRILQKHWSTWITEQDFKQISNLGLNFVRI 3.TXT YMTPSLFEPFQNGNDQS.GVPVDEYHWTQTLGKEAASRILQKHWSTWITEQDFKQISNLGLNFVRI 4.TXT 5.TXT FITPSLFEAFENQGQD...VPVDEYHYTKALGKDLAKERLDQHWSSWIVEADFQSIAGAGLNFVRI YITPSLFEAFRTNPYNDDGIPVDEYHYCEQLGEQEARNRLEYHWSTFYTEQDFADIKSKGFNLVRI 6.TXT .................................................................. 7.TXT Consensus

93 118 131 131 118 131 0

1.TXT PIGYWAFQLLDNDPYVQG.QVQYLEKALGWARKNNIRVWIDLHGAPGSQNGFDNSGLRDSYNFQNG 2.TXT PIGYWAFQLLDNDPYVQG.QESYLDQALEWAKKYDIKVWIDLHGAPGSQNGFDNSGLRDSYEFQNG 3.TXT PIGYWAFQLLDNDPYVQG.QVQYLEKALGWARKNNIRVWIDLHGAPGSQNGFDNSGLRDSYNFQNG PIGYWAFQLLDNDPYVQG.QVQYLEKALGWARKNNIRVWIDLHGAPGSQNGFDNSGLRDSYNFQNG 4.TXT PIGYWAFQLLDNDPYVQG.QESYLDQALEWAKKYDIKVWIDLHGAPGSQNGFDNSGLRDSYEFQNG 5.TXT PIGYWAFKDMPNDPYVKGSQEYYLDQAIQWAENNGLKVWVDLHGAVGSQNGFDNSGLRDSIDFLAD 6.TXT ...............................................SQNGFDNSGLRDSYEFQNG 7.TXT sqngfdnsglrds f Consensus

158 183 196 196 183 197 19

DNTQVTLNVLNTIFKKYGGNEYSDVVIGIELLNEPLGPVLNMDKLKQFFLD.GYNSLR.QTGSVTP 1.TXT DNTQVALDVLQYISNKYGGSDYGDVVIGIELLNEPLGSVLDMGKLNDFWQQ.GYHNLR.NTGSSQN 2.TXT DNTQVTLNVLNTIFKKYGGNEYSDVVIGIELLNEPLGPVLNMDKLKQFFLD.GYNSLR.QTGSVTP 3.TXT 4.TXT DNTQVTLNVLNTIFKKYGGNEYSDVVIGIELLNEPLGPVLNMDKLKQFFLD.GYNSLR.QTGSVTP DNTQVALDVLQYISNKYGGSDYGDVVIGIELLNEPLGSVLDMGKLNDFWQQ.GYHNLR.NTGSSQN 5.TXT ENLQNTKEILKYVLQKYSQQQYLNTVIGVELINEPLGPVIDMDKMKEQYIKPAYEYLRNELQSIQD 6.TXT NNTQITLDVLQQIFDKYGSSDYDDVIIGLELLNEPLGPVLDMAKLNEFWET.AYWNLR.NSNSTQT 7.TXT l ky y ig el neplg v m k y lr s Consensusn q

222 247 260 260 247 263 83

VIIHDAFQVFGYWNNFLTVAEGQWNVVVDHHHYQVFSGGELSRNINDHISVACNWGWDAKKESHWN 1.TXT VIIHDAFQTWDYFNDKFHT.PDYWNVVIDHHHYQVFSPGELSRSVDEHVKVACEWGANSTKENHWN 2.TXT VIIHDAFQVFGYWNNFLTVAEGQWNVVVDHHHYQVFSGGELSRNINDHISVACNWGWDAKKESHWN 3.TXT VIIHDAFQVFGYWNNFLTVAEGQWNVVVDHHHYQVFSGGELSRNINDHISVACNWGWDAKKESHWN 4.TXT VIIHDAFQTWDSFNDKFHT.PDYWNVVIDHHHYQVFSPGELSRSVDEHVKVACEWGANSTKENHWN 5.TXT IIVHDAFQPFHYWDDFMTVDTGYWGVVIDHHHYQVFSTGELQRDMGQHIQVACEWGSGILTESHWT 6.TXT VVIHDAFTASGYFNDKFQLNQGYWGLVIDHHHYQVFSQQEVQRSIDEHVEVACQWGKDSKGENLWN 7.TXT w v dhhhyqvfs e r e w Consensus hdaf h vac wg

288 312 326 326 312 329 149

VAGEWSAALTDCAKWLNGVNRGARYEGAYD.....NAPYIGSCQPLLDISQWSDEHKTDTRRYIEA 1.TXT LCGEWSAAMTDCTKWLNGVGRGSRYDQTFDYDPSQNQNYIGSCQGSQDISTWDDDKKSNYRRYIEA 2.TXT VAGEWSAALTDCAKWLNGVNRGARYEGAYD.....NAPYIGSCQPLLDISQWSDEHKTDTRRYIEA 3.TXT VAGEWSAALTDCAKWLNGVNRGARYEGAYD.....NAPYIGSCQPMLDISQWSDEHKTDTRRYIEA 4.TXT LCGEWSAAMTDCTKWLNGVGRGSRYDQTFDYDPSQNQNYIGSCQGSQDISTWDDDKKSNYRRYIEA 5.TXT VAGEWSAALTDCTKWLNGVGIGARYDGSFWKN.GVSSSFIGSCANNEDIYSWSEERKENTRKYIEA 6.TXT LCGEWSAALTDCAKWLNGVGKGARYDQTFG.....NSQYTGSCTNSQDISTWSSDVKANYRRYIEA 7.TXT di w gsc r yiea k Consensus gewsaa tdc kwlngv g ry

349 378 387 387 378 394 210

QLDAFEYTGGWVFWSWKTENAPEWSFQTLTYNGLFPQPVTDRQFPNQCGF 1.TXT QLDAFEKRSGWIFWTWKTETTLEWDFQKLSYYGIFPSPLNSRQYPGQCD. 2.TXT QLDAFEYTGGWVFWSWKTENAPEWSFQTLTYNGLFPQPVTDRQFPNQCGF 3.TXT QLDAFEYTGGWVFWSWKTENAPEWSFQTLTYNGLFPQPVTDRQFPNQCGF 4.TXT QLDAFEKRSGWIFWTWKTETTLEWDFQKLSYYGIFPSPLTSRQYPGQCD. 5.TXT QLDAFEKRGGWIFWCYKTETNIEWDASRLIEYGMFPQPLTDRRYPGQCA. 6.TXT QLDAFEKR.......................................... 7.TXT Consensuqldafe

399 427 437 437 427 443 218

7.5 (Fig. 5a). The results were in agreement with the observation that the stability of fungal β-1,3-glucanases is commonly between pH 3 and 8.0 (Wang et al. 2007; Xu et al. 2006). Contrary to this, stability of the exo-β-1, 3-glucanases of R. solani was reported to be at pH 10 (Vijayendra and Kashiwagi 2009). The optimal temperature of the purified exo-β-1,3glucanases was 40°C and the enzyme was stable in the temperature range of 20°C to 50°C (Fig. 5b). Exo-β-1,3glucanases in general show optimum temperature mostly between 30°C and 55°C (Lachance et al. 1977; Molina et al. 1989; Xu et al. 2006; Wang et al. 2007).

The activity of the purified β-1,3-glucanases from W. saturnus WC91-2 was greatly stimulated by Li+, Ni2+, and Ba2+. However, Mn2+, Ca2+, K+, Na+, Mg2+, Co2+, Zn2+, Hg2+, Ag+, Cu2+, Fe3+, and Fe2+ inhibited the enzyme activity (Table 2). It was found that Ca2+, Co2+, K+, Na+, and Mg2+ activated the activity of β-1,3-glucanases produced by P. anomala YF07b (Wang et al. 2007). However, Fe2+, Fe3+, Hg2+, Cu2+, Mn2+, Zn2+, and Ag+ acted as inhibitors in decreasing β-1,3-glucanase activity produced by P. anomala YF07b, with Fe2+ and Cu2+ showing the lowest rank (Wang et al. 2007). This means that some biochemical and physical properties of the β-1,

94

3-glucanases produced by W. saturnus WC91-2 used in this study were greatly different from those of β-1,3-glucanases produced by P. anomala YF07b. EDTA, EGTA, 1, 10-phenanthroline, PMSF, and iodoacetic acid were found to inhibit the activity of the β-1, 3-glucanases produced by both P. anomala YF07b (Wang et al. 2007) and W. saturnus WC91-2 (Table 3). However, EDTA had no effects on the activity of the β-1, 3-glucanases (the killer toxin from P. anomala 434 K5 type; Izgu et al. 2005). The apparent Km and Vmax values of the purified β-1, 3-glucanases produced by W. saturnus WC91-2 for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively (Fig. 6). In contrast, apparent Km value of the β-1,3-glucanases produced by P. anomala YF07b for laminarin was 1.17 g/l (Wang et al. 2007) and the apparent Km of the β-1,3-glucanases from P. anomala NCYC 432 and P. anomala NCYC 434 K5 type was 0.3 and 0.25 g/l, respectively (Izgu et al. 2005, 2006). However, at the optimal pH of 6.0, the β-1,3-glucanases from P. pastoris X-33 shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 µmol glucose produced/min/mg protein; Xu et al. 2006). This suggests that the β-1,3-glucanases produced by W. saturnus WC91-2 displayed lower affinity for laminarin than that produced by P. anomala, but higher than that produced by P. pastoris X-33. One peptide fragment of the purified β-1,3-glucanases contained the amino acid sequence YIEAQLDAFEKR (Fig. 7). At the same time, the amino acid sequence deduced from the partial gene cloned from W. saturnus WC91-2 also contained YIEAQLDAFE of all the exoβ-1,3-glucanases (Fig. 9). This means that the cloned partial DNA sequence indeed encoded exo-β-1,3glucanases and the gene encoding exo-β-1,3-glucanases indeed existed in the producers. Acknowledgment This research was supported by the National Natural Science Foundation of China, grant number 30670058.

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