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Original Research Paper
Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat bran as carbon source Cynthia Barbosa Rustiguel, João Atílio Jorge, Luis Henrique Souza Guimarães n Departamento de Biologia – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, 14040-901 Ribeirão Preto, São Paulo, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 5 February 2015 Received in revised form 9 April 2015 Accepted 22 May 2015
The filamentous fungus Aspergillus phoenicis (Aspergillus saitoi) produced high levels of mycelial β-Dfructofuranosidase (invertase) when cultivated under submerged fermentation using Khanna medium with wheat bran as the carbon source for 72 h at 40 °C, under orbital agitation (100 rpm). The mycelial invertase was purified 20-fold with 24% recovery through two chromatographic steps (DEAE-cellulose and Sephacryl S-200). The enzyme was characterized as a monomeric glycoprotein with 2% carbohydrate content and a native molecular mass of 131 kDa comprising two 70-kDa subunits. The optimal temperature and pH for activity were 65 °C and 4.5, respectively. The enzyme was resistant to temperatures of 50 °C and 60 °C and stable at pH 4.0–7.0. Activity increased in the presence of different ions, especially Mn2 þ ( þ177 %), and Ag þ increased the invertase activity by 91%. The mycelial invertase hydrolyzed sucrose, raffinose, and inulin, with greater specificity for the former. The K1/2 and Vmax values for sucrose were 22.5 mM and 124.9 U mg 1, respectively. & 2015 Published by Elsevier Ltd.
Keywords: Invertase Fructooligosaccharide Enzyme purification Sucrose
1. Introduction Invertases (β-D-fructofuranosidase, EC 3.2.1.26) are enzymes that catalyze the breakdown of β-2,1 glycosidic bonds from sucrose molecule to obtain an equimolar mixture of D-glucose and D-fructose known as invert sugar (Álvaro-Benito et al., 2007). These enzymes can be grouped as acid, neutral, and alkaline invertases based on the pH of maximal activity (Winter and Huber, 2000). Neutral and alkaline invertases have been reported in plants such as Arabidopsis thaliana (Xiang et al., 2011) and cyanobacteria such as Anabaena sp. (Vargas and Salerno, 2010). Comparatively, acid invertases are found in plants and microorganisms such as bacteria and fungi (BRENDA – The Comprehensive Enzyme Information System). Invertase production by filamentous fungi has been described in Cladosporium cladosporioides (Almeida et al., 2005), Aspergillus niger (Reddy et al., 2010), Aspergillus caesiellus (Novaki et al., 2010), Termitomyces clypeatus (Chowdhury et al., 2009), and A. phoenicis (Rustiguel et al., 2010), among others. Fungal invertases have attracted interest for various industrial applications, including those in the food and beverage industry. The invert sugar syrup obtained from sucrose hydrolysis by fungal
invertases is sweeter than sucrose and does not crystallize at a high concentrations (Bayramoglu et al., 2003). In addition, some microbial fructofuranosidases catalyze the transfructosylating reaction at high sucrose concentrations ( Z20%) to obtain fructoligosaccharides (FOS) such as 1-kestose, nystose, and fructosyl nystose (Guimarães, 2012). The first step to determine the potential applications of a specific enzyme is to investigate its physico-chemical properties. If important and unique properties are observed, further genetic engineering studies are conducted to improve the production and/ or enzyme properties. The extracellular fructofuranosidase from A. phoenicis was previously characterized and showed interesting and intriguing properties (Rustiguel et al., 2010). However, to our knowledge, the mycelial fructofuranosidase has not been previously investigated. Herein, this manuscript describes the production and characterization of a mycelial fructofuranosidase produced by A. phoenicis under submerged fermentation using wheat bran as the carbon source.
2. Material and methods 2.1. Microorganism and culture conditions
n
Corresponding author. Fax: þ 55 16 3602 3668. E-mail address:
[email protected] (L.H.S. Guimarães).
The filamentous fungus A. phoenicis (A. saitoi) was maintained
http://dx.doi.org/10.1016/j.bcab.2015.05.004 1878-8181/& 2015 Published by Elsevier Ltd.
Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i
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2
at 4 °C in oatmeal slants and subcultured at 20-day intervals. The submerged cultures were obtained by inoculating a spore suspension (105 spores/mL) in 25 mL Khanna medium (Khanna et al., 1995) in 125-mL Erlenmeyer flasks with different carbon sources (2% monosaccharides: glucose and raffinose; 1% oligosaccharides: starch and sucrose; or complex sources: sugar cane bagasse, crushed corncob, rice straw, oatmeal, wheat bran, and cassava flour) at initial pH 6.0 (Rustiguel et al., 2010). The media were sterilized at 120 °C and 1.5 atm for 25 min. The inoculated media were kept at 40 °C for 72 h.
Table 1 Influence of carbon sources on the production of fructofuranosidase by A. phoenicis.
No carbon source 19.81 7 1.8 Starch 7.157 2.7 Oatmeal 6.83 7 2.7 Sugar cane bagasse 57.337 7.9 Wheat bran 109.87 7 10.3 Cassava 8.62 7 3.1 Glucose 0.417 0.1 Rice straw 53.5 7 3.3 Raffinose 86.417 2.3 Crushed corncob 6.9 7 1.6 Sucrose 6.277 4.0
2.2. Obtainment of mycelial crude extract After cultivation, each medium was harvested using a vacuum pump with Whatman No. 1 filter paper. The free cell filtrate was stored for extracellular enzyme quantification. The mycelia obtained were washed three times with distilled water, pressed through the filter paper, macerated with sand sea in a porcelain mortar, and resuspended in 100 mM sodium acetate buffer, pH 5.0. The suspension was centrifuged at 23,000g for 15 min, and the free cell extract, termed the mycelial crude extract, was used to determine enzyme activity and for purification.
The microorganism was grown in 25 mL Khanna medium at 40 °C under orbital agitation (100 rpm) for 72 h. Total U ¼ U/mL extracellular extract volume.
Table 2 Purification of mycelial fructofuranosidase produced by A. phoenicis. Steps
2.3. Determination of enzyme activity and quantification of proteins and carbohydrates The enzyme activity was determined using 1% sucrose as the substrate in 100 mM sodium acetate buffer, pH 4.5. The reaction was conducted at different temperatures (30–80 °C) and pH values (3.0–8.0) using McIlvaine buffer. The hydrolysis products were estimated using DNS (3′,5′-dinitrosalicylic acid), as described by Miller (1959). One unit of enzyme activity (U) was defined as the amount of enzyme necessary to produce 1 μmol glucose per minute under the assay conditions. The protein was quantified as described by Lowry et al. (1951) using bovine serum albumin (BSA) as the standard and expressed as mg protein per mL sample. The specific activity was defined as U mg 1. The carbohydrate content was determined according to DuBois et al. (1956) using mannose as the standard. 2.4. Purification The mycelial crude extract was precipitated with 45% ammonium sulfate, resuspended in 10 mM Tris–HCl, pH 7.5, dialyzed in this same buffer overnight at 4 °C, and loaded onto DEAE-cellulose chromatographic columns (10.0 2.0 cm2) previously equilibrated with 10 mM Tris–HCl, pH 7.5. Fractions containing invertase activity were eluted using a linear gradient of NaCl (0–1 M) in 10 mM Tris–HCl, pH 7.5. Fractions (3.0 mL) were collected at a flow rate of 1.9 mL/min. The invertase activity for each fraction was determined as described above in 100 mM sodium acetate buffer, pH 4.5, at 60 °C. Active fractions were pooled, dialyzed in distilled water overnight at 4 °C, lyophilized, suspended in 50 mM Tris–HCl buffer, pH 7.5, with 50 mM NaCl and loaded onto Sephacryl S-200 chromatographic columns (80.0 2.0 cm2) previously equilibrated in this same buffer. Fractions (1.0 mL) containing invertase activity were eluted in this same buffer at a flow rate of 0.38 mL/min. Fractions with activity were pooled, dialyzed in distilled water overnight at 4 °C, and used for electrophoresis and enzyme characterization. 2.5. Electrophoresis The purified fraction was submitted to non-denaturing electrophoresis (7% PAGE) as described by Davis (1964) and denaturing electrophoresis (7% SDS-PAGE) as described by Laemmli (1970).
Specific Activity (U mg 1)
Carbon source
Activity (Total U)
Crude extract 806.4 DEAE-cellulose 872.6 Sephacryl S-200 196.4
Protein (Total mg)
Specific activity (U mg 1)
Yield (%)
Purification (X)
24.6 8.6 0.3
32.8 101.5 654.7
100.0 108.2 24.3
1.0 3.1 19.9
Proteins were separated at 120 V and 40 mA for 2 h. After running, the gels were stained using silver nitrate using the method described by Blum et al. (2005). To determine invertase activity in 7% PAGE, the gel was washed with 0.5 M sodium acetate buffer, pH 4.5, three times for 30 min each then with 0.1 M sodium acetate buffer, pH 4.5, three times for 20 min each. The washed gel was maintained in a solution containing 0.2 mg/mL phenazine methyl sulfate, 0.4 mg/mL nitroblue tetrazolium, 30 U/gel glucose oxidase, and 1% sucrose in the dark until protein was visible. 2.6. Molecular mass determination The native molecular mass was determined in a gel-filtration chromatographic column (Sephacryl S-200) as described above. Alcohol dehydrogenase (150 kDa), BSA (66 kDa), egg albumin (43 kDa), and carbonic anhydrase (29 kDa) were used as molecular mass markers. The molecular mass under denaturing conditions was determined with 7% SDS-PAGE as described above. α-2 macroglobulin (168 kDa), β-galactosidase (112 kda), lactoferrin (91 kDa), pyruvate kinase (67 kDa), and lactic dehydrogenase (36 kDa) were used as molecular mass markers. 2.7. Temperature and pH stabilities Thermal stability of the purified enzyme was determined in an aqueous solution at different temperatures (50–70 °C) for 1 h. Aliquots were taken at pre-determined times, stored in an ice bath, and used to determine the enzyme activity as described above. The pH stability was determined by incubating enzyme samples at different pH values (100 mM sodium acetate buffer, pH 3.5 and 4.5; 50 mM MES buffer, pH 6.0; 50 mM Tris–HCl buffer, pH 7.0 and 8.0; 50 mM CAPS buffer, pH 9.0 and 10.0) for 1 h before measuring activity. The best temperature for enzyme storage was also investigated by maintaining enzyme samples at 20 °C, 4 °C, and 27 °C for 200 h.
Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i
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NaCl (0-1 M)
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Fig. 1. DEAE-cellulose (A) and Sepacryl S-200 (B) chromatographic profiles for the mycelial fructofuranosidase produced by A. phoenicis under submerged fermentation using wheat bran as the carbon source for 72 h at 40 °C. Invertase activity was eluted from the DEAE-cellulose column with 205 mM NaCl in 10 mM Tris–HCl buffer, pH 7.5. Symbols: () invertase activity; (○) Abs. 280 nm.
2.9. Hydrolysis of different substrates and kinetic parameters Activity of the purified enzyme for 1% (m/v) sucrose, 1% (m/v) inulin, 1% (m/v) raffinose, and 1:1 (or 1:1:1) mixtures of these substrates was determined. All substrates were prepared in 100 mM sodium acetate buffer, pH 4.5, and the enzyme activity was determined as described above. Kinetic parameters (K1/2, Vmax, and Vmax/K1/2) were determined for sucrose (0.5 –300 mM) in the presence and absence of 1 mM AgNO3. The K1/2 and Vmax values were determined as described by Leone et al. (1992) using the SIGRAF software. 2.10. Analysis of the hydrolysis products from sucrose and raffinose The hydrolysis products for mycelial β-D-fructofuranosidase reaction with sucrose and raffinose were monitored using highperformance liquid chromatography (HPLC) equipped with an EC250/4.6 nucleosyl-100.5 NH2 column (30 0.75 cm2) (Shimadzu) at 60 °C using 82% aqueous acetonitrile as the mobile phase. Sucrose, glucose, fructose, kestose, and nystose were used as standards.
3. Results 3.1. Production of mycelial
Fig. 2. 7% SDS-PAGE (A and B) and 7% PAGE (C) analysis of purified mycelial fructofuranosidase from A. phoenicis. The enzyme was purified through two chromatographic steps: DEAE-cellulose and Sephacryl S-200. (A) Molecular mass markers.
2.8. Influence of different compounds on enzyme activity The influence of salts (AgNO3, BaCl2, CaCl2, CoCl2, CuSO4, FeSO4, HgCl2, MgCl2, MgSO4, MnCl2, NaCl, NH4Cl, and ZnCl2) and EDTA at 1 mM and 10 mM on mycelial invertase activity was determined by adding each to the enzymatic reaction as described above. The results were presented as relative activities (%).
β-D-fructofuranosidase
The fungus A. phoenicis produced high levels of mycelial β-Dfructofuranosidase using wheat bran (109.87 U mg 1) and raffinose (86.41 U mg 1) as carbon sources (Table 1). These values were 5.5-fold and 4.4-fold higher than the values obtained for medium without a carbon source. Furthermore, enzyme production increased 17.5-fold with wheat bran instead of sucrose as the carbon source. 3.2. Purification The mycelial β-D-fructofuranosidase from A. phoenicis was purified to electrophoretic homogeneity using two chromatographic steps, DEAE-cellulose (Fig. 1A) and Sephacryl S-200 (Fig. 1B), with a purification index of 19.9-fold and recovery of
Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i
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4
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Relative enzyme activity (%)
Relative enzyme activity (%)
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Fig. 3. Optimal temperature (A) and pH (C) and thermal (B) and pH (D) stabilities for the mycelial fructofuranosidase produced by A. phoenicis under submerged fermentation using wheat bran as the carbon source for 72 h at 40 °C. Symbols: (∇) 50 °C; (Δ) 60 °C; (○) 65 °C; (□) 70 °C. Inset: Influence of temperature storage at 20 °C (), 4 °C (■) and 27 °C (▲) on mycelial fructofuranosidase activity.
24.3 % (Table 2). The native molecular mass for purified β-Dfructofuranosidase from A. phoenicis was estimated as 131 kDa by gel filtration (Sephacryl S-200), with two identical 70-kDa subunits estimated by 7 % SDS-PAGE (Fig. 2) and 2.14 % carbohydrate content. 3.3. Influence of temperature and pH on activity
β-D-fructofuranosidase
The optimal temperature and pH for mycelial β-D-fructofuranosidase produced by A. phoenicis were 65 °C and 4.5, respectively (Fig. 3A and C). The A. phoenicis mycelial β-D-fructofuranosidase retained 90 % of its initial activity after incubation at 50 °C for 1 h, and the activity was reduced to 62 % when incubated
at 60 °C. Increasing the incubation temperature to 65 °C and 70 °C reduced enzyme stability with half-lives (t1/2) of 9 min and 3.5 min, respectively (Fig. 3B). The mycelial invertase was stable at pH 3.5–7.0 for 1 h, maintaining 72–78% of its activity (Fig. 3D). However, at alkaline pH values, enzyme activity was drastically reduced. The influence of storage temperature on enzymatic activity was also investigated. The mycelial enzyme maintained high levels of activity over 200 h at 20 ºC and 4 °C but decreased at 27 °C with a t1/2 of 76 h (Fig. 3B, inset). 3.4. Effect of different compounds on β-D-fructofuranosidase activity
β-D-fructofuranosidase activity increased in the presence of 1 mM metal ions, especially for Mn2 þ (177%) (Table 3).
Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i
C.B. Rustiguel et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 3 Influence of ions and other compounds on A. phoenicis mycelial fructofuranosidase activity. Compounds
No addition AgNO3 BaCl2 CaCl2 CoCl2 CuCl2 CuSO4 EDTA FeSO4 HgCl2 MgCl2 MgSO4 MnCl2 NaCl NH4Cl ZnCl2
5
K1/2 value was estimated as 22.5 mM with a Vmax of 129.4 U mg 1 in the absence of Ag þ and 70.8 mM with a Vmax of 152.0 U mg 1 in the presence of Ag þ .
Relative Fructofuranosidase Activity (%) 1 mM
10 mM
4. Discussion
100 94.3 7 4.3 51.0 7 3.5 186.17 2.1 82.17 6.1 204.57 7.8 200.6 7 8.2 189.8 7 6.1 37.4 7 2.7 63.6 7 5.8 193.2 7 3.0 200.17 6.5 277.8 7 7.4 200.9 7 7.8 85.4 7 2.2 86.5 7 4.2
100 191.6 7 5.3 48.97 7.2 37.6 7 1.3 83.4 7 2.0 26.2 7 4.2 60.8 7 0.1 78.0 7 0.5 18.6 7 3.2 4.6 7 1.9 87.2 7 5.5 34.4 7 6.8 82.3 7 1.8 84.7 7 6.7 30.17 1.5 82.4 7 1.8
A. phoenicis produced high levels of mycelial β-D-fructofuranosidase using wheat bran, an agro-industrial residue/product, as a carbon source. The wheat bran was acquired in a local market, and the composition was determined as 25% carbohydrates, 18% proteins, and 36% fibers. This composition is very interesting for fungal growth for its low cost and the availability of carbon and nitrogen sources and minerals. Agro-industrial residues and products such as wheat bran, sugar cane bagasse, and rice straw are interesting alternatives for microbial cultivation and obtainment of molecules with biotechnological importance such as enzymes and other products with high aggregate values. According to BCC Research (www.bccresearch.com), the global market for industrial enzymes will be US$7.1 billion by 2018. Complex carbon sources from agro-industrial residues have also been used for β-D-fructofuranosidase production by filamentous fungi such as A. niveus (Guimarães et al., 2009) and A. caespitosus (Alegre et al., 2009). Therefore, wheat bran was chosen for the fermentation process with the goal of enzyme purification and characterization. Moreover, this use of agro-industrial residues can reduce their accumulation and, consequently, their environmental impact. In the presence of glucose, sucrose, starch, oatmeal, and crushed corncob, enzyme production by A. phoenicis was extremely reduced. Sucrose was reported to be a good carbon source for invertase production by A. niger (Nguyen et al., 2005), A. japonicus (Chen and Liu, 1996), and C. cladosporioides (Almeida et al., 2005), among others. Glucose is an important inhibitor of β-D-fructofuranosidase production by A. phoenicis since it is quickly assimilated by the microorganism. The homodimeric mycelial β-D-fructofuranosidase was purified to electrophoretic homogeneity and its molecular mass and carbohydrate content were consistent with the values obtained for the extracellular enzyme (Rustiguel et al., 2010), suggesting minimal differences between the two preparations. Homodimers have also been reported for invertases produced by A. parasiticus (Lucca et al., 2013), A. niveus (Guimarães et al., 2009), and Rhodotorula glutinis (Rubio et al., 2002). However, monomers (Ishimoto and Nakamura, 1997) and homotetramers (Gutiérrez-Alonso et al., 2009) have also been reported. Different invertase isoforms were observed for Ashbya gossypii (Aguiar et al., 2014) and A. nidulans (Chen et al., 1996). Mycelial β-D-fructofuranosidase from A. phoenicis has a higher temperature of activity than that observed for the extracellular enzyme (Rustiguel et al., 2010), reinforcing their minimal differences. The activity reported in this article was also higher than that obtained for extracellular enzymes from Aspergillus sp. 27H (Su et al., 1991), A. niger AS0023 (L'Hocine et al., 2000), and Saccharomyces cerevisiae GCB-K5 (Shafiq et al., 2002). However, the thermal stability for the mycelial enzyme was similar to that of the extracellular enzyme (Rustiguel et al., 2010). This stability was higher than that observed for the enzyme from Aureobasidium pullulans DSM 2404, stable for 1 h at 45 °C (Yoshikawa et al., 2007), and for the enzyme from R. glutinis with a t1/2 of 30 min at 60 °C (Rubio et al., 2002). The fructofuranosidase produced by A. niger AS0023 retained 80 % of its activity after incubation at 65 °C for 2 h (L'Hocine et al., 2000), whereas the mycelial enzyme from A. phoenicis presented a low t1/2 (9 min) at the same temperature, and that for enzyme produced by Fusarium solani was 2.42 min 1 (Bhatti et al., 2006). In addition, enzymatic activity was maintained for long periods when the mycelial β-D-fructofuranosidase
Table 4 Hydrolysis of different substrates by the A. phoenicis fructofuranosidase. Substrate
Fructofuranosidase activity (U)
Sucrose Inulin Raffinose Sucrose þ inulin Sucrose þ raffinose Inulin þ raffinose
34.2 7 1.7 5.6 7 0.0 10.3 7 2.0 23.2 7 2.3 21.6 7 0.0 9.17 1.3
Interestingly, Ag þ also increased the enzymatic activity. Copper, Mg2 þ , and Na þ each increased activity 100%. Fructofuranosidase activity was decreased 37–96% by other ions, especially Fe þ and Hg þ . 3.5. Substrate hydrolysis The mycelial β-D-fructofuranosidase from A. phoenicis hydrolyzed sucrose, inulin, raffinose, and mixtures of these substrates (Table 4). The highest activity was observed for sucrose, which was 3.4- and 6-fold higher than those for raffinose and inulin, respectively. The enzymatic activities for 1:1 mixtures of sucrose and inulin or raffinose were similar but were 2.5-fold higher than that for the mixture of inulin and raffinose. The values obtained for the sucrose mixtures were between those obtained for each separate substrate, indicating that the same catalytic domain is used for hydrolysis of all three substrates. The presence of different catalytic domains for each substrate would predict experimental values corresponding to the sum of the values for hydrolysis of each substrate alone. The hydrolysis products from sucrose and raffinose were analyzed by HPLC (Fig. 4). The results show a drastic reduction in sucrose and a corresponding increase in glucose and fructose. Furthermore, hydrolysis of raffinose, a saccharide comprising fructose, galactose, and glucose, showed the hydrolysis products fructose, galactose, and lactose (glucose þgalactose). The raffinose concentration slowly decreased, demonstrating that the enzyme preferentially hydrolyzes sucrose. 3.6. Kinetic parameters The kinetic parameters for hydrolysis of sucrose by mycelial βD-fructofuranosidase from A. phoenicis are provided in Table 5. The
Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i
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6
fructose
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Fig. 4. HPLC profiles for hydrolysis of sucrose (A and B) and raffinose (C and D) by the mycelial fructofuranosidase produced by A. phoenicis under submerged fermentation using wheat bran as the carbon source for 72 h at 40 °C.
Table 5 Kinetic parameters of A. phoenicis mycelial fructofuranosidase. Parameters
– AgNO3
þ AgNO3
K1/2 (mM) Vmax (U mg 1) Vmax/K1/2 (U mg 1 mM 1) n (Hill's coefficient)
22.5 124.9 5.0 0.7
70.8 152.0 2.0 0.9
from A. phoenicis was stored at low temperatures. Most fungal β-D-fructofuranosidases present optimal activity at acidic pH (4.0–6.0), as was determined herein for mycelial β-Dfructofuranosidase from A. phoenicis and previously for enzymes from Paecylomyces variotii (Giraldo et al., 2011), Rhodotorula glutinis (Rubio et al., 2002), and A. japonicus (Wang and Zhou, 2006), among others. The pH stability was similar to that reported by Rubio et al. (2002) for the enzyme from R. glutinis. Stability to
alkaline pH was reported for β-D-fructofuranosidases from A. pullulans DSM 2404 and A. niger ATCC 20611 (Yoshikawa et al., 2007). The high temperature of activity in combination with the temperature and pH stability indicates the potential of the A. phoenicis mycelial β-D-fructofuranosidase for future biotechnological application. Mycelial β-D-fructofuranosidase activity was increased by Mn2 þ , as previously reported for that from A. ochaceus (Guimarães et al., 2007), while the positive influences of Cu2 þ , Mg2 þ , and Na þ were observed for the enzyme from F. solani (Bhatti et al., 2006). As previously reported for the extracellular enzyme from A. phoenicis (Rustiguel et al., 2010), we found that 10 mM Ag þ increased the mycelial enzyme activity by 91%. Ag þ was reported to inhibit β-D-fructofuranosidase activity in A. japonicus (Duan et al., 1993) and F. oxysporum (Nishizawa et al., 1980). Metal ions can change the overall charge of proteins, thereby affecting their properties. Further, metal ions can interact with the catalytic
Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i
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domains of enzymes to reduce or increase their activities. The mycelial β-D-fructofuranosidase hydrolyzed different substrates, indicating its multifunctional aspect, but showed the greatest activity for sucrose. The hydrolytic activity from A. gossypii β-D-fructofuranosidase was 10-fold higher for sucrose than raffinose, but no hydrolytic activity for inulin was observed (Aguiar et al., 2014). The enzyme produced by A. japonicus JN19 also hydrolyzes raffinose (Wang and Zhou, 2006). In fact, appropriate methods for characterization of invertase and inulinase activity are somewhat controversial: some authors use the sucrose-to-inulin (S/I) ratio to define both activities, while others disagree with this calculation because the S/I ratio can change based on the inulin source. Hence, the kinetic parameters should also be considered to elucidate this question. Accordingly, A. phoenicis mycelial β-D-fructofuranosidase has high potential for use in obtaining invert sugar sirup, which does not crystallize and it is sweeter than sucrose. Invert sugar is extensively used in the food industry, especially for candies, and can be applied in the beverage industry. Enzymes that hydrolyze raffinose are attractive for industrial applications, for example, to digest raffinose in sugar beet juice, as an ingredient in cocoa formulation, and to remove raffinose from soybean milk. Raffinose hydrolysis can also assist in obtaining sucrose from molasses by reducing the negative effect of raffinose (Kumar and Garg, 2009). Considering the intriguing activation by Ag þ , the kinetic parameters for sucrose hydrolysis were determined in the presence and absence of AgNO3. The effect of Ag þ on Vmax was not significant, whereas Ag þ increased the K1/2. Therefore, the enzyme acts more efficiently in the absence of silver. This result is inconsistent with that from the extracellular enzyme (Rustiguel et al., 2010), indicating the importance of minimal differences in the enzyme properties. The affinity of the A. phoenicis mycelial β-Dfructofuranosidase for sucrose was higher than that observed for enzymes produced by S. cerevisiae (Rashad and Nooman, 2009), R. glutinis (Rubio et al., 2002), and A. niger (Wallis et al., 1997).
5. Conclusion The filamentous fungus A. phoenicis produced thermo-tolerant mycelial β-D-fructofuranosidase using wheat bran, a low-cost carbon source, under submerged fermentation. The enzyme is active at elevated temperature and acidic pH. The mycelial β-Dfructofuranosidase is multifunctional, hydrolyzing different substrates to obtain important products for food industries such as invert sugar. Minor differences in the mycelial enzyme structure compared to that of the extracellular form can affect its properties. These physico-chemical properties highlight the biotechnological potential of A. phoenicis mycelial β-D-fructofuranosidase for future industrial application.
Acknowledgments We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Process 09/01283-0) and CAPES (Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior) for financial support and Maurício de Oliveira for technical assistance.
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