2020 年 26 巻 2 号 p. 265-280
The optimization of fermentation medium for α-galactosidase production by Trametes versicolor was investigated by using orthogonal design in shaker flask fermentation. The optimal liquid medium for α-galactosidase production by T. versicolor was consist of 1.0% soybean cake power, 0.60% galactose, 0.15% KH2PO4, 0.09% MgSO4. Then, the α-galactosidase from T. versicolor (TVG) was purified and characterized. The purified enzyme, a monomeric protein with a molecular weight of 70 kDa, was purified 332-fold by means of ion exchange chromatography and gel filtration. The optimal pH and temperature of TVG with p-nitro-phenyl α-D-galactopyranoside (pNPG) as substrate were 3.0 and 60 °C, respectively. The activity of TVG was inhibited by N-bromosuccinimide (NBS), constituting evidence for the essentiality of tryptophan residue(s) at or in the vicinity of the active centre. The α-galactosidase presented a broad substrate specificity, which included pNPG, melibiose, raffinose, and stachyose with Km values of 0.651, 3.66, 15.1, and 4.47 mM, correspondingly. Galactose acted as a noncompetitive inhibitor with Ki and Kis of 2.88 and 0.132 mM, respectively. A synergistic acceleration in guar gum degradation was found when TVG and mannanase were combined.
Trametes versicolor belongs to the family Basidiomycotina that has been used as a traditional medicinal mushroom (Jhan et al. 2016). The fungus, which looks like a Chinese folding fan with radial plications and shorthair on the pileus, is commonly distributed in a moist environment (Cui and Chisti 2003). Numerous bioactive substances have been isolated and characterized from T. versicolor, such as polysaccharides, laccases, sterols, polysaccharopeptides and carboxylate reductase. These substances are conducive to protect the environment and human health (Chen et al. 2016; Leliebre-Lara et al. 2016; Liu et al. 2019). However, no studies have appeared concerning the isolation and characterization of α-galactosidases from T. versicolor.
α-galactosidase (melibiase; α-D-galactoside galactohydrolase; EC 3.2.1.22) is an exo-acting glycosidase that catalyzes the hydrolysis of α-1,6-linked terminal galactopyranosyl moieties from various substrates, such as linear and branched galacto-oligosaccharides, polymeric galactomannans and synthetic substrates including pNPG (Ademark et al. 2001; Manzanares et al. 1998). Raffinose family oligosaccharides (RFO), which are a group of oligosaccharides of solubility, non-structure and irreducibility, are widely distributed in soy products. However, food rich in RFO arouses belching, abdominal distension, borborygmus and stomachache in human and other monogastric animals lacking an α-galactosidase (Gangola et al. 2016; Steggerda 1968). The α-galactosidase has been used to degrade RFO in various industrial applications, mainly in the food and feed businesses (Kotiguda et al. 2007; Singh and Kayastha 2013). The α-galactosidase has been used in several industrial applications, mostly in the sugar-making industry. Treatment of beet molasses containing raffinose with α-galactosidase would improve the production and quality of sucrose (Du et al. 2014). α-galactosidases are common enzymes in nature and are widely found in plants, mammals, bacteria, fungi and microorganisms (Table 1). In plants, α-galactosidases are maximally distributed in seeds, fruits and leaves. The α-galactosidases have been identified and purified from the bacteria Bacillus stearothermophilus (Pederson and Goodman 1980), fungi such as Aspergillus parasiticus (Nakai et al. 2010), archaeon such as Sulfolobus solfataricus (Brouns et al. 2006), and the mushroom Termitomyces eurrhizus (Zhang et al. 2015).
| Species | References |
|---|---|
| Plants | |
| Lycopersicon esculentum Mill | (Feurtado et al. 2001) |
| Cucumis sativus | (Itoh et al. 1986) |
| Saccharum officinarum | (Chinen et al. 1981) |
| Lablab purpureus (Linn.) Sweet | (Celem et al. 2009) |
| Coffee beans | (Malhotra and Singh 1976) |
| Cicer arietinum | (Singh and Kayastha 2012a) |
| Glycine max (Linn.) Merr. | (Porter et al. 1991) |
| Cyamopsis tetragonobola | (Bulpin et al. 1990) |
| Phaseolus coccineus | (Du et al. 2014) |
| Animals | |
| Homo sapiens | (Andreotti et al. 2010; Xu et al. 2015) |
| Sus scrofa liver | (Aguilera et al. 2012) |
| Ovis aries | (Bajwa and Sastry 1974) |
| Muroidea | (Valbuena et al. 2011) |
| Gallus domestiaus | (Davis et al. 1993) |
| Chinise Cricetidae | (Chang 1978) |
| Bacterials | |
| Bacillus stearothermophilus | (Pederson and Goodman 1980) |
| Escherichia coli | (Hantzopoulos and Calhoun 1987) |
| Thermus thermophilus | (Fridjonsson and Mattes 2001) |
| Bacillus megaterium | (Patil et al. 2010) |
| Lactobacillus reuteri | (Tzortzis et al. 2004) |
| Filamentous Fungi | |
| Streptomyces coelicolor | (Kondoh et al. 2005) |
| Talaromyces flavus | (Simerska et al. 2007) |
| Phlebia radiata | (Prendecka et al. 2003) |
| Aspergillus nidulans | (Nakai et al. 2010) |
| Aspergillus parasiticus | (Shivam and Mishra 2010) |
| Rhizomucor miehei | (Katrolia et al. 2012a) |
| Gibberella | (Cao et al. 2009) |
| Macro Fungi | |
| Lenzites elegans | (Sampietro et al. 2012) |
| Pleurotus florida | (Ramalingam et al. 2007) |
| Ganoderma lucidum | (Sripuan et al. 2003) |
| Tricholoma matsutake | (Geng et al. 2015) |
| Coriolus versicolor | (Du et al. 2014) |
| Pseudobalsamia microspora | (Yang et al. 2015) |
| Termitomyces eurrhizus | (Zhang et al. 2015) |
| Pleurotus ostreatus | (Brechtel et al. 2001) |
| Archaeon | |
| Sulfolobus solfataricus | (Brouns et al. 2006) |
In the present study, increased α-galactosidase activity was detected from T. versicolor in submerged culture. Thus we provide detailed information on the production and purification of the α-galactosidases from T. versicolor, which has great thermostability and stability under acidic conditions, and also exhibits high hydrolytic activity towards guar gum when combined with mannanase.
Materials A fungal strain of T. versicolor (ACCC 51345) was purchased from Agricultural Culture Collection of China (Beijing, China). It was grown on Potato Dextrose Agar (PDA) slant culture medium and stored at 4 °C in a refrigerator. DEAE-cellulose and CM-cellulose were purchased from Sigma Chemical Company (St. Louis, MO, USA). Q-Sepharose, Superdex 75 HR 10/30 and AKTA Purifier were obtained from GE Healthcare (Uppsala, Sweden). Galactose, glucose, sucrose, raffinose and pNPG were purchased from Sigma Chemical Company (St. Louis, MO, USA). Soybean cake powder and corn flour were purchased from Kang Mingwei Culture Media Company (Beijing, China). All other chemicals used were of analytical grade unless otherwise stated.
Culture medium Liquid induction culture medium contained 2.0% tryptone, and 0.5% KH2PO4. The basal liquid culture medium was composed of 2.0% soybean cake powder, 0.1% KH2PO4, and 0.1% MgSO4. Erlenmeyer flasks containing 50 mL of culture medium were autoclaved at 121 °C for 20 min. These flasks were inoculated with 3% inoculum (V/V) and then incubated at 25 °C on a rotary shaker (180 rpm).
Enzyme extraction Fermentation broth was filtered through four layers of wet gauze and then the filtrate was centrifuged at 8000 g for 10 min. The supernatant acquired was used for α-galactosidase test.
Enzyme activity assay For routine analysis and monitoring of activity in the process of fermentation, α-galactosidase activity was assayed in test tubes on the basis of Cao et al. (2009) with a slight modification. The substrate pNPG (10 mM) was dissolved in 0.2 M acetate buffer (pH 4.6), which was stored at 4 °C. Reaction mixture containing 50 µL suitably diluted enzyme and 50 µL pNPG was incubated at 50 °C for 15 min. The reaction was terminated by adding 400 µL of 0.5 M Na2CO3 and the released p-nitrophenol was determined spectrophotometrically at 405 nm. Reactions with the heat-treated enzyme samples (100 °C, 5 min) were used as controls. One unit of α-galactosidase activity was defined as the amount of enzyme that brings about the release of 1 µmol of p-nitrophenol released per minute under the conditions described above.
Screening the inducer for α-galactosidase production To study the influence of various inducers (galactose, glucose, sucrose, raffinose, soybean cake powder and corn flour) on α-galactosidase production; the inducers were mixed with the liquid induction culture medium at a concentration of 2.0% (w/v) and detected for production of extracellular α-galactosidase.
Optimization of culture conditions for maximal enzyme production The experimental design for this study was divided into three major parts. Firstly, the preferred nutrient (carbon sources, nitrogen sources, and inorganic salt) for α-galactosidase production was determined by varying one factor at a time while keeping the others constant (Li et al. 2013). In this experimental set up, different carbon sources like glucose, galactose, sucrose, raffinose and corn flour were added in basal liquid culture medium whereas the other parameters were unaltered and α-galactosidase activity was assessed. Effect of different nitrogen sources on enzyme production was evaluated using organic (peptone, yeast powder) and inorganic (sodium nitrate, ammonium nitrate, ammonium sulfate) compounds as a nitrogen source. To investigate the effect of different metal ions on α-galactosidase production, original metal ions from the basal liquid culture medium were replaced with different metal ions (Mg2+, K+, Ca2+, Fe2+) and by keeping rest of the media composition constant. Secondly, single factor experiments were performed to determine the appropriate range of culture conditions of T. versicolor. Four key factors influencing the α-galactosidase production were investigated, including soybean cake power, galactose, K+ and Mg2+ (Li et al. 2013). Thirdly, the optimum culture conditions for α-galactosidase production were obtained by using orthogonal design (L9(3)4) based on single factor experiments. In this study, with the concentrations of soybean cake power, galactose, K+ and Mg2+ as independent variables, and the activity of α-galactosidase as the index, the culture conditions of T. versicolor were optimized by conducting nine experiments, as shown in Tables 1 and 2 (Zhu et al. 2014).
| Levels | Factors | |||
|---|---|---|---|---|
| Soybean cake power (g/100 mL, A) |
Galactose (g/100 mL, B) |
K+ (g/100 mL, C) |
Mg2+ (g/100 mL, D) |
|
| 1 | 1.0 | 0.50 | 0.10 | 0.08 |
| 2 | 2.0 | 0.60 | 0.15 | 0.09 |
| 3 | 3.0 | 0.70 | 0.20 | 0.10 |
Effect of incubation time on α-galactosidase production To study the effect of different durations of incubation on α-galactosidase production, fermentation was performed at 25 °C on a rotary shaker and samples were prepared at every 24 h interval (continuously for 9 days) for enzyme assay.
Purification of α-galactosidase from the fermentation broth The fermentation was incubated for 7 days at 25 °C using the above optimized conditions. The culture filtrate was dialyzed against deionized water at 4 °C and subsequently adjusted to pH 4.6. Afterward, the filtrate was applied to a DEAE-cellulose (2.5 cm × 20 cm) column equilibrated with 10 mM NaAc-HAc buffer (pH 4.6). α-galactosidase was eluted with the buffer containing 200 mM NaCl. The active fractions were pooled and dialyzed in distilled water. Then, the dialyzed fraction was put on a column of CM-cellulose (pH 3.0), and a stepwise elution was done by increasing the NaCl concentration. The active fraction was collected by 50 mM NaCl in the starting buffer. Using the same methods, the active fraction was passed on a Q-Sepharose (pH 4.6) column to yield the active fraction Q2. Fraction Q2 was finally purified by fast protein liquid chromatography (FPLC) on a Superdex 75 HR 10/30 gel filtration column (pH4.6).
Determination of molecular mass and amino acid sequence The purified α-galactosidase from T. versicolor (TVG) was subjected to SDS-PAGE as described by Laemmli and Favre (1973). The native molecular mass was estimated by gel filtration on an FPLC Superdex 75 HR10/30 column. The standard molecular weight of protein marker used in gel filtration included 100, 80, 70, 60, 50, 40, 30, 25 kDa. The purified protein sample was digested by trypsin and analyzed by MALDI-TOF/TOF. The m/z values obtained from the MALDI-TOF/TOF spectra that corresponded to the peptides of TVG were matched to galactosidases through Mascot (www.matrixscience.com) and the National Center for Biotechnology (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The identified criteria were digestion with trypsin and one missed cleavage allowed; the rest were set by default. During the search, all peptide masses were assumed to be monoisotopic with a mass accuracy of ± 0.2 Da.
Biochemical properties of the enzyme The enzyme activity at different pH values was assayed under standard assay conditions in the pH range of 2.0–8.0 using 100 mM Na2HPO4-citric acid buffer. The pH stability was also investigated using the same buffer solutions which were pre-incubated with the enzyme at room temperature for 120 min.
To obtain the optimal temperature for TVG, the activity of the α-galactosidase was detected over the temperature range of 4–90 °C at the optimal pH. For determining the thermal stability of TVG, residual enzyme activity was determined under standard conditions following incubation of the enzyme over the temperature range 4–80 °C for 120 min.
The effects of various metal ions and chemical reagents (Fe2+, K+, Ca2+, Cd2+, Cu2+, Hg2+, Mg2+, Mn2+, Pb2+, Zn2+, Al3+, Fe3+, NBS, DTT, DIC, EDC, DEPC) on the activity of TVG were examined. The α-galactosidase was preincubated with different concentrations of metal ions and chemical reagents at 4 °C for 120 min and the residual activity toward pNPG was detected as described above.
Substrate specificity and enzyme kinetics Substrate specificity was studied using synthetic compounds (pNPG, p-nitrophenyl β-D-galacto-pyranoside, p-nitrophenyl a-D-glucopyranoside) and natural compounds (melibiose, raffinose, stachyose, locust bean gum, guar gum) as substrate. All the substrates were dissolved in NaAc–HAc buffer (pH 4.6, 100 mM). The substrate specificities of TVG with synthetic compounds were determined under the standard activity assay condition as described above. For natural compounds, the reaction mixture contained 50 µL of α-galactosidase and 200 µL of substrates such as raffinose (50 mM), stachyose (50 mM), locust bean gum (1.0%) and guar gum (1.0%). The enzyme activities were measured at 50 °C, 1 h by measuring the amount of reducing sugar produced using the 3,5-dinitrosalicylic acid (DNS) method of Miller (1959). When melibiose was used as the substrate, the amount of glucose released was measured with a glucose oxidase kit (Beijing BHKT Clinical Reagent Co., Ltd.). The reaction system containing 50 µL of α-galactosidase and 200 µL of melibiose (50 mM) was kept at 50 °C for 10 min. One unit of α-galactosidase activity was defined as the amount of enzyme that released 1 µmol of reducing sugar equivalent to galactose per minute under the assay conditions.
The Km and Vmax values for substrate hydrolysis were calculated using the Lineweaver–Burk plot. The substrate concentrations ranged from 0.1–1.0 mM for pNPG and 2–20 mM for melibiose, raffinose and stachyose, respectively. When galactose was used as an inhibitor, the inhibition constants (Ki and Kis) were also obtained by using the Lineweaver-Burk plot. pNPG concentrations ranged from 0.1–1.0 mM. The concentrations of galactose concentrations were 0, 2.5, 5, and 10 mM.
Hydrolysis of guar gum by the combination of α-galactosidase and mannanase All degradation reactions were performed at 40 °C and pH 4.6. Reactions containing 0.5% guar gum and either 10 U α-galactosidase (group 1) or 10 U mannanase (group 2) (1:4, V/V) or both enzymes (group 3) (10 U each) were incubated for 12 h, boiled for 5 min, and then incubated under the same conditions for 1 h; released reducing sugars in the mixture were measured using the DNS method.
Sequential addition of α-galactosidase and mannanase to guar gum substrate was performed as described (Wang et al. 2010). Briefly, reactions and conditions were the same as the reactions described for simultaneous enzyme addition, except that the initial reaction mixture containing α-galactosidase (group 4 and 5) or mannanase (group 6 and 7) alone was incubated for 5 h or 12 h; the reactions were boiled for 5 min to inactivate the enzyme and then cooled to the original reaction temperature. The other enzyme was then added to the reaction and incubated under the same conditions for another 7 h or 5 h. DNS method was used to determine the released reducing sugar (Miller 1959). The crude enzyme from fermentation broth was incubated with 0.5% guar gum at 40 °C for 7 h (group 8). Guar gum was replaced by water as the blank control at the same reaction conditions, and then the released reducing sugar was detected.
The group 7 with a good degradation ability of guar gum was mixed with three volumes of chilled ethanol (4 °C, 4h) in order to remove high molecular weight oligosaccharides and enzyme resistant fractions of guar gum, and then centrifuged at 7000 rpm for 20 min. The supernatant was flash evaporated, and subjected to high-performance anion exchange chromatography (HPAEC, Dionex ICS-3000) for the analysis of sugars.
Statistical analysis All of the experiments were performed in triplicate. The data were expressed as the mean ± standard deviation (SD) and were statistically analyzed by one-way analysis of variance (ANOVA). P values < 0.05 were considered as statistically significant.
Galactose, glucose, sucrose, raffinose, soybean cake powder and corn flour were added in the fermentation medium, respectively. Then the activity of α-galactosidase in the fermentation broth was determined every day. Fig. 1 showed the effect of inducers on the production of the enzyme by T. versicolor. Evidently soybean cake powder was the optimum inducer for α-galactosidase production by T. versicolor, as it gave the maximal enzyme activity (210 U/mL) when the incubation time was 14 days, and the enzyme activity was generated the earliest among six substrates (7 days). Corn flour and raffinose had a little effect on enzyme production, exhibiting low enzyme activity and requiring long incubation time. Other inducers such as glucose, galactose and sucrose had no effect on enzyme production. So soybean cake powder was a constant component in the fermentation medium for α-galactosidase production in the following studies (P < 0.001).
Effect of different inducers on α-galactosidase production. The error bars indicate the standard deviation calculated from three independent parallel experiments. (***) Significantly different from control at P < 0.001 by one-way ANOVA. Control: the liquid induction culture medium.
Glucose, galactose, sucrose, raffinose and corn flour at different concentrations were individually added in basal liquid culture medium, and the activity of α-galactosidase produced by T. versicolor was assayed. After fermentation for 8 days, the level of enzyme activity was observed when 0.5% galactose was used as a carbon source (256.4 U/mL), compared with the control group (219.5 U/mL) (P < 0.05). The effect of 0.5% sucrose on the enzyme activity was not obvious, and other carbon sources had negative effect on enzyme production (Fig. 2a).
The optimization of fermentation medium. (a) Effect of different carbon sources on α-galactosidase production. (b) Effect of different nitrogen sources on α-galactosidase production. (c) Effect of different metal ions on α-galactosidase production. The error bars indicate the standard deviation calculated from three independent parallel experiments. (***) Significantly different from control at P < 0.001; (**) Significantly different from control at P < 0.01; (*) significantly different from control at P < 0.05 by one-way ANOVA. Control: the basal liquid culture medium.
The effect of organic and inorganic nitrogen sources (peptone, yeast powder, sodium nitrate, ammonium nitrate, ammonium sulfate) on the enzyme production was tested, the results are showed in Fig. 2b. The highest level of α-galactosidase was observed in the control medium (222.9 U/mL). Significant repression of enzyme activity was observed in the presence of additional nitrogen sources (P < 0.05). In the process of fermentation, 2% soybean cake power in the control medium could be used as organic nitrogen source for the enzyme production, and addition of other organic nitrogen source is no longer required.
As is shown in Fig. 2c, Mg2+ (0.05%) and K+ (0.1%) ions were favorable for the enzyme production comparing with the control group (P < 0.05), while Ca2+ and Fe2+ ions have a negative effect on the α-galactosidase production.
An orthogonal L9(3)4 test design was used for optimization the culture conditions (Table 3). In this study, nine experiments were carried out at the concentration of soybean cake power 1.0%, 2.0% and 3.0%, galactose 0.50%, 0.60% and 0.70%, K+ 0.10%, 0.15% and 0.20%, Mg2+ 0.08%, 0.09% and 0.10% on the basis of the single-factor test (Fig. 1S). According to the value of Range, Mg2+ concentration (factor D) exerted the most significant effect on α-galactosidase production, and the order of importance that influenced α-galactosidase production was found to be D (Mg2+ concentration) > A (soybean cake power concentration) > B (galactose concentration) > C (K+ concentration). And the group of A1B2C2D2 could obtain the highest level of enzyme activity (288 U/mL). The optimal combination of the fermentation medium for α-galactosidase production was 1.0% soybean cake power, 0.60% galactose, 0.15% KH2PO4 and 0.09% MgSO4.
| Run | Factors | α-galactosidase activity (U/mL) | |||
|---|---|---|---|---|---|
| Soybean cake power (g/100 mL, A) |
Galactose (g/100 mL, B) |
K+ (g/100 mL, C) |
Mg2+ (g/100 mL, D) |
||
| 1 | A1 | B1 | C1 | D1 | 189 ± 3.56 |
| 2 | A1 | B2 | C2 | D2 | 288 ± 1.49 |
| 3 | A1 | B3 | C3 | D3 | 158 ± 2.12 |
| 4 | A2 | B1 | C2 | D3 | 194 ± 1.02 |
| 5 | A2 | B2 | C3 | D1 | 146 ± 3.74 |
| 6 | A2 | B3 | C1 | D2 | 185 ± 2.08 |
| 7 | A3 | A1 | C3 | D2 | 187 ± 1.15 |
| 8 | A3 | A2 | C1 | D3 | 123 ± 2.47 |
| 9 | A3 | A3 | C2 | D1 | 54.3 ± 1.23 |
| AVG 1 | 188 ± 2.86 | 190 ± 1.69 | 166 ± 1.88 | 130 ± 2.85 | |
| AVG 2 | 176 ± 1.45 | 163 ± 1.55 | 155 ± 2.86 | 198 ± 4.23 | |
| AVG 3 | 122 ± 1.23 | 133 ± 2.18 | 163 ± 2.86 | 158 ± 3.98 | |
| Ra | 66.3 | 56.7 | 10.6 | 67.9 | |
The effect of different incubation time on α-galactosidase production using the optimum liquid medium is displayed in Fig. 3. Sampling per one day was prepared for test. From day 4 onward there was no significant change in the activity of α-galactosidase. After 4 days, the activity begined to increase and obtained its peak value at 7 days (300 U/mL) under the optimum liquid medium.
Effect of incubation time on α-galactosidase production.
The α-galactosidase was purified through a multistep procedure as represented earlier (Fig. 4). As illustrated in Table 4, the α-galactosidase from T. versicolor was purified 322-fold, with a average recovery of 2.60% after enzyme purification. The specific activity against pNPG was 561 U·mg protein−1. According to FPLC and SDS-PAGE results, the molecular mass of monomeric TVG was 70 kDa (Fig. 5). The amino acid sequences of 3 inner peptides of TVG as acquired by ESI-MS/MS were FLYDLWGKD, GLKLTPQMGW and TSLANNQFVFA.
Elution profile of TVG on a (a) DEAE-cellulose column, (b) CM-cellulose column, (c) Q-sepharose column and (d) Superdex 75 HR 10/30 gel filtration. Black curves indicate protein content; red curves indicate α-galactosidase activity, the α-galactosidase activities were enriched in fractions D3, CM2, Q2 and SU1; blue curve indicate elution volume of standard proteins, from left to right: bovine serum albumin (67.0 kDa), ovalbumin (43.3 kDa), RNase A (13.7 kDa), aprotinin (6.51 kDa) and vitamin B12 (1.36 kDa).
| Chromatographic fractiona | Yield (mg) |
Total activity (U)b |
Special activity (U/mg)c |
Recovery activity (%) |
Purification foldd |
|---|---|---|---|---|---|
| Crude extract | 1.20×104 | 2.08×104 | 1.74 | 100 | 1.00 |
| D3 | 494 | 5.74×103 | 11.6 | 27.6 | 6.70 |
| CM2 | 8.25 | 2.55×103 | 309 | 12.3 | 177 |
| Q2 | 3.64 | 1.45×103 | 399 | 6.97 | 229 |
| SU1 | 0.98 | 550 | 561 | 2.60 | 322 |
SDS-PAGE analyse of TVG. Lanes: M, molecular mass standards; 1, Superdex 75 fraction.
As illustrated in Fig. 6, TVG showed the maximal activity at pH 3.0 in 100 mM Na2HPO4-citric acid buffer, whereas only 10% of its activity was discoverable at pH 7.0. Furthermore, TVG was comparatively stable over an acidic pH range of 2.0–5.0. The activity of TVG increased steadily from 20 °C and reached its maximum value at 60 °C, and declined from 60 °C until it reached residual level at 80 °C. Moreover, TVG had good thermal tolerance; only 12.1% of its activity was lost after 120 min incubation at 50 °C.
pH and temperature profiles of TVG. Effects of pH on the (a) activity and (b) stability of TVG; Effects of temperature on the (c) activity and (d) stability of TVG.
As shown in Table 5, Fe2+, K+, Ca2+, Mg2+, Mn2+, Zn2+ and Al3+ ions had negligible effect on enzyme activity when the concentration was increased from 1.25 mM to 10 mM. The modification of different amino acid functional groups by chemical reagents was shown in Fig. 7. In this study, N-bromosuccinimide (NBS) thoroughly suppressed the activity of TVG, while diethylpyrocarbonate (DEPC), diacetyl (DIC), carbodiimide (EDC) and dithiothreitol (DTT) did not affect the activity of TVG (data not shown).
| Metal ion concentration | Relative activity (%)a | |||
|---|---|---|---|---|
| 10 mM | 5 mM | 2.5 mM | 1.25 mM | |
| Fe2+ | 106 ± 2.12 | 91.5 ± 1.03 | 99.1 ± 2.06 | 92.1 ± 2.98 |
| K+ | 95.2 ± 2.11 | 94.3 ± 3.26 | 94.2 ± 2.88 | 83.2 ± 3.23 |
| Ca2+ | 105 ± 2.93 | 104 ± 2.68 | 109 ± 1.06 | 83.1 ± 3.93 |
| Cd2+ | 56.5 ± 1.31 | 69.7 ± 1.98 | 76.9 ± 1.89 | 78.5 ± 2.56 |
| Cu2+ | 45.2 ± 1.23 | 52.9 ± 4.58 | 58.5 ± 2.56 | 64.3 ± 1.37 |
| Hg2+ | 52.3 ± 1.81 | 65.1 ± 2.11 | 75.1 ± 1.98 | 80.1 ± 3.28 |
| Mg2+ | 106 ± 2.63 | 105 ± 1.85 | 105 ± 1.41 | 101 ± 1.20 |
| Mn2+ | 87.1 ± 1.01 | 104 ± 1.97 | 96.1 ± 2.13 | 110 ± 1.25 |
| Pb2+ | 13.9 ± 1.21 | 25.3 ± 2.45 | 44.2 ± 2.99 | 48.2 ± 1.98 |
| Zn2+ | 107 ± 2.56 | 103 ± 2.98 | 100 ± 4.54 | 90.1 ± 1.91 |
| Al3+ | 97.5 ± 4.45 | 96.1 ± 1.65 | 91.8 ± 1.89 | 94.6 ± 3.87 |
| Fe3+ | 0.00 ± 0.00 | 2.10 ± 0.00 | 15.6 ± 2.69 | 68.7 ± 2.98 |
Effect of chemical modification reagents NBS on the activity of TVG.
As shown in Table 6, TVG revealed the highest efficiency to pNPG, while had no detectable activity against p-nitrophenyl β-D-galactopyranoside and p-nitrophenyl a-D-glucopyranoside. Of the natural substrates tested, TVG was most active on disaccharide melibiose, followed by the tetrasaccharide stachyose and the trisaccharide raffinose. The polysaccharide, such as guar gum, was also a good substrate for TVG. The enzyme displayed meager activity on locust bean gum with the hydrolysis rate of 12.7%. As shown in Table 7, The Km values of TVG for hydrolysis of pNPG, melibiose, raffinose and stachyose were 0.651 mM, 3.66 mM, 15.1 mM, and 4.47 mM, respectively. This was in keeping with the above results showing that TVG displayed a higher affinity (lower Km) toward pNPG than natural oligosaccharides as substrates. The catalytic efficiency expressed by kcat/Km showed that the substrate pNPG was used most efficiently by the enzyme. Galactose was proved to be a noncompetitive (uncompetitive and noncompetitive mixed) inhibitor of TVG when pNPG was used as tested substrate (Fig. S2). The inhibition constants (Ki and Kis) were determined by Dixon plot to be 2.88 and 0.132 mM, correspondingly.
| Substrate | Concentration | Relative activity (%)a |
|---|---|---|
| pNPG | 10 mM | 100 ± 0.210 |
| p-Nitrophenyl β-D-galactopyranoside | 10 mM | nd |
| p-Nitrophenyl α-D-glucopyranoside | 10 mM | nd |
| Melibiose | 50 mM | 54.6 ± 2.51 |
| Rafnose | 50 mM | 38.4 ± 2.04 |
| Stachyose | 50 mM | 43.4 ± 1.11 |
| Locust bean gum | 1.00% | 12.7 ± 1.24 |
| Guar gum | 1.00% | 35.2 ± 3.82 |
| Substrate | Km (mM) | Vmax (mM·min−1) | kcat (s−1) | kcat/Km (mM−1·s−1) |
|---|---|---|---|---|
| pNPG | 0.651 | 0.0624 | 27.5 | 42.2 |
| Melibiose | 3.66 | 0.118 | 5.36 | 1.46 |
| Raffinose | 15.1 | 0.215 | 9.77 | 0.647 |
| Stachyose | 4.47 | 0.153 | 6.96 | 1.56 |
As shown in Table 8, guar gum was treated with α-galactosidase alone for 12 h but the least reducing sugar content (79.8 µg). Fermentation broth (group 8) has the best degradation effect by 1.93 fold compared to hydrolysis in the presence of α-galactosidase alone. More reducing sugar was released (154 µg) with shortest degradation time (7 h). The effect of simultaneous and sequential action was more remarkable than the effect of α-galactosidase or mannanase alone. The group 7 showed a comparatively good degradation ability of guar gum, and the released sugars were assayed by HPAEC (Fig. 3S). After 12 h of treatment, the ratio of released galactose and mannose in group 7 was about 3.87.
| Run | First enzyme | Reaction Time (h) | Second enzyme | Reaction Time (h) | Reducing sugar content (µg) | Degree of synergy (fold increase in activity) |
|---|---|---|---|---|---|---|
| 1 | Mannanase | 5 | No | 7 | 93.9 ± 3.34 | 1.18 |
| 2 | α-galactosidase | 5 | No | 7 | 79.8 ± 2.05 | 1.00 |
| 3 | Man/Gal | 5 | No | 7 | 103 ± 1.05 | 1.29 |
| 4 | α-galactosidase | 5 | Mannanase | 7 | 106 ± 2.33 | 1.33 |
| 5 | α-galactosidase | 12 | Mannanase | 5 | 106 ± 1.00 | 1.33 |
| 6 | Mannanase | 12 | α-galactosidase | 5 | 116 ± 0.870 | 1.45 |
| 7 | Mannanase | 5 | α-galactosidase | 7 | 117 ± 0.961 | 1.46 |
| 8 | Fermentation broth | 7 | No | 0 | 154 ± 3.34 | 1.93 |
It has been amply reported that α-galactosidase in microbiology was an inducible enzyme. Generally the inducer can be substrate, substrate analogue or degradation product of α-galactoside. In this study, soybean cake powder was the optimum inducer for α-galactosidase production by T. versicolor. The by-product of nature plant always acts as substrate for α-galactosidase production. The α-galactosidase was produced from Lactobacillus agilis by submerged fermentation using soybean vinasse as substrate (Sanada et al. 2009). In another study, red gram plant waste served as the best substrate for α-galactosidase production by Aspergillus oryzae as it gave the highest enzyme activity compared with other possible substrate (Shankar and Mulimani 2007). Some monosaccharides and oligosaccharides could also induce the enzyme activity. The α-galactosidase from Talaromyces flavus was obtained under induction of quinovose, and the role of this deoxysugar on the induction of the production of α-galactosidase seemed to be quite specific, as it was not obtained in the presence of raffinose, a common α-galactosidase inducer (Simerska et al. 2007).
The carbon source involved in microbial enzyme production is one of the most important factors (Hsu et al. 2005; Liu et al. 2007). In our study, galactose was chosen as the carbon source for subsequent experiments for α-galactosidase production (P < 0.05). This result was similar with other reports, the media containing galactose was effective for α-galactosidase production by Aspergillus foetidus ZU-G1 (Liu et al. 2007). The maximum α-galactosidase activity from A. parasiticus MTCC-2796 was achieved in the presence of galactose followed by melibiose and raffinose (Mishra et al. 2009). In other studies, α-galactosidase production needs different carbon sources. Thermomyces lanuginosus CBS395.62/b with a good growth on galactomannan-based medium was declared to synthesize a good deal of α-galactosidase (Svastits-Dücső et al. 2009). In the present study, the α-galactosidase activity decreased along with the increase of carbon source concentration. Because the “glucose effect”, a common phenomenon in microbial enzyme production, exists in the process of fermentation (Liu et al. 2007). Available carbon source was first consumed for microbial growth in the presence of galactose and without α-galactosidase production. In the case of exhaustion of available carbon source, bean cake power as carbon source and inducer made the enzyme production.
Apart from the carbon source and nitrogen source, inorganic salt was an indispensable inorganic element for the cell life (Crichton et al. 2008). In our study, four metal ions (Mg2+, K+, Ca2+, Fe2+) were chosen to take the place of two salt ions in the basic medium and the control group containing no metal ion. Considering the results, Mg2+ and K+ ions were chosen as the metal ions for subsequent experiments on α-galactosidase production. Many researches demonstrated that Mg2+ and K+ ions were indispensable elements in the process of microbial growth and enzyme production, which can regulate pH of the culture medium, REDOX potential and osmotic adjustment (Mishra et al. 2009). The involvement of Mg2+ ions in membrane permeabilization and acting as ion channels has been well established (Raol et al. 2015).
Orthogonal design has been applied in the study of optimization fermentation combination, because the best formula ratio and the degree of factor influence can be obtained while only a small amount of calculation of the experimental results is needed (Huang and Zhang 2011; Tian et al. 2014). The present study demonstrated that the significant factors and the optimal condition for α-galactosidase production were identified by the orthogonal design with only nine experiments. If a traditional full factorial design is employed to examine the effects of four factors, each at three levels, a total of 64 (43) experiments have to be conducted (Chen et al. 2010). It is obvious that the orthogonal design can significantly reduce the experimental work load. AVG1–AVG3 was the average α-galactosidase production under the various investigated conditions, and the maximum value was the optimum value. In additition, according to the largest donating rule, the factor with the largest Range value (AVGmax-AVGmin) have the greatest effect on α-galactosidase production (Guo et al. 2013). In our study, the values of Range on soybean cake power, galactose, K+ and Mg2+ were 66.3, 56.7, 10.6 and 67.9, respectively. The order of importance that influenced α-galactosidase production was found to be Mg2+ concentration > soybean cake power concentration > galactose concentration > K+ concentration. And the optimal combination of the fermentation medium for α-galactosidase production was 1.0% soybean cake power, 0.60% galactose, 0.15% KH2PO4, 0.09% MgSO4. It is therefore feasible to employ the orthogonal design to determine the significance of different environmental factors and identify the optimal condition for α-galactosidase production.
The maximum activity of TVG was found on the 7 days of fermentation.With extending cultivation time, the yield of α-galactosidase had decreasing trend. The decline in total α-galactosidase activity might be due to inhibition of cellular functions which results from depletion of nutritional factors from the growth medium, deactivation of the enzyme due to pH change or due to inducer exclusion (Liu et al. 2007; Mishra et al. 2009).
The molecular mass of TVG was 70 kDa, which was similar to other α-galactosidases acquired from natural materials. The α-galactosidase from Thielavia terrestris had a molecular weight of 82 kDa (Saad and Fawzi 2012), and its counterparts from Bifidobacterium bifidum and A. oryzae showed a molecular weight of 82.8 kDa (Goulas et al. 2009) and 64 kDa (Dhananjay and Mulimani 2008), respectively. Amino acid sequences of three internal peptide fragments of the enzyme were FLYDLWGKD, GLKLTPQMGW and TSLANNQFVFA. Database search using BLAST revealed that TVG showed homology in partial sequence with α-galactosidase from Trametes pubescens (84.6%) and Aspergillus mulundensis (61.9%).
After incubation at pH 2.0–5.0 for 120 min, only 19.8% of its activity was lost, signifying that TVG is an acid-tolerant protein. Generally, α-galactosidases from fungi were often characterized by a low optimum pH (pH 4.0–6.0), which makes them potentially useful in several applications occurring at acidic pH conditions (Zhang et al. 2015). The acidic pH of TVG is very favorable in keeping with the physicochemical conditions of the stomachs of monogastric animals. Therefor it can be used in degrading the oligosaccharides of the gastrointestinal tracts of the animals (Du et al. 2013). TVG had good thermal tolerance, approximately 88.1% of its activity remained after 120 min incubation at 50 °C. This temperature property was analogous to α-galactosidase from Rhizomucor miehei and Gibberella sp. F75 (Cao et al. 2009; Katrolia et al. 2012b).
The effects of various metal ions and other chemicals on TVG activity were assayed and the results suggest that TVG was very sensitive to some metal ions. The activity of TVG was strongly affected by Cu2+, Cd2+ and Hg2+ ions and almost undetectable in the presence of Pb2+ ions. Hg2+ ions could inhibit the enzyme activity due to its reaction with amino and imidazolium residues of histidine, thiol groups, and peptide linkages (Geng et al. 2015; Singh and Kayastha 2012a). The decrease of the α-galactosidases activity by Cu2+, Cd2+ and Hg2+ ions was consistent with previous researches including α-galactosidases from Pleurotus djamor and T. eurrhizus (Hu et al. 2016b; Zhang et al. 2015). Moreover, Fe3+ ions can strongly inactivated the activity of TVG at the concentration of 10 mM. This results agree with finding represented for the α-galactosidases isolated from Tricholoma matsutake and Pleurotus citrinopileatus (Geng et al. 2015; Hu et al. 2016a). The amino groups of tryptophan, histidine and arginine were chemically modified by NBS, DEPC, and DIC, respectively (Geng et al. 2015; Gote et al. 2007). The carboxyl groups and disulfide bonds were modified by EDC and DTT, respectively (Huang et al. 1991; Zhu et al. 1995). In this study, chemical modification reagents (DEPC, EDC, DIC, DTT) did not affect the activity of TVG, which manifested that the residues of tryptophan, histidine and arginine, carboxyl groups and disulfide bonds were absent at or near by the active center. As the tryptophan modifying agent, NBS completely inhibited the activity of TVG. Pretreatment of TVG with 0.2 mM NBS for 0.5 h resulting in almost exhaustive destruction of α-galactosidase activity, which displayed that tryptophan was essential for the activity of TVG.
Substrate specificity of TVG was examined by employing oligosaccharides, polysaccharides and synthetic substrates. TVG was devoid of activity toward synthetic substrates p-Nitrophenyl β-D-galactopyranoside and p-Nitrophenyl α-D-glucopyranoside. The result was similar with α-galactosidase from T. matsutake and Irpex lacteus (Geng et al. 2015; Guo et al. 2016). The enzyme showed the highest hydrolysis activity toward pNPG, which suggested that probably the para configuration of the substrate pNPG facilitated its access to the active site of enzyme (Du et al. 2014). The calculated Km of TVG (0.651 mM) toward pNPG was lower than that of α-galactosidase from T. matsutake (0.99 mM), P. djamor (0.76 mM) and Neosartorya fischer (1.52 mM), but much higher than that of the α-galactosidase from P. citrinopileatus (0.21 mM) (Geng et al. 2015; Hu et al. 2016a; Hu et al. 2016b; Wang et al. 2014). TVG is noncompetitively (uncompetitively and non-competitively mixed) inhibited by galactose; a similar result has been reported by Gao et al. (2016) and Jang et al. (2019). The inhibition mode of galactose on TVG was different from those on many other counterparts which were competitively inhibited by galactose (Mutra et al. 2018; Singh and Kayastha 2012b). The inhibition constant Ki of TVG was determined to be 2.88 mM, which was higher than that of α-galactosidase from Aspergillus terreus (0.76 mM) , indicating that TVG is more tolerant to galactose than this enzyme (Ferreira et al. 2011).
The main composition of guar gum is galactomannan, which belongs to the form of polysaccharide of galactoside, complete degradation of guar gum need mixture enzyme to act (Roberts 2011; Wang et al. 2000). In the present study, the effect of simultaneous and sequential action was more remarkable than the effect of galactosidase or mannanase alone. Synergistic action having good degradation effect ascribes to the differentence of mechanistic properties of mannanase and α-galactosidase. Mannanase hydrolyzes internal β-1,4-linkages of the mannose backbone, and debranching of galactose side chains by α-galactosidase increased the degradation rate of guar gum (Moreira and Filho 2008; Mudgil et al. 2014). Another, mannanase and galactosidase working together with guar gum only need 5 h which can generate the same amount of reducing sugar with the effect of the two enzymes sequential work 12 h. This is because synergistic degradation of guar gum by TVG and mannanase can accelerate the mannose backbone rupture and release the sugar chain of galactose. Mahammad (2006) first revealed a synergetic degradation of guar gum by sequential or simultaneous action. Along with the debranching of guar gum by α-galactosidase, the rate of viscosity reduction was enhanced by β-mannanase. Sequential addition of α-galactosidase (r-AgalB) and mannanase (MEY-1) remarkabley improved the hydrolysis efficiency of guar gum comparing with α-galactosidase or mannanase lonely effecting on guar gum. Furthermore, the degradation of guar gum by the enzyme combination was promoted when the reaction time increased from 5 h to 12 h, or temperature increased from 37 °C to 55 °C (Wang et al. 2010). It is very important to degrade guar gum in industry, which can decrease the viscosity of solution or change the molecular structure of guar gum for better application in food, cosmetic, medicine and other fields (Mudgil et al. 2012). For example, partially hydrolyzed guar gum produced from guar gum by enzymatic process has been widely used in food formulation such as beverages and breads, food binder, and source of dietary fiber (Roberts 2011; Yoon et al. 2008).
In this study, the optimizing production, purification and characterization of TVG were studied in detail. The optimal liquid medium for TVG production was composed of 1.0% soybean cake power, 0.60% galactose, 0.15% KH2PO4 and 0.09% MgSO4. TVG displays distinctive traits including thermal and pH stabilities, and effective degradation on guar gum. In future researchs, high levels of TVG can be cloned and heterologously expressed for purification. Immobilization technology is also an effective measure to enhance the enzymatic propertie. These findings indicate that TVG is a promising product with potential applications in industry and research.
Acknowledgements This work was financially supported by Special Foundation for Agro-scientific Research in the Public Interest (No. 201303080) and Special Foundation for Outstanding Youth of Henan Academy of Agricultural Sciences (No. 2018YQ13).
