1. Introduction
α-galactosidase (EC 3.2.1.22) is an exoglycosidase that specifically catalyzes the hydrolysis of α-1,6-linked D-galactosyl residues of α-galactooligosaccharides and galactomannans [
1]. α-galactosidase has been widely applied in clinical medicine, food processing, animal feed, and pulp and paper industries [
2,
3,
4,
5,
6]. Raffinose family oligosaccharides (RFOs) have been identified as antinutritional factors in soybean because they cannot be decomposed in humans or other monogastric animals and are prone to induce flatulence and gastrointestinal disturbance [
7]. The enzymatic processing of soybean products by α-galactosidases has been reported to be one of the most effective techniques to reduce RFOs levels in soybean products, thus increasing their nutritional values [
4,
8]. At present, the yield, activity, and heat resistance of α-galactosidase are increased mainly by genetic engineering bacteria construction and amino acid mutation, but these two methods may not guarantee the safety of the enzyme. Therefore, it is particularly urgent to develop natural and safe food-derived α-galactosidase. Plants, animals, and microorganisms are known sources of α-galactosidase. The edible fungi are preferred enzyme sources in food industries because of their high nutritional value and unique characteristics. Moreover, the enzymes isolated from edible fungi were mild, safe, green, and environmentally friendly. Some α-galactosidases have been confirmed to possess the potential to hydrolyze RFOs, and they are derived from edible fungi such as
Irpex lacteus [
7],
Pleurotus citrinopileatus [
9],
Leucopaxillus tricolor [
10],
Coriolus versicolor [
11],
Tremella aurantialba [
12],
Termitomyces eurrhizus [
13],
Hericium erinaceus [
14],
Tricholoma matsutake [
15],
Pleurotus djamor [
16], and
Agaricus bisporus [
17].
Oudemansiella radicata (belonging to the
Oudemansiella genus,
Physalacriaceae family, Agaricomycetes class) is a kind of precious edible medicinal mushroom farmed in China, and it is known as “Edible Queen” for its high nutritious and economic value [
18].
O. radicata is rich in polysaccharides, enzymes, amino acids, vitamins, triterpenes, ergosterols, and other nutrients, and it is a healthy, green, and safe food-derived enzyme source [
19]. Recently, the isolation and characterization of some active enzymes from
O. radicata, such as ribonuclease, metalloprotease, laccases, and cellulase, have attracted researchers’ attention [
19,
20,
21]. However, to our knowledge, there is little research on the characterization of α-galactosidase from
O. radicata.
Nowadays, the increasing harsh conditions of industrial processes and the need to reduce costs have made immobilized enzymes very attractive for researchers. Not only can the enzyme immobilization technique improve the stability of the enzyme and prolong the reaction time, but it can also simplify the downstream processing and improve the operation stability. Although there are many newly developed immobilization carriers, sodium alginate (Na-alginate) is still widely used owing to its excellent biocompatibility, low cost, non-toxicity, high stability, and good spheroidization [
22]. Chitosan, a rare alkaline polysaccharide with a positive charge in nature, is incorporated as a dopant into alginate gels [
23]. In this study, the sodium alginate–chitosan co-immobilization method was adopted to immobilize α-galactosidase from
O. radicata. This method can not only solve the problems with the Na-alginate embedding method, such as high leakage of biomolecules, low mechanical strength, and large pore size, but also retain the enzyme activity to the maximum extent, and, thus, it exhibits great application potential and development prospects [
24]. Moreover, the carboxylate part of Na-alginate could ionically interact with the protonated amino group of its chitosan counterpart to form a physically cross-linked hydrogel, thus effectively avoiding enzyme molecule leakage [
25]. Meanwhile, chitosan outside the Na-alginate can also be cross-linked with glutaraldehyde to form schiff base in the immobilization process.
In this study, a novel α-galactosidase (ORG) was first purified and characterized from O. radicata by using ion-exchange chromatography and gel filtration. Then, the purified enzyme was immobilized by the sodium alginate–chitosan co-immobilization method. The characterization of free and immobilized enzymes and their potential application in the removal of the RFOs from soymilk were investigated. The stability of temperature, pH, and storage were evaluated and compared between free and immobilized enzymes. In addition, the reusability of the immobilized enzyme was examined. This study laid a foundation for the application of O. radicata-derived α-galactosidase in the food industry, and provided a new perspective for the intensive processing of functional substances in edible fungi.
2. Materials and Methods
2.1. Materials
The dry fruiting bodies of O. radicata were purchased from Yunnan Hanlu Fungus Industry Co., Ltd. (Yunnan, China). DEAE-Sepharose, CM-Sepharose, and Q-Sepharose were obtained from Sigma Chemical Co., Ltd. (Saint Louis, MO, USA). Superdex75 10/300 GL and AKTA purifier were purchased from GE Healthcare, Chicago, IL, USA. The protein molecular weight standards (Blue Plus Protein Marker) were supplied by Beijing TransGen Biotech Co., Ltd. (Beijing, China). All the substrates (such as 4-nitrophenyl-α-D-galactopyranoside (pNPG)) and proteases were purchased from Beijing Solarbio Life Sciences Co., Ltd. (Beijing, China). The commercial glucose oxidase (GOD) kit was purchased from Beijing Solarbio Life Sciences Co., Ltd. The reagents (sodium hydroxide, glacial acetic acid, calcium chloride, pentanediol, citric acid, dinitrosalicylic acid (DNS), and sodium acetate (NaAc)) were obtained from Tianjin Kaitong Chemical Reagent Co., Ltd. (Tianjin, China), and they were analytically pure.
2.2. Enzyme Activity Standard Assay and Protein Content Determination
α-galactosidase activity was determined with
pNPG as a substrate. As previously reported [
26], the 40 μL diluted enzyme solution was mixed evenly with 40 μL NaAc-HAc buffer (100 mM, pH 4.6) containing 10 mM
pNPG and reacted for 10 min at 40 °C. Then, the reaction was terminated by adding 320 μL Na
2CO
3 solution (500 mM), and the absorbance was determined at 405 nm. The 0.1 g immobilized enzyme was mixed evenly with 0.1 mL NaAc-HAc buffer (100 mM, pH 4.6) containing 10 mM
pNPG and reacted for 10 min at 40 °C. Then, the reaction was terminated by adding 0.8 mL Na2CO3 solution (500 mM), and the absorbance was determined at 405 nm. The amount of enzyme that released 1 μmol
p-nitrophenol from
pNPG per min at 40 °C was defined as one unit (U) of enzyme. Protein content was measured by the Bradford method with bovine serum albumin as the standard protein [
27]. Specific enzyme activity was expressed as U/mg protein.
2.3. Purification of α-Galactosidase
The dry fruiting bodies of O. radicata were mixed with normal saline at the ratio of 1:10 (w/v), homogenized using a wall-breaking machine, and extracted at 4 °C for 4 h. The extract was centrifuged at 6500 rpm for 15 min at 4 °C. The supernatant was dialyzed overnight (3500 Da molecular interception) to obtain the crude enzyme solution. The crude enzyme solution was subjected to anion exchange chromatography on DEAE-Sepharose column (5 cm × 20 cm) pre-equilibrated with 10 mM NaAc-HAc buffer (pH 4.4) at a flow rate of 10 mL/min. After removal of unabsorbed proteins, adsorbed proteins were eluted with NaAc-HAc buffer (pH 4.4) containing 50 mM, 150 mM, and 1 M NaCl, respectively. After the dialysis with distilled water, the active fraction with high α-galactosidase activity was chromatographed on cation exchange CM-Sepharose column (2.5 × 30 cm) pre-equilibrated with 10 mM NaAc-HAc buffer (pH 4.0) at a flow rate of 4 mL/min. After removal of unadsorbed proteins, adsorbed proteins were obtained by elution with NaAc-HAc buffer (pH 4.0) containing 50 mM, 150 mM, and 1 M NaCl. After dialysis, the fractions with the high α-galactosidase activity were pooled and then loaded onto Q-Sepharose column (1.5 cm × 10 cm) which was equilibrated with 10 mM NaAc-HAc buffer (pH 4.0) at a flow rate of 1 mL/min in advance. After removal of unadsorbed protein, adsorbed proteins were eluted with NaAc-HAc buffer (pH 4.0) containing a linear gradient NaCl (0~1 M). The fractions with the high α-galactosidase activity were pooled, dialyzed, and freeze-dried. Subsequently, Superdex75 10/300 gel filtration column (25.6 mL) was pre-equilibrated with 10 mM NaAc-HAc buffer (pH 4.0) containing 100 mM NaCl. The dry protein powder was homogenized in 200 μL deionized water and centrifuged at 10,000 rpm for 5 min at 4 °C. After being loaded onto the gel filtration column, the supernatant was subjected to fast protein liquid chromatography (FPLC) using an AKTA Purifier at a flow rate of 0.8 mL/min. The active fractions were pooled, concentrated by ultrafiltration with Amicon Ultra-15 centrifugal filter devices (50,000 MWCO), and loaded onto Superdex75 10/300 gel filtration column again, followed by FPLC. The active fractions were pooled, concentrated by ultrafiltration (50,000 MWCO), and freeze-dried for further analysis. The obtained enzyme powder was named ORG.
2.4. Determination of Molecular Mass
The molecular mass of the ORG was detected using gel filtration in combination with SDS-PAGE. A standard curve based on elution volume and molecular mass standards (GE Healthcare) was obtained, and then the native molecular mass of the α-galactosidase was calculated based on the elution volume. Blue Plus Protein Marker was used as the molecular mass standard with the range of 14 kDa~100 kDa. In SDS-PAGE, a 12% resolving gel and a 5% stacking gel were used following the standard procedure.
2.5. Analysis of Amino Acid Sequence
The purified ORG band was excised, digested, and then dissolved in 0.1% formic acid and 2% acetonitrile for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The inner amino acid sequences of ORG were compared with those of α-galactosidases from other sources by Mascot. Sequence homologues were obtained from NCBI database.
2.6. Immobilization of ORG
The ORG was immobilized using a previously reported method with minor modification [
24]. The ORG was dissolved in 10 mM NaAc-HAc solution (pH 4.6). The dissolved enzyme solution and 0.04 g/mL. Na-alginate solution was mixed evenly at the ratio of 3:1. The chitosan was dissolved in 2% acetic acid to prepare 10 mL 5% chitosan–acetic acid transparent solution, and then the transparent solution was mixed with 4% CaCl
2 solution at the ratio of 1:1. The mixture was stirred evenly by a magnetic stirrer, adjusted to pH 5.0, and stood. The Na-alginate solution of embedded enzyme was dropped evenly into chitosan–CaCl
2 mixture solution though a 5 mL syringe to generate the immobilized beads with a diameter of about 0.15 cm, and then the resultant beads were cooled on ice. The immobilized enzyme was stood at room temperature (20~25 °C) for 1 h, cleaned, and put into 2% glutaraldehyde for cross-linking reaction at 4 °C for 2 h. After cross-linking and cleaning, the final obtained enzyme was the immobilized α-galactosidase from
O. radicata, and it was named iORG. The enzyme activity and protein content of iORG were measured. The enzyme immobilization efficiency (also called immobilization yield) was defined as the difference between the activity of the free enzyme and the residual activity in the supernatant at the end of the immobilization period, multiplied by 100 and divided by the activity of the free enzyme [
24]. The activity recovery (also called recovered activity or expressed activity) of the immobilized α-galactosidase is the percentage of enzyme activity that is maintained in the immobilized enzyme compared to the offered enzyme activity [
28]. The specific activity yield (%) was defined as the specific activity of immobilized enzyme, multiplied by 100 and divided by the specific activity of the free enzyme [
22].
2.7. Biochemical Characterization
2.7.1. Effects of Temperature and pH on Activity of ORG and iORG
The optimal pH of ORG and iORG was determined within the pH range of 2.2~8.0 with 100 mM Na2HPO4-citric acid buffer. ORG or iORG solution was mixed with pH buffers (2.2~8.0) and incubated at 4 °C for 2 h. Afterwards, the pH stability was detected by the residual enzyme activities. The optimal temperature of ORG and iORG was determined over the temperature range (4~90 °C). The thermostability of ORG and iORG was measured by the residual activities after 2 h incubation at each temperature (4, 10, 20, 30, 40, 50, 60, 70, 80, and 90 °C). In order to determine the effects of the incubation time on thermostability of ORG and iORG, these two enzymes were incubated separately at 50 °C and 60 °C each for 180 min. At 30 min interval, the enzymes were withdrawn, cooled immediately, and tested for residual enzyme activities by standard assay.
2.7.2. Effects of Metal Ions, Chemical Reagents, and Side Modification Reagents on ORG
Various metal ions (2.5, 5, 10, and 20 mM), chemical reagents (2, 20, and 200 mM), or side modification reagents (0.4, 0.8, 1.2, 1.6, and 2.0 mM) were mixed with ORG at the ratio of 1:1 and incubated at 37 °C for 2 h to investigate their influences on ORG. After incubation, the relative activity of ORG was detected by standard assay.
2.7.3. Effects of Proteases on Activity of ORG
The ORG was incubated at 37 °C with 2, 10, or 20 mg/mL seven proteases, separately at the ratio of 1:1 for 1 h (including pepsin, pH 4.0; acid protease, pH 4.0; alkaline protease, pH 8.0; neutral protease, pH 7.0; trypsin, pH 7.0; α-chymotrypsin, pH 7.0; or papain, pH 7.0). ORG incubated under the same conditions without proteases was used as control. After incubation, the residual activity of ORG was measured by standard assay.
2.7.4. Substrate Specificity Determination
The specificity of ORG towards 4 synthetic substrates (
pNPG, oNPG, 4-nitrophenyl β-D-glucuronide, and 4-nitrophenyl α-D-glucopyranoside) was measured by standard assay. The activity of the ORG towards natural substrates including oligosaccharides (such as raffinose and stachyose) and polysaccharides (locust bean gum and guar gum) was determined using the DNS method [
29] with minor modifications. Enzyme solution was mixed with natural substrates at the ratio of 1:1. After 30 min incubation at 50 °C, the reducing sugar content was determined. The amount of enzyme that released 1 µmol of reducing sugar from natural substrates per min at 50 °C was defined as one unit (U) of α-galactosidase activity. The activity of the ORG towards melibiose and maltose substrates was determined by using a glucose oxidase (GOD) kit.
2.7.5. Inhibitors Kinetics
The inhibition mode of ORG by galactose and melibiose was determined by the Lineweaver–Burk plot, respectively. The pNPG concentrations were 0.5, 1, 2, 4, and 8 mM. The concentrations of the inhibitor melibiose were 0, 1, 3, 5, and 10 mM, and the inhibitor galactose concentrations were 0, 1, 5, 10, and 20 mM. The inhibition constant Ki was determined from Dixon plot, and enzymatic reactions were performed by standard assay with pNPG (1.5~3 mM) as substrate and with galactose or galactose as an inhibitor.
2.7.6. Enzymatic Hydrolysis of Raffinose and Stachyose
The reaction system NaAc-HAc buffer (1 M, pH 4.6) contained 1 mL ORG (2 U/mL), 0.5 mL raffinose (50 mM), and 0.5 mL stachyose (50 mM). The mixture was incubated at 40 °C. Aliquots of the reaction mixtures were withdrawn at various time intervals and boiled for 5 min to stop the reaction. The release amount of reducing sugar was determined by the DNS method. The hydrolysis products were analyzed by thin-layer chromatography (TLC).
2.7.7. Kinetic Constants of ORG and iORG
The Michaelis–Menten plot was drawn in OriginPro 2021 software, and based on it, the kinetic constants (Km and Vmax) of substrate hydrolysis were computed. The enzyme activity was determined by standard assay with pNPG (0.5~4 mM) as substrate and by DNS method mentioned in 2.7.4 with raffinose (2~40 mM) and stachyose (2~40 mM) as substrates. The catalytic efficiency constant (Kcat/Km) was calculated based on Km and Vmax.
2.7.8. Storage Stability and Reusability
ORG and iORG were stored for 10 days at 4 °C and room temperature (20~25 °C). Enzyme activity was determined by standard assay every 2 days. The enzyme activity determined on the first day was used as control (100%), based on which enzyme activity at other time points was measured. The enzyme activity was determined by standard assay with pNPG as substrate and by DNS method mentioned in 2.7.4 with raffinose as substrate to investigate the reusability of iORG. After the reaction was completed, iORG was washed with deionized water and NaAc-HAc buffer (10 mM, pH 4.6) successively and used again in a new reaction system. The above experiments were repeated 6 times. The activity of iORG in the first reaction was used as control (100%) for calculating the relative activity of iORG after each use.
2.8. Elimination of RFOs from Soymilk by ORG and iORG
The soymilk was prepared using soybeans purchased from the local market according to the method described previously [
30]. Soymilk (5 mL) was added with (0.2~1.2 U/mL) ORG or iORG and incubated at 50 °C for 5 h in a shaking water bath at 100 rpm to determine the optimal enzyme dosage. After incubation, the samples were boiled for 5 min and centrifuged (10,000 rpm, 10 min). After degradation of soymilk samples, the released reducing sugar content was determined by DNS method [
29]. In addition, soymilk (5 mL) was incubated with ORG or iORG (1 U/mL) at 50 °C for 7 h in a shaking water bath at 100 rpm. The soymilk treated with distilled water instead of the enzyme solution by the same method that was used as a blank control. Aliquots of the reaction mixtures were withdrawn every hour and boiled for 5 min to terminate the reaction. The reducing sugar content released from the withdrawn aliquots at different incubated times was determined by DNS method to determine the optimal incubated time. When soymilk was incubated with iORG, aliquots of the reaction mixtures were withdrawn every hour, and the hydrolysates of reaction mixtures were detected by the TLC method. The reaction mixtures were spotted onto a silica gel 60 plate (Merck, Germany), and developed using solution with the n-butanol/methanol/water ratio of 6:4:3 (
v/v/v). The sugar spots were visualized by heating the plate at 95 °C for 10~15 min after being sprayed with the chromogenic reagent (consisting of 1 g diphenylamine, 1 mL aniline, 50 mL acetone, and 5 mL85% phosphoric acid).
2.9. Statistical Analysis
OriginPro 2021 software (OriginLab Corporation, Northampton, MA, USA) was used for plotting. All experiments were performed in triplicate. The data were expressed as mean ± standard deviations (SD). One-way analysis of variance (ANOVA) was performed using SPSS 20.0 software (IBM Inc., Chicago, IL, USA). p < 0.05 was considered as statistically significant.
4. Discussion
ORG, as the native purified enzyme, might be a heterodimer, and its molecular mass is 74 kDa. It has been reported that α-galactosidases from
A. squamosa seeds [
35] and
P. citrinopileatus [
9] are also heterodimer, whereas those from
L. elegans [
40] and
Thielavia terrestris [
43] are homodimer. The α-galactosidases exist in both monomeric and multimeric forms, exhibiting structural diversity [
7,
36,
45,
47]. Based on sequence similarity, the α-galactosidases are divided into glycoside hydrolase (GH) family 4, 27, 36, 57, 97, and 110 [
7,
17,
33,
40]. Most reported fungal α-galactosidases belonged to GH family 27 and 36. ORG shared considerably high sequence similarity to the reported GH family 27 α-galactosidases, suggesting that ORG should be a new α-galactosidase member of the GH family 27.
Similar to α-galactosidases from
C. versicolor [
11],
A. bisporus [
17], and
A. squamosa seeds [
35], ORG is an acidic α-galactosidase. The optimal temperature at which ORG exhibited the maximum activity was 50 °C, and this optimal temperature of ORG was the same with that of α-galactosidases from
P. citrinopileatus [
9],
L. tricolor [
10], and
Aspergillus oryzae YZ1 [
47], and it was higher than that of other α-galactosidases reported in previous studies [
36,
39] (
Table 7). Although ORG displayed higher thermostability than some natural mushroom α-galactosidases [
10,
15,
17], its thermostability was lower than some recombinant α-galactosidases from thermophilic fungi [
43,
45] and other sources [
7,
36], which would limit its industrial application to some extent. Nowadays, various methods have been developed to improve the thermostability and pH stability of α-galactosidases [
8,
23,
33]. In the present study, the sodium alginate–chitosan co-immobilized method was adopted to increase the thermostability and pH stablity of ORG. The pH profiles illustrated that the immobilization increased the pH stablity of ORG and shifted the optimal pH of ORG to the alkaline side (
Figure 3a,b). Previous literature showed that the alteration of enzyme microenvironment due to immobilization or support is the main reason for the change of optimum pH value [
48]. The carboxylate part of Na-alginate could ionically interact with the protonated amino group of the chitosan counterpart to form a net anionic charge. The concentration of [H
+] within the microenvironment of immobilized enzyme is higher than that in the bulk of the medium, which causes the pH-activity curve to shift to the alkaline. The thermostability assay results showed that iORG demonstrated significantly higher thermostability than ORG within the temperature range of 4~70 °C (
Figure 3d–f). This might be due to the immobilization of the enzyme to the support, providing stability and resulting in formation of the enzyme–substrate complex without any hindrance for the access of substrates to the active site [
39,
49]. Besides, the calcium alginate–chitosan matrix absorbs a large amount of heat and preserves the enzyme against denaturation, which is an important reason for the improved thermal stability of the immobilized enzyme [
25]. It is worth noting that iORG exhibited higher catalytic efficiency towards stachyose (6.88 mM
−1·s
−1) and raffinose (7.83 mM
−1·s
−1) than some reported α-galactosidases [
14,
32,
33,
36,
45] (
Table 6).
RFOs are anti-nutritional factors in soybean and other legumes, and they can cause flatulence. So far, many α-galactosidases have been reported to remove RFOs from soymilk [
31,
33,
37,
45,
46,
50]. However, some α-galactosidases exhibited defects such as low enzymatic activity and poor RFO removal capacity, reflected by slow and incomplete hydrolysis of RFOs, which might be due to their low catalytic efficiencies or low stability. For example, after soymilk was treated with α-galactosidase from
Aspergillus terreus for 12 h, the RFOs were incompletely hydrolyzed [
50]. Chen et al. (2015) reported that after soymilk was treated with 10 U/mL α-galactosidase from
R. miehei for 8 h, the RFOs were completely hydrolyzed [
44]. In this study, iORG completely hydrolyzed soybean milk in only 3 h, which could be attributed to its high catalytic efficiency and high stability. The degradation effect of iORG (within 3 h) on soymilk RFOs has been reported to be better than that of α-galactosidases from
B. megaterium [
36],
B. thetaiotaomicron [
33] and
A. oryzae YZ1 [
46], which is consistent with the results of catalytic efficiency. Among the reported enzymes, α-galactosidase from
I. lacteus showed the rapidest removal of RFOs from soymilk and complete hydrolysis towards stachyose and raffinose within 30 min [
7], which might be ascribed to a high-level expression of recombinant enzyme.
Furthermore, previous reports showed that the RFO degradation effects in soybean products (soymilk, soybean meal, etc.) were closely related to the enzyme dosage [
37,
45,
46]. One previous study reported that it took 6, 4, and 2 h for RFOs in soybean meal to be completely (>95%) hydrolyzed at the doses of 1, 2, and 5 U/mL of α-galactosidase from
Paecilomyces thermophila, respectively [
45]. Another study demonstrated that after soymilk was treated for 3 h at the doses of 5, 10, and 15 U/mL of α-galactosidases from
A. oryzae YZ1, 87.1%, 88.0%, and 97.8% of RFOs were hydrolyzed, respectively [
46]. These findings confirmed that RFO degradation was enzyme dosage-dependent. This study determined the optimal enzyme dosage as 1 U/mL. Consistent with the reports by Baffa et al. [
8], Çelem et al. [
22], and Bayraktar et al. [
39], this study showed that immobilization can improve the stability of the enzyme and its degradation ability towards RFOs. In addition, iORG retained 51.43 ± 1.05% relative enzyme activity towards raffinose after being reused six times. In conclusion, iORG exhibited excellent stability, high ability to remove RFOs from soymilk, and great reusability and, thus, it has a great potential to be used to eliminate RFOs from soymilk as well as other soybean products.