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Good hydrolysis activity on raffinose family oligosaccharides by a novel α-galactosidase from Tremella aurantialba
Journal Pre-proofs Good hydrolysis activity on raffinose family oligosaccharides by a novel αgalactosidase from Tremella aurantialba Xueran Geng, Dongxue Yang, Qiaoyi Zhang, Mingchang Chang, Lijing Xu, Yanfen Cheng, Hexiang Wang, Junlong Meng PII: DOI: Reference:
S0141-8130(19)33363-X https://doi.org/10.1016/j.ijbiomac.2019.10.136 BIOMAC 13637
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
7 May 2019 12 October 2019 14 October 2019
Please cite this article as: X. Geng, D. Yang, Q. Zhang, M. Chang, L. Xu, Y. Cheng, H. Wang, J. Meng, Good hydrolysis activity on raffinose family oligosaccharides by a novel α-galactosidase from Tremella aurantialba, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.136
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© 2019 Published by Elsevier B.V.
Good hydrolysis activity on raffinose family oligosaccharides by a novel α-galactosidase from Tremella aurantialba Xueran Genga,b,c, Dongxue Yangc, Qiaoyi Zhangd, Mingchang Changa,b, Lijing Xua,b, Yanfen Chenga,b, Hexiang Wangc,*[email protected], Junlong Menga,b*[email protected] aCollege
of Food Science and Engineering, Shanxi Agricultural University, Taigu, Shanxi 030801, China
bCollaborative
Innovation Center of Advancing Quality and Efficiency of Loess Plateau Edible Fungi,
Taigu, Shanxi 030801, China cState
Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural
University, Beijing 100193, China dOrient
Science & Technology College of Hunan Agricultural University
*Corresponding
author.
Abstract An α-galactosidase designated as TAG was purified from the dried fruit bodies of Tremella aurantialba with 182.5-fold purification. The purification procedure involved ion exchange chromatography on Q-sepharose, DEAE-Cellulose, and Mono Q and gel filtration by FPLC on Superdex 75. The purified α-galactosidase was a monomeric protein with a molecular mass of 88 kDa. The optimal pH of TAG was 5.0 and more than 60% of the original enzyme activity remained at pH 2.0 and 3.0. Its optimal temperature was 54 °C with good thermo-stability, 30.8% of the original activity was retained after exposure to a temperature of 70 °C for 1 h. The metal ions Hg2+, Cu2+, Fe3+ and Mg2+ strongly inhibited the enzyme activity. The enzyme activity was found to be inhibited by N-bromosuccinimide indicating that tryptophan was essential to the catalytic activity of α-galactosidase. The enzyme completely hydrolysed stachyose and partially hydrolysed raffinose to 1
galactose at 50 °C within 6 h as detected by thin layer chromatography and the dinitrosalicylic acid method and the content of reducing sugar reached 4.36 mg/mL. Key words: mushroom, Tremella aurantialba, α-galactosidase, oligosaccharides 1 Introduction Tremella aurantialba, belonging to the so-called “jelly mushrooms” group that forms a large brain-shaped, golden basidiocarp, is an edible and medicinal mushroom. In recent years, researchers have attempted to find new functional compounds from higher basidiomycetous fungi. Among the numerous mushrooms investigated, the genus Tremella showed various pharmacological properties including anti-tumour [1], anti-diabetic [2] and anti-hyperlipidemia [3] activities. Since polysaccharides from mushrooms exhibited anti-tumour and antioxidant functions, a series of reports on mushroom polysaccharides have been published. So, in previous studies, greater emphasis on T. aurantialba polysaccharides from its mycelium and fruiting bodies is evident. Chao et al. investigated the physio-chemical and biological properties of crude mycelium polysaccharide (CMCP) and purified mycelium polysaccharide (MCP) from the T. aurantialba and revealed that CMCP and MCP demonstrated antioxidant and immunostimulatory activities [4]. Both polysaccharide (TAPA1) from T. aurantialba fruiting bodies and its three chemically modified derivatives exhibited significant antioxidant activities and oxidative injury protection effects [5]. Due to the reported pharmacological properties, T. aurantialba has attracted growing interest among those involved in biomedical science. α-Galactosidase (EC 3.2.1.22) is used to catalyse the hydrolase of α-1,6-linked terminal galactose residues from different raffinose family oligosaccharides (RFOs), such as stachyose, raffinose, melibiose, and polysaccharides such as guar gum and locust bean gum [6]. These RFOs were identified as anti-nutritional factors that cannot be digested and accumulate in the large intestine of humans and 2
other monogastric animals deficient in α-galactosidase and subsequently fermented by microbial and then caused flatus. α-Galactosidases are widespread among plants, animals and microorganisms. These enzymes have considerable potential for diverse applications in food and feed industries, pulp and paper, sugar production, pharmaceutical industries and other industries [7]. In the food industry, the ability of α-galactosidase to eliminate RFOs can improve the nutritional value of soybean products and make them easily digestible. In the pulp and paper industry, α-galactosidase can improve the bleaching efficiency when processing softwood kraft pulp [8]. In sugar production, due to the release of galactosyl residues from raffinose, α-galactosidase promotes the crystallisation of sucrose and increases the yield of sucrose in the production of beet sugar [9]. In the pharmaceutical industry, α-galactosidase is used to convert blood from group B to group O and in the treatment of Fabry’s disease [10]. In recent years, the discovery of novel and natural source of enzymes with beneficial properties has generated a significant amount of research activity: there have been several α-galactosidases purified from mushrooms, including Hericium erinaceus [11], Lentinula edodes [12], Leucopaxillus tricolor [13], Pleurotus citrinopileatus [14], Tricholoma matsutake [15], Coriolus versicolor [16], Ganoderma lucidum [17], Lenzites elegans [18], Pleurotus florida [19], Termitomyces eurrhizus [20], Agaricus bisporus [21], and Pleurotus djamor [22]. α-Galactosidase purified from Lentinula edodes showed strong resistance to protease pepsin, papain, acid protease and neutral protease [12]. α-Galactosidase from Hericium erinaceus was stable across a broader pH range (pH 2.0–7.0) than other fungal α-galactosidases [11]. These features of the enzyme from mushroom make it possible to break through application bottlenecks. Due to the safety of α-galactosidases and absence of side effects therewith, the popularity of using edible fungi as functional foods has been escalating. As one of the medical edible fungi, T. aurantialba is seen as a good source of α-galactosidases with safety and 3
absence of side effects ensured. In the present study, the purification and characterisation of a monomeric α-galactosidase from the fruiting bodies of T. aurantialba were demonstrated. The enzymatic features of this enzyme make it valuable in the food and feed industries. 2 Materials and methods 2.1 Materials Fresh fruiting bodies of T. aurantialba were acquired from Yunnan Province (China). The fruiting bodies were dried naturally outdoors. Q-Sepharose and DEAE-cellulose were purchased from Sigma Chemical Co., USA. Superdex G-75 HR 10/30 and AKTA Purifier were obtained from GE Healthcare, USA. The substrates 4-nitro-phenyl α-D-galactopyranoside (pNPGal), o-nitrophenyl α-D-galactoside (oNPGal), 4-nitrophenyl β-D-glucuronide, locust bean gum, guar gum, melibiose, galactose, lactose, sucrose, glucose, xylose, fructose, stachyose and raffinose were purchased from Sigma Chemical Company (St. Louis, MO, USA). Unless otherwise stated, other chemical reagents used were of analytical grade. 2.2 Purification of α-galactosidase The flowchart followed in the purification of α-galactosidase is shown in Fig. 1. The dried fruiting bodies of T. aurantialba were homogenised using a Waring blender in distilled water (w:v=1:30) and extracted overnight at 4 °C. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. A 1 M pH 4.2 sodium acetate buffer was added to the clear supernatant to 10 mM. Subsequently, the supernatant obtained was subjected to anion exchange chromatography on Q-Sepharose (gel volume, 400 mL) previously equilibrated in 10 mM sodium acetate buffer (pH 4.2). After removal of unadsorbed proteins, adsorbed proteins were eluted with 50 mM, 200 mM and 1000 4
mM NaCl added to the same buffer, respectively. After being dialysed against 10 mM Tris-HCl buffer at pH 7.5, the active fraction (with its enriched α-galactosidase activity) was then chromatographed on another anion exchange DEAE-Cellulose column (2.5×20 cm) in Tris-HCl buffer (10 mM, pH 7.5) at a flow rate of 2.5 mL/min. Unbound protein was eluted with the starting buffer and bound proteins were desorbed by addition of 100 mM, 150 mM, 200 mM and 500 mM NaCl successively in the same buffer. After being dialysed against 10 mM pH 7.4 Tris-HCl buffer, the fraction with the highest α-galactosidase was pooled and then loaded on a column of Mono Q (0.46 × 10 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.4). Following removal of unadsorbed protein, adsorbed proteins were eluted with a linear gradient of 0-500 mM NaCl in the starting buffer. The active fraction was finally purified by fast protein liquid chromatography (FPLC) using an AKTA Purifier on a Superdex G-75 HR10/300 gel filtration column (10 mM Tris-HCl 7.4) at a flow rate of 0.8 mL/min. The active fraction was pooled, dialysed, and freeze-dried for further analysis. All purification steps were undertaken at room temperature. 2.3 Assay of α-galactosidase activity and protein determination The standard assays of α-galactosidase activity were executed by measuring the amount of p-nitrophenol released from the substrate pNPGal following a published method [15] with slight modification. Diluted enzyme (100 μL) was mixed with 100 μL 10 mM pNPGal solution (in 100 mM sodium acetate buffer, pH 4.6) at 50 °C for 10 min, followed by the addition of 500 mM Na2CO3 (800 μL) to end the reaction. The amount of p-nitrophenol released was determined spectrophotometrically at 405 nm. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute under the aforementioned reaction conditions. Protein concentration was determined by the Bradford method using bovine serum albumin as the 5
standard [23]. Fractions obtained during chromatographic purification were screened for proteins by measurement of absorbance at 280 nm. 2.4 Determination of molecular mass The molecular mass of the purified α-galactosidase was assessed using FPLC-gel filtration and combined with SDS-PAGE. A standard curve based on elution volume and molecular mass standards (GE Healthcare) was obtained, and the native molecular mass of the α-galactosidase was then calculated based on its elution volume. As molecular weight standards for gel filtration, the following proteins were used: albumin (66.0 kDa), lactate dehydrogenase (140.0 kDa), catalase (232.0 kDa), ferritin (440.0 kDa), and thyroglobulin (669.0 kDa). In SDS-PAGE, a 12% resolving gel and a 5% stacking gel were used following the standard procedure [24]. According to the standard curve of relative electrophoretic mobility and unstained molecular mass standards (Thermo Fisher), the inactive monomeric protein or sub-units of the purified α-galactosidase could be calculated based on its electrophoretic mobility. 2.5 Peptide sequence analysis The single protein stripe of purified α-galactosidase on SDS-PAGE was recovered and digested by trypsin, then dissolved in 0.1% formic acid and 2% acetonitrile for liquid chromatography-tandem mass spectrometry system coupled to an LTQ-Orbitrap mass spectrometer (Thermo Electron). Original data were transformed by BIWORKS and the data were searched against the NCBI database using MASCOAT software. 2.6 Biochemical properties of the enzyme Biochemical properties of TAG (α-galactosidase purified from Tremella aurantialba) including the effects of temperature, pH, metal ions and chemical modification reagents on the enzyme activity 6
were determined using the method of Geng et al. [15] with modifications thereto. Temperature optimum and stability of TAG were determined by using pNPGal as substrate at pH 4.6. To determine the optimal temperature, standard activity assay was measured at different temperatures from 20 °C to 70 °C instead of 50 °C as in the standard assay. The purified α-galactosidase was diluted to 10 U/mg in distilled water to assess the thermo-stability. The thermo-stability was measured after incubation at 30-70 °C for 1 h and subsequent determination of the residual α-galactosidase activity under standard conditions. In the optimum pH assay, a series of pNPGal solutions in various Na2HPO4-citric acid buffers (200 mM) ranging from pH 2.0 to 8.0 were used. The optimal pH for the enzyme could thus be ascertained. The stabilities of the enzyme to different concentrations of metal ions (K+, Ca2+, Cu2+, Mg2+, Cd2+, Mn2+, Hg2+, Pb2+, Zn2+, Fe2+, Al3+, and Fe3+ ions) and chemical modification reagents including diethyl pyrocarbonate
(DEPC),
2,4,6-trinitrophenol
(TNBS),
diacetyl
(DIC),
dithiothreitol
(DTT),
N-bromosuccinimide (NBS), and carbodiimide (EDC) were investigated. The isolated enzyme was incubated with the aforementioned reagents at 4 °C for 1 h. The residual α-galactosidase activity was measured in triplicate using the standard assay. The activity of the enzyme without any additive was taken as 100%. 2.7 Substrate specificity The substrate preference of TAG toward different types of substrates including synthetic substrates, oligosaccharides and polysaccharides was determined by measuring hydrolytic activity under standard assay conditions. In the case of synthetic substrates, the amount of pNP released by pNPGal, oNPG and 4-nitrophenyl β-D-glucuronide was measured in the standard assay. When other 7
galacto-oligosaccharides (raffinose and stachyose) and polysaccharides (locust bean gum and guar gum) were used as substrates, the amount of reducing sugar produced was determined using the 3,5-dinitrosalicylic acid reagent method as described by Miller [25] with modifications thereto. The reaction velocities of the enzyme were calculated as the amount of reducing sugar produced per minute at 50 °C. While a glucose-oxidase-peroxidase (GOD-POD) kit (Beijing BHKT Clinical Reagent Co., Ltd.) was used to estimate the glucose produced by melibiose as described by Dwevedi and Kayastha [26]. The reaction velocities of the enzyme were calculated as the amount of glucose produced per minute at 50 °C. 2.8 Enzymatic hydrolysis of RFOs The reaction mixture consisted of 50 μL diluted enzyme, 30 μL distilled water, and 20 μL raffinose (60 mM) and 20 μL stachyose (60 mM) in 0.1 M sodium acetate buffer (pH 4.6). The mixture was incubated for different durations at 50 °C. Aliquots were boiled for 5 min at predetermined time-intervals (0 min, 30 min, 1 h, 3 h, 6 h, and 12 h) to terminate the reaction and centrifuged to remove the denatured protein. Then the reaction products were analysed by TLC using silica gel G plates (10 cm × 10 cm) and developed using a three-phase solvent system of n-propanol: acetic acid: water at 10:15:1 (v/v/v). The silica gel plates were incubated with a chromogenic reagent (diphenylamine 1 g, aniline 1 mL, acetone 50 mL), and then the plates were heated in an oven at 105 °C for 15 min to detect the saccharides. The hydrolysates of raffinose or stachyose were also detected using the DNS method. 3 Results 3.1 Purification of TAG TAG was purified from T. aurantialba by employing a protocol that comprised, sequentially, 8
chromatographic separation on anion exchanger Q-Sepharose, DEAE-cellulose, and the stronger anion exchanger Mono-Q, the purification scheme for TAG is summarised in Table 1. Since the crude extract of T. aurantialba was too sticky to subject to a Q-Sepharose column, a suction filtration was used. After filtration, only that fraction eluted with 200 mM NaCl demonstrated α-galactosidase activity. The viscosity of the solution decreased greatly, which lays the foundation for subsequent purification process. Fractions eluted by DEAE-cellulose with buffer, 100 mM NaCl, 150 mM NaCl, 200 mM NaCl and 500 mM NaCl were defined as D1, D2, D3, D4 and D5, respectively. Fraction Q was resolved into four bound fractions (D2 to D5) by anion exchange column chromatography on DEAE-cellulose. Only fraction D3 manifested α-galactosidase activity (Fig. 2a). Fraction D3 was further applied to a column of Mono Q. Three fractions eluted by Mono Q with 0-500 mM NaCl were designated as Mono Q1, Mono Q2 and Mono Q3, respectively. α-Galactosidase activity was pooled in fraction Mono Q3 (Fig. 2b). Five fractions eluted by gel filtration chromatography with 10 mM Tris-HCl buffer (pH 7.4) were defined as SU1, SU2, SU3, SU4 and SU5 respectively. The final step involved by gel filtration chromatography on Superdex 75 and the resulting active fraction was SU2 (Fig. 2c). The native molecular mass estimated by FPLC on Superdex 75 was 91 kDa, as judged from the comparison of its elution volume from the Superdex 75 column with those of molecular mass standards (Fig. 2c). A major band of 88 kDa was observed on SDS-PAGE (Fig. 2d), which was similar to the native molecular mass determined by FPLC. This indicated that TAG was a monomeric protein with a molecular mass of 88 kDa. The purification efficiency of each step is summarised in Table 1. TAG showed a specific activity of 26300 U/mg against pNPGal and the purification was surprisingly high, attaining 182.5-fold purification. 3.2 Amino acid sequence of TAG 9
According to the data from LC-MS/MS, the amino acid sequences of six inner peptides obtained through protein BLAST searches, closely matched previously reported α-galactosidases (Table 2). Peptide 1, peptide 2, peptide 4, and peptide 6 shared high identity with α-galactosidases from bacteria sources. Peptide 2 also exhibited 83% identity to alkaline alpha galactosidase family protein from Eukaryota Populus trichocarpa (XP_002322710.1). Peptide 3 showed 75% identity to a glycoside hydrolase family 27 protein and 60% identity to a putative alpha-galactosidase from Penicillium oxalicum 114-2. Peptide 5 shared 55% identity with α-galactosidases from Fusarium. 3.3 Biochemical properties of TAG In the present study, the activity of TAG increased over the temperature range of 20-50 °C and decreased gradually when the temperature exceeded 54 °C (Fig. 3a). Thus, the optimum temperature for TAG activity was deemed to be 54 °C. When the temperature was increased to 70 °C, the activity of TAG to hydrolysis of pNPGal disappeared. Almost all of the enzyme activity was retained after 1 h of incubation at a temperature range from 30 to 50 °C (Fig. 3b). More than 80% of the initial activity was conserved after 1 h of incubation at 60 °C, after incubation at 70 °C for 1 h, only 30% of the original activity was preserved. TAG achieved its maximal hydrolysis towards to pNPGal at pH 5.0 (Fig. 4). The acidic conditions did not significantly affect the enzymatic activity. TAG maintained at least 60% activity at pH 2.0 and pH 3.0. The activity of TAG decreased when the pH was increased from 5.0 to 8.0. There was no residual, significant, α-galactosidase activity at pH > 8.0. TAG showed different sensitivities to various metal ions (Table 3). Several metal ions including Hg2+, Cd2+, Cu2+, Ca2+, Mg2+, Fe3+, and Al3+ ions exerted strong inhibitory effects on enzyme activity, whereas no metal ions showed positive effects on the enzyme. Mn2+, K+, and Pb2+ ions partially 10
inhibited enzyme activity at the concentration of 1.25 mM. As the concentrations increased, the activity of TAG was significantly inhibited by these three ions. When the concentration of Zn2+ ions was low, there was no evident effect on TAG. Otherwise Zn2+ ions could strongly inhibit the activity of TAG. 3.4 Side residue modification of TAG The effects of six chemical modification reagents including NBS, DEPC, DTT, EDC, DIC and TNBS on the activity of TAG were tested (Table 4). The activity of TAG was completely inactivated at NBS concentrations from 0.2 to 1 mM: this indicates that tryptophan residue modification could be critical to the stability of TAG and was an indispensable group for TAG enzymatic activity [27]. No effects were observed when incubation at all the concentrations of DEPC, EDC, and TNBS. DTT and DIC showed slightly negative effects on the activities of TAG. 3.5 Substrate specificity of TAG To investigate the substrate specificity of TAG, synthetic substrates (pNPGal, oNPG, and 4-nitrophenyl β-D-glucuronide) and natural substrates (melibiose, raffinose, stachyose, locust bean gum and guar gum) were examined (Table 5). Among the synthetic substrates, TAG exhibited the highest activity with pNPGal (100%), much higher than oNPG (18.9%) and 4-nitrophenyl β-D-glucuronide (9.2%), indicating this enzyme showed high specificity for anomeric carbon. Natural substrates were also good substrates for this enzyme. Reduction of 38.3% stachyose, 37.2% raffinose, and 29.4% melibiose was observed after treatment with TAG, suggesting that TAG delivered remarkable degradative efficiency toward oligosaccharides: however, the activity of TAG (1500 U/mg) towards locust bean gum and guar gum was not determined in this study. 3.6 Hydrolysis of RFOs by TAG The hydrolysis products of the substrate raffinose and stachyose were analysed by thin layer 11
chromatography with silica gel plates. As shown in Fig. 5a, a digest of RFOs (10 mM stachyose and 10 mM raffinose) by TAG at 0.5, 1, 2, 3, and 6 h was performed. After 0.5 h, both raffinose and stachyose began to be hydrolysed by TAG. TAG effectively hydrolysed stachyose to sucrose and galactose within 6 h. While, after treatment with TAG for 6 h, a faint dot of raffinose remained. During the hydrolysis of raffinose and stachyose, the amount of reducing sugar was also monitored (Fig. 5b). The content of reducing sugar increased gradually after treatment with TAG during hydrolysis, indicating that stachyose and raffinose were hydrolysed to yield galactose and sucrose, which was in accordance with the results of TLC. 4 Discussion Raffinose-family oligosaccharides, which are widely distributed in soy, are the main anti-nutritional components [28]. α-Galactosidase is capable of catalysing the hydrolysis of α-1,6-linked terminal galactose residues from galactooligosaccharide such as raffinose and stachyose [29]. So α-galactosidases were used to hydrolyse anti-nutritional carbohydrates such as raffinose-family oligosaccharides in many applications, particularly in the food and feed industries [30, 31]. In the present study, an α-galactosidase (TAG) with a molecular mass of 88 kDa from the fruiting bodies of T. aurantialba was assessed. Although the optimal pH and temperature of TAG resemble those of the other α-galactosidases, there are also distinctive features and remarkable differences including molecular mass, substrate specificity and hydrolysis of RFOs [12, 13]. Moreover, its sequence demonstrated low homology to some other sources α-galactosidases, thus signifying that TAG is a novel α-galactosidase. The specific activity of TAG toward pNPGal after 182-fold purification was 26,300 U/mg, which exceeded those of α-galactosidases from mushrooms, for example, Tricholoma matsutake (909 U/mg) [15], Pleurotus citrinopileatus (7.92 U/mg) [14], Coriolus 12
versicolor (398.6 U/mg) [16], Ganoderma lucidum (23.2 U/mg) [17], and Lenzites elegans (27.7 U/mg) [18]. Although the yield was low, combining the fermentation of TAG or the cloning of the enzyme to produce higher enzyme amounts can make it easier to purify [32-34] and may convert this promising enzyme into a suitable product with potential industrial applications. The yield of α-galactosidases isolated from mushrooms ranged from 0.36% to 28% [11-15, 18, 21, 22]. The α-galactosidase purified from Lenzites elegans showed the highest yield, however, the yield of α-galactosidase from Pleurotus djamor was the lowest [18, 22]. The yield of TAG (2.5%) is similar to that of α-galactosidase purified from Leucopaxillus tricolor (2.7%) [13]. The yield of α-galactosidases from plants and microorganisms varies by specimen. The yields of plant sources of α-galactosidases from hemp seeds [35], Vigna mungo [36], and Phaseolus coccineus seeds [37] were 1.4%, 5.8%, and 0.6%, respectively. The yields of microorganisms from Fusarium oxysporum and Neosartorya fischeri P1 were 1.1% and 3.7%, respectively. Compared with other α-galactosidase from plants and microorganisms, the yield of most α-galactosidases from mushrooms did not show any significant advantages: however, in other regards, α-galactosidases from mushrooms exhibited significant advantages. Mushrooms grow faster than most plants, so such sources of α-galactosidases are easier to obtain. The properties such as safety and lack of apparent side effects to human make α-galactosidases from mushrooms superior to α-galactosidase from other microorganisms. TAG is a monomeric protein akin to other fungal α-galactosidases and has a molecular mass of 88 kDa which fell within the reported range of molecular masses of most fungal α-galactosidases (40-249 kDa) [11, 13]. The monomeric α-galactosidase TAG differed from those of other heterodimeric α-galactosidases from Pleurotus citrinopileatus [14], Aspergillus niger, and tetrameric α-galactosidase 13
from Aspergillus niger [38]. Hence, there are large variations in the molecular mass and the exit forms of α-galactosidases from different microorganisms. Many reported fungal α-galactosidases have been classified into GH families 27 and 36 [39], but there was only one peptide sharing 75% of its identity with a glycoside hydrolase family 27 protein. So, we can only propose preliminarily that TAG may be a novel member of the glycoside hydrolase family 27. Moreover, only by knowing the whole acid sequence of TAG can we ensure to which glycoside hydrolase family TAG belongs. In view of the use in production, it is crucial to understand the characteristics of TAG. According to reported findings, the optimum temperature of α-galactosidases from bacteria was between 37 and 40 °C, and those from mycelial fungi and Saccharomyces was a little higher at 50 to 60 °C. TAG required a moderate ambient temperature of 54 °C for maximal activity and it manifested considerably high thermo-stability and maintained most of its activity when the temperature was between 30 to 60 °C. Many α-galactosidases purified from fungi are classified as mesophilic enzymes and showed poor thermo-stability. α-Galactosidase from T.matsutake showed poor thermo-stability at higher temperatures and was completely inactivated after incubation at 50 °C for 30 min [15]. α-Galactosidase from Agaricus bisporus lost 90% of its activity within 40 min at 50 °C [21]. α-Galactosidase from P. citrinopileatus maintained about 70% of its original activity at 40 °C after incubation for 1 h, but most of the activity was lost at 50 °C. So it is worth noting that the good thermo-stability of TAG differentiated it from those α-galactosidases purified from fungi. The pH of an enzyme reaction is pivotal as it regulates the ionisation state of both enzyme and substrate. It is noteworthy that TAG was an acidic enzyme and the optimum pH was 5.0, which was in agreement with those α-galactosidases reported elsewhere, such as Leucopaxillus tricolor (pH 5.0) 14
[13], Pseudobalsamia microspore (pH 5.0) [40], Termitomyces eurrhizus (pH 5.0) [20], and Pleurotus djamor (pH 5.0) [22]. The optimum pH of α-galactosidases from mushroom was between 4.4 and 6.0 [11]. In an acidic atmosphere (at pH 2.0 or pH 3.0) more than 60% of its original activity was preserved, which is beneficial to application of the enzyme in acidic conditions. In short, the properties including moderate optimal reaction temperature, better thermo-stability, and higher activity in acidic conditions, made TAG easier to apply in food and feed industries. The effects of metal ions on the activity of α-galactosidases from different sources are different. Metal ions Ag+ and Hg2+ strongly inhibited the activity of Ganoderma lucidum α-galactosidase [17]. Ali reported that the activity of α-galactosidase from Penicillium politans NRC-510 was drastically inhibited by Hg2+, Fe3+, Ag+, and Co2+ ions [41]. As reported by Ramadevi [42], in the presence of Hg2+ ions the bulk of enzyme activity of α-galactosidase from black gram ceased. The Hg2+ ions completely eliminated the activity of α-galactosidases from Cannabis sativa seeds [35], Irpex lacteus [43], and Bacillus coagulans [44]. Since α-galactosidases from several sources were all strongly inhibited by Hg2+ ions. It is supposed that Hg2+ ions may interact with thiol groups, imidazole groups and peptide bonds in these α-galactosidases [42, 45-47]. It has been also speculated that the strong interaction of Hg2+ with the free cysteines present in the catalytic pocket interferes with substrate binding [42, 46]. It was interesting that, not only did different metal ions show different effects on α-galactosidases, but the same metal ions exhibited different effects on these α-galactosidases. TAG was severely inhibited by Mg2+ ions. In the presence of Mg2+, the activity of α-galactosidase from Thielavia terrestris NRRL 8126 was partially inhibited, whereas Mg2+ ions had no effect on the enzymatic activity of α-galactosidases from Aspergillus terreus [48, 49]. TAG not only acted on synthetic substrates such as pNPGal, oNPG, and 4-nitrophenyl 15
β-D-glucuronide, it also showed good degradation on raffinose-family oligosaccharides such as stachyose, raffinose, and melibiose. Soy products are one of the main sources of protein, but they contain some undigested oligosaccharides such as stachyose and raffinose (RFOs). RFOs are not digested by normal human carbohydrases and instead are fermented by intestinal microflora, leading to flatulence. There have been some reports on use of fungal α-galactosidases for removal of RFOs from soy milk. α-Galactosidases are versatile and find many applications in the pharmaceutical, nutraceutical, and food and feed industries. As with the results exhibited by TLC, TAG could effectively hydrolyse the two anti-nutritional galactooligasaccharides raffinose and stachyose. This finding further demonstrated that TAG has great potential in the hydrolysis of RFOs in soy products and elimination of anti-nutrient factors.
Author contributions X.G. and D.Y. performed the experiments. Q.Z. collected and analysed the data. M.C., L.X. and Y.C. provided the materials and reagents. J.M. wrote the manuscript. H.W. conceived and designed the study. Acknowledgments This work was financially supported by Higher Education Technology Innovation Project in Shanxi (2019L0373); Doctoral Science Foundation of Shanxi Agricultural University (2017YJ33, 2015YJ04 and 2014YJ09 ); Reward to Outstanding Doctor Working in Shanxi (No. SXYBKY201722); Applied Basic Research Project in Shanxi (201801D221300, 201701D221156). No conflict of interest exits in this manuscript.
Reference 16
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Fig. 1. Purification flow chart. Fig. 2. Elution profiles of the α-galactosidase from Tremella aurantialba and SDS-PAGE analysis of TAG. (a) DEAE-Cellulose column (2.5 × 20 cm); (b) Mono-Q column (0.46 × 10 cm); (c) Superdex 75 10/300 GL gel filtration column and molecular mass standards for calibrating Superdex 75 column; (d) SDS-PAGE of the purified α-galactosidase from Tremella aurantialba. Fig. 3. Optimum temperature (a) and temperature stability (1-h incubation) of α-galactosidase 20
from Tremella aurantialba (b). Fig. 4. Optimum pH of α-galactosidase from Tremella aurantialba. Fig. 5. TLC analysis of hydrolysis products of oligosaccharide by α-galactosidase from Tremella aurantialba (a) and reducing sugar content after treatment with α-galactosidase from Tremella aurantialba (b).
Table 1. Purification efficiency of α-galactosidase from Tremella aurantialba (assay conducted at 50 C)
Chromatographic
Protein
Total activity
Specific activity
Recovery of activity
Purification
fraction
(mg)
(U)
(U/mg)
(%)
fold
Crude extract
3880.0
5.6×105
144.4
100.0
1.0
Q
281.6
4.4×105
1578.3
78.5
10.9
D3
25.9
1.4×105
5388.3
25.0
37.3
monoQ3
4.4
3.4×104
8175.4
6.1
56.6
SU2
0.5
1.4×104
26348.8
2.5
182.5
Table 2. Sequence comparison of TGA with other α-galactosidases
source
Sequencea
Peptide1
NIIEKGKR
Bacteria
Bacteroides reticulotermitis JCM 0512
Bacteria
Vibrio sp. ER1A
Organism
Identity/%
Accession No.
IVEKGKR
86
GAE83815
NIREKGK
86
KFA98918.1
21
Bacteria
Vibrio shilonii
NIREKGK
86
WP_006075129.1
Bacteria
Bacteroides finegoldii
IVEKGK
83
WP_007747800.1
Peptide2
ADDANVVR
Bacteria
Novosphingobium nitrogenifigens
DDADVV
83
WP_008071841
Eukaryota
Populus trichocarpa (alkaline alpha
EDANVV
83
XP_002322710.1
galactosidase family protein ) Bacteria
Amycolatopsis balhimycina
DDALVVR
86
WP_020644360.1
Bacteria
Amycolatopsis rifamycinica
DDALVVR
86
KDN21246.1
Peptide3
DLIIAITSDK
Halanaerobium hydrogeniformans
DLISAIYNDK
70
YP_003993988.1
Bacteria
(glycoside hydrolase family rotein) bacteria
Flavobacterium soli
IIAINQDK
75
WP_026705712.1
fungi
Pleurotus ostreatus PC15
IIAINQDK
75
KDQ32138.1
(glycoside hydrolase family 27 protein) bacteria
Lechevalieria aerocolonigenes
IIAINQDK
75
WP_030473288.1
fungi
Penicillium oxalicum 114-2
ELVIAAKSDK
60
EPS30642.1
(putative alpha-galactosidase) Peptide4
NVVSNLPK
bacteria
Bacillus clausii
NVISNLP
86
KFE59438.1
bacteria
Sphingobacterium sp. 21
VVSKLPK
86
YP_004319270.1
bacteria
Dictyoglomus thermophilum H-6-12
NLITNLPK
63
YP_002250158.1
Peptide5
NVSFYFHSDNTVLLNEYYKS 22
LSK fungi
Fusarium oxysporum Fo47
NVSYRFHVDNTTGDLINDHY
55
EWZ46264.1
fungi
Fusarium oxysporum f. sp. raphani
NVSYRFHVDNTTGDLINDHY
55
EXK83468.1
NVSYRFHVDNTTGDLINDHY
55
ENH66755.1
54005 fungi
Fusarium oxysporum f. sp. cubense race 1 Peptide6
LEKLITTAER
bacteria
Paraprevotella clara CAG:116
LENLIQTADR
70
WP_021980806.1
bacteria
Propionibacterium
LDKLITLADR
70
WP_016666042.1
bacteria
Paraprevotella clara
LENLIQTADR
70
WP_008618213.1
aIdentical
amino acids are bold
Table 3. The effects of different metal ions on the activity of α-galactosidase (results represent mean ± SD, N=3)
Metal ion
Relative galactosidase activity (%)
concentration
1.25 mM
2.5 mM
5 mM
10 mM
Fe3+
10.3 ± 1.33l
10.0 ± 0.31lm
6.1 ± 0.11m
0 ± 0.00n
Fe2+
62.3 ± 3.35g
66.2 ± 2.53f
76.5 ± 1.72d
94.4 ± 5.74b
Zn2+
101.0 ± 2.99a
86.8 ± 2.19c
16.4 ± 1.27jk
19.6 ± 0.63ij
K+
71.2 ± 2.53e
14.2 ± 0.72kl
12.2 ± 0.42kl
8.8 ± 0.24lm
Mn2+
66.0 ± 3.32f
14.4 ± 1.20k
13.4 ± 1.57kl
19.5 ± 1.33ij
23
Cd2+
22.5 ± 1.04i
11.3 ± 0.63kl
10.7 ± 0.72l
11.2 ± 0.72l
Al3+
15.6 ± 1.04jk
15.1 ± 0.24jk
17.6 ± 0.48j
20.0 ± 0.48ij
Mg2+
9.8 ± 0.63lm
10.3 ± 0.48l
9.3 ± 0.48lm
9.1 ± 0.72lm
Pb2+
70.9 ± 2.09e
41.5 ± 1.87h
11.0 ± 0.63l
10.7 ± 0.72l
Cu2+
7.1 ± 1.10m
9.3 ± 0.48lm
6.1 ± 0.42m
5.8 ± 0.24m
Ca2+
12.2 ± 0.72kl
8.3 ± 0.63lm
11.7 ± 0.41kl
13.0 ± 1.04kl
Hg2+
1.0 ± 0.72n
0 ± 0.00n
0.0 ± 0.00n
0.0 ± 0.00n
Statistical significance is indicated by different letters as assessed by two-way ANOVA (p< 0.05).
Table 4. The effects of different chemical modification reagents on the activity of α-galactosidase (results represent mean ± SD, N=3)
Chemical modification reagent
Relative galactosidase activity (%)
concentration
0.2 mM
0.4 mM
0.6 mM
0.8 mM
1 mM
NBS
0 ± 0.00
0 ± 0.59
0 ± 0.00
0 ± 0.59
0 ± 0.59
DTT
82 ± 1.63
83 ± 1.63
74 ± 2.69
66 ± 1.63
55 ± 2.23
DIC
104 ± 2.40
104 ± 0.04
105 ± 2.75
97 ± 2.61
88 ± 1.04
DEPC
96 ± 2.26
97 ± 2.59
99 ± 2.47
97 ± 3.71
98 ± 3.71
EDC
106 ± 1.43
103 ± 1.43
106 ± 2.71
105 ± 3.30
97 ± 2.87
TNBS
95 ± 3.32
96 ± 3.41
96 ± 2.38
96 ± 1.89
100 ± 1.89
24
Table 5. The substrate diversity of α-galactosidase (1500 U/mg) from Tremella aurantialba
Substrate
Concentration
Relative activity (%)
pNPGal
5 mM
100 ± 0.03
oNPGal
5 mM
18.9 ± 0.01
4-nitrophenyl β-D-glucuronide
5 mM
9.2 ± 0.00
Raffinose
20 mM
37.2 ± 0.06
Melibiose
20 mM
29.4 ± 0.23
Stachyose
20 mM
38.3 ± 0.06
Locust bean gum
0.1%
-
Guar gum
0.1%
-
“-“: not detected
25
S0141-8130(19)33363-X https://doi.org/10.1016/j.ijbiomac.2019.10.136 BIOMAC 13637
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
7 May 2019 12 October 2019 14 October 2019
Please cite this article as: X. Geng, D. Yang, Q. Zhang, M. Chang, L. Xu, Y. Cheng, H. Wang, J. Meng, Good hydrolysis activity on raffinose family oligosaccharides by a novel α-galactosidase from Tremella aurantialba, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.136
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© 2019 Published by Elsevier B.V.
Good hydrolysis activity on raffinose family oligosaccharides by a novel α-galactosidase from Tremella aurantialba Xueran Genga,b,c, Dongxue Yangc, Qiaoyi Zhangd, Mingchang Changa,b, Lijing Xua,b, Yanfen Chenga,b, Hexiang Wangc,*[email protected], Junlong Menga,b*[email protected] aCollege
of Food Science and Engineering, Shanxi Agricultural University, Taigu, Shanxi 030801, China
bCollaborative
Innovation Center of Advancing Quality and Efficiency of Loess Plateau Edible Fungi,
Taigu, Shanxi 030801, China cState
Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural
University, Beijing 100193, China dOrient
Science & Technology College of Hunan Agricultural University
*Corresponding
author.
Abstract An α-galactosidase designated as TAG was purified from the dried fruit bodies of Tremella aurantialba with 182.5-fold purification. The purification procedure involved ion exchange chromatography on Q-sepharose, DEAE-Cellulose, and Mono Q and gel filtration by FPLC on Superdex 75. The purified α-galactosidase was a monomeric protein with a molecular mass of 88 kDa. The optimal pH of TAG was 5.0 and more than 60% of the original enzyme activity remained at pH 2.0 and 3.0. Its optimal temperature was 54 °C with good thermo-stability, 30.8% of the original activity was retained after exposure to a temperature of 70 °C for 1 h. The metal ions Hg2+, Cu2+, Fe3+ and Mg2+ strongly inhibited the enzyme activity. The enzyme activity was found to be inhibited by N-bromosuccinimide indicating that tryptophan was essential to the catalytic activity of α-galactosidase. The enzyme completely hydrolysed stachyose and partially hydrolysed raffinose to 1
galactose at 50 °C within 6 h as detected by thin layer chromatography and the dinitrosalicylic acid method and the content of reducing sugar reached 4.36 mg/mL. Key words: mushroom, Tremella aurantialba, α-galactosidase, oligosaccharides 1 Introduction Tremella aurantialba, belonging to the so-called “jelly mushrooms” group that forms a large brain-shaped, golden basidiocarp, is an edible and medicinal mushroom. In recent years, researchers have attempted to find new functional compounds from higher basidiomycetous fungi. Among the numerous mushrooms investigated, the genus Tremella showed various pharmacological properties including anti-tumour [1], anti-diabetic [2] and anti-hyperlipidemia [3] activities. Since polysaccharides from mushrooms exhibited anti-tumour and antioxidant functions, a series of reports on mushroom polysaccharides have been published. So, in previous studies, greater emphasis on T. aurantialba polysaccharides from its mycelium and fruiting bodies is evident. Chao et al. investigated the physio-chemical and biological properties of crude mycelium polysaccharide (CMCP) and purified mycelium polysaccharide (MCP) from the T. aurantialba and revealed that CMCP and MCP demonstrated antioxidant and immunostimulatory activities [4]. Both polysaccharide (TAPA1) from T. aurantialba fruiting bodies and its three chemically modified derivatives exhibited significant antioxidant activities and oxidative injury protection effects [5]. Due to the reported pharmacological properties, T. aurantialba has attracted growing interest among those involved in biomedical science. α-Galactosidase (EC 3.2.1.22) is used to catalyse the hydrolase of α-1,6-linked terminal galactose residues from different raffinose family oligosaccharides (RFOs), such as stachyose, raffinose, melibiose, and polysaccharides such as guar gum and locust bean gum [6]. These RFOs were identified as anti-nutritional factors that cannot be digested and accumulate in the large intestine of humans and 2
other monogastric animals deficient in α-galactosidase and subsequently fermented by microbial and then caused flatus. α-Galactosidases are widespread among plants, animals and microorganisms. These enzymes have considerable potential for diverse applications in food and feed industries, pulp and paper, sugar production, pharmaceutical industries and other industries [7]. In the food industry, the ability of α-galactosidase to eliminate RFOs can improve the nutritional value of soybean products and make them easily digestible. In the pulp and paper industry, α-galactosidase can improve the bleaching efficiency when processing softwood kraft pulp [8]. In sugar production, due to the release of galactosyl residues from raffinose, α-galactosidase promotes the crystallisation of sucrose and increases the yield of sucrose in the production of beet sugar [9]. In the pharmaceutical industry, α-galactosidase is used to convert blood from group B to group O and in the treatment of Fabry’s disease [10]. In recent years, the discovery of novel and natural source of enzymes with beneficial properties has generated a significant amount of research activity: there have been several α-galactosidases purified from mushrooms, including Hericium erinaceus [11], Lentinula edodes [12], Leucopaxillus tricolor [13], Pleurotus citrinopileatus [14], Tricholoma matsutake [15], Coriolus versicolor [16], Ganoderma lucidum [17], Lenzites elegans [18], Pleurotus florida [19], Termitomyces eurrhizus [20], Agaricus bisporus [21], and Pleurotus djamor [22]. α-Galactosidase purified from Lentinula edodes showed strong resistance to protease pepsin, papain, acid protease and neutral protease [12]. α-Galactosidase from Hericium erinaceus was stable across a broader pH range (pH 2.0–7.0) than other fungal α-galactosidases [11]. These features of the enzyme from mushroom make it possible to break through application bottlenecks. Due to the safety of α-galactosidases and absence of side effects therewith, the popularity of using edible fungi as functional foods has been escalating. As one of the medical edible fungi, T. aurantialba is seen as a good source of α-galactosidases with safety and 3
absence of side effects ensured. In the present study, the purification and characterisation of a monomeric α-galactosidase from the fruiting bodies of T. aurantialba were demonstrated. The enzymatic features of this enzyme make it valuable in the food and feed industries. 2 Materials and methods 2.1 Materials Fresh fruiting bodies of T. aurantialba were acquired from Yunnan Province (China). The fruiting bodies were dried naturally outdoors. Q-Sepharose and DEAE-cellulose were purchased from Sigma Chemical Co., USA. Superdex G-75 HR 10/30 and AKTA Purifier were obtained from GE Healthcare, USA. The substrates 4-nitro-phenyl α-D-galactopyranoside (pNPGal), o-nitrophenyl α-D-galactoside (oNPGal), 4-nitrophenyl β-D-glucuronide, locust bean gum, guar gum, melibiose, galactose, lactose, sucrose, glucose, xylose, fructose, stachyose and raffinose were purchased from Sigma Chemical Company (St. Louis, MO, USA). Unless otherwise stated, other chemical reagents used were of analytical grade. 2.2 Purification of α-galactosidase The flowchart followed in the purification of α-galactosidase is shown in Fig. 1. The dried fruiting bodies of T. aurantialba were homogenised using a Waring blender in distilled water (w:v=1:30) and extracted overnight at 4 °C. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. A 1 M pH 4.2 sodium acetate buffer was added to the clear supernatant to 10 mM. Subsequently, the supernatant obtained was subjected to anion exchange chromatography on Q-Sepharose (gel volume, 400 mL) previously equilibrated in 10 mM sodium acetate buffer (pH 4.2). After removal of unadsorbed proteins, adsorbed proteins were eluted with 50 mM, 200 mM and 1000 4
mM NaCl added to the same buffer, respectively. After being dialysed against 10 mM Tris-HCl buffer at pH 7.5, the active fraction (with its enriched α-galactosidase activity) was then chromatographed on another anion exchange DEAE-Cellulose column (2.5×20 cm) in Tris-HCl buffer (10 mM, pH 7.5) at a flow rate of 2.5 mL/min. Unbound protein was eluted with the starting buffer and bound proteins were desorbed by addition of 100 mM, 150 mM, 200 mM and 500 mM NaCl successively in the same buffer. After being dialysed against 10 mM pH 7.4 Tris-HCl buffer, the fraction with the highest α-galactosidase was pooled and then loaded on a column of Mono Q (0.46 × 10 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.4). Following removal of unadsorbed protein, adsorbed proteins were eluted with a linear gradient of 0-500 mM NaCl in the starting buffer. The active fraction was finally purified by fast protein liquid chromatography (FPLC) using an AKTA Purifier on a Superdex G-75 HR10/300 gel filtration column (10 mM Tris-HCl 7.4) at a flow rate of 0.8 mL/min. The active fraction was pooled, dialysed, and freeze-dried for further analysis. All purification steps were undertaken at room temperature. 2.3 Assay of α-galactosidase activity and protein determination The standard assays of α-galactosidase activity were executed by measuring the amount of p-nitrophenol released from the substrate pNPGal following a published method [15] with slight modification. Diluted enzyme (100 μL) was mixed with 100 μL 10 mM pNPGal solution (in 100 mM sodium acetate buffer, pH 4.6) at 50 °C for 10 min, followed by the addition of 500 mM Na2CO3 (800 μL) to end the reaction. The amount of p-nitrophenol released was determined spectrophotometrically at 405 nm. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute under the aforementioned reaction conditions. Protein concentration was determined by the Bradford method using bovine serum albumin as the 5
standard [23]. Fractions obtained during chromatographic purification were screened for proteins by measurement of absorbance at 280 nm. 2.4 Determination of molecular mass The molecular mass of the purified α-galactosidase was assessed using FPLC-gel filtration and combined with SDS-PAGE. A standard curve based on elution volume and molecular mass standards (GE Healthcare) was obtained, and the native molecular mass of the α-galactosidase was then calculated based on its elution volume. As molecular weight standards for gel filtration, the following proteins were used: albumin (66.0 kDa), lactate dehydrogenase (140.0 kDa), catalase (232.0 kDa), ferritin (440.0 kDa), and thyroglobulin (669.0 kDa). In SDS-PAGE, a 12% resolving gel and a 5% stacking gel were used following the standard procedure [24]. According to the standard curve of relative electrophoretic mobility and unstained molecular mass standards (Thermo Fisher), the inactive monomeric protein or sub-units of the purified α-galactosidase could be calculated based on its electrophoretic mobility. 2.5 Peptide sequence analysis The single protein stripe of purified α-galactosidase on SDS-PAGE was recovered and digested by trypsin, then dissolved in 0.1% formic acid and 2% acetonitrile for liquid chromatography-tandem mass spectrometry system coupled to an LTQ-Orbitrap mass spectrometer (Thermo Electron). Original data were transformed by BIWORKS and the data were searched against the NCBI database using MASCOAT software. 2.6 Biochemical properties of the enzyme Biochemical properties of TAG (α-galactosidase purified from Tremella aurantialba) including the effects of temperature, pH, metal ions and chemical modification reagents on the enzyme activity 6
were determined using the method of Geng et al. [15] with modifications thereto. Temperature optimum and stability of TAG were determined by using pNPGal as substrate at pH 4.6. To determine the optimal temperature, standard activity assay was measured at different temperatures from 20 °C to 70 °C instead of 50 °C as in the standard assay. The purified α-galactosidase was diluted to 10 U/mg in distilled water to assess the thermo-stability. The thermo-stability was measured after incubation at 30-70 °C for 1 h and subsequent determination of the residual α-galactosidase activity under standard conditions. In the optimum pH assay, a series of pNPGal solutions in various Na2HPO4-citric acid buffers (200 mM) ranging from pH 2.0 to 8.0 were used. The optimal pH for the enzyme could thus be ascertained. The stabilities of the enzyme to different concentrations of metal ions (K+, Ca2+, Cu2+, Mg2+, Cd2+, Mn2+, Hg2+, Pb2+, Zn2+, Fe2+, Al3+, and Fe3+ ions) and chemical modification reagents including diethyl pyrocarbonate
(DEPC),
2,4,6-trinitrophenol
(TNBS),
diacetyl
(DIC),
dithiothreitol
(DTT),
N-bromosuccinimide (NBS), and carbodiimide (EDC) were investigated. The isolated enzyme was incubated with the aforementioned reagents at 4 °C for 1 h. The residual α-galactosidase activity was measured in triplicate using the standard assay. The activity of the enzyme without any additive was taken as 100%. 2.7 Substrate specificity The substrate preference of TAG toward different types of substrates including synthetic substrates, oligosaccharides and polysaccharides was determined by measuring hydrolytic activity under standard assay conditions. In the case of synthetic substrates, the amount of pNP released by pNPGal, oNPG and 4-nitrophenyl β-D-glucuronide was measured in the standard assay. When other 7
galacto-oligosaccharides (raffinose and stachyose) and polysaccharides (locust bean gum and guar gum) were used as substrates, the amount of reducing sugar produced was determined using the 3,5-dinitrosalicylic acid reagent method as described by Miller [25] with modifications thereto. The reaction velocities of the enzyme were calculated as the amount of reducing sugar produced per minute at 50 °C. While a glucose-oxidase-peroxidase (GOD-POD) kit (Beijing BHKT Clinical Reagent Co., Ltd.) was used to estimate the glucose produced by melibiose as described by Dwevedi and Kayastha [26]. The reaction velocities of the enzyme were calculated as the amount of glucose produced per minute at 50 °C. 2.8 Enzymatic hydrolysis of RFOs The reaction mixture consisted of 50 μL diluted enzyme, 30 μL distilled water, and 20 μL raffinose (60 mM) and 20 μL stachyose (60 mM) in 0.1 M sodium acetate buffer (pH 4.6). The mixture was incubated for different durations at 50 °C. Aliquots were boiled for 5 min at predetermined time-intervals (0 min, 30 min, 1 h, 3 h, 6 h, and 12 h) to terminate the reaction and centrifuged to remove the denatured protein. Then the reaction products were analysed by TLC using silica gel G plates (10 cm × 10 cm) and developed using a three-phase solvent system of n-propanol: acetic acid: water at 10:15:1 (v/v/v). The silica gel plates were incubated with a chromogenic reagent (diphenylamine 1 g, aniline 1 mL, acetone 50 mL), and then the plates were heated in an oven at 105 °C for 15 min to detect the saccharides. The hydrolysates of raffinose or stachyose were also detected using the DNS method. 3 Results 3.1 Purification of TAG TAG was purified from T. aurantialba by employing a protocol that comprised, sequentially, 8
chromatographic separation on anion exchanger Q-Sepharose, DEAE-cellulose, and the stronger anion exchanger Mono-Q, the purification scheme for TAG is summarised in Table 1. Since the crude extract of T. aurantialba was too sticky to subject to a Q-Sepharose column, a suction filtration was used. After filtration, only that fraction eluted with 200 mM NaCl demonstrated α-galactosidase activity. The viscosity of the solution decreased greatly, which lays the foundation for subsequent purification process. Fractions eluted by DEAE-cellulose with buffer, 100 mM NaCl, 150 mM NaCl, 200 mM NaCl and 500 mM NaCl were defined as D1, D2, D3, D4 and D5, respectively. Fraction Q was resolved into four bound fractions (D2 to D5) by anion exchange column chromatography on DEAE-cellulose. Only fraction D3 manifested α-galactosidase activity (Fig. 2a). Fraction D3 was further applied to a column of Mono Q. Three fractions eluted by Mono Q with 0-500 mM NaCl were designated as Mono Q1, Mono Q2 and Mono Q3, respectively. α-Galactosidase activity was pooled in fraction Mono Q3 (Fig. 2b). Five fractions eluted by gel filtration chromatography with 10 mM Tris-HCl buffer (pH 7.4) were defined as SU1, SU2, SU3, SU4 and SU5 respectively. The final step involved by gel filtration chromatography on Superdex 75 and the resulting active fraction was SU2 (Fig. 2c). The native molecular mass estimated by FPLC on Superdex 75 was 91 kDa, as judged from the comparison of its elution volume from the Superdex 75 column with those of molecular mass standards (Fig. 2c). A major band of 88 kDa was observed on SDS-PAGE (Fig. 2d), which was similar to the native molecular mass determined by FPLC. This indicated that TAG was a monomeric protein with a molecular mass of 88 kDa. The purification efficiency of each step is summarised in Table 1. TAG showed a specific activity of 26300 U/mg against pNPGal and the purification was surprisingly high, attaining 182.5-fold purification. 3.2 Amino acid sequence of TAG 9
According to the data from LC-MS/MS, the amino acid sequences of six inner peptides obtained through protein BLAST searches, closely matched previously reported α-galactosidases (Table 2). Peptide 1, peptide 2, peptide 4, and peptide 6 shared high identity with α-galactosidases from bacteria sources. Peptide 2 also exhibited 83% identity to alkaline alpha galactosidase family protein from Eukaryota Populus trichocarpa (XP_002322710.1). Peptide 3 showed 75% identity to a glycoside hydrolase family 27 protein and 60% identity to a putative alpha-galactosidase from Penicillium oxalicum 114-2. Peptide 5 shared 55% identity with α-galactosidases from Fusarium. 3.3 Biochemical properties of TAG In the present study, the activity of TAG increased over the temperature range of 20-50 °C and decreased gradually when the temperature exceeded 54 °C (Fig. 3a). Thus, the optimum temperature for TAG activity was deemed to be 54 °C. When the temperature was increased to 70 °C, the activity of TAG to hydrolysis of pNPGal disappeared. Almost all of the enzyme activity was retained after 1 h of incubation at a temperature range from 30 to 50 °C (Fig. 3b). More than 80% of the initial activity was conserved after 1 h of incubation at 60 °C, after incubation at 70 °C for 1 h, only 30% of the original activity was preserved. TAG achieved its maximal hydrolysis towards to pNPGal at pH 5.0 (Fig. 4). The acidic conditions did not significantly affect the enzymatic activity. TAG maintained at least 60% activity at pH 2.0 and pH 3.0. The activity of TAG decreased when the pH was increased from 5.0 to 8.0. There was no residual, significant, α-galactosidase activity at pH > 8.0. TAG showed different sensitivities to various metal ions (Table 3). Several metal ions including Hg2+, Cd2+, Cu2+, Ca2+, Mg2+, Fe3+, and Al3+ ions exerted strong inhibitory effects on enzyme activity, whereas no metal ions showed positive effects on the enzyme. Mn2+, K+, and Pb2+ ions partially 10
inhibited enzyme activity at the concentration of 1.25 mM. As the concentrations increased, the activity of TAG was significantly inhibited by these three ions. When the concentration of Zn2+ ions was low, there was no evident effect on TAG. Otherwise Zn2+ ions could strongly inhibit the activity of TAG. 3.4 Side residue modification of TAG The effects of six chemical modification reagents including NBS, DEPC, DTT, EDC, DIC and TNBS on the activity of TAG were tested (Table 4). The activity of TAG was completely inactivated at NBS concentrations from 0.2 to 1 mM: this indicates that tryptophan residue modification could be critical to the stability of TAG and was an indispensable group for TAG enzymatic activity [27]. No effects were observed when incubation at all the concentrations of DEPC, EDC, and TNBS. DTT and DIC showed slightly negative effects on the activities of TAG. 3.5 Substrate specificity of TAG To investigate the substrate specificity of TAG, synthetic substrates (pNPGal, oNPG, and 4-nitrophenyl β-D-glucuronide) and natural substrates (melibiose, raffinose, stachyose, locust bean gum and guar gum) were examined (Table 5). Among the synthetic substrates, TAG exhibited the highest activity with pNPGal (100%), much higher than oNPG (18.9%) and 4-nitrophenyl β-D-glucuronide (9.2%), indicating this enzyme showed high specificity for anomeric carbon. Natural substrates were also good substrates for this enzyme. Reduction of 38.3% stachyose, 37.2% raffinose, and 29.4% melibiose was observed after treatment with TAG, suggesting that TAG delivered remarkable degradative efficiency toward oligosaccharides: however, the activity of TAG (1500 U/mg) towards locust bean gum and guar gum was not determined in this study. 3.6 Hydrolysis of RFOs by TAG The hydrolysis products of the substrate raffinose and stachyose were analysed by thin layer 11
chromatography with silica gel plates. As shown in Fig. 5a, a digest of RFOs (10 mM stachyose and 10 mM raffinose) by TAG at 0.5, 1, 2, 3, and 6 h was performed. After 0.5 h, both raffinose and stachyose began to be hydrolysed by TAG. TAG effectively hydrolysed stachyose to sucrose and galactose within 6 h. While, after treatment with TAG for 6 h, a faint dot of raffinose remained. During the hydrolysis of raffinose and stachyose, the amount of reducing sugar was also monitored (Fig. 5b). The content of reducing sugar increased gradually after treatment with TAG during hydrolysis, indicating that stachyose and raffinose were hydrolysed to yield galactose and sucrose, which was in accordance with the results of TLC. 4 Discussion Raffinose-family oligosaccharides, which are widely distributed in soy, are the main anti-nutritional components [28]. α-Galactosidase is capable of catalysing the hydrolysis of α-1,6-linked terminal galactose residues from galactooligosaccharide such as raffinose and stachyose [29]. So α-galactosidases were used to hydrolyse anti-nutritional carbohydrates such as raffinose-family oligosaccharides in many applications, particularly in the food and feed industries [30, 31]. In the present study, an α-galactosidase (TAG) with a molecular mass of 88 kDa from the fruiting bodies of T. aurantialba was assessed. Although the optimal pH and temperature of TAG resemble those of the other α-galactosidases, there are also distinctive features and remarkable differences including molecular mass, substrate specificity and hydrolysis of RFOs [12, 13]. Moreover, its sequence demonstrated low homology to some other sources α-galactosidases, thus signifying that TAG is a novel α-galactosidase. The specific activity of TAG toward pNPGal after 182-fold purification was 26,300 U/mg, which exceeded those of α-galactosidases from mushrooms, for example, Tricholoma matsutake (909 U/mg) [15], Pleurotus citrinopileatus (7.92 U/mg) [14], Coriolus 12
versicolor (398.6 U/mg) [16], Ganoderma lucidum (23.2 U/mg) [17], and Lenzites elegans (27.7 U/mg) [18]. Although the yield was low, combining the fermentation of TAG or the cloning of the enzyme to produce higher enzyme amounts can make it easier to purify [32-34] and may convert this promising enzyme into a suitable product with potential industrial applications. The yield of α-galactosidases isolated from mushrooms ranged from 0.36% to 28% [11-15, 18, 21, 22]. The α-galactosidase purified from Lenzites elegans showed the highest yield, however, the yield of α-galactosidase from Pleurotus djamor was the lowest [18, 22]. The yield of TAG (2.5%) is similar to that of α-galactosidase purified from Leucopaxillus tricolor (2.7%) [13]. The yield of α-galactosidases from plants and microorganisms varies by specimen. The yields of plant sources of α-galactosidases from hemp seeds [35], Vigna mungo [36], and Phaseolus coccineus seeds [37] were 1.4%, 5.8%, and 0.6%, respectively. The yields of microorganisms from Fusarium oxysporum and Neosartorya fischeri P1 were 1.1% and 3.7%, respectively. Compared with other α-galactosidase from plants and microorganisms, the yield of most α-galactosidases from mushrooms did not show any significant advantages: however, in other regards, α-galactosidases from mushrooms exhibited significant advantages. Mushrooms grow faster than most plants, so such sources of α-galactosidases are easier to obtain. The properties such as safety and lack of apparent side effects to human make α-galactosidases from mushrooms superior to α-galactosidase from other microorganisms. TAG is a monomeric protein akin to other fungal α-galactosidases and has a molecular mass of 88 kDa which fell within the reported range of molecular masses of most fungal α-galactosidases (40-249 kDa) [11, 13]. The monomeric α-galactosidase TAG differed from those of other heterodimeric α-galactosidases from Pleurotus citrinopileatus [14], Aspergillus niger, and tetrameric α-galactosidase 13
from Aspergillus niger [38]. Hence, there are large variations in the molecular mass and the exit forms of α-galactosidases from different microorganisms. Many reported fungal α-galactosidases have been classified into GH families 27 and 36 [39], but there was only one peptide sharing 75% of its identity with a glycoside hydrolase family 27 protein. So, we can only propose preliminarily that TAG may be a novel member of the glycoside hydrolase family 27. Moreover, only by knowing the whole acid sequence of TAG can we ensure to which glycoside hydrolase family TAG belongs. In view of the use in production, it is crucial to understand the characteristics of TAG. According to reported findings, the optimum temperature of α-galactosidases from bacteria was between 37 and 40 °C, and those from mycelial fungi and Saccharomyces was a little higher at 50 to 60 °C. TAG required a moderate ambient temperature of 54 °C for maximal activity and it manifested considerably high thermo-stability and maintained most of its activity when the temperature was between 30 to 60 °C. Many α-galactosidases purified from fungi are classified as mesophilic enzymes and showed poor thermo-stability. α-Galactosidase from T.matsutake showed poor thermo-stability at higher temperatures and was completely inactivated after incubation at 50 °C for 30 min [15]. α-Galactosidase from Agaricus bisporus lost 90% of its activity within 40 min at 50 °C [21]. α-Galactosidase from P. citrinopileatus maintained about 70% of its original activity at 40 °C after incubation for 1 h, but most of the activity was lost at 50 °C. So it is worth noting that the good thermo-stability of TAG differentiated it from those α-galactosidases purified from fungi. The pH of an enzyme reaction is pivotal as it regulates the ionisation state of both enzyme and substrate. It is noteworthy that TAG was an acidic enzyme and the optimum pH was 5.0, which was in agreement with those α-galactosidases reported elsewhere, such as Leucopaxillus tricolor (pH 5.0) 14
[13], Pseudobalsamia microspore (pH 5.0) [40], Termitomyces eurrhizus (pH 5.0) [20], and Pleurotus djamor (pH 5.0) [22]. The optimum pH of α-galactosidases from mushroom was between 4.4 and 6.0 [11]. In an acidic atmosphere (at pH 2.0 or pH 3.0) more than 60% of its original activity was preserved, which is beneficial to application of the enzyme in acidic conditions. In short, the properties including moderate optimal reaction temperature, better thermo-stability, and higher activity in acidic conditions, made TAG easier to apply in food and feed industries. The effects of metal ions on the activity of α-galactosidases from different sources are different. Metal ions Ag+ and Hg2+ strongly inhibited the activity of Ganoderma lucidum α-galactosidase [17]. Ali reported that the activity of α-galactosidase from Penicillium politans NRC-510 was drastically inhibited by Hg2+, Fe3+, Ag+, and Co2+ ions [41]. As reported by Ramadevi [42], in the presence of Hg2+ ions the bulk of enzyme activity of α-galactosidase from black gram ceased. The Hg2+ ions completely eliminated the activity of α-galactosidases from Cannabis sativa seeds [35], Irpex lacteus [43], and Bacillus coagulans [44]. Since α-galactosidases from several sources were all strongly inhibited by Hg2+ ions. It is supposed that Hg2+ ions may interact with thiol groups, imidazole groups and peptide bonds in these α-galactosidases [42, 45-47]. It has been also speculated that the strong interaction of Hg2+ with the free cysteines present in the catalytic pocket interferes with substrate binding [42, 46]. It was interesting that, not only did different metal ions show different effects on α-galactosidases, but the same metal ions exhibited different effects on these α-galactosidases. TAG was severely inhibited by Mg2+ ions. In the presence of Mg2+, the activity of α-galactosidase from Thielavia terrestris NRRL 8126 was partially inhibited, whereas Mg2+ ions had no effect on the enzymatic activity of α-galactosidases from Aspergillus terreus [48, 49]. TAG not only acted on synthetic substrates such as pNPGal, oNPG, and 4-nitrophenyl 15
β-D-glucuronide, it also showed good degradation on raffinose-family oligosaccharides such as stachyose, raffinose, and melibiose. Soy products are one of the main sources of protein, but they contain some undigested oligosaccharides such as stachyose and raffinose (RFOs). RFOs are not digested by normal human carbohydrases and instead are fermented by intestinal microflora, leading to flatulence. There have been some reports on use of fungal α-galactosidases for removal of RFOs from soy milk. α-Galactosidases are versatile and find many applications in the pharmaceutical, nutraceutical, and food and feed industries. As with the results exhibited by TLC, TAG could effectively hydrolyse the two anti-nutritional galactooligasaccharides raffinose and stachyose. This finding further demonstrated that TAG has great potential in the hydrolysis of RFOs in soy products and elimination of anti-nutrient factors.
Author contributions X.G. and D.Y. performed the experiments. Q.Z. collected and analysed the data. M.C., L.X. and Y.C. provided the materials and reagents. J.M. wrote the manuscript. H.W. conceived and designed the study. Acknowledgments This work was financially supported by Higher Education Technology Innovation Project in Shanxi (2019L0373); Doctoral Science Foundation of Shanxi Agricultural University (2017YJ33, 2015YJ04 and 2014YJ09 ); Reward to Outstanding Doctor Working in Shanxi (No. SXYBKY201722); Applied Basic Research Project in Shanxi (201801D221300, 201701D221156). No conflict of interest exits in this manuscript.
Reference 16
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Fig. 1. Purification flow chart. Fig. 2. Elution profiles of the α-galactosidase from Tremella aurantialba and SDS-PAGE analysis of TAG. (a) DEAE-Cellulose column (2.5 × 20 cm); (b) Mono-Q column (0.46 × 10 cm); (c) Superdex 75 10/300 GL gel filtration column and molecular mass standards for calibrating Superdex 75 column; (d) SDS-PAGE of the purified α-galactosidase from Tremella aurantialba. Fig. 3. Optimum temperature (a) and temperature stability (1-h incubation) of α-galactosidase 20
from Tremella aurantialba (b). Fig. 4. Optimum pH of α-galactosidase from Tremella aurantialba. Fig. 5. TLC analysis of hydrolysis products of oligosaccharide by α-galactosidase from Tremella aurantialba (a) and reducing sugar content after treatment with α-galactosidase from Tremella aurantialba (b).
Table 1. Purification efficiency of α-galactosidase from Tremella aurantialba (assay conducted at 50 C)
Chromatographic
Protein
Total activity
Specific activity
Recovery of activity
Purification
fraction
(mg)
(U)
(U/mg)
(%)
fold
Crude extract
3880.0
5.6×105
144.4
100.0
1.0
Q
281.6
4.4×105
1578.3
78.5
10.9
D3
25.9
1.4×105
5388.3
25.0
37.3
monoQ3
4.4
3.4×104
8175.4
6.1
56.6
SU2
0.5
1.4×104
26348.8
2.5
182.5
Table 2. Sequence comparison of TGA with other α-galactosidases
source
Sequencea
Peptide1
NIIEKGKR
Bacteria
Bacteroides reticulotermitis JCM 0512
Bacteria
Vibrio sp. ER1A
Organism
Identity/%
Accession No.
IVEKGKR
86
GAE83815
NIREKGK
86
KFA98918.1
21
Bacteria
Vibrio shilonii
NIREKGK
86
WP_006075129.1
Bacteria
Bacteroides finegoldii
IVEKGK
83
WP_007747800.1
Peptide2
ADDANVVR
Bacteria
Novosphingobium nitrogenifigens
DDADVV
83
WP_008071841
Eukaryota
Populus trichocarpa (alkaline alpha
EDANVV
83
XP_002322710.1
galactosidase family protein ) Bacteria
Amycolatopsis balhimycina
DDALVVR
86
WP_020644360.1
Bacteria
Amycolatopsis rifamycinica
DDALVVR
86
KDN21246.1
Peptide3
DLIIAITSDK
Halanaerobium hydrogeniformans
DLISAIYNDK
70
YP_003993988.1
Bacteria
(glycoside hydrolase family rotein) bacteria
Flavobacterium soli
IIAINQDK
75
WP_026705712.1
fungi
Pleurotus ostreatus PC15
IIAINQDK
75
KDQ32138.1
(glycoside hydrolase family 27 protein) bacteria
Lechevalieria aerocolonigenes
IIAINQDK
75
WP_030473288.1
fungi
Penicillium oxalicum 114-2
ELVIAAKSDK
60
EPS30642.1
(putative alpha-galactosidase) Peptide4
NVVSNLPK
bacteria
Bacillus clausii
NVISNLP
86
KFE59438.1
bacteria
Sphingobacterium sp. 21
VVSKLPK
86
YP_004319270.1
bacteria
Dictyoglomus thermophilum H-6-12
NLITNLPK
63
YP_002250158.1
Peptide5
NVSFYFHSDNTVLLNEYYKS 22
LSK fungi
Fusarium oxysporum Fo47
NVSYRFHVDNTTGDLINDHY
55
EWZ46264.1
fungi
Fusarium oxysporum f. sp. raphani
NVSYRFHVDNTTGDLINDHY
55
EXK83468.1
NVSYRFHVDNTTGDLINDHY
55
ENH66755.1
54005 fungi
Fusarium oxysporum f. sp. cubense race 1 Peptide6
LEKLITTAER
bacteria
Paraprevotella clara CAG:116
LENLIQTADR
70
WP_021980806.1
bacteria
Propionibacterium
LDKLITLADR
70
WP_016666042.1
bacteria
Paraprevotella clara
LENLIQTADR
70
WP_008618213.1
aIdentical
amino acids are bold
Table 3. The effects of different metal ions on the activity of α-galactosidase (results represent mean ± SD, N=3)
Metal ion
Relative galactosidase activity (%)
concentration
1.25 mM
2.5 mM
5 mM
10 mM
Fe3+
10.3 ± 1.33l
10.0 ± 0.31lm
6.1 ± 0.11m
0 ± 0.00n
Fe2+
62.3 ± 3.35g
66.2 ± 2.53f
76.5 ± 1.72d
94.4 ± 5.74b
Zn2+
101.0 ± 2.99a
86.8 ± 2.19c
16.4 ± 1.27jk
19.6 ± 0.63ij
K+
71.2 ± 2.53e
14.2 ± 0.72kl
12.2 ± 0.42kl
8.8 ± 0.24lm
Mn2+
66.0 ± 3.32f
14.4 ± 1.20k
13.4 ± 1.57kl
19.5 ± 1.33ij
23
Cd2+
22.5 ± 1.04i
11.3 ± 0.63kl
10.7 ± 0.72l
11.2 ± 0.72l
Al3+
15.6 ± 1.04jk
15.1 ± 0.24jk
17.6 ± 0.48j
20.0 ± 0.48ij
Mg2+
9.8 ± 0.63lm
10.3 ± 0.48l
9.3 ± 0.48lm
9.1 ± 0.72lm
Pb2+
70.9 ± 2.09e
41.5 ± 1.87h
11.0 ± 0.63l
10.7 ± 0.72l
Cu2+
7.1 ± 1.10m
9.3 ± 0.48lm
6.1 ± 0.42m
5.8 ± 0.24m
Ca2+
12.2 ± 0.72kl
8.3 ± 0.63lm
11.7 ± 0.41kl
13.0 ± 1.04kl
Hg2+
1.0 ± 0.72n
0 ± 0.00n
0.0 ± 0.00n
0.0 ± 0.00n
Statistical significance is indicated by different letters as assessed by two-way ANOVA (p< 0.05).
Table 4. The effects of different chemical modification reagents on the activity of α-galactosidase (results represent mean ± SD, N=3)
Chemical modification reagent
Relative galactosidase activity (%)
concentration
0.2 mM
0.4 mM
0.6 mM
0.8 mM
1 mM
NBS
0 ± 0.00
0 ± 0.59
0 ± 0.00
0 ± 0.59
0 ± 0.59
DTT
82 ± 1.63
83 ± 1.63
74 ± 2.69
66 ± 1.63
55 ± 2.23
DIC
104 ± 2.40
104 ± 0.04
105 ± 2.75
97 ± 2.61
88 ± 1.04
DEPC
96 ± 2.26
97 ± 2.59
99 ± 2.47
97 ± 3.71
98 ± 3.71
EDC
106 ± 1.43
103 ± 1.43
106 ± 2.71
105 ± 3.30
97 ± 2.87
TNBS
95 ± 3.32
96 ± 3.41
96 ± 2.38
96 ± 1.89
100 ± 1.89
24
Table 5. The substrate diversity of α-galactosidase (1500 U/mg) from Tremella aurantialba
Substrate
Concentration
Relative activity (%)
pNPGal
5 mM
100 ± 0.03
oNPGal
5 mM
18.9 ± 0.01
4-nitrophenyl β-D-glucuronide
5 mM
9.2 ± 0.00
Raffinose
20 mM
37.2 ± 0.06
Melibiose
20 mM
29.4 ± 0.23
Stachyose
20 mM
38.3 ± 0.06
Locust bean gum
0.1%
-
Guar gum
0.1%
-
“-“: not detected
25