1. Introduction
Trehalose consists of two glucose molecules connected by an α-(1,1)-glycosidic bond. Trehalose is dispersed widely in microorganisms, plants, and animals. In fungi, this compound is related to significant biological phenomena; for instance, trehalose is possibly provided as a carbon source to germinating spores and growing cells (Francois & Parrou, 2001; Jorge, Polizeli, Thevelein, & Terenzi, 1997), and it may work to be stabilized cellular membranes and proteins (Simola, HaÅNnninen, Stranius, & Makarow, 2000; Singer & Lindquist, 1998).
Trehalose is contained about 2%-20% in mushrooms. It was reported that trehalose is used as carbohydrate substrates in the mycelial and fruit-body growth of Agaricus bisporus (Wood & Goodenough, 1977), Flammulina velutipes (Kitamoto & Gruen, 1976), and Favolus arcularius (Kitamoto et al., 1978). Trehalose is hydrolyzed by two enzymes: trehalose phosphorylase and trehalase. Trehalose phosphorylase generates either glucose and β-glucose-1-phosphate (β-G1P) or α-glucose-1-phosphate (α-G1P) from trehalose. In mushrooms, the α-type trehalose phosphorylases were detected in and purified from F. velutipes (Kitamoto, Akashi, Tanaka, & Mori, 1988), Schizophyllum commune (Eis & Nidetzky, 1999), A. bisporus (Wannet et al., 1998), and Pleurotus ostreatus (Kitamoto, Osaki, Tanaka, Sasaki, & Mori, 2001). Moreover, there are the two trehalose-hydrolyzing enzymes which are known as acid and neutral trehalases. The optimum pH of a typical neutral trehalase is 6.5-8.0, whereas that of an acidic trehalase is 4.0-5.0 (Schomburg, Chang, & Schomburg, 2002). It was reported that the two trehalases have been found in several fungal species (Parrou, Jules, Beltran, & Francois, 2005). It is different in their subcellular localization and various biochemical properties.
Trehalases have been classified as a member of the glycoside hydrolase (GH) family 37 and 65 in the Carbohydrate-Active enZYmes database (CAZY; http://www.cazy.org/). In yeasts and filamentous fungi, acid trehalases were belonging to GH family 65 and neutral trehalases were belonging to GH family 37 (Parrou et al., 2005). The acid trehalase (GH family 65) from Saccharomyces cerevisiae is an extracellular enzyme. Moreover, this protein is localized in the periplasmic space (Parrou et al., 2005). On the other hand, the neutral trehalase (GH family 37) is a cytosolic enzyme that rapidly hydrolyzes endogenous trehalose in response to developmental programs such as during spore germination.
In the case of mushroom, Liu, Shang, Liu, and Tan (2016) reported that the trehalose content and trehalase activities change during fruit-body formation in F. velutipes. The acid trehalase from a culture filtrate of Lentinus edodes ‘Mori 465’ was purified and characterized by Murata et al. (2001). Previously, we studied the variation in trehalose content and trehalase activity during fruit-body development and autolysis in Pleurotus sp. (Arastoo et al., 2018). The trehalose content of whole fruit bodies decreased sharply during the autolysis process, whereas the trehalase activity increased toward the inner region from the outer region of the pilei. We clarified that trehalases play an important role during the autolysis process. However, there are few reports on the structure and function of trehalases in mushrooms. In this paper, we found that novel acid trehalase belonging to GH family 37 in Pleurotus sp. To understand the enzymatic properties of trehalases from Pleurotus sp., we cloned and expressed the acid trehalase from Pleurotus sp. This is the first report about the novel GH family 37 acid trehalase from Pleurotus sp.
2. Materials and methods
2.1. Chemicals
Trehalose was purchased from FUJIFILM Wako Pure Chemicals Co. (Osaka, Japan). Soluble starch was purchased from Kanto Chemical Co., INC. (Tokyo, Japan). All other chemicals used here were of molecular biology grade.
2.2. Mushroom strain, cultivation, and homogenization
The novel strain MH00604 of Pleurotus sp. was supplied to us by Hokuto Co. (Nagano, Japan) (Azumi et al., 2016; Ishikawa et al., 2016). The strain was cultivated to obtain fruit bodies according to a procedure reported previously (Azumi et al., 2016). To purify acid trehalase from the fruit bodies of Pleurotus sp., we used 19-d-old fruit bodies (fresh weight, 270 g) after scratching the external aerial mycelia. The fruit bodies were homogenized using a mixer in 20 mM acetate buffer (pH 5.0) containing 1 mM EDTA and 1 mM PMSF. The homogenization solution was centrifuged at 9,000 × g for 30 min. The supernatant was filtered through 3.0, 0.8, and 0.45 μm filters serially. After the filtration, the solution was concentrated using an ultrafiltration unit (10 kDa cut off, Cole-Parmer Instrument Co., Bunker Court Vernon Hills, IL, USA). The supernatant was used as a crude enzyme solution.
2.3. Enzymatic assay and protein determination
Trehalase activity was assayed by measuring the glucose released from trehalose. The reaction mixture contained 20 mM trehalose in 20 mM acetate buffer (pH 5.0) with a suitable amount of the enzyme solution in a total volume of 110 μL. The reducing sugars produced by this reaction were determined according to the Somogyi-Nelson method (Somogyi, 1952) (standard assay method). One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar per min.
The protein concentration was determined using a Micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, USA) and bovine serum albumin as the standard. In the case of recombinant acid trehalase, the protein concentration of acid trehalase was calculated using an absorbance of 280 nm and the protein extinction coefficient, according to the method of Gill and von Hippel (1989).
2.4. Purification of acid trehalase
All purification steps were carried out at 4 ºC, unless otherwise noted.
Step 1. The crude enzyme solution was loaded onto a TOYOPEARL DEAE-650M ion-exchange column [2.5 cm (inner diameter) × 15 cm] equilibrated with 20 mM acetate buffer (pH 5.0). The bound enzyme was eluted with 20 mM acetate buffer (pH 5.0) containing 0.3 M NaCl. The active fractions were dialyzed against 20 mM acetate buffer (pH 5.0).
Step 2. The dialyzed enzyme solution was loaded onto a HiTrap Q FF column (column volume, 5 mL) equilibrated with 20 mM acetate buffer (pH 5.0). The bound enzyme was eluted with 20 mM acetate buffer (pH 5.0) containing 0.5 M NaCl. The active fractions were dialyzed against 20 mM phosphate buffer (pH 6.0).
Step 3. Ammonium sulfate was added to the dialyzed enzyme solution to 30% saturation. The solution was kept at 4 ºC overnight and then centrifuged at 10,000 x g for 30 min at 4 ºC. The supernatant was loaded onto a HiTrap Phenyl FF column (column volume, 1 mL) equilibrated with 20 mM phosphate buffer (pH 6.0) containing 30% saturated ammonium sulfate. The enzyme was eluted with 20 mM phosphate buffer (pH 6.0).
Step 4. The active fractions obtained after HiTrap Phenyl FF column chromatography were loaded onto a SuperdexTM200 10/300 GL column equilibrated with 20 mM phosphate buffer (pH 6.0). The active fraction was collected.
2.5. N-terminal amino acid sequence analysis
The proteins separated by SDS-PAGE were transferred onto a PVDF membrane. The membrane was washed extensively with water; stained with a mixture of 0.25% Coomassie Brilliant Blue R-250, 5% aqueous methanol, and 7.5% acetic acid for 5 min; and then destained with 90% aqueous methanol for 10 min. The area of the membrane containing the desired protein band was cut out, the protein was extracted from the membrane, and the N-terminal amino acid sequence of the protein was determined using a Procise® 491HT automated protein sequencer (Applied Biosystems, Foster City, CA, USA). The chemical process employed by the protein sequencer to determine the amino acid sequence was derived from the degradation method developed by Edman.
2.6. Isolation of total RNA and cDNA synthesis
Total RNA was extracted from the freeze-dried mushroom powder using Isogen II (Nippon Gene, Japan), according to the manufacturer's instructions. First-strand cDNA was synthesized using an oligo(dt)20 adapter primer (5´-CGCCAGGGTTTTCCCAGTCACGACTTTTTTTTTTTTTTTTTTTT-3´) and PrimeScript II reverse transcriptase (Takara Bio, Japan), according to the manufacturer's instructions.
2.7. cDNA cloning and sequencing of the acid trehalase gene from Pleurotus sp.
We obtained data on the trehalase gene from P. ostreatus PC15 v2.0 (Protein ID: 1094192) and P. eryngii ATCC 90797 v1.0 (Protein ID: 595636) from the Joint Genome Institute (JGI). PCR amplification was performed using a P. ostreatus forward primer (5´-ATGCTGACTTCGTTGGCGGCGACGTT-3´) and a P. eryngii reverse primer (5´-GTCTTGCTGCCTTCGTGGCGCTATGA-3´). In this case, we used a P. eryngii reverse primer. Because the nucleotide sequence of 3'-region of trehalase gene from Pleurotus sp. was more likely to resemble the sequence of trehalase gene from P. eryngii.
PCR was carried out in a reaction mixture (20 μL) containing the Pleurotus sp. cDNA, 0.5 μM each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio, Shiga, Japan), using the following thermocycling conditions: one cycle at 98 ºC for 1 min; followed by 30 cycles of 98 ºC for 10 s, 60 ºC for 5 s, and 72 ºC for 60 s; and one final cycle at 72 ºC for 5 min. To determine the entire sequence, we created several primers using the decoded sequence. DNA sequencing was determined by the method of Sanger (1981). The sequence data reported in the present paper have been submitted to the DDBJ, EMBL, and NCBI databases under the accession number LC558355.
2.8. Expression of acid trehalase in Pichia pastoris
Forward (5´-AGAGAGGCTGAAGCTGAATTCCAAACTTCATCCGCGACGGCGCCAG-3´, EcoRI site underlined) and reverse (5´-GAGTTTTTGTTCTAGAAATAGCGCCACGAAGGCAGCAAGACCG-3´, XbaI site underlined) primers were synthesized on the region corresponding to amino acid residues 20-29 and 742-749 of Pleurotus sp. acid trehalase, respectively. PCR was carried out in a reaction mixture (20 μL) containing the Pleurotus sp. cDNA, 0.5 μM each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio), using the following thermocycling conditions: one cycle at 98 ºC for 1 min; followed by 30 cycles of 98 ºC for 10 s, 60 ºC for 5 s, and 72 ºC for 60 s; and one final cycle at 72 ºC for 5 min. The nucleotides of the amplified fragment were confirmed by sequencing. The amplified fragment was inserted into the EcoRI/XbaI sites of the pPICZαA vector (Invitrogen, Waltham, MA, USA). The expression plasmid was linearized by SacI and transformed into competent Pi. pastoris GS115 cells by electroporation. Cells were spread on YPDS medium (1% yeast extract, 2% peptone, 2% dextrose,1 M sorbitol, and 1.5% agar) containing 100 μg/mL of Zeocin and incubated at 28 ºC for 2-4 d. Colonies were picked and spread onto separate YPDS plates containing 100, 500, 1000, and 2000 μg/mL of Zeocin. Colonies that exhibited growth in the presence of a high concentration of Zeocin were then selected and cultured in a 500 mL Erlenmeyer flask containing 25 mL of BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × 10-5% biotin, and 1% glycerol) at 28 ºC at 250 rpm for 48 h. The culture medium was centrifuged at 3,000 × g for 5 min and the resulting cell pellets were resuspended in BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × 10-5% biotin, and 0.5% methanol). The cell suspension was then added to 0.2 L of BMMY medium and grown at 17 ºC for 7 d at 230 rpm, with 0.5% methanol added daily.
2.9. Effects of pH and temperature on acid trehalase activity and enzyme stability
The acid trehalase activity of the purified recombinant enzyme (0.12 U/mL) was measured using trehalose as a substrate at 37 ºC in 0.1 M glycine-HCl buffer (pH 2.5-3.0), 0.1 M citrate-phosphate buffer (pH 3.0-4.0), 0.1 M sodium acetate buffer (pH 4.0-6.0), 0.1 M phosphate buffer (pH 6.0-8.0), 0.1 M Tris-HCl buffer (pH 8.0-9.0), and glycine-NaOH buffer (pH 9.0-11.0). The effect of temperature on trehalase activity was examined at 20-80 ºC.
The effect of pH on enzyme stability was analyzed by incubating the enzyme for 24 h at 4 ºC in a wide-range buffer (A solution: 0.2 M borate and 0.05 M citric acid; B solution: 0.1 M Na3PO4•12H2O, adjusting the pH after mixing the A and B solutions). After incubation, the remaining activity was measured using trehalose. To examine the effect of temperature on enzyme stability, the purified enzyme (0.12 U/mL) was incubated in 20 mM sodium acetate buffer (pH 5.0) for 30 min at various temperatures in the range 20-80 ºC. After incubation, the remaining activity was measured using trehalose at 37 ºC.
2.10. SDS-PAGE
Protein samples were treated with an equal volume of 2 × SDS sample buffer [0.125 M Tris-HCl (pH 6.8), 14% glycerol, 4% SDS, 0.01% bromophenol blue, and 10% 2-mercaptoethanol], then separated via SDS-PAGE using the method of Laemmli (Laemmli, 1970). A 10% acrylamide gel for SDS-PAGE was used as the separating gel. Unstained protein molecular-weight markers were also run, to calibrate the molecular mass of the sample proteins. Protein bands were detected by staining with Coomassie Brilliant Blue R-250.
2.11. Bottom-up peptide analysis using LC-MS
In the bottom-up peptide analysis using LC-MS, excised polyacrylamide gel pieces were destained with 100 μL of 50% acetonitrile containing a 25 mM ammonium bicarbonate solution for 1 h at room temperature with gentle agitation. Destained gel pieces were treated for reduction and alkylation using 100 μL of 10 mM DTT in 25 mM ammonium bicarbonate for 45 min at 56 ºC and 100 mL of freshly prepared 10 mM iodoacetamide in 25 mM ammonium bicarbonate in the dark for 30 min at 37 ºC, respectively. After the gel plugs were dried, 400 ng of sequencing-grade trypsin (Trypsin Gold, Promega, Madison, WI, United States) in 20 μL of 25 mM ammonium bicarbonate was added and the plugs were incubated for 12 h at 37 ºC. Digested peptides were recovered from the gel plugs using 50 μL of 50% acetonitrile in 5% formic acid (FA) for 30 min at 25 ºC. The extracted peptides were concentrated in a speed-vacuum concentrator and added to 20 μL of 5% acetonitrile in 0.1% FA.
A Nano-HPLC system (nanoADVANCE, Bruker-Michrom, Billerica, MA, United States) was used to identify proteins automatically using a micro-column switching device coupled to an autosampler and a nanogradient generator. The peptide solution (5 μL) was loaded onto a C18 reversed-phase capillary column (100 mm ID × 30 cm, Zaplous aPep C18; AMR, Tokyo, Japan) in conjunction with a Magic AQ C18 trapping column (300 mm ID × 10 mm; Bruker-Michrom). The peptides were separated using a nanoflow linear acetonitrile gradient of buffer A (0.1% FA) and buffer B (0.1% FA, 99.9% acetonitrile), going from 5% to 45% buffer B over 50 min at a flow rate of 500 μl/min. The column was then washed in 95% buffer B for 5 min. The Hystar 3.2 system control software (Bruker Daltonics Inc., Billerica, MA, United States) was used to control the entire process. The eluted peptides were ionized through a CaptiveSpray source (Bruker Daltonics) and introduced into a Maxis 3G Q-TOF mass spectrometer (Bruker Daltonics) set up in a data-dependent MS/MS mode, to acquire full scans. The four most-intense peaks in any full scan were selected as precursor ions and were fragmented using collision energy. MS/MS spectra were interpreted, and peak lists were generated using the DataAnalysis 4.1 and BioTools 3.2 (Bruker Daltonics Inc) software for detailed bottom-up characterization of proteins and peptides.
Fixed modification was set on cysteine with carbamidomethylation. Variable modification was based on methionine with oxidation and asparagine/glutamine with deamidation. The maximum missed cleavage was set to two and limited to trypsin cleavage sites. Precursor mass tolerance (MS) and fragment mass tolerance (MS/MS) were set to 100 ppm and ± 0.6 Da, respectively. Proteins that were identified using a threshold of 0.05 were used. Peptides scoring < 20 were automatically rejected, to ensure that all protein identifications were based on reliable peptide identifications.
2.12. Substrate specificity
The activities of the purified acid trehalase were tested using 10 mM trehalose, 10 mM maltose, 10 mM lactose, 10 mM sucrose, 0.1% soluble starch, and 0.1% glycogen. In the case of sucrose, trehalose, soluble starch, and glycogen, the breakdown of each substrate was measured as described above. The enzymatic activities against maltose and lactose were assayed based on the hydrolysis products obtained using high-performance anion exchange chromatography (HPAEC). After the reaction, each sample solution was centrifuged at 17,700 × g for 5 min, and the supernatant was used for HPAEC with a CarboPac PA-1 column (4 × 250 mm, Thermo Fisher Scientific). Elution was performed using 0.1 M NaOH (0-5 min), 0.1 M NaOH, 0-0.15 M sodium acetate (5-15 min), 0.1 M NaOH, 0.8 M sodium acetate (15-20 min), and 0.1 M NaOH (20-35 min) at a flow rate of 1.0 mL/min, and the substrates and products were monitored using a pulsed amperometric detector.
2.13. Effect of metal ions on enzymatic activity
Enzymatic activities were determined via an enzymatic assay using trehalose after the preincubation of the enzyme in 0.1 M Tris-HCl (pH 8.0) containing each compound at 4 ºC for 24 h. The concentration of metal ions and EDTA was 1 mM. All metal ions were added as chloride salts.
2.14. Effect of inhibitors on enzymatic activity
The recombinant acid trehalase and validamycin A (FUJIFILM wako chemicals, Osaka, Japan) and 1-deoxynojirimycin (FUJIFILM wako chemicals) solutions (10-2 - 104 μM) were preincubated in 20 mM acetate buffer (pH 5.0) at 37 ºC for 30 min. After preincubation, the enzymatic activities were measured using a standard assay method.
2.15.Endo-H treatment of the recombinant acid trehalase
Purified recombinant acid trehalase was treated with Endo-H in denaturing buffer at 4 ºC for 24 h. After treatment, the sample was applied onto a SuperdexTM200 10/300 GL column equilibrated with 20 mM phosphate buffer (pH 6.0). The endo-H treated sample was analyzed using SDS-PAGE.
3. Results and discussion
3.1. Partial purification, gene cloning and sequencing of the acid trehalase from Pleurotus sp.
After Superdex 200 gel filtration column chromatography, the peak fraction of acid trehalase from Pleurotus sp. was applied to SDS-PAGE (data not shown). The molecular masses of the main bands were estimated at 90 and 50 kDa. The N-terminal amino acid sequence of the Pleurotus sp. 90 kDa protein was determined to be LPXQVPLPP- (X: unidentified), which is in good agreement with the corresponding sequence of the GH family 37 protein from P. ostreatus PC 15 v2.0 (GenBank: KDQ26031).
Using gene cloning, the length of the Pleurotus sp. acid trehalase gene was determined to be 2247 bp, encoding for a protein of 749 amino acids. The predicted molecular mass of the protein was 81.2 kDa. The amino acid sequence of Pleurotus sp. acid trehalase was similar to those of the trehalases of P. ostreatus PC 15 v2.0 (98%, GenBank: KDQ26031), P. eryngii (95%, JGI protein ID: 595636), and Crucibulum laeve (69%, GenBank: TFK40489), as well as that of the hypothetical protein of Panaeolus cyanescenes (67%, GenBank: PPQ63598.1) (Fig. 1). The acid trehalase (GH 37 protein) from Pleurotus sp. also shared the homology with that of neutral trehalase (GH 37 protein, 27%, Gene Bank: X65925.1) from S. cerevisiae. It was reported that GH37 neutral trehalase from S. cerevisiae is activated by 14-3-3 protein and Ca2+ ion (Alblova et al., 2017). 14-3-3 protein controls cell cycle, cell growth, differentiation, survival, apoptosis, migration and spreading (Mhawech, 2005). In future work, we intend to elucidate the activation mechanism of acid trehalase from Pleurotus sp.
Fig. 1 Sequence alignment of trehalases from Pleurotus sp., P. eryngii (95%, JGI Protein ID595636), P. ostreatus (98%, KDQ26031), C. laeve (69%, TFK40489), and Pa. cyanescenes (67%, PPQ63598.1). The percentages indicated in parentheses represent the sequence identities with the amino acid sequence of the protein from Pleurotus sp. The asterisk indicates the conserved amino acids among the trehalases. The colon and period indicate a site belonging to a group exhibiting strong and weak similarity, respectively. All sequences are numbered from the Met-1 residue of the peptide.
Phylogenetic analysis was performed based on the amino acid sequence homologies of acid trehalase and GH family 37 from other species. The phylogenetic analysis revealed that Pleurotus sp. acid trehalase is more closely related to P. ostreatus trehalase than to P. eryngii trehalase (Fig. 2).
Fig. 2 Phylogenetic tree of trehalase from Pleurotus sp. and other species from the GH family 37. The enzyme names and accession numbers were as follows: P. eryngii trehalase (JGI Protein ID: 595636), P. ostreatus trehalase (KDQ26031), Tricholoma matsutake trehalase (JGI Protein ID: 1283800), Agaricus bisporus trehalase (XP_006462478), Coprinopsis cinerea trehalase (XP_001835625), Lentinula edodes trehalase (GAW08978.1), Shizophyllum commune trehalase (XP_003034277), Mus musculus trehalase (Q9JLT2.1), Arabidopsis thaliana trehalase (Q9SU50.1), and Saccharomyces cerevisiae trehalase (CAA58961.1). We used Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) to construct the phylogenetic tree. A phylogenetic tree was created following the protocol on the Clustal Omega site. This is a Neighbour-joining tree without distance corrections.
The mature active form (amino acid residues 19 to 749) of Pleurotus sp. acid trehalase was expressed in Pi. pastoris GS 115, and the acid trehalase activity of the crude solution of the recombinant enzyme was 0.25 U/mL. The present recombinant enzyme was purified from Pi. pastoris GS115 harboring pPICZαA-Pleurotus sp. acid trehalase and its molecular mass was estimated to be 117 kDa using SDS-PAGE (Fig. 3, lane 2). The protein band was very broad. The acid trehalase from Pleurotus sp. contained eighteen N-glycosylation sites; thus, it was considered that the recombinant acid trehalase was glycosylated. When the recombinant acid trehalase was treated with Endo-H, the protein band shifted from 117 kDa to 80 and 70 kDa (Fig. 3, lane 3). The protein band of 29 kDa was derived from Endo-H (Fig. 3, lane 3). We found that the recombinant enzyme had an N-glycosylated sugar chain. The specific activity of recombinant acid trehalase was 0.475 U/mg protein. The specific activity of Endo-H-treated recombinant enzyme was 0.387 U/mg protein. The deglycosylated recombinant enzyme also retained the activity.
Fig. 3 SDS-PAGE of the recombinant purified trehalase and Endo-H-treated trehalase. The Precision Plus Protein All Blue Standard was used as a marker (Bio-Rad, Hercules, CA, USA). Lane 1: marker, lane 2: recombinant purified acid trehalase, Lane 3: Endo-H-treated acid trehalase.
We tried to perform the bottom-up analysis using the deglycosylated recombinant enzyme (80 kDa). The protein sequence coverage of Endo-H treated recombinant acid trehalase was 46.5% (Fig. 4). The recombinant protein was identified as acid trehalase from Pleurotus sp.
Fig. 4 Recombinant acid trehalase sequence coverage. Sequence coverage of recombinant acid trehalase was calculated after identification of peptides detected in the peptide map. The amino acid sequence is shown with red letters to indicate the identified regions. The amino acid sequences of blue letters were derived from pPICZalpha vector. Sequence coverage does not include vector-derived sequences.
The activity and stability of recombinant acid trehalase at various temperatures and pH values were determined in enzymatic assays using trehalose as the substrate. The optimum pH of recombinant acid trehalase was 4.5 (Fig. 5A), and its activity was stable between pH 3.0 and 7.0 (Fig. 5B). The mushroom trehalases from Tricholoma matsutake (pH 5.0), and L. edodes (pH 5.0) have similar reported optimum pH values (Kusuda et al., 2010; Murata et al., 2001). Fungi and yeast trehalases also exhibit optimal activity around pH 5.0 (Biswas & Ghosh, 1996; Bharadwaj & Maheshwari, 1999). Arastoo et al. (2018) reported that the pH in the fruit body is weakly acidic during fruit-body development (Arastoo et al., 2018). It is expected that acid trehalase exists in a stable state in fruit bodies.
Fig. 5 Functional properties of purified recombinant acid trehalase. All reactions were conducted with purified enzyme using trehalose as the substrate. A; Effect of pH on enzymatic activity at 37 ºC in the following buffers (0.1 M): glycine-HCl (pH 2.5-3.0); citrate-phosphate (pH 3.0-4.0); CH3COOH-NaOH (pH 4.0-6.0); KH2PO4-NaOH (pH 6.0-8.0); Tris-HCl (pH 8.0-9.0); and glycine-NaOH (pH 9.0-11.0). B; Effect of pH on enzymatic stability. Assays were conducted at 37 ºC (pH 5.0) after a 24 h incubation in the broad-range buffer (pH 2.0-12.0). The pH was adjusted to the mix of solution A (0.2 M boric acid and 0.05 M citrate) and solution B (0.1 M Na3PO4•12H2O). C; Effect of temperature on enzymatic activity measured from 20-80 ºC. D; Effect of temperature on enzyme stability. Assays were performed at 37 ºC after a 30 min incubation in 20 mM sodium acetate buffer (pH 5.0) at 20-80 ºC. Each data point represents the average of triplicate measurements. Error bars show standard deviation of measurements.
The optimal temperature for recombinant trehalase was 40 ºC (Fig. 5C), which was similar to those observed for T. matsutake (40 ºC), L. edodes (40 ºC), S. cerevisiae (40-45 ºC), and Bombyx mori (40-45 ºC) (Kusuda et al., 2010; Arastoo et al., 2018; Biswas & Ghosh, 1996; Huang, Furusawa, Sadakane, & Sugimura, 2006). In contrast, the optimal temperature (60 ºC) for the trehalase from Humicola grisea was higher than that of Pleurotus sp. acid trehalase (Lúcio-Eterovic, Jorge, Polizeli, & Terenzi, 2005), and recombinant trehalase was stable at 40 ºC (Fig. 5D).
3.5. Substrate specificity
Maltose, lactose, sucrose, soluble starch, and glycogen were examined as substrates and compared with trehalose. Recombinant acid trehalase from Pleurotus sp. showed high activity exclusively toward trehalose. This result was similar to that obtained for the trehalase from L. edodes (Murata et al., 2001).
3.6. Effects of metal ions and inhibitors on enzymatic activity
The effects of various metal ions and EDTA on enzymatic activity are shown in Table 1. The enzymatic activity was strongly inhibited by Al3+, Fe2+, and Hg2+. The addition of Cu2+ and Zn2+ resulted in moderate inhibition. The effects of Al3+, Fe2+, and Hg2+ on enzymatic activity were similar to those of the trehalase from L. edodes (Murata et al., 2001). The acid trehalase from Pleurotus sp. was shown the almost no effect of Mn2+ addition. Mn2+ was stimulated of the trehalase activities from H. grisea (Lúcio-Eterovic et al., 2005), S. serevisiae (Maicas, Guirao-Abad, & Argüelles, 2016), and Chaetomium thermophilum (Lúcio-Eterovic et al., 2005).
Table 1 Effects of metal ions and EDTA against the activity of acid trehalase
The enzyme activities were determined with the enzyme assay using trehalose following the pre-incubation of enzyme in 0.1 M Tris-HCl buffer (pH 8.0) containing each compound at 4 ºC for 24 h. The concentration of metal ions and EDTA were used in 1 mM. All the metal ions were added as chloride salts. The amount of reducing sugar contained in the sample was then determined according to the standard assay method. Data are shown as means ± SD of triplicate measurements.
| Added substances |
Residual activity (%) |
| Control |
100 ± 3.27 |
| EDTA |
110 ± 25.0 |
| Mg2+ |
84.8 ± 3.91 |
| Al3+ |
13.5 ± 2.57 |
| Ca2+ |
86.0 ± 5.39 |
| Mn2+ |
79.8 ± 4.38 |
| Fe2+ |
8.61 ± 0.490 |
| Cu2+ |
46.3 ± 1.19 |
| Zn2+ |
56.4 ± 6.11 |
| Ag+ |
98.6 ± 10.0 |
| Ba2+ |
91.9 ± 4.51 |
| Hg2+ |
5.63 ± 0.992 |
The inhibition of recombinant acid trehalase by validamycin A (IC50 = 0.98 μM) was stronger than that of 1-deoxynojirimycin (IC50 = 16 μM) (Fig. 6). The IC50 of trehalases by validamycin A were 11.0 μM and 7.6 μM in Rhizoctonia oryzae and Sclerotium fumigatum (Shigemoto, Okuno, & Matsuura, 1989). We found that validamycin A had a high specificity against the recombinant acid trehalase. It was reported that validamycin A acted as competitive inhibitor of the trehalases from Amanita muscaria (Wisser, Guttenberfer, Hampp, & Nehls, 2000) and Dictostelium discoideum (Temesvari & Cotter, 1997).
Fig. 6 Effects of inhibitors on the recombinant acid trehalase. The recombinant acid trehalase and inhibitor solution (10-2 to 104 μM) were preincubated for 30 min at 37 ºC. After preincubation, the enzymatic activities were measured using a standard assay. The average value of triplicate measurements was used for each activity value.
To understand the function of acid trehalase during the growth of fruit bodies and autolysis, we intend to investigate the expression level of the enzyme gene using real-time PCR. We have been also planning to elucidate the structure of acid trehalase using X-ray crystallography.
