28
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The purified enzyme hydrolyzed soluble starch into glucose at 13.2 units/mg under conditions. The purified TmGLA gave a single protein band on SDS-PAGE, with a molecular mass of approximately 61.5 kDa (Fig. 2). The molecular mass of the native enzyme as determined by high-performance gel chromatography on a TSKgel G3000SWXL column was 63.9 kDa, indicating that the native enzyme was active as a monomer.
Fig. 2. SDS-PAGE of purified TmGLA.
Proteins were separated on 12.5% polyacrylamide gel in the presence of 0.01% SDS.
Lane1, molecular weight standards; Lane 2, Purified TmGLA.
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To identify the purified TmGLA, the internal amino acid sequences of the protein were analyzed. After SDS-PAGE of the purified TmGLA, it was digested by trypsin and then peptide fragments were obtained. The tryptic peptide fragments, G-1 to G-7, were sequenced by MALDI-TOF/MS analysis (Table 2).
Table 2. Tryptic fragments matching the theoretical digested fragments of glucoamylase (TmGLA) from Tricholoma matsutake a.
No. Measured mass (Da)
Predicted mass (Da)
Δmass
(Da) MC b Tryptic fragment sequence Position (start-end)
G-1 2307.022 2306.206 -0.816 0 AGVVIASPSTTNPNYLFTWIR 55—75
G-2 1991.847 1990.965 -0.882 1 FNIDETAFTGPWGRPQR 130—146
G-3 2999.786 2999.049 -0.030 0 DANTVLASIHTFDAAAGCDATTFQPCSDR 269—297
G-4 1466.704 1465.793 -0.911 0 ALANLLTYVDAFR 298—310
G-5 1907.833 1906.826 -1.007 0 NIYAINSGIATNQGSATGR 311—329
G-6 1527.646 1526.737 -0.909 0 YTPIGGGLSEQYSR 415—428
G-7 1085.528 1084.520 -1.007 0 QGIAPASWGAK 454—464
a Peptide mass spectrometric analysis was performed on a MALDI-TOF/MS system; autoflex speed KN2 and the MALDI-TOF/MS data were analyzed using Proteinscape.
b MC, number of miscleavages in tryptic digestion.
NCBInr BLAST search of the protein database indicated that the peptide sequences of the enzyme were identical to that of glycoside hydrolase family 15 protein TmGlu1 from Tricholoma matsutake NBRC30773 [21]. Attempted analysis of the N-terminal amino acid sequence of TmGLA did not detect the sequence. Yoon et al. reported the purification and molecular cloning of GH family 15 glucoamylase of carbohydrate-active enzymes (CAZymes; CAZY Carbohydrate-Active enZYmes.: http://www.cazy.org/) from the brown-rot basidiomycete Fomitopsis palustris (FpGLA). The N-terminal glutamine of the native FpGLA might have been changed to pyroglutamate, leading to a failure of Edman degradation to function [48]. Because the amino acid sequence of FpGLA showed 62% homology with that of TmGLA and the N-terminal amino acid of TmGLA is glutamine, it is suggested that the glutamine changes to pyroglutamate in native TmGLA. Therefore, we detected the internal amino acid sequence of TmGLA by peptide mass fingerprinting using MALDI-TOF/MS.
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Enzymatic properties of TmGLA.
The enzymatic properties of TmGLA were determined using soluble starch as a substrate. The enzyme activity was determined using the Glucose Assay Kit (BioVision).
The effects of chemical compounds on TmGLA activity were assayed. The enzyme was slightly activated by the presence of the Ca2+ and Mn2+. Among the Ba2+, K+, Al3+, Mg2+, Fe3+, Na+, Ni+, Li+, Cu2+, Co2+, EDTA and DTT had no effect on enzyme activity. The activity of the enzyme was slightly inhibited by the presence of 5 mM Hg2+, Fe2+, Zn2+, and Pb2+. In contrast, 5 mM Ag+ was strongly inhibited the glucoamylase activity. There is a result indicating that this enzyme was not a divalent cation (Ca2+, Mg2+, Mn2+) requiring protein, suggesting the stabilization of the higher structure of TmGLA. The effects of pH and temperature on enzyme activity are shown in Fig. 3. The enzyme was active between 4°C and 50°C, and was stable pH 4.0-6.0, with maximum activity at 60°C and pH 5.0.
Enzymatic characteristics of glucoamylase from basidiomycetes have been investigated and reported; however, few reports have been published on species such as Lyophyllum shimeji (LyGLA) [34], Lentinula edodes (LeGLA; gla1) [49], Schizophyllum commune (ScGLA) [50], and Hypocrea peltata (HyGLA) [51]. Except for LyGLA, the enzymatic properties of these enzymes are similar to TmGLA with regard to active temperature and pH range, the effect of metal ions, and molecular mass. LyGLA was purified from extracellular L. shimeji strain MH01721. The purified enzyme showed a molecular mass of approximately 25 kDa on SDS-PAGE, and was most active at around 40 °C and pH 5.0. LyGLA was remarkably activated by the presence of Ca2+ ions. These properties of LyGLA differ from those of other basidiomycetes, including TmGLA.
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Fig. 3. Effect of temperature and pH on purified TmGLA enzyme activity. A and B:
Effect of temperature on enzyme stability and optimal activity, respectively. A: Purified enzyme was incubated in 50 mM sodium acetate buffer at pH 5.0 for 60 min; temperature range, 4°C-80°C. B: The effect of temperature (4°C-80°C) on enzyme activity. C and D:
Effect of pH on enzyme stability and activity, respectively. C: Enzyme was incubated for 30 min at 30°C in 50 mM sodium acetate buffer (pH 2.0-5.0, black square), 50 mM sodium phosphate buffer (pH 5.0-8.0, black circle), or 50 mM Tris-HCl buffer (pH 8.0-10.0, black triangle). D: The activity of purified enzyme on soluble starch in 50 mM sodium acetate buffer (pH 2.0-5.0, black square), 50 mM sodium phosphate buffer (pH 5.0-8.0, black circle), or 50 mM Tris-HCl buffer (pH 8.0-10.0, black triangle). Data are presented as the mean of three trials ± SD.
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The substrate specificity of TmGLA was assayed using carbohydrates with different polysaccharides (Table 3). TmGLA readily hydrolyzed amylose A (MW, 2,900;
146%) and showed equal activity against pullulan (95%), amylopectin (92%), and amylose B (MW, 16,000; 90%). Activity against glycogen and corn starch was weak (49%
and 35%, respectively). In addition, the enzyme also acted against malto-oligosaccharides (maltose, 15%; maltotriose, 52%; maltotetraose, 65%; and maltopentaose, 74%) and isomaltose (7%). No enzyme activity was observed with the substrates trehalose (α, α-1,1-linkage), kojibiose (α-1,2-linkage), nigerose (α-1,3-linkage), α-cyclodextrin, and β-cyclodextrin. These results indicate that TmGLA hydrolyzes α-1,4 and α-1,6 linkages of glycosidic saccharides. Soluble starch was hydrolyzed by TmGLA, and the final products were analyzed by TLC (Fig. 4). It was shown that the purified glucoamylase gradually hydrolyzed to glucose units during 24 hours after the start of the reaction.
Glucoamylase (α-1,4-glucan glucohydrolase, amyloglucosidase, EC 3.2.1.3) belongs to GH family 15; it is an exo-acting enzyme that removes the glucose units from the non-reducing ends of amylose, amylopectin, and glycogen by hydrolyzing α-1,4-linkages in a consecutive manner, producing D-glucose as the sole product. It also hydrolyzes α-1,6-linkages, although at a much slower rate [52]. TmGLA showed broad specific activity toward and α-1,6-glycosidic substrates. Amylose A with α-1,4-glycosidic polysaccharide could be the best substrate of the enzyme, and low-molecular-weight amylose A (average M.W. 2,900) was better than the high-molecular-low-molecular-weight form (amylose B, average M.W. 16,000) in this regard. Hydrolyzing activity toward several malto-oligosaccharides was also examined. The enzyme could hydrolyze maltopentaose, maltotetraose, maltotriose, maltose, and isomaltose, although the activity was lower toward smaller oligosaccharides.
However, the enzyme did not hydrolyze the cyclic polysaccharides. When the enzyme hydrolyzed low-molecular-weight amylose A, the produced saccharide was glucose. From these results, we conclude that TmGLA is a common glucoamylase belonging to GH family 15.
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Fig. 4. Hydrolysis product of soluble starch by the purified enzyme.
TmGLA was incubated in 50mM sodium acetate buffer (pH 5.0) at 37 ℃ for 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h and 24 h. Hydrolysates were subject with TLC in ethyl acetate/methanol/water (8:4:3, v/v) and stained with 50% H2SO4.
Table 3. Substrate specificity of TmGLA.
Substrate a Relative activity
(%)
Specific activity (units/mg protein) Polysaccharides (1.0%)
Soluble starch 100 13.9
Amylose A (MW, 2,900) 139 19.3
Amylose B (MW, 16,000) 90 12.5
Amylopectin 92 12.8
Pullulan 95 13.2
Glycogen 49 6.81
Corn starch 35 4.86
Oligosaccharides (10 mM)
Maltopentaose 74 10.3
Maltotetraose 65 9.02
Maltotriose 52 7.22
Maltose 16 2.22
Isomaltose 7 1.04
β-cyclodextrin ND ND
α-cyclodextrin ND ND
Trehalose ND ND
Kojibiose ND ND
Nigerose ND ND
a Assays were performed at 37°C and pH 5.0. The activities on
polysaccharides and oligosaccharides are based on released D-glucose as determined using the Glucose assay kit.
ND, not detected
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Cloning and sequencing of TmGlu1 gene.
The de novo genome sequence of the genomic DNA T. matsutake NBRC 30605 was determined using Illumina NextSeq 500 paired-end technology provided in Kindai University, Faculty of Agriculture. The final assembled data contained 88,884 contigs of total length 131,718,925 bp, with an N50 contig length of 2909 bp. The gene prediction and functional annotation were carried out by AUGUSTUS 2.5.5 [53] and the Microbial Genome Annotation Pipeline [MiGAP; 54]. It predicted a total of 23,546 protein coding genes. The deduced amino acid sequences of seven trypsin-digested peptides from native TmGLA of TmGlu1 from T. matsutake NBRC30773 [accession no. AB604354; 21] were mapped. A 2,186 bp DNA fragment starting at ATG and terminating at the TAG codon was found in the nucleotide sequences encoding TmGlu1 on the whole genome sequence data of T. matsutake NBRC 30605. Alignment with the TmGlu1 cDNA indicated that this genomic sequence contained the complete protein-coding region. The coding region of the TmGlu1 gene (1,731 bp) was interrupted by eight introns (58, 59, 59, 59, 56, 52, 56, and 56 bp). The open reading frame encoded a polypeptide of 576 amino acids (Fig. 5).
The first 19 amino acid residues in the N-terminal region were assigned as a signal peptide using the online program SignalP 4.1 server. The result of NCBI Conserved Domain Search suggested that this protein has a two-domain structure consisting of an N-terminal domain with the GH family 15 signature and a C-terminal carbohydrate-binding module (CBM) 20 region. Six N-glycosylation sites were predicted as an Asn-Xaa-Thr/Ser region [55]. The alignment of the TmGlu1 protein sequence compared with the T. matsutake NBRC 30773 [21], Pleurotus ostreatus PC15 [56], Phanerochaete carnosa HHB-10118-sp [57], Pholiota microHHB-10118-spora [58], Lentinula edodes L-54 [59], F. palustris FFPRI 0507 [48] and glucoamylase gene encoding sequences, deduced from the individual cDNAs were shown in Table 4. The primary structure of TmGlu1 was found to have 100%, 73%, 70%, 69%, 67%, 66%, and 62% sequence homology with the sequences of these glucoamylase genes encoding amino acid respectively.
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Fig. 5. Nucleotide sequence and deduced amino acid sequence of TmGLA. The potential signal peptide sequence is double-underlined. Tryptic fragments from the purified enzyme are outlined. The box indicates potential N-glycosylation sites (Asn-Xaa-Ser/Thr). The carbohydrate binding module (CBM) 20 domain is underlined.
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Table 4. Homology between Tricholoma matsutake glucoamylase and that from several other fungi.
Microorganism [Strain] Homology (%) GenBank accession no.
Tricholoma matsutake [NBRC 30773] 100 BAL43555.1 Pleurotus ostreatus [PC15] 73 KDQ28034.1 Phanerocheate carnosa [HHB-10118-sp.] 70 XP_007399504.1 Pholiota microspora [NGW19-6] 69 BAM72726.1 Pholiota microspora [NGW19-6] 67 BAU78332.1 Lentinula edodes [L-54] 66 AAF75523.1 Fomitopsis palustris [FFPRI 0507] 62 BAE47183.1
Expression of TmGlu1 gene in P. pastoris X-33 and purification of the recombinant enzyme.
Based on the gene sequence, gene cloning of TmGLA from T. matsutake NBRC30605 was performed. The deduced amino acid sequence of the gene was identical to that of TmGlu1 from T. matsutake NBRC 30773. The putative glucoamylase gene sequence from T. matsutake was detected and characterized as TmGlu1, which was cloned by using degenerate primers designed based on the conserved amino acid sequences of glucoamylases in several basidiomycetes, and when T. matsutake mycelia were cultured in medium containing starch as a carbon source, the gene showed a correspondingly high transcription level [21]. However, whether the product of the TmGlu1 gene has glucoamylase activity was not confirmed. To elucidate whether the translated TmGlu1 gene had TmGLA activity, the gene was expressed in P. pastoris X-33, although E. coli expression systems were doing not work at all as inclusion body (data not shown).
Synthetic TmGlu1 gene, cloned in-frame with the S. cerevisiae α-mating factor secretory signal under the control of the methanol-inducible AOX1 promoter, was integrated into the genome of P. pastoris X-33 using a zeocin selection strategy.
Recombinant TmGlu1 was purified from the culture supernatant of the P.
pastoris X-33 transformant, as described in “Materials and Methods,” and was used for glucoamylase assay. The time course of the glucoamylase activity with secretory expression in Pichia pastoris is shown in Fig. 6A. The glucoamylase activity was
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approximately 8.8-fold greater in the presence of casamino acids. Purified enzyme (3.63 mg) was obtained from one liter of the culture supernatant. The enzyme showed a specific activity of 10.3 unit/mg for soluble starch. On SDS-PAGE analysis, a band of glycosylated recombinant TmGlu1 appeared at about 65.0 kDa, as determined by comparison with the molecular mass marker. After endoglycosidase H treatment, the band appeared at about 59.0 kDa in the non-glycosylated state; when the same treatment was carried out using the native enzyme, bands of the same molecular weight were observed (Fig. 6B). Native TmGLA underwent the same shift in mobility in SDS-PAGE analysis.
Fig. 6. Time course of glucoamylase activity expressed in Pichia pastoris X-33 and SDS-PAGE analysis of the purified recombinant enzyme. A: Black dot, X-33/TmGlu1 (BMMH with 1.0% casamino acid); black triangle, X-33/control (BMMH with 1.0% casamino acids); white dot, X-33/TmGlu1 (BMMH without casamino acids); and white triangle, X-33/control (BMMH without casamino acids). Data are presented as the means ± SD (n = 3). B: SDS-PAGE of purified native TmGLA and recombinant enzyme with endoglycosidase H treatment. Lane 1, purified TmGLA from T. matsutake NBRC 30605; Lane 2, purified TmGLA treated with endoglycosidase H; Lane 3, purified recombinant enzyme from culture supernatant from Pichia pastoris; Lane 4, purified recombinant enzyme treated with endoglycosidase H; Lane 5, molecular weight standards.
A B
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Also, the recombinant protein showed almost the same enzymatic properties as native TmGLA with regard to the effects of temperature, pH, metal ions, and substrate specificity (data not shown). The internal amino acid sequence from recombinant TmGlu1 was detected by MALIDI-TOF/MS. The peptide sequences of G-4 and G-5 (Table 2) matched the native TmGLA peptide sequences.
These results suggested that glycosylation of the recombinant protein is similar to that of native TmGLA from T. matsutake NBRC30605, although which type of carbohydrate molecules attached to both proteins requires further investigation. P.
pastoris utilizes most of the post-translational modification pathway typically associated with higher eukaryotes. Thus, the secreted proteins carry the appropriate sites for N-linked glycosylation of core oligosaccharides on asparagine residues of the nascent polypeptide in the recognition sequence Asn-Xaa-Ser/Thr [55]. In the case of TmGlu1, the enzyme has 6 potential glycosylation sites (Fig. 5). A very low level of the recombinant protein was induced when standard BMMH medium was used for cultivation (Fig. 6A). The addition of casamino acid into the culture medium was critically effective for inducing expression of the recombinant protein. For example, Kaushik et al. reported that casamino acid supplementation increased the secretion of recombinant dengue virus serotype-3 envelope domain III (EDIII) [60]. The experiment clearly indicated that the supplementation increased the secretory titer of recombinant EDIII by reducing the intercellular retention of recombinant protein. Then TmGlu1 expression, the same phenomenon occurred because glucoamylase activity occurs intercellularly when cultivation methods without casamino acid were applied, although lower activity was detected in culture with casamino acid. However, there are no reports providing a concrete explanation for this unique effect of casamino acid for the P. pastoris expression system.
In the present study, the author report that T. matsutake NBRC 30605 strain produces active glucoamylase similar to that of other basidiomycetes such as L. edodes, S. commune, P. microspora, L. shimeji and H. paltata, each of which develops fruiting bodies in artificial media. It might be expressed amount of mycelial growth of the fungus by inducing of the enzyme in the cultivation with starch substrate. There are some researches regarding the relationship of fruiting body formation and glucoamylase activity in the ectomycorrhizal mushroom L. shimeji and the saprophytic mushrooms L.
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edodes and P. microspore. The glucoamylase activity of these basidiomycetes showed markedly increasing in fruiting body development stage. In L. shimeji, glucoamylase activity was 4.6 times higher in the fruiting body than in the mycelia [34]. On the other hands, glucoamylase activity from L. edodes was 4 - 5 times higher in the fruiting body than the mycelia [59]. Furthermore, Li et al. found that the gene expression level, activity and glucose contents in the medium were dramatically increased until fruiting body formation on P. microspore [61]. In the future research, it is necessary to establish detailed analysis of the growth condition of solid-state medium consisting mainly of starch and the role of glucoamylase in T. matsutake.
SUMMARY
An extracellular glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake NBRC 30605 strain (TmGLA) was purified homogeneity with a single band on SDS-PAGE. The purified enzyme is a monomer with a molecular mass of approximately 64 kDa. Its maximum activity was observed at 60°C and pH 5.0. The enzyme remains active at 50°C and in the pH range of 4.0-6.0. The enzyme activity is strongly inhibited by Ag+. The enzyme degrades α-1,4- and α-1,6-glycosidic linkages in various oligosaccharides and polysaccharides. The internal amino acid sequences (seven peptides) are identical to the glucoamylase from T. matsutake NBRC 30773 (TmGlu1) belonging to the glycoside hydrolase family 15 protein. Pichia pastoris transformed with the TmGlu1 gene secreted the active enzyme in glycosylated form, and its enzymatic characteristics were the same as those of the native enzyme. In this chapter, demonstrates that TmGLA is a potentially valuable agent for starch degradation in the vegetative mycelial growth of T. matsutake.
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