[Show abstract][Hide abstract] ABSTRACT: The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.
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[Show abstract][Hide abstract] ABSTRACT: We previously reported on a β-N-acetylhexosaminidase, LeHex20A, belonging to glycoside hydrolase family 20 (GH20), from the fruiting body of Lentinula edodes (shiitake mushroom). In this study, we purified, cloned, and characterized another β-N-acetylhexosaminidase, LeHex20B, from L. edodes fruiting bodies. The cDNA of LeHex20B includes an open reading frame of 1,686 bp encoding a 20 amino acid signal peptide and a 541 amino acid mature protein. The amino acid sequence identity of LeHex20A and LeHex20B was 57 %, and LeHex20B had high sequence identity to GH20 proteins; thus, LeHex20B belongs to GH family 20. LeHex20B showed β-N-acetylhexosaminidase activity and catalyzed degradation of chitooligosaccharides (GlcNAc2-6) exolytically with N-acetylglucosamine (GlcNAc) production. The maximum LeHex20B activity was observed at pH 5.0 and at 60 °C. LeHex20B had highest catalytic efficiency (k
m) for GlcNAc3 and showed high affinity for GlcNAc3-6. The transcript level of LeHex20A was significantly increased in fruiting bodies after harvest, suggesting that LeHex20A is mainly involved in fruiting body autolysis. On the other hand, LeHex20B was highly expressed in young fruiting bodies and mycelia. Therefore, LeHex20B seems to be mainly involved in elongation of fruiting bodies and mycelia.
No preview · Article · Apr 2015 · Journal of Wood Science
[Show abstract][Hide abstract] ABSTRACT: Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [β-d-Glcp-(1→6)-d-Glc] and gentianose [β-d-Glcp-(1→6)-d-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of β-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.
[Show abstract][Hide abstract] ABSTRACT: Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable β-glucan for medical purposes based on its anti-cancer activity and immunomodulating activity. However, lentinan content in fruiting bodies decreases after harvesting and storage due to an increase in glucanase activity. In this study, we downregulated the expression of an exo-β-1,3-glucanase, exg2, in L. edodes using RNA interference. In the wild-type strain, β-1,3-glucanase activity in fruiting bodies remarkably increased after harvesting, and 41.7% of the lentinan content was lost after 4 days of preservation. The EXG2 downregulated strain showed significantly lower lentinan degrading activity (60-70% of the wild-type strain) in the fruiting bodies 2-4 days after harvesting. The lentinan content of fresh fruiting bodies was similar in the wild-type and EXG2 downregulated strains, but in the downregulated strain, only 25.4% of the lentinan was lost after 4 days, indicating that downregulation of EXG2 enables keeping the lentinan content high longer.
No preview · Article · Jul 2014 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Three genes encoding glycoside hydrolase family 12 (GH12) enzymes from Lentinula edodes, namely Lecel12A, Lecel12B, and Lecel12C, were newly cloned by PCR using highly conserved sequence primers. To investigate enzymatic properties, recombinant enzymes encoded by L. edodes DNAs and GH12 genes from Postia placenta (PpCel12A and PpCel12B) and Schizophyllum commune (ScCel12A) were prepared in Brevibacillus choshinensis. Recombinant LeCel12A, PpCel12A, and PpCel12B, which were grouped in GH12 subfamily 1, preferentially hydrolyzed 1,3-1,4-β-glucan. By contrast, LeCel12B, LeCel12C, and ScCel12A, members of the subfamily 2, exhibited specific hydrolysis of xyloglucan. These results suggest that two subfamilies of GH12 are separated based on the substrate specificity. Transcript levels of L. edodes genes increased 72 h after growth of L. edodes mycelia cells in the presence of plant cell wall polymers such as xyloglucan, 1,3-1,4-β-glucan, and cellulose. These results suggest that L. edodes GH12 enzymes have evolved to hydrolyze 1,3-1,4-β-glucan and xyloglucan, which might enhance hyphal extension and nutrient acquisition.
No preview · Article · Jul 2013 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Polyporus brumalis ibrc05015 is a strain capable of high laccase (Lac) production. Among several inducers, 0.25 mM copper was most effective for Lac production. One of the Lacs induced by copper was PbLac1, and its transcription was induced within 60 min after copper addition. The promoter region of pblac1 contained six putative metal response elements and one Ace1 consensus cis-element. We cloned the P. brumalis PbAce1 transcription factor, a homologue of Saccharomyces cerevisiae transcription factor Ace1, which regulates metallothionein genes in response to excess copper. PbAce1 complemented the function of Ace1 in an S. cerevisiae Δace strain. The conserved N-terminal copper-fist DNA binding domain of PbAce1 was required for complementation. In the PbAce1 complemented Δace1 strain, the pblac1 promoter was constitutively expressed at a high level, independent of copper concentration. PbAce1 has two Cys-rich repeat motifs (PbC1 and PbC2), which are similar to the Cys-rich repeat domain in metallothionein proteins, and are uniquely conserved in the C-terminal domain of basidiomycetous Ace1 sequences. These C-terminal domains could be involved in copper sensing and concentration-dependent Lac production in basidiomycetous fungi.
[Show abstract][Hide abstract] ABSTRACT: Many carbohydrates are involved in the biofilm formation and activities of glucosyltransferases (Gtfs) of Streptococcus mutans, and the effects of various disaccharides and polysaccharides were investigated in this study, including the hot water-extracted glucan fraction of the Lentinula edodes fruiting body (HWG). HWG was found to inhibit the initial adhesion of S. mutans to saliva-coated hydroxyapatite (sHA), and also laminarin to inhibit glucan synthesis by Gtfs. However, sucrose-dependent biofilm formation by S. mutans was not inhibited by these materials. Interestingly, dextran was found to have an inhibitory effect on the sucrose-dependent biofilm formation. The data suggest that the presence of such an edible glucan as dextran in daily foods would act to some degree on S. mutans for suppressing the cariogenic activity.
No preview · Article · Dec 2012 · Bioscience Biotechnology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC 22.214.171.124. The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.
[Show abstract][Hide abstract] ABSTRACT: The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
[Show abstract][Hide abstract] ABSTRACT: A β-1,6-glucanase, LePus30A, was purified and cloned from fruiting bodies of the basidiomycete Lentinula edodes. β-1,6-glucanases degrade β-1,6-glucan polysaccharides, a unique and essential component of fungal cell walls. The complementary DNA of LePus30A includes an open reading frame of 1,575 bp encoding an 18 amino acid signal peptide and the 506 amino acid mature protein. Sequence analysis indicated that LePus30A is a member of glycoside hydrolase family 30, and highly similar genes are broadly conserved among basidiomycetes. The purified LePus30A catalyzed depolymerization of β-1,6-glucan endolytically and was highly specific toward β-1,6-glucan polysaccharide. It is known that the cell walls of fruiting bodies of basidiomycetes are autodegraded after harvesting by means of enzymatic hydrolysis. The transcript level of LePus30A gene (lepus30a) was significantly increased in fruiting bodies after harvesting. Moreover, LePus30A showed hydrolyzing activity against the cell wall components of L. edodes fruiting bodies. These results suggest that LePus30A is responsible for the degradation of the cell wall components during fruiting body autolysis after harvest.
No preview · Article · Apr 2011 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Enzymatic degradation of amylouronate (α-(1 → 4)-linked polyglucuronic acid sodium salt, α-(1 → 4)-linked glucuronan), which was prepared from water-soluble starch by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation, was investigated. A bacterial strain TH501b capable of degrading amylouronate was isolated from soil samples collected in the natural environment. Molecular analysis of the 16S rRNA gene showed that TH501b belongs to the genus Paenibacillus. A hydrolytic enzyme responsible for the degradation of amylouronate, amylouronate hydrolase-I (AUH-I), was detected in the cell-free extract of TH501b. AUH-I was purified by four steps of column chromatography and some properties were characterized. The molecular mass of the native AUH-I was estimated to be approximately 115 kDa by size exclusion chromatography (SEC), whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed two major bands at 80 kDa and 46 kDa, respectively. The enzyme was most active at pH 6.0–7.0 and 30 °C. The SEC analysis of reaction products revealed that AUH-I liberated glucuronate as a sole product from amylouronate, indicating that AUH-I hydrolyzed amylouronate exolytically, and thus, was classified as α-glucuronidase.
No preview · Article · May 2009 · Carbohydrate Polymers
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of endo-beta-(1-->4)-glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8A resolution as the first three-dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical beta-jelly roll fold, which is similar to glycoside hydrolase family 16 and PL7 enzymes. A calcium ion is bound to the site far from the cleft and appears to contribute to the stability. There are several completely conserved residues in the cleft. Possible catalytic residues are predicted based on structural comparison with PL7 alginate lyase A1-II'.
[Show abstract][Hide abstract] ABSTRACT: The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on β-(1→4)-polyglucuronate (cellouronate) as a sole carbon source. The
cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature
protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins,
indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization
of cellouronate endolytically by β-elimination and was highly specific for cellouronate. The enzyme was most active at pH
6.5 and 50°C, and its activity and thermostability increased in the presence of Ca2+, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.
Full-text · Article · Oct 2008 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: A bacterial strain, Brevundimonas sp. SH203, has an ability to degrade cellouronate, β-(1→4)-linked polyglucuronic acid sodium salt, which is artificially
prepared from regenerated cellulose by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation. In a previous
paper, an endo-type cellouronate lyase (CUL-I) has been isolated from the strain. In this paper, we purified another cellouronate
lyase, CUL-II, from cell-free extracts of Brevundimonas sp. SH203. CUL-II was a monomeric protein with a molecular mass of 56kDa by size exclusion chromatography and 62kDa by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and most active at pH 7.5. CUL-II formed monomers in a small quantity
from cellouronate without forming any intermediate oligomers, whereas it degraded C4′–C5′ unsaturated cellouronate dimer more
easily. Thus, CUL-II behaves as an exo-type lyase in degradation of cellouronate. When CUL-I and CUL-II were simultaneously
treated to cellouronate, it was degraded to monomers more efficiently than treatment with one enzyme alone, CUL-I or CUL-II.
Hence, cellouronate is synergistically degraded to monomers by Brevundimonas sp. SH203 by endo- and exo-type lyases, CUL-I and CUL-II, respectively.
[Show abstract][Hide abstract] ABSTRACT: Biodegradation of cellouronate (β-1,4-linked polyglucuronic acid sodium salt, β-1,4-linked glucuronan), which was prepared from regenerated cellulose by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation, was investigated. A bacterial strain with the ability to degrade cellouronate was isolated from soil collected in a natural environment, and identified as Brevundimonas sp. SH203 by comparing the nucleotide sequences of its 16S rDNA with those registered in the GenBank database. Cellouronate lyase-I (CUL-I), being responsible for the depolymerization of cellouronate, was purified to homogeneity from cell-free extracts. CUL-I was a monomeric protein with the molecular mass of 39 kDa by SDS–PAGE and 37 KDa by size exclusion chromatography (SEC). The enzyme activity was optimum at pH 7.5 and was inhibited by some divalent metal ions such as Mg2+, Fe2+ and Mn2+. The enzymatic reaction products were analyzed by SEC, TLC and 13C NMR. The results indicated that CUL-I catalyzed to depolymerize cellouronate endolytically to oligocellouronates and monomeric uronate.
No preview · Article · Jun 2006 · Carbohydrate Polymers