Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes shiitake mushroom)

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 3.2.1.52. 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 (k(cat)/K(m)) for chitotetraose, and the K(m) values for GlcNAc(6) 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

Crystal structure of polysaccharide lyase family 20 endo-β-1,4-glucuronan lyase from the filamentous fungus Trichoderma reesei

AbstractThe crystal structure of endo-β-(1→4)-glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8Å resolution as the first three-dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical β-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′

Cloning of the Trichoderma reesei cDNA Encoding a Glucuronan Lyase Belonging to a Novel Polysaccharide Lyase Family▿ †

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

Extracellular enzymes secreted in the mycelial block of Lentinula edodes during hyphal growth

Abstract Lentinula edodes (shiitake mushroom) is one of the most widely cultivated edible mushrooms and is primarily cultivated using sawdust medium. While there have been improvements in the cultivation technology, the mechanism of mycelial block cultivation, such as mycelial growth and enzymatic sawdust degradation, has not been clarified. In this study, the mycelium was elongated longitudinally in the bottle sawdust culture for 27 days, and the cultivated sawdust medium was divided into three sections (top, middle, and bottom parts). To determine spatial heterogeneity in the enzyme secretion, the enzymatic activities of each part were analyzed. Lignocellulose degradation enzymes, such as endoglucanase, xylanase, and manganese peroxidase were highly secreted in the top part of the medium. On the other hand, amylase, pectinase, fungal cell wall degradation enzyme (β-1,3-glucanase, β-1,6-glucanase, and chitinase), and laccase activities were higher in the bottom part. The results indicate that the principal sawdust degradation occurs after mycelial colonization. Proteins with the laccase activity were purified from the bottom part of the medium, and three laccases, Lcc5, Lcc6 and Lcc13, were identified. In particular, the expression of Lcc13 gene was higher in the bottom part compared with the level in the top part, suggesting Lcc13 is mainly produced from the tip region and have important roles for mycelial spread and nutrient uptake during early stage of cultivation

Lentinan Degradation in the <i>Lentinula edodes</i> Fruiting Body during Postharvest Preservation Is Reduced by Downregulation of the <i>exo</i>-β-1,3-Glucanase EXG2

Lentinan from <i>Lentinula edodes</i> fruiting bodies (shiitake mushrooms) is a valuable β-glucan for medical purposes based on its anticancer 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 <i>exo</i>-β-1,3-glucanase, <i>exg2</i>, in <i>L. edodes</i> 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

Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns

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

MOESM1 of Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns

Additional file 1. Additional information of L. edodes multicopper oxidases, such as signal peptide prediction, enzyme purificaition, expression patterns, primer design, data set for phylogenetic analysis, and sequence infromation