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

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 (ß-1,3-glucanase, ß-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.

Pectin decomposition at the early stage of brown-rot decay by Fomitopsis palustris.

The sapwood of Japanese cedar (Cryptomeria japonica D. Don) was decayed by the brown-rot fungus Fomitopsis palustris under bright and dark conditions. Scanning electron microscopy revealed the presence of mycelia inside the wood even after 1 week from the start of fungal exposure. Moreover, holes were observed in the torus after fungal exposure. Ruthenium red staining revealed that the pectin in pits was largely absent for 3 weeks. These events occurred before the mass loss of wood samples was confirmed at the early stage. Moreover, FpPG28A was more highly expressed at the hyphal front on a pectin-containing medium under dark conditions compared with bright conditions. This up-regulation under dark conditions indicated that the pectin decomposition ability was promoted inside the wood where light could not reach. In conclusion, we suggest that the brown-rot fungus completed its hyphal expansion within the wood via pectin decomposition in pits before holocellulose decomposition.

Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris.

Brown-rot fungi preferentially degrade softwood and cause severe breakdown of wooden structures. At the initial stage of the brown-rot decay, penetrating hyphae of the fungi are observed in ray parenchyma. Since starch grains are known to be present in the ray parenchyma of sapwood, investigation of the functions and roles of the starch-degrading enzymes is important to understand the initial stage of brown-rot decay. We purified and characterized two starch-degrading enzymes, an α-amylase (FpAmy13A) and a glucoamylase (FpGLA15A), from the brown-rot fungus, Fomitopsis palustris, and cloned the corresponding genes. The optimal temperature for both enzymes was 60 °C. FpAmy13A showed higher activity at a broad range of pH from 2.0 to 5.0, whereas FpGLA15A was most active at pH 5.0-6.0. Notable thermal stability was found for FpGLA15A. Approximately 25% of the activity remained even after treatment at 100 °C for 30 min in sodium phosphate buffer at pH 7.0. These different characteristics imply the different roles of these enzymes in the starch degradation of wood.

A bacterial endo-ß-1,4-glucuronan lyase, CUL-I from Brevundimonas sp. SH203, belonging to a novel polysaccharide lyase family.

Cellouronate is a (1,4)-ß-D-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose. We have previously isolated a cellouronate-degrading bacterial strain, Brevundimonas sp. SH203, that produces a cellouronate lyase (ß-1,4-glucuronan lyase, CUL-I). In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized ß-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family.

A GH family 28 endo-polygalacturonase from the brown-rot fungus Fomitopsis palustris: Purification, gene cloning, enzymatic characterization and effects of oxalate.

Brown-rot fungi are the wood-decay basidiomycetes and have ability to break down plant cell wall carbohydrates. It has been suggested that degradation of pectin is important for the initial stages of brown rot. We purified an endo-polygalacturonase (FpPG28A) from the brown-rot fungus Fomitopsis palustris, analysis of the predicted amino acid sequence indicated that FpPG28A belongs to GH family 28. The highest activity of purified FpPG28A was observed at 60 °C in 50 mM sodium acetate buffer (pH 5.0); this activity was highly specific for polygalacturonic acid chains. However, calcium polygalacturonate gel was not degraded by FpPG28A under those optimal conditions. We observed that calcium polygalacturonate gel was readily degraded by the enzyme in the oxalate buffer. Furthermore, the thermostability of FpPG28A was elevated in oxalate buffer at pH 3.0. These results indicated that oxalate has an important role in the degradation of woody pectin by FpPG28A.

Draft Genome Sequence of Brevundimonas sp. Strain SH203, Producing Cellouronate (ß-1,4-Linked Polyglucuronate) Lyase.

In this study, we report the draft genome sequence of Brevundimonas sp. strain SH203, which was previously isolated from natural soil and has the ability to degrade ß-1,4-polygluculonate (cellouronate). This genomic information may provide new insight into the mechanisms by which cellouronate is degraded.

Preparation of completely C6-carboxylated curdlan by catalytic oxidation with 4-acetamido-TEMPO.

Pure (1→3)-ß-polyglucuronic acid sodium salt was prepared from curdlan by oxidation with 4-acetamido-TEMPO/NaClO/NaClO2 in water at pH 4.7 and 35°C. The oxidation conditions, including the reaction time and amounts of reagents added, were optimized for the preparation of (1→3)-ß-polyglucuronic acids with high molecular weights. The primary C6 hydroxyl groups of curdlan were completely oxidized to the corresponding C6-carboxylates using a one- or two-step reaction process by controlling the oxidation conditions, thus providing pure (1→3)-ß-polyglucuronic acids consisting only of D-glucuronosyl units. Unfortunately, however, the increased amounts of reagents and long reaction time led to significant depolymerization of the curdlan during the oxidation process, and the resulting (1→3)-ß-polyglucuronic acids had weight-average degrees of polymerization of 340-360. The (13)C and (1)H NMR chemical shifts of the products were successfully assigned using pure (1→3)-ß-polyglucuronic acid.

Stability of (1→3)-ß-polyglucuronic acid under various pH and temperature conditions.

Regioselective oxidation of C6 primary hydroxyls to carboxyls was applied to curdlan, to prepare a water-soluble (1→3)-ß-polyglucuronic acid Na salt [(1,3)-ß-PGluA], using a 4-acetamido-TEMPO/NaClO/NaClO2 oxidation system at pH 4.7. Changes in the chemical structure and degree of polymerization of (1,3)-ß-PGluA treated in water at various temperatures and pHs were studied to evaluate the stability of (1,3)-ß-PGluA to these treatments. This polyuronic acid was found to be stable, without any depolymerization, to treatment in water at pHs 1-9 and room temperature for up to 128 h; slight depolymerization was observed at pHs 11 and 13. When heated in water at pH 1 and high temperatures (1,3)-ß-PGluA molecules were randomly depolymerized by hydrolysis, primarily forming glucuronic acid. In contrast, dicarboxylic-acid-type monomers containing ethylene carbons were formed from the C1-carboxyl or C1 reducing ends of (1,3)-ß-PGluA molecules by treatment under alkaline conditions; this was initiated by ß-alkoxy elimination, similar to the peeling-off reaction of cellulose.

Biodegradation of (1→3)-ß-polyglucuronate prepared by TEMPO-mediated oxidation.

2,2,6,6-Tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation was applied to curdlan to prepare water-soluble sodium (1→3)-ß-polyglucuronate, and its biodegradation behavior was then investigated. A bacterial strain (EH621) having the ability to degrade (1→3)-ß-polyglucuronate was isolated from soil, and identified as Paenibacillus sp. Strain EH621 cultured with (1→3)-ß-polyglucuronate decreased the initial total carbon in the supernatant by approximately 60% within 3 days, showing that (1→3)-ß-polyglucuronate can be readily degraded and metabolized by microorganisms present in the natural environment. Hydrolytic enzyme activity was detected in the cell-free extract of EH621, which was highly specific to (1→3)-ß-polyglucuronate. Analyses of the enzymatic degradation products revealed that (1→3)-ß-polyglucuronate was endolytically degraded to its monomeric unit, glucuronate, via some oligomers and dimers.

Genetic replacement of tesB with PTE1 affects chain-length proportions of 3-hydroxyalkanoic acids produced through ß-oxidation of oleic acid in Escherichia coli.

Acyl-CoA thioesterase II (TesB), which catalyzes hydrolysis of acyl-CoAs to free fatty acids and CoA, is involved in 3-hydroxyalkanoic acid production in Escherichia coli. Effects of genetic replacement of tesB with Saccharomyces cerevisiae acyl-CoA thioesterase gene PTE1 on 3-hydroxyalkanoic acid production from oleic acid through ß-oxidation were examined. Kinetic analyses using ß-oxidation intermediates showed that hydrolyses of C4-acyl substrates are more efficient by PTE1 than by TesB. Deletion of tesB in E. coli decreased 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and hexanoic acid in medium after cultivation with oleic acid as a sole carbon source. Hexanoic acid concentration was much lower than those of 3-hydroxyacids. In genetic complementation of tesB deletion, use of PTE1, instead of tesB, affected proportions of the 3-hydroxyalkanoic acids. Proportion of 3-hydroxybutyric acid was higher in a PTE1-complemented strain than in a tesB-complemented strain, while proportions of 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid markedly increased in the tesB-complemented strain. Proportion of 3-hydroxyoctanoic acid did not significantly increase in the PTE1-complemented strain. These data indicate possibilities of 3-hydroxyalkanoic acid production from oleic acid through ß-oxidation and customization of their chain-length proportions by genetic replacement of tesB with a gene encoding acyl-CoA thioesterase with a different kinetic property.

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

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'.

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 beta-(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 beta-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50 degrees 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.