Effect of vanillin on the production of wood-decomposing enzymes from a wood-rotting fungus, <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

To examine the effect of vanillin on the production of the wood-decomposing enzymes of a wood-rotting fungus, vanillin was added as a model of lignin-related phenols to Coriolus versicolor cultures containing cellulosic and xylan substrates. Among five conditions tested, cellobiose alone was the most effective inducer of cellulolytic and xylanolytic enzymes. Addition of vanillin enhanced the effect of cellobiose on enzyme production. However, vanillin did not act as greatly in other cultures, except for cellobiose. Analytical isoelectric focusing and active staining of endo--1,4-glucanase demonstrated that isozyme patterns in the presence of vanillin were the same as those in absence of vanillin, indicating that vanillin does not induce novel isozymes but rather enhances enzyme production. On the other hand, vanillin, which enhanced production of phenol-oxidizing enzymes, was not always determined in all cultures, suggesting that the action of vanillin depends on the kinds of carbohydrates. Therefore, the effect of a monolignol vanillin on enzyme production was associated with coexistent carbohydrates.     

Production of acetyl esterase during wood biodegradation byCoriolus versicolor

A role of acetyl esterase in wood biodegradation byCoriolus versicolor was examined by the assay of enzyme production and the chemical analysis of decayed wood meal of Japanese beech (Fagus crenata). Enzyme assay demonstrated that the degradation proceeded in two stages and acetyl esterase production was correlated with the cellulolytic and xylanolytic enzyme production in the second stage, not with the production of phenol-oxidizing enzymes. From the results of chemical analysis, acetyl and xylose contents in wood meal were observed to decrease simultaneously in the second stage. In contrast, rapid decrease of lignin was recognized during the initial three wk of incubation, and it was closely related with the production of phenol-oxidizing enzymes in the first stage. These results show that acetyl esterase ofC. versicolor participates in the degradation of acetylxylan and acts with the cellulolytic and xylanolytic systems, not with the ligninolytic system.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune

To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

Tamarind kernel powder co-induces xylanase and cellulase production during submerged fermentation of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Tamarind kernel powder (TKP), a soluble agro-residue, was used to examine the production of both cellulolytic and xylanolytic enzymes in a submerged culture of Termitomyces clypeatus, an edible mushroom. Soluble TKP containing xyloglucan as the major polysaccharide induced all cellulolytic and xylanolytic enzymes, and enzyme production increased up to 3% (w/v) TKP with culture filtrate consisting of xylanase and CMCase at a ratio of 4: 1 app. Strong catabolic repression of enzyme production was also observed with the soluble substrate, although fed-batch addition of soluble substrate at late growth phase modified the enzyme kinetics by improving the yield by 30%. The results indicate that inducers were possibly released from TKP by cellulose and xylan fractions of the lignocellulosic polymer. Therefore, the present study reports the successful economic utilization of TKP, an abundantly available soluble agro-residue, for the production of both cellulolytic and xylanolytic enzymes in a single fermentation method.     

Anaerobic fungi and their cellulolytic and xylanolytic enzymes

Anaerobic fungi are the inhabitants of the digestive tract of herbivorous mammals, ruminants as well as non-ruminants. One of the major characteristics of all anaerobic fungi examined thus far, is their production and secretion of a range of polysaccharide-degrading enzymes, including cellulases, xylanases and glucoside-hydrolases. The cellulolytic enzymes of the anaerobic fungusNeocallimastix frontalis have been shown to possess a high activity. Therefore anaerobic fungi and/or their enzymes could be interesting for many biotechnological applications including saccharafication of lignocellulosic residues, production of polysacchari-dehydrolysing enzymes. This review summarizes the present knowledge of anaerobic fungi with special emphasis on their cellulolytic and xylanolytic enzymes. Further, a comparison with aerobic fungi is made.Abbreviations G₂ cellobiose - G₃ cellotriose - G₄ cellotetraose - G₅ cellopentaose - HMM-complex high molecular mass complex - PNPF p-nitrophenyl--fucopyranoside - PNPG p-nitrophenyl--glucopyranoside     

Enzymatic release of ferulic acid fromIpomoea batatas L. (sweet potato) stem

Ferulic acid is a phenolic compound that serves as a major biosynthetic precursor of vanillin in higher plants. We investigated the ability of the 3 commercial enzymes—Ultraflo L, Viscozyme L, and α-Amylase—to induce the release ferulic acid from theIpomoea batatas L. (sweet potato) stem. The rate of release for ferulic acid was optimal when Ultraflo L (1.0%) was used compared with the other enzymes, whereas Viscozyme L was most effective for the release of vanillic acid and vanillin. Thus, these enzymes may be useful for the large-scale production of ferulic acid and other phenolic compounds from sweet potato stem.     

Lignin biodegradation and ligninolytic enzyme studies during biopulping of Acacia mangium wood chips by tropical white rot fungi

White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20–30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02–26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Saccharophagus degradans 2-40 is a marine gamma proteobacterium that can produce polyhydroxyalkanoates from lignocellulosic biomass using a complex cellulolytic system. This bacterium has been annotated to express three surface-associated β-glucosidases (Bgl3C, Ced3A, and Ced3B), two cytoplasmic β-glucosidases (Bgl1A and Bgl1B), and unusual for an aerobic bacterium, two cytoplasmic cellobiose/cellodextrin phosphorylases (Cep94A and Cep94B). Expression of the genes for each of the above enzymes was induced when cells were transferred into a medium containing Avicel as the major carbon source except for Bgl1B. Both hydrolytic and phosphorolytic degradation of cellobiose by crude cell lysates obtained from cellulose-grown cells were demonstrated and all of these activities were cell-associated. With the exception of Cep94B, each purified enzyme exhibited their annotated activity upon cloning and expression in E. coli. The five β-glucosidases hydrolyzed a variety of glucose derivatives containing β-1, (2, 4, or 6) linkages but did not act on any α-linked glucose derivatives. All but one β-glucosidases exhibited transglycosylation activity consistent with the formation of an enzyme-substrate intermediate. The biochemistry and expression of these cellobiases indicate that external hydrolysis by surface-associated β-glucosidases coupled with internal hydrolysis and phosphorolysis are all involved in the metabolism of cellobiose by this bacterium.     

Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan

The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.     

Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing α-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and β-glucoside hydrolyzing activities. All strains synthesized β-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on α-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10–13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. β-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and β-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi. These studies were supported byPakistan Atomic Energy Commission. Some chemicals were purchased from funds allocated byUnited States Agency for International Development, Washington (DC, USA), under PSTC proposal 6.163.     

Production of Cellulases by Aspergillus fumigatus and Characterization of One β-Glucosidase

Aspergillus fumigatus produces substantial extracellular cellulases on several cellulosic substrates including simple sugars. Low glucose potentiates enzyme production, but most cellulose-induced cellulases are repressed by high glucose. As production of cellulase in a wide substrate range is unusual, the cellulolytic complex of this thermophilic fungus was investigated. A β-glucosidase was separated by gel filtration and ion-exchange chromatography. It migrated in native polyacrylamide gel as a single protein (130 kDa), which split under denaturing conditions into two smaller proteins having molecular masses of 90 kDa and 45 kDa. However, only the 90-kDa protein was active. Conventional chromatographic procedures were unsuccessful for the separation of these two proteins. Therefore, the 130-kDa protein was studied for its kinetic properties. It hydrolyzed p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose, but not β-glucans, laminarin, and p-nitrophenyl-β-D-xilopyranoside. The optimal pH and temperature of p-NPG and cellobiose hydrolysis were 5.0 and 4.0, and 65°C and 60°C, respectively. The K _m values, determined for cellobiose and p-NPG of hydrolysis, were 0.075 mM and 1.36 mM, respectively. Glucose competitively inhibited the hydrolysis of p-NPG. The K_i was 3.5 mM.     

A semi-continuous culture system for production of cellulolytic and xylanolytic enzymes by the anaerobic fungus Piromyces sp. strain E2

Summary A system was developed for the semi-continuous cultivation of an anaerobic fungus, Piromyces sp. strain E2 (isolated from an Indian elephant), on Avicel (microcrystalline cellulose). The fungus was grown in a semi-continuous culture system: solids and fungal biomass was retained by means of a simple filter construction whereas the culture fluid was removed continuously. The production of fermentation products (acetate, ethanol, formate, lactate, hydrogen or methane), cellulolytic and xylanolytic enzymes, and protein by the fungus in monoculture or co-culture with Methanobacterium formicicum during growth on Avicel was monitored up to 45 days. These productions stabilized after an adaptation period of 24 and 30 days in the semi-continuous co-culture and monoculture, respectively. After this period the average (±SD) avicelase, -glucosidase, endoglucanase, and xylanase production in the semi-continuous monoculture were 27±6, 140±16, 1057±120 and 5012±583 IU.l^–1.dya^–1, respectively. Co-culture with the methanogen caused a shift in fermentation products to more acetate, and less ethanol and lactate. Furthermore, the production of all cellulolytic enzymes increased (40%) and xylanolytic enzyme production decreased (35%).Correspondence to: H. J. M. Op den Camp     

Effect of glycosylation on the biochemical properties of <Emphasis Type="Italic">β</Emphasis>-xylosidases from <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>

Aspergillus versicolor grown on xylan or xylose produces two β-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these β-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R_f. Temperature optimum of xylan-induced and xylose-induced β-xylosidases was 45°C and 40°C, respectively, and 35°C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55°C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.