Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana

Summary The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^–1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p-nitrophenyllactoside (p-NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p-NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone. Correspondence to: G. Canevascini     

Evidence that Cellulolysis by an Anaerobic Ruminal Fungus Is Catabolite Regulated by Glucose, Cellobiose, and Soluble Starch

A Piromyces-like ruminal fungus was used to study preferential carbohydrate utilization of [U-¹⁴C]cellulose, both alone and in combination with several soluble sugars. For cells grown on cellulose alone, cellulolytic activity was immediate and, initially, greater than that observed in the presence of added carbohydrate. Cellulolytic activity remained minimal in cultures containing cellulose plus glucose or cellobiose until the soluble sugar was depleted. Soluble starch also regulated cellulose activity but to a lesser extent. The results presented suggest that some fungal cellulases are susceptible to catabolite regulatory mechanisms.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Kinetics of the utilization of different C sources and the cellulose formation by Acetobacter xylinum

Different methylated glucose derivatives and cellobiose were examined as the carbon sources for growth and cellulose formation by Acetobacter xylinum. HPLC studies were carried out to gain information about the kinetics of the utilization of the C sources used. The type and yields of the synthesized cellulose were described. Besides glucose, cellobiose was a substrate for the synthesis of this polysaccharide by the bacteria. Other methylated derivatives of glucose were not accepted for a comparable synthesis of this polymer. An estimation of citrate in an unmodified culture liquid (SH medium) showed utilization in a late phase of cultivation. The influence of this organic acid on the pH value, cellulose synthesis and growth is described. By the application of citric acid as a sole carbon source “gel-like” forms of cellulose were formed generally.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Studies on Cellulose Synthesis by a Cell-free Oat Coleoptile Enzyme System: Inactivation by Airborne Oxidants

Particulate cell wall polysaccharide synthetase from oat coleoptiles could use either guanosine diphosphate glucose or uridine diphosphate glucose; the latter was a much more effective glucose donor. The neutral polymer derived from uridine diphosphate glucose utilization yielded, after cellulase digestion, mostly cellobiose and to a lesser extent a substance tentatively identified as a mixed-linkage β1,4 = β1,3-trisaccharide; only cellobiose was found after guanosine diphosphate glucose utilization. The uridine diphosphate glucose utilizing system was inactivated by peroxyacetyl nitrate treatment of intact tissue and to a lesser extent by ozone treatment suggesting that this system is a possible site of interference with cellulose and non-cellulosic glucan biosynthesis in vivo. Direct treatment of the enzyme in vitro by peroxyacetyl nitrate, iodoacetamide or p-chloromercuribenzoate also inactivated the enzyme, indicating that the mechanism of inactivation possibly involves reaction with sulfhydryl groups.     

Growth properties of Cellulomonas flavigena mutants affected in cellulose utilization.

The role of cellobiose metabolism in cellulose utilization by Cellulomonas flavigena was investigated by studying mutants unable to grow on cellobiose or cellulose. The results show that the ability to utilize cellulose is strictly dependent on the ability to utilize cellobiose.     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.     

Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of the genes is starch hydrolysis, esculin hydrolysis, cellobiose utilization, chitin hydrolysis, carboxymethyl cellulose hydrolysis, and polygalacturonic acid hydrolysis. A restriction endonuclease cleavage map was constructed, and the genes for starch, carboxymethyl cellulose, cellobiose, chitin, and pectin hydrolysis were subcloned.     

Yersinia kristensenii: A new species of enterobacteriaceae composed of sucrose-negative strains (formerly called atypicalYersinia enterocolitica orYersinia enterocolitica-Like)

Induction of cellulase was observed inFusarium sp. with reduction in lag period by lactose-pregrown cells as compared with glucose-pregrown cells. Insoluble cellulose (Sigmacell) induced maximum cellulase production in the induction medium. Supplementation of the culture growing on cellulose by cellobiose or glucose resulted in increased cellular growth and decreased cellulase production. Stepfeeding of cellobiose to the culture growting on carboxymethyl cellulose resulted in decreased cellulase production. Significant cellulase activity was detected in the culture filtrate of cells growing on Sigmacell supplemented with glucose, only when the glucose disappeared from the medium. This suggests that cellulase production may in part be regulated by catabolite repression.     

Fermentation of cellodextrins by cellulolytic and noncellulolytic rumen bacteria.

Water-soluble cellodextrins were prepared from microcrystalline cellulose by using fuming hydrochloric acid and acetone precipitation. This cellodextrin preparation contained only trace amounts of glucose and cellobiose and was primarily composed of cellotetraose and cellopentaose. When various species of cellulolytic and noncellulolytic bacteria were cultured with cellodextrins, their growth rates and maximal optical densities were in most cases similar to those observed with cellobiose. Time course samplings and analyses of cellodextrins by high-pressure liquid chromatography indicated that longer-chain cellodextrins were hydrolyzed extracellularly to cellobiose and cellotriose. Cellodextrin utilization by noncellulolytic rumen bacteria and extracellular hydrolysis of cellodextrins increase the possibility that cross-feeding occurs in the rumen and help to explain the high numbers of noncellulolytic bacteria in ruminants fed fibrous diets.     

Fermentation of cellodextrins by cellulolytic and noncellulolytic rumen bacteria

Water-soluble cellodextrins were prepared from microcrystalline cellulose by using fuming hydrochloric acid and acetone precipitation. This cellodextrin preparation contained only trace amounts of glucose and cellobiose and was primarily composed of cellotetraose and cellopentaose. When various species of cellulolytic and noncellulolytic bacteria were cultured with cellodextrins, their growth rates and maximal optical densities were in most cases similar to those observed with cellobiose. Time course samplings and analyses of cellodextrins by high-pressure liquid chromatography indicated that longer-chain cellodextrins were hydrolyzed extracellularly to cellobiose and cellotriose. Cellodextrin utilization by noncellulolytic rumen bacteria and extracellular hydrolysis of cellodextrins increase the possibility that cross-feeding occurs in the rumen and help to explain the high numbers of noncellulolytic bacteria in ruminants fed fibrous diets.     

The oxidation of pipecolic acid in preincubated soils

Chernozem soil was preincubated with 0.1% glucose, glucose plus ammonium nitrate, hydrolyzed casein or amino acids for five days and with 1% wheat straw, pectin, peptone or cellulose for ten days. The soil was modified by this treatment so that the pipecolic acid was oxidized in two steps, as was shown by two peaks on the graphic plot of the oxygen uptake. In the control soil sample preincubated with water, the oxygen consumption curve formed one peak. In brown soil it was necessary to increase the concentration of glucose and hydrolyzed casein to 1% to obtain the oxidation of pipecolic acid in two phasos. Approximately a third of the oxygen for the complete oxidation of pipecolic acid was consumed in the first phase of oxidation, another third in the second phase of oxidation. A relation was found between the occurrence of the second period of oxidation of pipecolic acid and the amount of organic carbon in the soil.     

Utilization of carbohydrates by Thermomonospora sp. Grown on glucose,cellobiose, and cellulose

Thermomonospora sp. was grown on glucose, cellobiose, and in order to study its growth characteristics with different carbohydrate substrates and to assess the validity of some of the assumptions made in a previously proposed model for the cellulose fermentation with this microorganism. It was observed that the nitrogen and protein contents of the cells are essentially constant during the fermentation and independent of the carbon source when glucose or cellobiose are utilized. Under oxygen starvation conditions it was shown that unidentification organic compound(s) accumulate(s) in the culture broth. Culture fluorescence was shown to be an excellent variable for monitoring and control of the fermentation process. This microorganism showed a preference for crystalline cellulose (Avicel) as substrate although it grows readily on a more amorphous cellulose (Solka Floc). The production of extra cellular protein is shown to be growth related. Data were obtained confirming the decrease in the number of active adsorption sites as the cause for the decrease in the cellulose digestion rate. It is suggested that a future model should account for the time change of surface characteristics of the cellulose particles.     

Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405

Cultures of Clostridium thermocellum ATCC-27405, maintained on cellulose and not adapted to grow on glucose utilize cellobiose preferentially over D-glucose, and are only able to initiate growth on D-glucose when the cellobiose has been exhausted from the growth medium. However, D-glucose is the carbon source preferentially utilized when cultures of this microorganism, previously adapted for growth on glucose, are transferred to a medium with equivalent concentrations of both sugars. One reason for the preferential utilization of glucose over that of cellobiose might be the competitive inhibition of cellobiose phosphorylase by intracellular glucose accumulation. When in the glucose-adapted cultures the pressure to grow on glucose as the sole carbon source is again released, both sugars can be simultaneously utilized.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Kinetics of cellobiose hydrolysis using cellobiase composites from Ttrichoderma reesei and Aspergillus niger

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary(1) to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.     

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01?? (1??=0.1?nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.     

Regulation of xylanase activity in cellulomonas sp. IIbc

The regulation mechanism governing the xylanolytic activity in the strain Cellulomonas sp. IIbc was studied. High levels of activity were detected during the cultivation on cellulose as the only carbon source. No activity was produced with glucose, xylose or cellobiose cultures, but in the last one, the synthesis was de-repressed when the sugar concentration dropped to 0.2%. The activity was not inhibited by glucose, cellobiose and xylose up to 1% concentration. A basal constitutive synthesis was detected in nutrient broth cultures. At the same time, xylose and cellobiose acted as inducers of the xylanase activity.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.