Regulation of β-1,4-Endoglucanase Synthesis in Thermomonospora fusca

In Thermomonospora fusca YX, endocellulase synthesis varies over a 100-fold range depending on the carbon source used. This study shows that the variation is caused by two regulatory mechanisms: an induction mechanism that increases the rate of endocellulase synthesis about 20-fold and a growth rate-dependent repression mechanism that changes the rate of synthesis over a 6-fold range in both induced and noninduced cells. In T. fusca, endocellulase synthesis can be induced by cellulose, cellobiose, or cellodextrin. Cellulase is involved in inducer generation from cellulose. Growth rate-dependent repression can be reversed by limiting cultures for carbon, nitrogen, or, to a lesser extent, phosphorus. Further evidence for two separate regulatory mechanisms is provided by the isolation of mutants (CC-1 and CC-2) whose endocellulases are synthesized constitutively but are still sensitive to growth rate-dependent repression. These conclusions about total endocellulase synthesis were extended to the individual endocellulases by showing that three T. fusca endocellulases are coordinately regulated.     

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Summary The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellobiose dehydrogenase remain to be elucidated.     

Regulation of beta-1,4-Endoglucanase Synthesis in Thermomonospora fusca

In Thermomonospora fusca YX, endocellulase synthesis varies over a 100-fold range depending on the carbon source used. This study shows that the variation is caused by two regulatory mechanisms: an induction mechanism that increases the rate of endocellulase synthesis about 20-fold and a growth rate-dependent repression mechanism that changes the rate of synthesis over a 6-fold range in both induced and noninduced cells. In T. fusca, endocellulase synthesis can be induced by cellulose, cellobiose, or cellodextrin. Cellulase is involved in inducer generation from cellulose. Growth rate-dependent repression can be reversed by limiting cultures for carbon, nitrogen, or, to a lesser extent, phosphorus. Further evidence for two separate regulatory mechanisms is provided by the isolation of mutants (CC-1 and CC-2) whose endocellulases are synthesized constitutively but are still sensitive to growth rate-dependent repression. These conclusions about total endocellulase synthesis were extended to the individual endocellulases by showing that three T. fusca endocellulases are coordinately regulated.     

Solubilization of Acid-swollen cellulose by an enzyme system from a species of alternaria

An unknown species of Alternaria, when grown on a medium containing carboxymethylcellulose as a carbon source produced a mixture of extracellular enzymes which solubilized acid-swollen cellulose. The product of the hydrolysis was a 1:2 molar mixture of cellobiose and glucose. The organism apparently produced no cellobiase. It is suggested that the mixture of cellulolytic enzymes contains at least two different enzymes which degrade cellulose in an endwise manner.     

Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose

Assay of cellulase enzymology on cellulose was investigated by two methods: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 μM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.     

Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol

Biofuel production from renewable resources can potentially address lots of social, economic and environmental issues but an efficient production method has yet to be established. Combinations of different starting materials, organisms and target fuels have been explored with the conversion of cellulose to higher alcohols (1-propanol, 1-butanol) being one potential target. In this study we demonstrate the direct conversion of untreated plant biomass to 1-propanol in aerobic growth conditions using an engineered strain of the actinobacterium, Thermobifida fusca. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase was added to T. fusca leading to 1-propanol production during growth on glucose, cellobiose, cellulose, switchgrass and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. These results represent the first demonstration of direct conversion of untreated lignocellulosic biomass to 1-propanol in an aerobic organism and illustrate the potential utility of T. fusca as an aerobic, cellulolytic bioprocess organism.     

On the mode of action of fungal cellulases

Summary The mode of action of the cellulolytic enzymes of two strong cellulose decomposing fungi, Penicillium oxalicum Curie et Thom and Helminthosporium cyclops Drechsler, was studied. The culture filtrates and enzyme preparations obtained from them showed high cellulase activity and very weak cellobiase activity. The cellulolytic system of both experimental organisms seems to be multicomponent. The cellulase component showed its activity mainly extracellulary and the cellobiase component, mainly intracellulary. It seems, therefore, that during growth of both fungi on a cellulose medium, the extracellular cellulase acts hydrolytically on the cellulose substrate forming cellobiose which is further acted upon by intracellular cellobiase to form glucose. Paper chromatographic assay of the products of the enzymatic reaction sub-stantiated this conclusion.     

A cellulase assay coupled to cellobiose dehydrogenase

An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented. Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result. The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition. The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity. With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated. An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out.     

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.     

Preparation of cellulase concoction using differential adsorption phenomenon

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.     

The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol⁻¹ on Avicel and 35.9 U μmol⁻¹ on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.     

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.     

Heterologous co-production of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> Cel9A with other cellulases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.     

Microcalorimetric study of aerobic growth of Cellulomonas sp. 21399 on various carbohydrates

Summary Microcalorimetry was used to study the energetic aerobic growth of Cellulomonas sp. 21399 on glucose, cellobiose and amorphous and crystalline cellulose. The thermochemical aspect of growth on glucose was established with regard to the anabolic contribution. The results obtained allowed the use of glucose as a reference substrate for cellulose degradation. The experimental enthalpy change and the maximum catabolic activity, calculated from the maximum power evolved by the culture, were, respectively,-1079 kJ/mol and 0.85 mmol glucose per hour per dry weight of cells. The growth response on amorphous cellulose was equivalent to that demonstrated on glucose. However, on crystalline cellulose media, Cellulomonas sp. 21399 exhibited eight times less power and the quantity of heat evolved during growth showed that 50% of the cellulose was degraded. Quantitative results and the shape of power-time curves achieved indicate that the structural features of cellulose strongly influence its microbial degradability.     

The Effect of Cellobiose,Glucose, and Cellulose on the Survival of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> A3C Cultures Grown Under Ammonia Limitation

The ruminal, cellulolytic bacterium, Fibrobacter succinogenes A3C, grew rapidly on cellulose, cellobiose, or glucose, but it could not withstand long periods of energy source starvation. If ammonia was limiting and either cellobiose or glucose was in excess, the viability declined even faster. The carbohydrate-excess, ammonia-limited cultures did not spill energy, but they accumulated large amounts of cellular polysaccharide. Cultures that were carbohydrate-limited had approximately 4 nmol ATP mg cell protein^–1, but ATP could not be detected in cultures that had an excess of soluble carbohydrates. However, if F. succinogenes A3C was provided with excess cellulose and ammonia was limiting, ATP did not decline, and the cultures digested the cellulose soon after additional nitrogen sources were added. From these results, it appears that excess soluble carbohydrates can promote the death of F. succinogenes, but cellulose does not.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.     

An Apparent Cellulase Complex in Tomato (Lycopersicon esculentum L.) Fruit

Four enzyme-containing fractions were separated by ammonium sulfate fractionation of 2-day, postbreaker tomato (Lycopersicon esculentum L. cv. Manhattan). The pH optima and substrate specificities are reported. The enzymes were identified as a nonspecific β-glucosidase, an exo-β-1,4-glucanase, and two endocellulases. Both endocellulase fractions were able to catalyze the hydrolysis of various insoluble cellulose materials.     

Comparison of growth and cellulolytic enzyme production inAspergillus chevalieri andPenicillium steckii from mouldy cacao beans

Aspergillus chevalieri andPenicillium steckii grew best at 30°C and at pH of 6.5–7.5. Among the carbon sources employed, sucrose supported maximum growth ofA. chevalieri while glucose was best forP. steckii. Growth of both organisms was optimal on ammonium tartrate as the sole source of nitrogen.A. chevalieri andP. steckii grew in synthetic media containing, respectively, soluble or insoluble cellulose as the sole carbon source, releasing a cellulolytic enzyme into the medium. The enzymes from each organism were separated and partially purified by molecular exclusion and ion-exchange chromatography into two components. There was synergism between the components of enzymes from each organism in that they together released more glucose units from insoluble cellulose than could be predicted from their activities alone. The molar mass of the enzymes estimated from the elution volume on Sephadex was approximately 110 kg/mol forA. chevalieri and 94 kg/mol forP. steckii.