OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.     

Kinetics and relative importance of phosphorolytic and hydrolytic cleavage of cellodextrins and cellobiose in cell extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of beta-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60 degrees C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35 degrees C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by > or = 20-fold for both cellobiose and cellopentaose over a 10-fold range of beta-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of beta-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V(max) values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K(m) for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).     

Fermentation of Cellulosic Substrates in Batch and Continuous Culture by Clostridium thermocellum

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with ~5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch cellobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting either in vitro or in vivo were comparable to those previously reported for Trichoderma reesei on the same substrates.     

Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

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.     

Organization and distribution of the cellulosome in Clostridium thermocellum.

The properties of the cellulosome (the cellulose-binding, multicellulase-containing protein complex) in Clostridium thermocellum were examined by comparing the cellulase systems derived from the wild type and an adherence-defective mutant. The growth conditions--specifically, growth either on cellulose (Avicel) or on cellobiose as insoluble or soluble carbon sources, respectively--were found to be critical to the distribution of the cellulosome in the mutant system: the cellobiose-grown mutant (in contrast to the wild type) lacked the cellulosome on its surface and produced only minor quantities of the extracellular cellulosome accompanied by other relatively low-molecular-weight cellulases. The polypeptide composition of the respective purified cellulosome was dependent on the nature of the carbon source and was similar for both wild-type and mutant cells. Ultrastructural analysis revealed the presence of novel polycellulosomal protuberances on the cell surface of the cellobiose-grown wild type which were absent in the mutant.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h^-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article no. 95-07-064.     

β-Glucanase expression by Ruminococcus flavefaciens FD-1

Abstract Ruminococcus flavefaciens has been hypothesized to produce cellulase constitutively. We have studied the effect of carbon source, either cellobiose or cellulose, on the production of cellulase in batch cultures of R. flavefaciens FD-1. Total CMCase and ¹⁴C-cellulase activity was approximately 2-fold higher in cellobiose grown cells than in cellulose grown cells, whereas p-nitrophenyl-β- d -cellobiosidase (PNPCase) activity was not affected by culture conditions. The addition of cellulose to cells growing on cellobiose did not alter the amount or rate of PNPCase and ¹⁴C-cellulase production. Northern blot analysis of mRNAs produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that two of the four β-glucanase genes cloned from R. flavefaciens FD-1 were only expressed in cells grown with cellulose as the substrate. Although the adherence of cells and cellulase enzyme to native cellulose can complicate interpretations of these data, the results indicate that cellulase synthesis by R. flavefaciens is differentially regulated by carbon source.     

Production and Catabolite Repression of the Constitutive Polygalacturonic Acid trans-Eliminase of Aeromonas liquefaciens

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.     

Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata.

Specific cellulase production rates (SCPR) were compared with intracellular cyclic AMP (cAMP) levels in the thermophilic actinomycete, Thermomonospora curvata, during growth on several carbon sources in a chemically defined medium. SCPR and cAMP levels were 0.03 U (endoglucanase [EG] units) and 2 pmol per mg of dry cells, respectively, during exponential growth on glucose. These values increased to about 6 and 25, respectively, during growth on cellulose. Detectable EG production ceased when cAMP levels dropped below 10. Cellobiose (usually considered to be a cellulase inducer) caused a sharp decrease in cAMP levels and repressed EG production when added to cellulose-grown cultures. 2-deoxy-D-glucose, although nonmetabolizable in T. curvata, depressed cAMP to levels observed with glucose, but unlike glucose, the 2DG effect persisted until cells were washed and transferred to fresh medium. SCPR values and cAMP levels in cells grown in continuous culture under conditions of cellobiose limitation were markedly influenced by dilution rate (D). The maxima for both occurred at D = 0.085 (culture generation time of 11.8 h). When D was held constant and cellobiose concentration was increased over a 14-fold range to support higher steady state population levels, SCPR values decreased about fivefold, indicating that extracellular catabolite accumulation may be a factor in EG repression. The role of cAMP in the mechanism of this repression appears to be neither simple nor direct, since large changes (up to 200-fold) in SCPR accompany relatively small changes (10-fold) in cellular cAMP levels.     

Cellulase biosynthesis during degradation of cellulose derivatives by Thermomonospora curvata

A variety of commercially used cellulose derivatives were compared with crystalline cellulose as substrates for induction of cellulase biosynthesis in the actinomycete Thermomonospora curvata. Cellulase induction during growth on uncoated cellophane was as rapid as that on crystalline cellulose, but on coated cellophanes, induction was delayed. Susceptibility to enzymatic attack determined the inductive potential of the substrate. Cellulose acetate was a poor substrate because of its extreme recalcitrance to attack. With other cellulose derivatives, soluble sugar accumulation caused a transient repression of cellulase biosynthesis, but the ratio of cellobiose (a cellulase inducer) to glucose (a cellulase repressor) was not a controlling factor. Crystalline cellulose yielded the lowest inducer/repressor sugar ratio (1.1:1 compared to 3.8–4.0:1 for cellulose derivatives), but supported the highest cellulase production. Glucose could not repress cellulase biosynthesis in the presence of cellobiose due to the strong preference for uptake of the disaccharide even by glucose-grown cells.     

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V_max values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K_m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.