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.     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

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     

Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects

The activities of six purified Thermomonospora fusca cellulases and Trichoderma reesei CBHI and CBHII were determined on filter paper, swollen cellulose, and CMC. A simple method to measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found to effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% to 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases to digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 mumol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 to 7.8. T. reesei CBHII and T. fusca E3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) of T. fusca endocellulases E2 and E5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a beta-glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHI to T. fusca crude cellulase increased its activity on filter paper 1.7-fold. (c) 1993 John Wiley & Sons, Inc.     

Chitin binding by Thermobifida fusca cellulase catalytic domains

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.     

Biochemistry and Genetics of Actinomycete Cellulases

Abstract

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from “T. curvata”. The T. fusca cellulase genes are expressed at a low level in Escherichia coli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Regulation of enzyme synthesis in the arginine biosynthetic pathway of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the synthesis of only two out of eight arginine biosynthetic enzymes tested was regulated. Comparisons were made between the specific activities of these enzymes in bacteria grown on arginine or on its precursor, glutamate. N2-Acetylornithine 5-aminotransferase (ACOAT), an enzyme involved in both the biosynthesis and catabolism of arginine, was induced about 14-fold during growth of the organism on arginine as the only carbon and nitrogen source, and the anabolic ornithine carbamoyltransferase (aOTC), a strictly biosynthetic enzyme, was repressed 18-fold. Addition of various carbon sources to the arginine medium led to repression of ACOAT and to derepression of aOTC. Fructose, which supported only slow growth of P. aeruginosa, had a weak regulatory effect on the synthesis of the two arginine enzymes while citrate, a good carbon source for this organism, had a strong effect. The repression of ACOAT by citrate was not relieved by adding cyclic AMP to the medium. Under a variety of growth conditions leading to different enzyme activities, a linear relationship between the reciprocal of the specific activity of ACOAT and the specific activity of aOTC was observed. This inverse regulation of the formation of the two enzymes suggested that a single regulatory system governs their synthesis. Such a view was supported by the isolation of citrate-resistant regulatory mutants which constitutively formed ACOAT at the induced level and aOTC at the repressed level.     

Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca.

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 micromol cellobiose.min-1.mL-1, more than 15-fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C. Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.     

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.     

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.     

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.     

Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass

Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.     

Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis

Nonlinear kinetics are commonly observed in the enzymatic hydrolysis of cellulose. This nonlinearity could be explained by any or all of the following three factors: enzyme inactivation, product inhibition, or substrate heterogeneity. In this study, four different approaches were applied to test the above hypotheses using two Thermomonospora fusca endocellulases, E2 and E5. The lack of stimulation of cellulase activity by beta-glucosidase rules out the possibility of product inhibition as a cause of the observed nonlinearity. The results from the other three approaches all provide strong evidence against enzyme inactivation and strong evidence for substrate heterogeneity as the cause of the nonlinear kinetics. The most direct evidence for substrate heterogeneity is that pretreatment of swollen cellulose with either E2cd or E5cd gave a product that was hydrolyzed at a much (3- to 4-fold) slower rate than untreated swollen cellulose even though the initial treatment degraded only 15-18% of the substrate. Furthermore, the activation energy of E2 catalyzed hydrolysis of swollen cellulose increased from 10 kcal/mol for the initial rate to 29 kcal/mol for hydrolysis after 24% digestion.     

The non-catalytic amino acid Asp446 is essential for enzyme activity of the modular endocellulase Cel9 from Myxobacter sp. AL-1

The modular endocellulase Cel9 of the bicistronic operon cel9-cel48 of Myxobacter sp. AL-1 shares not only amino acid sequence similarity but also biochemical properties similar to those of Thermobifida fusca endo/exocellulase E4. Amino acid alignments of a T. fusca E4 cellulase subfamily of family 9 cellulases revealed that Asp(446) of Myxobacter sp. AL-1 Cel9, a putatively noncatalytic residue, is highly conserved in one of the catalytic domains of this subfamily. Directed mutagenesis of residue aspartate (Asp(446)) to alanine generated a Cel9 mutant that lost more than 99% of its activity, suggesting that Asp(446) plays an essential structural role in Cel9 during cellulose degradation. Owing to its high degree of conservation and essential role, we propose that Asp(446) of Myxobacter sp. AL-1 Cel9 is a good landmark that distinguishes members of the E4 subfamily of family 9 cellulases.     

Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans.

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.