Cellulose digestion in termites and cockroaches: What role do symbionts play?

• 1.1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.
• 2.2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.
• 3.3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO₂ in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.
• 4.4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.
• 5.5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.
• 6.6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.     

Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce extracellular cellulase [see 1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Purification and characterization of cellulolytic enzymes fromTrichoderma harzianum

Two forms of filter paper activity (filter paper activity; cellulose 1,4-β-cellobiosidase, EC 3.2.1.91) and single forms of CM-cellulase (carboxymethyl cellulase; endo-l,4-β-glucanase, EC 3.2.1.4) and β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) from the culture filtrate ofTrichoderma harzianum were separated and partially purified by (NH₄)₂SO₄ precipitation, ion-exchange chromatography and gel filtration. The final preparation was purified about 12-, 20- and 27-fold for FP-activity, CM-oellulase and β-glueosidase, respectively. The pH and temperature optima, stability, kinetic parameters, effeet of metal ions and molar mass of each was determined. A distinct type of synergistic action between cellulase components was observed for efficientin vitro saccharification of dewaxed cotton.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Column cellulose hydrolysis reactor: Cellulase adsorption profile

Summary A column cellulose hydrolysis reactor was set up using a single passage of cellulase enzyme which was followed with a continuous percolation of buffer. Hydrolysis rates were found to decline precipitously upon the removal of the non-adsorbed cellulase components. By comparing specific activities of the cellulase before and after adsorption on the cellulose column, it was concluded that the adsorption efficiencies for the cellulase components decreased from exoglucanase (1,4--d-glucan cellobiohydrolase EC 3.2.1.91) to endoglucanase [1,4-(1,3;1,4)--d-glucan 4-glucanohydrolase, EC 3.2.1.4] to -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.21). Of the adsorbed cellulase components, the rate of endoglucanase leaching from the cellulose column was 20 times that for the exoglucanase despite the greater adsorption efficiency of the latter. By analysing the cellulase components which were bound and not bound by the cellulose column and comparing them with a purified exoglucanase enzyme on sodium dodecyl sulfate polyacrylamide gels, it was confirmed that the major cellulase component adsorbed to the cellulose column was an exoglucanase component. The resultant loss of other cellulase components from the reactor was probably the cause for the much reduced rate of cellulose hydrolysis when these components were flushed out of the column.     

Comparison of batch and semicontinuous cultures for production of protein from mesquite wood by Brevibacterium sp. JM98A

The production of protein by a Brevibacterium sp. JM98A usingmesquite wood as the substrate was compared in batch and semicontinuous cultures. A 14 liter glass fermentor with automatic pH, temperature, and foam control was used for the study. A pH range of 6.6 to 7.2 was optimum for the growth of JM98A. The batch and semicontinuous cultures were compared on the basis of viable cell counts, protein production, CMC-Ase (β-1,4-glucanase) activity, and filter paper cellulase (β-1,4-glucan cellobiohydrolyase) activity. Total hexose, cellulose, and reducing sugar consumption were measured. The semicontinuous process yielded 2.97 times as much protein in 72 hr as the batch cultures. Most of the biomass resulted from the utilization of soluble sugars rather than from the degradation of cellulose during the semicontinuous process.     

Zum Einfluß der physikalischen Struktur des Substrates auf die Hydrolyse von Cellulose durch verschiedene Enzymsysteme und Enzymkomponenten

Starting from cellulose samples prepared from cotton lintes and differing in lattice type, crystallinity and fibrillar morphology, enzymatic hydrolysis of fibre cellulose has been studied employing complete enzyme systems from Trichoderma, Sporotrichum, Gliocladium and Penicillium as well as isolated endo- and exo-1,4-β-glucanases from Trichoderma reesei and Sporotorichum pulverulentum. The effect of hydrolysis was characterized by content of reducing sugars (RS) and of glucose in the hydrolyzate as well as by DP and X-ray diffraction pattern of the residues. With all the complete enzyme systems investigated about the same order of degradability was found with a series of substrates differing in physical structure. The hydrolysis effect of cellulase from S. pulverulentum proved to be sensitive to the gas atmosphere above the system (N₂ or O₂), probably due to the interaction of an O₂-atmosphere with the activity of the cellubiose-oxydase existent in the system. Isolated endoglucanase from S. pulverulentum and T.reesei still led to a considerable formation of RS and glucose, a corrosion of the fibre surface and a significant descrease in DP. Influence of substrate physical structure was rather small with regard to RS, but still considerable with regard to residue-DP. The effect of isolated exoglucanases depends largely on the chemical structure of the cellobiohydrolase in question, as demonstrated with the two samples “CBH I” and “CBH II” from T. reesei. With CBH I, rather resembling endo-glucanase behaviour, a considerable formation of RS and a significant corrosion of the fibre surface has been observed. On the other hand, only negligibly small amounts of RS were formed by CBH II. Results are discussed with regard to the complex mechanism of cellulase action on fibrous cellulose and with regard to the relevance of different parameters of physical structure of cellulose in connection with enzymatic hydrolysis. A remarkable acceleration of the Cellulose III → Cellulose I lattice transition due to chain fragmentations in the presence of cellulase can be concluded the experiments.     

Production of xylanolytic enzymes in association with the cellulolytic activities of Penicillium funiculosum

Penicillium funiculosum produced 16 and 0.4 units ml^?1 of d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37), respectively, in shake flasks. Both enzymes were 100% stable when heated at 50°C for 30 min and on prolonged heating d-xylanase and β-d-xylosidase showed 46 and 20% loss, respectively. Maximum hydrolysis (75%) of d-xylan was obtained when the end products were removed. The addition of β-d-xylosidase markedly influenced the degree of hydrolysis of d-xylan. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylose, d-xylobiose, d-xylotriose, d-xylotetraose, d-xylopentose and l-arabinose. The fractionation of culture filtrate of Penicillium funiculosum grown on cellulose powder or in a combination of cellulose powder and wheat bran indicated the presence of two d-xylanases. The role of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase on the overall hydrolysis of pure cellulose and lignocellulosic substrates is discussed.     

The surface structure of well-ordered native cellulose fibrils in contact with water

CP/MAS ¹³C NMR spectroscopy was used in combination with spectral fitting to examine the surface structure of hydrated cellulose I fibrils from Halocynthia and Gluconoacetobacter xylinus. To increase the spectral intensities and minimize signal overlap, G. xylinus celluloses site-specifically enriched in ¹³C either on C4 or on both C1 and C6 were examined. The experimental data showed multiple C4 and C6 signals for the water accessible fibril surfaces in the highly crystalline celluloses. These signal multiplicities were attributed to structural features in the surface layers induced by the fibril interior, and could not be extracted by spectral fitting in celluloses with a lower degree of crystallinity such as cellulose from cotton.     

Synergism between enzymes of Sclerotium rolfsii involved in the solubilization of crystalline cellulose

Synergism between (1→4)-β-d-glucan cellobiohydrolase, endo-(1→4)-β-d-glucanases, and β-d-glucosidases of Sclerotium rolfsii for solubilization of native and amorphous celluloses is discussed. Besides synergism between cellobiohydrolase and endo-β-glucanases of S. rolfsii, a synergistic effect between endo-β-glucanases and β-glucosidases [which behaved rather as (1→4)-β-d-glucan glucohydrolases] was observed for solubilization of crystalline and amorphous celluloses. It seems that a cellobiohydrolase initiates the attack on crystalline cellulose and an endo-β-d-glucanase the attack on amorphous cellulose.     

Crystalline index change in cellulose during aerobic and anaerobic Cellulomonas uda growth

Summary Cultures of Cellulomonas uda were monitored under both aerobic and anaerobic conditions using three commercially available celluloses with varying degrees of crystallinity. In all cases, a high level of cellulose was metabolized and the same maximum carboxymethylcellulase activity (2.6 IU/mg of cellular protein) was observed. Measurement of the crystalline index of celluloses during cellulose growth revealed that the amorphous and crystalline regions were solubilized simultaneously. Investigation of the solubilization rate showed that a decline occurred when a considerable amount of cellulose still remained in the medium. Hypotheses were suggested to explain the biphasic pattern of the kinetics obtained.     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Biochemical and Immunological Relationships Between Endo-β-1,4-Glucanases from Cockroaches

The cellulase from Geoscapheus dilatatus consisted of two major and four minor endo-β-1,4-glucanase components. Two major and one minor component were purified to homogeneity. The major endo-β-1,4-glucanase components, named GD1 and GD2, were similar to EG1 and EG2 from Panesthia cribrata in terms of M_r and kinetic properties. The purified minor component, named GD3, was distinct from GD1 and GD2 because of a lower M_r and a lower specific activity. Polyclonal antibodies raised against the two major endo-β-1,4-glucanase components, EG1 and EG2, of the cellulase from P. cribrata cross-reacted with each other and with pure GD1 and GD2 from the foregut and midgut of the related cockroach G. dilatatus but did not cross-react with GD3. Endo-β-1,4-glucanase components were partially purified from the foregut and midgut of four other cockroaches. These comprised three other Australian cockroaches also from the superfamily Blaberoidea and one American cockroach, Cryptocercus punctulatus, from the superfamily Blattoidea. The endogenous cellulases from all cockroaches examined consisted of either two or three major endo-β-1,4-glucanase components. The amino acid sequence of the N-terminus region of the two major endo-β-1,4-glucanase components from P. cribrata were determined and are homologous with those belonging to glycosyl hydrolase family 9 (cellulase family E).     

Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β-d-glucosidase (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β-d-glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. d-Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. d-Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while d-fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β-d-glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Russian Article pp. 191–195

Three cellulase components and one xylanase of Trichoderma sp. M-17 have been immobilzed on a soluble high molecular weight polymer (PVA), using carbodiimide. The immobilized enzymes retained about 80% of the cellulase, cellulose 1,4-β-cellobiosidase, β-glucosidase and 60% endo-1,4-β-xylanase activities. The bound enzymes catalyzed the hydrolysis of alkali-treated cornstalks with a higher efficiency than the free cellulase. The potential for reutilization of the immobilized enzymes was studied using membrane filters and the system was found to be active for three cycles.     

Molecular characterization of SCO0765 as a cellotriose releasing endo-<Emphasis Type="Italic">β</Emphasis>-1,4-cellulase from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A(3)

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50–60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.     

Comparative study of samples of powdered and microcrystalline celluloses of different natural origins: Supermolecular structure and the chemical composition of powdered samples

Methods of wide-angle X-ray scattering (WAXS), high resolution solid-state ¹³C NMR, and Fourier transform IR-spectroscopy are applied to study supermolecular structures and functional compositions of lignocellulose samples of wood and grass origins and powdered celluloses (PC) obtained from them under identical hydrolysis conditions. It was shown by WAXS that the structure of cellulose I is preserved in samples of powdered celluloses, however, an increased degree of crystallinity and cross-section sizes of crystallites are observed in PC samples. Specific features of changes in the supermolecular structure of cellulose occurred after the hydrolysis, i.e., an increase in the content of cellulose I_β in PC compared to the initial samples, are established by ¹³C NMR method. It was shown by means of ¹³C NMR and Fourier transform IR-spectroscopy that the functional chemical composition of lignocelluloses is weakly affected by the hydrolysis. The presence of residual lignin functional groups in the samples is confirmed.     

Major characteristics of the cellulolytic system of Clostridium thermocellum coincide with those of the purified cellulosome

The properties of the cellulosome (a cellulose-binding, multiple cellulase-containing protein complex isolated from Clostridium thermocellum) have been compared with the previously reported characteristics for crude cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations. Similar to the crude enzyme system, true cellulolytic activity was demonstrated for the purified cellulosome on the basis of extensive solubilization of microcrystalline cellulose. The cellulolytic activity of the purified cellulosome was enhanced both by calcium ions and by thiols, and was inhibited by cellobiose (the major end product of the cellulosome-mediated cellulose degradation). In addition, at low ionic strength, cellulose-adsorbed cellulosome was detached intact from the cellulose matrix. Using controlled conditions, maximum enzymatic activity was shown to correspond to suboptimal conditions of cellulosome adsorption to cellulose. The results suggest that previous data accumulated for the crude cellulase system in C. thermocellum essentially reflect the contribution of the cellulosome.