Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

Increases in endo-1,4-β-d-glucanase titres produced by Aspergillus fumigatus through application of simple statistical methods

Yields of endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] produced by Aspergillus fumigatus IMI 255091 in shake flask cultures have been improved through application of a form of evolutionary operation incorporating a standard factorial design. This approach gave considerable improvements in yield, up to the point at which the limitations of the shake flask technique were noticeable. Further improvements then resulted from use of a 5 litre disc-turbine agitated fermenter.     

Comparison of the 3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and determining cellulase activity

A comparison has been made between the 3,5-dinitrosalicylic acid (DNS) and alkaline copper methods of assaying for reducing sugars released during the enzymatic hydrolysis of cellulose by culture filtrates from Trichoderma harzianum E58. The DNS method was shown to be more readily influenced by the incubation conditions and by components derived from lignocellulosic substrates. The endo-1,4-β-d-glucanase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] values obtained with the DNS assay were always considerably higher than those obtained with the alkaline copper method and did not give reducing values that were proportional to the actual number of hemiacetal reducing groups. The alkaline copper assay was not affected by the degree of polymerization of the substrate. Although this latter method appeared to be superior to the DNS assay it was still affected by the incubation conditions, nature of the substrate and the influence of other cellulase components on each of the specific enzyme assays.     

Occurrence of a procellulase in the culture filtrates of Penicillium janthinellum

The endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3:1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] activity of two-day old culture filtrates of Penicillium janthinellum has been enhanced four-fold by incubating with a 10-day old culture filtrate of Penicillium funiculosum grown on the same medium. An inactive protein isolated by fractionation of two-day old culture filtrate of P. janthinellum using preparative isoelectric focusing, showed 30- to 50-fold enhancement of endo-1,4-β-d-glucanase activity. This fraction has been designated the ‘procellulase’ in the present paper. The purity of the procellulase was confirmed by analytical isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It had a molecular weight of 68 000 and an isoelectric point of pH 3.7.     

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.     

Isolation of new anaerobic,thermophilic and cellulolytic bacteria-JT strains and their cellulase production

Six microbial strains (JT) of endospore-forming, anaerobic, thermophilic and cellulolytic bacteria were isolated from camel feces, compost, soil and hot spring water in Japan. These strains are gram negative and classified as the genus Clostridium. Strains JT3-1, JT3-2 and JT3-3 can digest starch. All of the strains produce a high activity of extracellular cellulases in cellobiose and cellulose media.Strain JT1 produced 1.36 units/ml of CMCase (endo-β-1,4-d-glucanase, EC 3.2.1.4), 66.2 units/ml of β-glucosidase (ED 3.2.1.21) and 39.9 units/ml of β-xylosidase (EC 3.2.1.37) in 1% cellobiose medium. Strain JT3-3 produced 1.87 units/ml of CMCase, 166.3 units/ml of β-glucosidase and 23.6 units/ml of β-xylosidase in 1% cellulose medium.     

Purification and properties of endo-(1→4)-β-d-glucanase from Ruminococcus albus

An enzyme active against O-(carboxymethyl)cellulose (CMC) was purified from a synthetic medium containing ball-milled cellulose wherein Ruminococcus albus had been cultivated for 70 h. After 570-fold purification, a homogeneous enzyme was obtained in a yield of 3%. The enzyme degraded CMC (molecular weight, 180,000; degree of substitution, 0.6) to a smaller polymer having a molecular weight of ∼20,000, and generated a small proportion of glucose, but negligible proportions of such cello-saccharides as cellobiose, cellotriose, cellotetraose, or cellopentaose. The fact that the enzyme could produce water-insoluble fragments was discovered by dissolving substrate and products in Cadoxen solution. No water-soluble cello-oligomers were detected by thin-layer chromatography after degradation of water-insoluble cellulose by the purified enzyme. Therefore, the enzyme was classified as an endo-(1→4)-β-d-glucanase.     

Functional characteristics of two d-xylanases purified from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) were purified from Trichoderma harzianum culture filtrates. From kinetic analyses, apparent V_max and K_m values of 580 U mg^?1 protein and 0.16% d-xylan were obtained for the 20 000 dalton endo-1,4-β-d-xylanase, while values of 100 U mg^?1 protein and 0.066% d-xylan were obtained for the 29 000 dalton endo-1,4-β-d-xylanase. Substrate levels >1% (w/v) d-xylan were found to be inhibitory to both enzymes. Both d-xylanases were highly active against d-xylans obtained from various sources. Of the polymeric sugars tested, carboxymethyl cellulose was the only substrate which was hydrolysed to any extent. Little or no activity was observed against cellulose. Analyses by h.p.l.c. demonstrated the absence of hydrolytic activity by both d-xylanases on d-xylobiose. d-Xylotriose was cleaved to a limited extent by the 29 000 dalton d-xylanase only, while d-xylotetraose was hydrolysed by both. In the presence of d-xylotetraose, the 20 000 dalton d-xylanase had an associated transxylosidase activity which was not observed with the 29 000 dalton enzyme. When the solubilization assay was used, neither of the d-xylanases was inhibited by high concentrations of d-xylose and xylobiose.     

Purification and properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 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).     

Inhibitor of β-d-glucosidase and endo-1,4-β-d-glucanase produced by Aspergillus fumigatus IMI 255091

Inconsistencies in assays of fermentation broths of Aspergillus fumigatus IMI 255091 were observed for endo-1,4-β-d-glucanase [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). Dilution of the original sample appeared to enhance activity. These enzymes were apparently not adsorbed by sintered microporous inorganic spheroids specially fabricated for protein adsorption. The adsorbents removed other proteins, including material shown to be of low molecular weight and assumed to be an inhibitor, permitting considerably enhanced activity.     

Biochemical characterization of a 27 kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The K_m and V_max values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml⁻¹ and 0.315 U min⁻¹, respectively. The enzyme was strongly inhibited by Hg²⁺ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database.     

4,6-Dimethoxy-1,3,5-triazine oligoxyloglucans: Novel one-step preparable substrates for studying action of endo-β-1,4-glucanase III from Trichoderma reesei

Two kinds of 4,6-dimethoxy-1,3,5-triazine (DMT) oligoxyloglucans, DMT-β-XXXG and DMT-β-XLLG, have been synthesized via one-step procedure starting from the corresponding unprotected oligoxyloglucans in water. The resulting DMT derivatives were found to be hydrolyzed by endo-β-1,4-d-glucanase III from Trichoderma reesei (EGIII) and utilized as substrates for determination of the kinetic parameters of EGIII. The present DMT-method would be a convenient analytical tool for studying the action of glycosyl hydrolases due to the extremely simple synthetic process of DMT-glycosides without using protecting groups.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for 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) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island,Indian Ocean

The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-β-d-glucosaminidase, the cellulases, endo-β-1,4-glucanase and β-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-β-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-β-1,4-glucanase, β-glucohydrolase and N-acetyl-β-d-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-β-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.     

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.

     

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.     

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-β-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-β-1,4-glucanase (34.234 U/ml) and endo-β-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.