Cellulase and Other Polymer-Hydrolyzing Activities of Trichomitopsis termopsidis, a Symbiotic Protozoan from Termites

Crude extracts of the anaerobic, cellulolytic protozoan Trichomitopsis termopsidis possessed endo-β-1,4-glucanase and cellobiase activities, as evidenced by hydrolytic action on carboxymethyl cellulose and cellobiose, respectively. Cell extracts also hydrolyzed microcrystalline cellulose. Hydrolysis of microcrystalline cellulose displayed optima at pH 5 and at 30°C, and glucose was the sole product liberated. Cellulolytic activities of T. termopsidis appeared to be entirely cell associated. Hydrolytic activity was also detected against Douglas fir wood powder, xylan, starch, and protein, but not chitin. The importance of these enzymes in the nutrition of T. termopsidis is discussed in terms of the natural habitat of this protozoan (the hindgut of wood-eating termites).     

Genomic insights into the carbohydrate catabolism of <Emphasis Type="Italic">Cairneyella variabilis gen. nov. sp. nov</Emphasis>., the first reports from a genome of an ericoid mycorrhizal fungus from the southern hemisphere

This paper describes a novel species of ericoid mycorrhizal fungus from Australia, Cairneyella variabilis, Midgley and Tran-Dinh, gen. nov. sp. nov. The genome of C. variabilis was sequenced and a draft genome assembled. The draft genome of C. variabilis is 52.4 Mbp in length, and to our knowledge, this is the first study to present a genome of an ericoid mycorrhizal fungus from the southern hemisphere. Using the SignalP and dbCAN bioinformatic pipelines, a study of the catabolic potential of C. variabilis was undertaken and showed genes for an array of degradative enzymes, most of which appear to be secreted from the hyphae, to access a suite of different carbon sources. Isolates of C. variabilis have been previously shown to utilise cellulose, carboxymethyl cellulose (CMC), cellobiose, xylan, pectin, starch and tannic acid for growth, and in the current study, putative enzymes for these processes were revealed. These enzymes likely play key roles in nutrient cycling and other edaphic processes in heathland environments. ITS phylogenetic analyses showed C. variabilis to be distinct from the fungi of the “Hymenoscyphus ericae aggregate”.     

Utilisation of plant cell-wall polysaccharides and organic phosphorus substrates by isolates of Aspergillus and Penicillium isolated from soil

The ability of seventy isolates (comprising 43 species) of Aspergillus and Penicillium, from soil and compost, to grow on sources of carbon and phosphate from plant remains was examined. Only two isolates from compost actively degraded crystalline cellulose, though most others grew on carboxymethyl cellulose. Most isolates produced biomass on cellobiose, and all on glucose, pectin and xylan. All fungi grew on phytic acid and most on DNA. If these data indicate utilisation of carbon and organic phosphorus in nature, then isolates of Trichocomaceae from soil have limited access to cellulose and considerably greater reliance on pectin and hemicellulose. The fungi may also gain their phosphorus from organic sources. The variation within species may indicate the existence of ecotypes.     

Genome Sequence of <Emphasis Type="Italic">Microbulbifer mangrovi</Emphasis> DD-13<Superscript>T</Superscript> Reveals Its Versatility to Degrade Multiple Polysaccharides

Microbulbifer mangrovi strain DD-13^T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, β-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.     

Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB from Thermotoga neapolitana

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high V_max value and low apparent K_m value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the V_max values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the K_m values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The K_i values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the K_i values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.     

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml⁻¹, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.     

Role of Aerobic Microbial Populations in Cellulose Digestion by Desert Millipedes

I examined the role of aerobic microbial populations in cellulose digestion by two sympatric species of desert millipedes, Orthoporus ornatus and Comanchelus sp. High numbers of bacteria able to grow on media containing cellulose, carboxymethyl cellulose, or cellobiose as the substrate were found in the alimentary tracts of the millipedes. Enzyme assays indicated that most cellulose and hemicellulose degradation occurred in the midgut, whereas the hindgut was an important site for pectin degradation. Hemicellulase and β-glucosidase in both species and possibly C_x-cellulase and pectinase in O. ornatus were of possible microbial origin. Degradation of [¹⁴C]cellulose by millipedes whose gut floras were reduced by antibiotic treatment and starvation demonstrated a reduction in ¹⁴CO₂ release and ¹⁴C assimilation and an increase in ¹⁴C excretion over values for controls. It appears that the millipede-bacterium association is mutualistic and makes available to millipedes an otherwise mostly unutilizable substrate. Such an association may be an important pathway for decomposition in desert ecosystems.     

A New Pectolytic Enzyme in Erwinia aroideae Formed in the Presence of Nalidixic Acid

Pectolytic enzyme formation by whole cells of Erwinia aroideae was markedly stimulated when nalidixic acid was added to a culture medium. The activity of pectolytic enzyme was markedly stimulated by nalidixic acid when the activity was measured by the decrease of viscosity of pectin, while activities of both polygalacturonic acid trans-eliminase and polygalacturonase which were measured respectively by the increase of optical density at 230 nm and the liberation of aldehyde groups, were not stimulated. The analysis of pectolytic enzyme by carboxymethyl cellulose column chromatography indicated that there was a significant difference in the elution profiles between the pectolytic enzyme induced by nalidixic acid and that synthesized under normal conditions. Therefore, we conclude that two enzymes are distinct protein species.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

Substrate specificity and mode of action of a cellulase from Aspergillus niger.

The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.     

Digestive ability of the freshwater crayfish Paranephrops zealandicus (White) (Parastacidae) and the role of microbial enzymes

SUMMARY. 1 Ingestion rate, assimilation efficiency and digestive enzyme activity were investigated in the New Zealand freshwater crayfish, Paranephrops zealandicus (White). Rates of ingestion of fresh and decaying Elodea canadensis Michx. were highly variable at 15°C and assimilation efficiency averaged 21%.
2.Hepatopancreas extracts showed enzyme activity towards each of nine substrates tested; microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), cellobiose, amylose, pectin, mannan, laminarin, chitin and'Azocoll' (a dye-collagen complex).
3. Three genera of Enterobacteriacae were isolated from digestive juices and hepatopancreas samples and microbial activity was implicated in the breakdown of MCC, laminarin and protein. Host-specific activity was not detected in the assays with MCC suggesting a solely microbial source for this enzyme.
4. Although cellulose cannot be broken down without some degree of prior conditioning, the polytrophic feeding strategy of P. zealandicus is indicated by the presence of host-specific enzymes that hydrolyse storage and structural sugars of algae, fungi and higher plants as well as animal protein.     

Isolation of a Cellodextrinase from Bacteroides succinogenes

An enzyme which released the cellobiose group from p-nitrophenyl cellobioside was isolated from the periplasmic space of Bacteroides succinogenes grown on Avicel crystalline cellulose in a continuous cultivation system and separated from endoglucanases by column chromatography. The molecular weight of the enzyme was approximately 40,000, as estimated by gel filtration. The enzyme has an isoelectric point of 4.9. The enzyme exhibited low hydrolytic activity on acid-swollen cellulose and practically no activity on carboxymethyl cellulose, Avicel cellulose, and cellobiose, but it hydrolyzed p-nitrophenyl lactoside and released cellobiose from cellotriose and from higher cello-oligosaccharides. These data demonstrate that the enzyme is a cellodextrinase with an exotype of function.     

A Cell Wall-degrading Endopolygalacturonase Secreted by Colletotrichum lindemuthianum

Cultures of Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner have been induced to secrete an endopolygalacturonase (polygalacturonide glycanohydrolase EC3.2. 1.15). This enzyme has been brought to a high state of purity by ion exchange, gel filtration, and agarose affinity chromatography. The enzyme has optimal activity at pH 5, has an apparent molecular weight as determined by gel filtration of about 70,000, and prefers polygalacturonic acid to pectin as its substrate. The enzyme, while hydrolyzing only 1% of the glycosidic bonds, reduces the viscosity of a polygalacturonic solution by 50%. Nevertheless, the initial as well as the final products of polygalacturonic acid hydrolysis are predominantly tri- and digalacturonic acid and, to a lesser extent, monogalacturonic acid. The purified enzyme catalyzes the removal of about 80% of the galacturonic acid residues of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus) as well as from the walls isolated from 8-day-old Red Kidney bean (Phaseolus vulgaris) hypocotyls.     

Isolation of mesophilic solvent-producing clostridia from Colombian sources: physiological characterization, solvent production and polysaccharide hydrolysis

One hundred and seventy-eight new butanol-acetone producing bacteria related to saccharolytic clostridia were isolated from agricultural sources in Colombia and their fermentation potential was evaluated. Thirteen isolates produced more total solvents from glucose than Clostridium acetobutylicum ATCC 824. The isolates with the highest single solvent production were IBUN 125C and IBUN 18A with 0.46 mol butanol and 0.96 mol ethanol formed from 1 mol glucose, yielding 25. 2 and 29.1 g l(-1) total solvents, respectively, which is close to the maximum values described to date. Most of the new isolates produced exoenzymes for the hydrolysis of starch, carboxymethyl cellulose, xylan, polygalacturonic acid, inulin and chitosan. Together with the high efficiency of solvent production, these hydrolytic isolates may be useful for the direct fermentation of biomass. According to their physiological profile, the most solvent-productive isolates could be classified as strains of C. acetobutylicum, Clostridium beijerinckii, and Clostridium NCP262.     

Cellobiose dehydrogenase from Volvariella volvacea and its effect on the saccharification of cellulose

A gene encoding cellobiose dehydrogenase (VvCDH) from Volvariella volvacea was successfully expressed in Pichia pastoris with codon optimization using its native signal sequence. VvCDH had optimum pH and temperature at 5.5 and 60 °C respectively and showed a broad range of pH stability between 5 and 8. Kinetic analysis showed that the best substrate is cellobiose and that the K_m for celloolgosaccharides increases with substrate length. Moreover, lactose is also efficiently oxidized, but glucose and maltose are poor substrates. A large amount of gluconic acid was generated and the overall hydrolysis yield was increased when adding VvCDH to Trichoderma reesei D-86271 enzymatic cocktail during hydrolysis of cellulose substrates, indicating VvCDH involved in the enzymatic cellulose saccharification. VvCDH shows some different enzymatic properties from basidiomycetous CDHs and can be supplemented to T. reesei cellulase cocktail for commercial application.     

Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.     

Evidence that Cellulolysis by an Anaerobic Ruminal Fungus Is Catabolite Regulated by Glucose, Cellobiose, and Soluble Starch

A Piromyces-like ruminal fungus was used to study preferential carbohydrate utilization of [U-¹⁴C]cellulose, both alone and in combination with several soluble sugars. For cells grown on cellulose alone, cellulolytic activity was immediate and, initially, greater than that observed in the presence of added carbohydrate. Cellulolytic activity remained minimal in cultures containing cellulose plus glucose or cellobiose until the soluble sugar was depleted. Soluble starch also regulated cellulose activity but to a lesser extent. The results presented suggest that some fungal cellulases are susceptible to catabolite regulatory mechanisms.     

Polygalacturonase-Mediated Solubilization and Depolymerization of Pectic Polymers in Tomato Fruit Cell Walls : Regulation by pH and Ionic Conditions

The hydrolysis of cell wall pectins by tomato (Lycopersicon esculentum) polygalacturonase (PG) in vitro is more extensive than the degradation affecting these polymers during ripening. We examined the hydrolysis of polygalacturonic acid and cell walls by PG isozyme 2 (PG2) under conditions widely adopted in the literature (pH 4.5 and containing Na⁺) and under conditions approximating the apoplastic environment of tomato fruit (pH 6.0 and K⁺ as the predominate cation). The pH optima for PG2 in the presence of K⁺ were 1.5 and 0.5 units higher for the hydrolysis of polygalacturonic acid and cell walls, respectively, compared with activity in the presence of Na⁺. Increasing K⁺ concentration stimulated pectin solubilization at pH 4.5 but had little influence at pH 6.0. Pectin depolymerization by PG2 was extensive at pH values from 4.0 to 5.0 and was further enhanced at high K⁺ levels. Oligomers were abundant products in in vitro reactions at pH 4.0 to 5.0, decreased sharply at pH 5.5, and were negligible at pH 6.0. EDTA stimulated PG-mediated pectin solubilization at pH 6.0 but did not promote oligomer production. Ca²⁺ suppressed PG-mediated pectin release at pH 4.5 yet had minimal influence on the proportional recovery of oligomers. Extensive pectin breakdown in processed tomato might be explained in part by cation- and low-pH-induced stimulation of PG and other wall-associated enzymes.     

Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-d-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.     

Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component.

The recombinant CelS (rCelS), the most abundant catalytic subunit of the Clostridium thermocellum cellulosome, displayed typical exoglucanase characteristics, including (i) a preference for amorphous or crystalline cellulose over carboxymethyl cellulose, (ii) an inability to reduce the viscosity of a carboxymethyl cellulose solution, and (iii) the production of few bound reducing ends on the solid substrate. The hydrolysis products from crystalline cellulose were cellobiose and cellotriose at a ratio of 5:1. The rCelS activity on amorphous cellulose was optimal at 70 degrees C and at pH 5 to 6. Its thermostability was increased by Ca2+. Sulfhydryl reagents had only a mild adverse effect on the rCelS activity. Cellotetraose was the smallest oligosaccharide substrate for rCelS, and the hydrolysis rate increased with the substrate chain length. Many of these properties were consistent with those of the cellulosome, indicating a key role for CelS.