Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Regulation of cellobiase secretion in Termitomyces clypeatus by co-aggregation with sucrase

Regulated secretory proteins are sorted via selective co-aggregation in eukaryotes. Cellobiase (C) of the filamentous fungus Termitomyces clypeatus remained co-aggregated with sucrase (S), and only one isoform of each of the enzymes was present in intra- and extracellular extracts. Kinetics of secretion of sucrase increased in vivo and in vitro in secreting (Sc) medium and decreased under non-secreting (NSc) conditions similar to those observed for cellobiase. In the Sc condition, total enzyme production and activity ratios of cellobiase and sucrase (C/S) in cell-bound, extra- and intracellular preparations increased with time and were significantly higher from those obtained in non-secretory media. It was concluded that secretion of sucrase in culture medium is under same cellular regulation as that of cellobiase, and sucrase is involved in regulating extracellular release of cellobiase through co-aggregation in the fungus. Received: 27 August 2001 / Accepted: 1 November 2001     

Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose

We have established an improved large deletion method in Escherichia coli genome using a combination of two different recombination systems, lambda Red and Cre/loxP. The loxP site could be rapidly and efficiently integrated in the genome by lambda Red and large deletions of both 117- and 165-kbp regions could be generated in 100% efficiency by Cre/loxP. Comparative genomic hybridization microarray experiments of deletion strains indicated that deletions were generated only in expected regions of the genome. These results have demonstrated that the method is useful for genome engineering in E. coli.     

Synergism among three purified cellulolytic components of Clostridium thermocellum

Abstract Three clostridial cellulases viz. a hydrophilic cellobiohydrolase (CBH3), a hydrophobic endoglucanase (EG1), and an aggregate-forming hydrophilic endoglucanase (EG5), all purified from recombinant strains of Escherichia coli , were used in different combinations to reconstitute the synergistic effect during cellulose hydrolysis. EG1 and EG5 were weakly active on crystalline cellulose, if added separately or together in the reaction mixture. However, when CBH3 was added to the reaction mixture, its hydrolytic activity was increased to 1.8-fold in the presence of EG1 and EG5. A further increase in the activity from 1.8 to 2.2-fold was observed when calcium and dithiothreitol were added to the reaction mixture containing all three enzymes and filter paper as substrate. The synergistic effect remained unaffected even when EG1 was replaced by its 33-amino acid C-terminal deleted variant BL35. BL35 was less active compared to EG1, but was equally hydrophobic as EG1. These results suggest that the hydrophobic interaction between cellulolytic components and/or with the crystalline substrate is important for positive synergistic effect.     

Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans

This paper reviews the properties of the cellulosome and noncellulosome cellulases produced by Clostridium cellulovorans, an anaerobic, mesophilic, spore-forming microorganism that produces copious amounts of cellulase. The three major subunits of the cellulosome, CbpA, exoglucanase S (ExgS), and P100, are described, as well as the properties of the functional domains of CbpA. The properties of two noncellulosomal endoglucanases, EngD and EngF, are compared. The functions of the cellulose-binding domain (CBD) of CbpA indicate its potential uses in biotechnology. Received: November 18, 1997 / Accepted: November 26, 1997     

The modular cellulase CelZ of the thermophilic bacterium Clostridium stercorarium contains a thermostabilizing domain

The non-catalytic region of the Clostridium stercorarium cellulase CelZ (Avicelase I) comprises two protein segments (C and C′) grouped into different subfamilies of cellulose-binding domain (CBD) family III. The C-terminally located family IIIb domain C was identified as a true cellulose-binding domain responsible for anchoring the CelZ enzyme to cellulose. The family IIIc domain C′ immediately adjacent to the catalytic domain was unable to mediate binding to cellulose. A deletion study revealed a lack of independence of this pair of domains: almost the entire C′ domain was required to maintain the catalytic activity and the thermostability of the enzyme.     

Molecular and biochemical markers for monitoring dynamic shifts of cellulolytic protozoa in Reticulitermes flavipes

Lower termites rely on cellulolytic protozoa to aid in the digestion of their wood-based diet. However, despite the major contribution of protozoa to the lower termite digestive system, few techniques have been developed to monitor shifts in protozoan populations. This study investigated whether quantitative real-time PCR (qRT-PCR) and/or cellulase enzyme assays can be used to monitor changes of cellulolytic protozoan populations in the lower termite, Reticulitermes flavipes (Kollar). Previously developed cellulase primer sets were used to test for changes in cellulase gene expression, while three different cellulase enzyme assays were used to assess changes in cellulase enzyme activity. The results from this study indicate that qRT-PCR is a reliable method to monitor shifts in cellulolytic protozoan populations. Specifically, qRT-PCR can serve as a useful monitoring technique during high-throughput screening of novel termite control agents such as cellulase inhibitors, and help to answer questions relating to whether or not such control agents impact cellulolytic protozoan populations.     

Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.     

Removal of carboxymethyl cellulase activity from the culture filtrate of Termitomyces clypeatus producing xylanase

Termitomyces clypeatus produced 450 IU xylanase ml^–1 in a medium containing starch-free wheat bran powder as the carbon source. Carboxymethyl cellulase (CMCase) activity in the culture filtrate was removed by keeping the filtrate at pH 10 for 60 min followed by a change to pH 6. Treatment of Kraft pulp (bamboo) with the filtrate at pH 7 decreased the kappa number from 10.5 to 5 with release of reducing groups equivalent to 0.15 mg glucose g^–1 pulp.     

Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-d-glucose (1 mg/ml), tunicamycin (30 μg/ml), 1-deoxynojirimycin (30 μg/ml) and d-glucono-δ-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except d-glucono-δ-lactone which released 60% of the total protein. 2-Deoxy-d-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected.     

Increased enzyme secretion by 2-deoxy-D-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-D-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-D-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-D-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-D-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.     

Synthesis and secretion of a heat-stable carboxymethylcellulose from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus

Summary The cellulase gene celA of Clostridium thermocellum coding for the thermostable endoglucanase A was transferred from Escherichia coli to Bacillus subtilis 168 and B. stearothermophilus CU21 using plasmids derived from the Bacillus vector pUB110. When the structural part of the gene was joined to a pUB110 promoter the recombinant plasmids (pSE102, pSE105) were stably maintained and expressed carboxymethylcellulase (CMCase) activity. In B. stearothermophilus CU21 (pSE105) the clostridial CMCase was produced over a wide temperature range up to the maximal growth temperature (68° C). In contrast to E. coli, all of the CMCase synthesized in bacilli was released into the culture medium. About 50% of the extracellular protein secreted by B. subtilis 168 (pSE102) carrying the celA gene consisted of endoglucanase A. These findings demonstrate the feasibility of producing cellulolytic enzymes from thermophilic anaerobes in bacilli.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Regulation of nitrate reduction in a cyanobacterium Phormidium uncinatum: Distinctive modes of ammonium-repression of nitrate and nitrite reductases

Abstract A 5.8 kbp DNA fragment from Clostridium cellulovorans (ATCC 35296) containing endo-β-1,4-glucanase (1,4-β- d -glucan glucanohydrolase, carboxymethylcellulase, CMCase; EC 3.2.1.4) gene, engD was cloned in Escherichia coli . The clone harboring a subcloned 3.8 kb fragment in plasmid, pEQ52V, produced an enzyme that showed both endo-β-1,4-glucanase activity as well as cellobiosidase activity. Zymograms with the engD encoded enzyme with carboxymethyl-cellulose as the substrate indicated that the molecular mass of the active protein was 50 000.     

Cellobiose dehydrogenase production by the mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus produces cellobiose dehydrogenase (CDHtc) in cellulose medium with the highest yield (55.88 U mL⁻¹) among all the reported fungal species. The enzyme has been isolated and purified from the culture filtrate.

CDHtc was found to be a very thermolabile enzyme with the temperature optimum at 30 °C, while it exhibited a wide range of pH stability from pH 2.0 to 8.0. Lactose was efficiently converted to lactobionic acid in presence of the enzyme. Addition of glucose in the cellulose medium on the first day of growth induced a lag period in enzyme production but ultimately facilitated earlier CDHtc production and the yield was also comparable to that achieved in the cellulose medium.     

Overproduction of a Clostridium cellulolyticum endoglucanase by mutant strains of Escherichia coli

We have studied the expression of an endoglucanase from Clostridium cellulolyticum in mutant strains of Escherichia coli that overproduce haemolysin. When these mutants were transformed with plasmids encoding the endoglucanase, they showed a significantly enhanced endoglucanase activity, compared to transformed parental strains. Among the mutants, strain Hha-2 showed the highest production. We have identified the endoglucanase gene product synthesized in E. coli Hha-2/pBP8 and detected an increased amount of the enzyme parallel to the increase of endoglucanase activity. This was mainly localized in the periplasm and only a small percentage of it was found in the culture fluid.     

Breakdown of crystalline cellulose by synergistic action between cellulase components from Clostridium thermocellum and Trichoderma koningii

Abstract Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Suprisingly, not all fungal cellulase components exhibit this property, and no such synergism has been observed so far between fungal and bacterial cellulases.
The cellulase complex of Clostridium thermocellum cannot effect the extensive breakdown of highly ordered cellulose unless Ca²⁺ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellum cellulase to effect the breakdown of cellulose in the absence of Ca²⁺ and DTT. enhanced activity is observed if Ca²⁺ and DTT are present.
This finding may have important applications in industry: it certainly has important implications for those interested in the basic mechanism of cellulase action in C. thermocellum .     

Evidence of an alternative route of cellobiase secretion in the presence of brefeldin A in the filamentous fungus Termitomyces clypeatus

Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 microgram/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50°C, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.     

Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): Identification of catalytic and cellulose-binding domains

Summary The nucleotide sequence of the celZ gene coding for a thermostable endo--1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 by which encodes a preprotein of 986 amino acids with an M_r of 109000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fuorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.