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PubMed1999

kdgREcc negatively regulates genes for pectinases, cellulase, protease, HarpinEcc, and a global RNA regulator in Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc- mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc- mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.

Amino Acid Sequence↗

PubMed2000

A celR mutation affecting transcription of cellulase genes in Thermobifida fusca.

Biosynthesis of extracellular cellulases in the cellulose-degrading actinomycete Thermobifida fusca is controlled by a transcriptional regulator, CelR, and cellobiose, which acts as an inducer interfering with the CelR-DNA interaction. We report the identification and characterization of a mutation in the celR gene that changes Ala(55) in the hinge helix of CelR to Thr. The wild-type and mutant celR genes were cloned in Escherichia coli, and their protein products were characterized. The CelR mutant protein bound DNA more weakly than the wild-type protein and formed a less stable complex with DNA in the presence of cellobiose. The results of Western analysis and gel retardation experiments suggest that CelR is produced constitutively and its DNA-binding activity is regulated through posttranslational modification.

Actinomycetales↗

PubMed2002

Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans.

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.

Bacterial Proteins↗

PubMed2003

Prevalence of sensitisation to cellulase and xylanase in bakery workers.

AIMS: To assess the prevalence of sensitisation to a range of exogenous fungal enzymes used in bakeries, and determine the relation between sensitisation and work related symptoms. METHODS: Serum samples (n = 135) from a previous cross sectional study investigating the prevalence of respiratory symptoms and sensitisation to dust components, were reanalysed for specific IgE to the mixed enzymes cellulase, hemicellulase, and xylanase. RESULTS: Eight (6%) of sera tested had detectable specific IgE to mixed enzymes (excluding fungal alpha-amylase) and 16 (12%) to fungal alpha-amylase. A significant increase (p = 0.03) in nasal symptoms was found in those workers sensitised to enzymes (including alpha-amylase and the mixed enzymes, but with or without sensitisation to wheat flour) when compared to those sensitised to wheat flour alone. Both groups had significantly greater levels of nasal symptoms in comparison to those with no evidence of sensitisation. CONCLUSIONS: The association between specific IgE to mixed enzymes, and an increased prevalence of nasal symptoms in individuals sensitised to enzymes, highlights the importance of measuring sensitisation to the full range of exogenous enzymes used in the baking industry, as well as to wheat flour.

Cellulase↗

PubMed1975

Release of cell-bound polygalacturonase and cellulase from mycelium of Rhizoctonia solani.

Propagules of Rhizoctonia solani grown in modified Czapek's medium containing sodium polypectate or carboxymethyl cellulose as a sole carbon source produced both extracellular and cell-bound polygalacturonase (PG), and cellulase (Cx), respectively. The cell-bound enzymes can be released to various extents by shaking the germinating propagules in solutions of NaCl, KCl, phosphate buffer, Na2EDTA (ethylenediaminetetraacetate), detergents such as Triton X-100 (octyl phenoxypolyethoxyethanol), Tween 80 (polyoxyethylene sorbitan monooleate), Celmusol, and distilled water. Sodium dodecyl sulfate (SDS) inactivated both PG and Cx but did no affect Cx activity in phosphate buffer solution. PG was more easily released by salts from the mycelium of R. solani than Cx. The release of both enzymes was a passive process and was not due to an osmotic effect. The amount of the cell-bound fraction was correlated with the total amount of the extracellular fraction rather than with the mycelial growth. At least one-third of the cell-bound fractions of both enzymes was found to be associated with the cell wall fraction of the mycelium.

Cell Wall↗

PubMed1975

Sequential production of polygalacturonase, cellulase, and pectin lyase by Rhizoctonia solani.

The sequence of appearance of cell wall degrading enzymes of Rhizoctonia solani propagules was followed. Polygalacturonase (PG; EC 3.2.1.15) was induced earlier by sodium polypectate (NaPP) as compared with the induction of cellulase (Cx; EC 3.2.1.4) by carboxymethyl cellulose (CMC), cellobiose, or fibrous cellulose powder. Increasing CMC concentration to 0.5% shortened the time of Cx appearance. In Czapek medium containing citrus pectin, pectin lyase (PL; EC 4.2.2.10) was produced faster and at higher amounts than in a medium containing NaPP as the sole carbon source. PG appearance also preceded that of PL in media simultaneously supplemented with their respective inducers. NaPP, which induced production of PG, repressed Cx production. Among the Cx inducers, only CMC and cellobiose repressed PG production to any extent. At pH 6.0, either in a synthetic medium or on autoclaved bean hypocotyl segments, a delay in PG production as compared with Cx and Pl production was observed. Optimal pH levels for enzyme production and activity were 4.0 and 5.0 for PG, and 5.5 for Cx, and 8.0 and 7.5 for PL. PG was less repressed than Cx by glucose, cellobiose, and monogalacturonic acid, while PL was not affected.

Carboxymethylcellulose Sodium↗

PubMed1994

Alkaline phosphatase and a cellulase reporter protein are not exported from the cytoplasm when fused to large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein.

Using a gene fusion approach, hybrid proteins were created by linking alkaline phosphatase (PhoA) or a cellulase reporter (delta CenA) to four large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein (RsaA; 1026 amino acids). Three of the sites (amino acids 189, 220, 315) were selected on the basis of TnphoA experiments that suggested the first 250-350 amino acids of RsaA could mediate export of PhoA from the cytoplasm while the fourth lay only 21 amino acids from the C-terminus. Expression of all fusions except rsaA(315):delta cenA and rsaA(315):phoA was toxic to C. crescentus. None of the gene fusions were toxic when expressed by Escherichia coli DH5 alpha, where all the hybrid proteins accumulated as inclusion bodies. The toxicity of hybrid proteins encoding 189, 220, and 1005 RsaA-derived amino acids was related to the nature of the hybrid protein itself because truncated RsaA peptides lacking their reporter domains were nontoxic. Further study of RsaA(delta C21) showed that this and presumably other truncated RsaA derivatives were neither secreted nor prone to intracellular accumulation. Although C. crescentus tolerated the expression of rsaA(315):delta cenA and rsaA(315):phoA, the encoded hybrid proteins were not exported in significant quantities from the cytoplasm. These results extend and confirm earlier work that large portions of the S-layer protein N-terminus cannot mediate export of passenger proteins from the cytoplasm and that the entire native S-layer protein may be required to properly interact with the RsaA secretion machinery.

Alkaline Phosphatase↗

PubMed2003

Expression and export of a Ruminococcus albus cellulase in Butyrivibrio fibrisolvens through the use of an alternative gene promoter and signal sequence.

Ruminococcal cellulase (Ruminococcus albus F-40 endoglucanase EgI) was successfully expressed in Butyrivibrio fibrisolvens OB156C, using the erm promoter from pAMbeta1. A newly identified signal peptide coding region of xynA from B. fibrisolvens 49 allowed efficient translocation of the foreign EgI into the extracellular fraction. First, B. fibrisolvens xynA with or without its own putative signal peptide (XynA SP) coding region was cloned into a shuttle vector to transform B. fibrisolvens OB156C. Both plasmids caused a 2- to 2.4-fold increase in xylanase activity. The transformant expressing XynA with the signal peptide showed a significantly higher proportion of activity in the extracellular fraction than the transformant with XynA lacking the signal peptide (75% vs. 19%), demonstrating the significance of XynA SP in the translocation of the expressed enzyme. Second, using the XynA SP coding region, secretion of EgI was attempted in B. fibrisolvens. Since the signal peptide of R. albus EgI did not function in B. fibrisolvens, it was replaced with the XynA SP. A high activity variant of EgI containing the XynA SP was transcribed using the erm promoter, resulting in a 27-fold increase in endoglucanase activity, most of which (>93%) was in the extracellular fraction of the B. fibrisolvens transformant. EgI without the XynA SP was scarcely detected in the extracellular fraction (<10%).

Bacterial Proteins↗

PubMed2004

Partial amino acid sequence of a cellulase-like component with IgE-binding properties from Stachybotrys chartarum.

BACKGROUND: The aim of this study was to characterize the amino acid sequence of a selected Stachybotrys chartarum component and to investigate human IgE reactivity against components of S. chartarum and nine other fungal species. METHODS: Human IgE reactivity against S. chartarum and nine other fungal extracts was investigated by the immunoblotting method. For automated amino acid sequencing analyses, the S. chartarum extract was purified by ion exchange chromatography prior to in-gel alkylation and digestion with modified trypsin. RESULTS: Human IgE reactivity was detected against eight components in the S. chartarum extract. Over 80% of the sera from the exposed subjects and less than 50% of the control sera recognized the 33-, 48- and 50-kD S. chartarum components. The human sera detected a 48- to 50-kD component from the extracts of eight fungal species. Nineteen peptide sequences were identified from the 48-kD component of S. chartarum. An analysis of the peptide sequences revealed homology with known fungal glycoside hydrolase enzymes (cellulases). CONCLUSIONS: The data showed human IgE reactivity against several S. chartarum components, including one at 48 kD. On the other hand, the human sera recognized 48- to 50-kD components from seven other fungal species, suggesting shared antigenic components (e.g. enolase) between the fungi. Thus, to our knowledge, this is the first antigen identified from S. chartarum.

Adult↗

PubMed1983

Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry.

Two components of the cellulase complex (E.C. 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level. Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae. Both enzymes were also present in the culture medium. Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium.

Cellulase↗

PubMed1992

Purification and characterization of an endoglucanase from the cellulosomes (multicomponent cellulase complexes) of Clostridium thermocellum.

A subunit with carboxymethyl cellulase (CMCase) activity was isolated from the cellulosomes of Clostridium thermocellum after dissociation of the cellulosomes by a mild sodium dodecyl sulfate (SDS) treatment. The subunit displayed only one protein band of 51 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but after boiling with SDS it had 3 bands of 60, 56, and 48 kDa. Prolonged incubation with SDS changed the subunit to display exclusively the 48-kDa band after boiling. The 51-kDa subunit was presumably a partially denatured form, and differentiated into 3 species with apparent M(r) of 60, 56, and 48 k through deglycosylation in SDS solution. Enzymatic properties of the 51-kDa subunit resembled those of the endoglucanase A which was purified from the culture fluid and from a E. coli clone with exceptions of temperature and pH optima.

Amino Acid Sequence↗

PubMed1994

Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Saccharomyces cerevisiae.

As a step to breed a Saccharomyces cerevisiae strain able to produce ethanol directly from cellulose, we combined cDNA for Aspergillus aculeatus FI-CMCase (FI-carboxymethyl cellulase) with the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae and used the resultant plasmid, pYEC91, to transform S. cerevisiae. The transformed cells produced active FI-CMCase within the cytoplasm. Western-blot analysis following SDS-polyacrylamide gel electrophoresis demonstrated that the cells contained a peptide having the same molecular mass and immunological identity as A. aculeatus FI-CMCase.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of a cellulase from the giant snail Achatina fulica.

A cellulase with activity as a cellobiohydrolase [EC3.2.1.91] was purified from the intestinal juice of the giant snail Achatina fulica. The enzyme (M(r) = about 23,000, and pI = about 5.3) also had a weak beta-glucosidase activity. The amino acid sequence of the N-terminal 20 amino acids was analyzed, and no similarity was noted for sequences of other known cellobiohydrolases.

Amino Acid Sequence↗

PubMed1997

Cloning, sequencing, and expression of a thermostable cellulase gene of Humicola grisea.

The egl2 gene coding a thermostable endoglucanase (EGL2) was cloned from Humicola grisea. The DNA sequence of egl2 predicted two putative introns in the coding region. The deduced amino acid sequence of EGL2 was 388 amino acids in length and showed 99.5% identity with the H. insolens CMC 3. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5' upstream region of the egl2 gene. The egl2 gene was expressed in Aspergillus oryzae, and EGL2 was purified. EGL2 produced by A. oryzae showed a high activity toward carboxymethyl cellulose. The optimal temperature of EGL2 was 75 degrees C, and EGL2 had more than 80% residual activity after heating up to 75 degrees C for 10 min. This is the first report of enzymatic properties of the EGL2-type thermostable cellulase homologs from Humicola.

Amino Acid Sequence↗

PubMed1999

Isolation and characterization of the actin gene from the cellulolytic fungus Humicola grisea and analysis of transcription levels of actin and cellulase genes.

An actin gene was isolated from the cellulolytic fungus Humicola grisea. The gene structure, which has 5 introns in the coding region, is similar to those of the so far cloned fungal actin genes. But there are some differences in intron sizes and codon usage. Transcription levels of actin and cellulase genes were also investigated.

5' Untranslated Regions↗

PubMed2000

Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an Alkaliphilic bacillus strain.

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.

Amino Acid Sequence↗

PubMed2003

Synthesis of glyceroyl beta-N-acetyllactosaminide and its derivatives through a condensation reaction by cellulase.

A condensation reaction between N-acetyllactosamine and glycerol was directly catalyzed by using a commercially available cellulase preparation from Trichoderma reesei. 1-O-beta-N-Acetyllactosaminyl-(R, S)-glycerols (1) were readily synthesized in a 5% yield based on the N-acetyllactosamine added and conveniently isolated by two-step column chromatographies. The use of a partially purified enzyme increased 2.3-fold the yield of 1, compared to that of the crude enzyme containing beta-D-galactosidase activity. When various alkanols (n:2-4) were used in the condensation reaction, the corresponding alkyl beta-N-acetyllactosaminides were obtained in yields of 0.3-1.1% of the desired compounds.

Cellulase↗

PubMed2005

Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs.

Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-beta-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5-96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-beta-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20-30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.

Amino Acid Sequence↗