Structure of a Bacillus subtilis endo-beta-1,4-glucanase gene.

The nucleotide sequence of the portion of a Bacillus subtilis (strain PAP115) 3 kb Pst I fragment which contains an endo-beta-1, 4-glucanase gene has been determined. This gene encodes a protein of 499 amino acid residues (Mr = 55,234) with a typical B. subtilis signal peptide. Escherichia coli which has been transformed with this gene produces an extracellular endoglucanase with an amino-terminus corresponding to the thirtieth encoded amino acid residue. The gene is preceded by a cryptic reading frame with a rho-independent terminator structure, and itself has such a structure in the immediate 3'-flanking region. We have also identified, in the 5'-flanking region, nucleotide sequences which resemble promoter elements recognized by Bacillus RNA polymerase E sigma 43. Comparison of the encoded amino acid sequence to other known beta-glucanases reveals a small region of similarity to the encoded protein of the Clostridium thermocellum celB gene. These similar regions may contain substrate-binding and/or catalytic sites.     

Bipartite organization of the Bacillus subtilis endo-beta-1,4-glucanase revealed by C-terminal mutations.

The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-beta-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate. CD spectra indicated that the bulk of the alpha-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and approximately 4- to 5-fold higher with carboxymethylcellulose (soluble cellulose). These results along with data which show that EG470 binding capacity to microcrystalline cellulose is approximately 11 times more than that of EG300, demonstrate the importance of residues 330-499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330-499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine-326 and serine-321, respectively. Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.     

celA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-beta-1,4-glucanase.

celA from the cellulolytic bacterium Bacillus lautus PL236 encodes EG-A, an endo-beta-1,4-glucanase. An open reading frame of 2,100 bp preceded by a ribosome-binding site encodes a protein with a molecular mass of 76,863 Da with a typical signal sequence. The NH2-terminal active domain of EG-A is not homologous to any reported cellulase or xylanase and may represent a new family of such enzymes. A 150-amino-acid COOH-terminal peptide is homologous to noncatalytic domains in several other cellulases (A. Meinke, N.R. Gilkes, D.G. Kilburn, R.C. Miller, Jr., and R.A.J. Warren, J. Bacteriol. 173:7126-7135, 1991). Upstream of celA, a partial open reading frame encodes a 145-amino-acid peptide which also belongs to the family mentioned. Zymogram analysis of extracts from Escherichia coli and supernatants of Bacillus subtilis and B. megaterium, including protease-deficient mutants thereof, which express celA, revealed two active proteins, EG-A-L and EG-A-S, with Mrs of 74,000 and 57,000, respectively. The proportion of EG-A-L to EG-A-S depends on the extracellular proteolytic activity of the host organism, indicating that EG-A-S arises from posttranslational proteolytic modification of EG-A-L. Since EG-A-S has an NH2 terminus corresponding to the predicted NH2-terminal sequence of EG-A, processing appears to take place between the catalytic and noncatalytic domains described. EG-A-L and EG-A-S were purified to homogeneity and shown to have almost identical characteristics with respect to activity against soluble substrates and pH and temperature dependency. EG-A-L binds strongly to cellulose, in contrast to EG-A-S, and has higher activity against insoluble substrates than the latter. We conclude that the COOH-terminal 17,000-Mr peptide of EG-A-L constitutes a cellulose-binding domain.     

Structural characterization of the Acetobacter xylinum endo-beta-1,4-glucanase CMCax required for cellulose biosynthesis

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis-related endo-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A resolution. CMCax falls into the glycoside hydrolase family 8 (GH-8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH-8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH-8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing-end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane-anchored endoglucanases that are required for cellulose biosynthesis.     

Stability of the endo-beta-1,4-glucanase and beta-1,4-glucosidase from Bacteroides succinogenes

The endo-beta-1,4-glucanase (carboxymethylcellulase) activity in cell extracts prepared from Bacteroides succinogenes S85 was almost unaffected by prolonged incubation at 39 degrees C in the presence of merthiolate, a sulfhydryl inhibitor. The beta-1,4-glucosidase (cellobiase) activity, however, was rapidly inactivated by the same treatment. The cellobiase was also inactivated by exposure to air, but was stabilized by dithiothreitol in a nitrogen atmosphere. These results suggest that the cellobiase required reduced sulfhydryl groups for activity.     

Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans.

Endo-beta-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subtilis PAP115 endo-beta-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, S1 nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ alpha-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum celB endoglucanase sequence but very little similarity to endoglucanases from other Bacillus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding.     

Structure of an endo-beta-1,4-glucanase gene from Clostridium acetobutylicum P262 showing homology with endoglucanase genes from Bacillus spp

The nucleotide sequence of an endo-beta-1,4-glucanase gene of Clostridium acetobutylicum contained two putative extended promoter consensus sequences, a Shine-Dalgarno sequence and a TTG initiation codon. The nucleotide sequence of the gene coding for the C-terminal region of this enzyme was not required for activity. Extensive homology in the nucleotide and amino acid sequences of the endoglucanase genes from C. acetobutylicum and Bacillus spp. was demonstrated.     

Structure of an endo-beta-1,4-glucanase gene from Clostridium acetobutylicum P262 showing homology with endoglucanase genes from Bacillus spp.

The nucleotide sequence of an endo-beta-1,4-glucanase gene of Clostridium acetobutylicum contained two putative extended promoter consensus sequences, a Shine-Dalgarno sequence and a TTG initiation codon. The nucleotide sequence of the gene coding for the C-terminal region of this enzyme was not required for activity. Extensive homology in the nucleotide and amino acid sequences of the endoglucanase genes from C. acetobutylicum and Bacillus spp. was demonstrated.     

Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.     

Mapping of the gene for endo-β-1,3-1,4-glucanase of Bacillus subtilis

Abstract An integrating plasmid has been used to mutagenise the gene coding for endo-β-1,3-1,4-glucanase of Bacillus subtilis . The gene, named bgl , has been mapped by PBS-1 transduction to the sacA-pureA region of the B. subtilis chromosome and is closely linked to the hutP 1 locus. The order of markers in this region is sacA 321- thiC 5- bgl - hutP 1- purA 16.     

Cloning and sequencing of a molluscan endo-beta-1,4-glucanase gene from the blue mussel, Mytilus edulis.

Using polymerase chain reaction, cloning and sequencing techniques, a complementary DNA encoding a low molecular mass cellulase (endo-1,4-beta-D-glucanase, EC 3.2.1.4) has been identified in the digestive gland of the marine mussel, Mytilus edulis. It contains a 5' untranslated region, a 633-nucleotide ORF encoding a 211 amino-acid protein, including a 17 amino-acid signal peptide and a complete 3' untranslated region. At the C-terminal end of the purified mature protein, a 13 amino-acid peptide is lacking in comparison to the protein sequence deduced from the ORF. This peptide is probably removed as a consequence of post-translational amidation of the C-terminal glutamine. The endoglucanase genes have been isolated and sequenced from both Swedish and French mussels. The coding parts of these two sequences are identical. Both genes contain two introns, the positions of which are conserved. However the length of the introns are different due to base substitutions, insertions or deletions showing the existence of interspecies length polymorphism. The percentage of similarity for the introns of the two gene sequences is 96.9%. This is the first time a molluscan cellulase is characterized at DNA level. Amino acid sequence-based classification has revealed that the enzyme belongs to the glycosyl hydrolase family 45 [B. Henrissat (Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France), personal communication]. There is no cellulose binding domain associated with the sequence.     

Molecular cloning,expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1

An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively. A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.     

Towards understanding the role of membrane-bound endo-beta-1,4-glucanases in cellulose biosynthesis

Recent studies have highlighted the involvement of membrane-anchored endo-beta-1,4-glucanases in cellulose biosynthesis in plants, suggesting that there are parallels with Agrobacterium tumefaciens and other bacteria which also require endo-beta-1,4-glucanases for cellulose synthesis. This review summarises recent literature on endo-beta-1,4-glucanases and their role in plant development and addresses the possible functions of membrane-anchored isoforms in the synthesis of cellulose.     

Distribution and properties of endo-beta-1,4-glucanase from a lower termite, Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-beta-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract. EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and Km were 48 kDa, 6.0, 50 degrees C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-beta-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-beta-1,4-glucanases of family 9.     

Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis.

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Secretion and processing of the Bacillus subtilis endo-β-1,3-1,4-glucanase in Escherichia coli

The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium. The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme. Lysis of E. coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein. However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E. coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane. The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B. subtilis. Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E. coli is processed at a site two amino acids distant from the processing site used in B. subtilis.