Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.     

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.     

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.     

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.     

Cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium xylanase C in Escherichia coli.

The nucleotide sequence of the Clostridium stercorarium F-9 xynC gene, encoding a xylanase XynC, consists of 3,093 bp and encodes a 1,031-amino acids with a molecular weight of 115,322. XynC is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family IX cellulose-binding domain, and two S-layer homologous domains. Immunological analysis indicated the presence of XynC in the culture supernatant of C. stercorarium F-9 and in the cells, most likely on the cell surface. XynC purified from a recombinant E. coli was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethylcellulose. XynC hydrolyzed xylan and xylooligosaccharides larger than xylotriose to produce xylose and xylobiose. This enzyme was optimally active at 85 degrees C and was stable up to 75 degrees C at pH 5.0 and over the pH range of 4 to 7 at 25 degrees C.     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.     

Molecular cloning and expression of a novel family A endoglucanase gene from Fibrobacter succinogenes S85 in Escherichia coli

A Fibrobacter succinogenes S85 gene that encodes endoglucanase hydrolysing CMC and xylan was cloned and expressed in Escherichia coli DH5 by using pUC19 vector. Recombinant plasmid DNA from a positive clone hydrolysing CMC and xylan was designated as pCMX1, harboring 2,043 bp insert. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The nucleotide sequence accession number of the cloned gene sequence in Genbank is U94826. The endoglucanase gene cloned in this study does not have amino sequence homology to the other endoglucanase genes from F. succinogenes S85, but does show sequence homology to family 5 (family A) of glycosyl hydrolases from several species. The ORF encodes a polypeptide of 654 amino acids with a measured molecular weight of 81.3 kDa on SDS-PAGE. Putative signal sequences, Shine-Dalgarno-type ribosomal binding site and promoter sequences (-10) related to the consensus promoter sequences were deduced. The recombinant endoglucanase by E. coli harboring pCMX1 was partially purified and characterized. N-terminal sequences of endoglucanase were Ala-Gln-Pro-Ala-Ala, matched with deduced amino sequences. The temperature range and pH for optimal activity of the purified enzyme were 55 approximately 65 degrees C and 5.5, respectively. The enzyme was most stable at pH 6 but unstable under pH 4 with a K(m) value of 0.49% CMC and a V(max) value of 152 U/mg.     

Xylanase and acetyl xylan esterase activities of XynA,a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation

The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.     

Cloning, characterization, and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli

Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.     

Structure and expression of genes coding for xylan-degrading enzymes of Bacillus pumilus

The complete nucleotide sequence of the beta-xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617-bp open reading frame for beta-xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N-terminal region and the molecular mass 62607 Da) of the beta-xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine-Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG. The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3' end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197-201]. The results of the Northern hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5' and 3' ends of the xynA and xynB gene were mapped with nuclease S1. The '-10' regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the '-35' regions were different from all the known promoters for B. subtilis RNA polymerases.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.     

Overproduction and characterization of xylanase B from Aspergillus niger

The xynB gene, which encodes endo-beta-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg x L(-1). The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 degrees C with stability in the pH range 4.0-7.0 and temperature up to 50 degrees C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg x mL(-1) and 3881 U x mg(-1), respectively.     

Properties of exgS,a gene for a major subunit of the Clostridium cellulovorans cellulosome

The nucleotide sequence of P70, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been determined. The gene designated as exgS (Genbank Accession No. U34793) consists of 2112 bp and encodes a protein containing 703 amino acids with a molecular mass of 77.7 kDa. ExgS has a putative signal peptide sequence of 32 amino acids. The N-terminal region is separated from the C-terminal region by a short Pro–Thr–Pro linker. The C-terminal region of ExgS contains a duplicated sequence (DS), each sequence consisting of 22 amino acids. exgS, located 67 bp downstream of cbpA in the chromosome, is immediately upstream of a gene encoding a family 9 type endoglucanase that we have designated as EngH. This gene cluster to date consists of regA–cbpA–exgS–engH. Recombinant ExgS (rExgS) containing no signal peptide was expressed in E. coli. The rExgS actively digested several forms of cellulose, including Avicel, Sigmacell101, crystalline cellulose, and xylan, but not carboxymethyl cellulose (CMC). Cellotetraose was the smallest oligosaccharide substrate for rExgS. The enzymatic studies indicated that ExgS was an exoglucanase and had some properties similar to that of CelS from C. thermocellum and CelF from C. cellulolyticum. An exoglucanase has now been found to be a component of the C. cellulovorans cellulosome as well as the previously reported endoglucanases.     

Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.     

Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan

A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.     

Characterization of an endoglucanase belonging to a new subfamily of glycoside hydrolase family 45 of the basidiomycete Phanerochaete chrysosporium

The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley beta-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.     

Sequencing and Expression of Additional Xylanase Genes from the Hyperthermophile Thermotoga maritima FjSS3B.1

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.     

Cloning of the xynB Gene from Dictyoglomus thermophilum Rt46B.1 and Action of the Gene Product on Kraft Pulp

A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from Dictyoglomus thermophilum Rt46B.1. Family 11 xylanase consensus fragments (GXCFs) were amplified from Rt46B.1 genomic DNA by using different sets of consensus PCR primers that exhibited broad specificity for conserved motifs within fungal and/or bacterial family 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (xynB) was identified. The entire gene sequence was obtained in the second step by using genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstream and downstream of the xynB GXCF region. The putative XynB peptide (M_r, 39,800) encoded by the Rt46B.1 xynB open reading frame was a multidomain enzyme comprising an N-terminal catalytic domain (M_r, 22,000) and a possible C-terminal substrate-binding domain (M_r, 13,000) that were separated by a short serine-glycine-rich 23-amino-acid linker peptide. Seven xylanases which differed at their N and C termini were produced from different xynB expression plasmids. All seven xylanases exhibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85°C. Pretreatment of Pinus radiata and eucalypt kraft-oxygen pulps with XynB resulted in moderate xylan solubilization and a substantial improvement in the bleachability of these pulps.