Biomedical subjects

M D Gibbs

Publications and source records attributed to M D Gibbs.

16 recordsLinked to original sources

PubMed2000

The thermostabilizing domain, XynA, of Caldibacillus cellulovorans xylanase is a xylan binding domain.

We show that the N-terminal 'thermostabilizing domain' (TSD) of the xylanase, XynA, from the thermophilic bacterium Caldibacillus cellulovorans also acts as a xylan binding domain. Affinity electrophoresis experiments show that this TSD selectively binds soluble xylan and binds weakly to hydroxyethylcellulose. Based on this, and previously reported evidence, we propose that xylanase-associated TSDs are xylan binding domains.

Amino Acid Sequence↗

PubMed2000

Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile Caldicellulosiruptor isolate Tok7B.1.

DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases. We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clusters are not closely linked, and each is different in its organization from any described for Cs. saccharolyticus. The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types IIIb, IIIc, or VI. A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE.

Amino Acid Sequence↗

PubMed2000

A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp.

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.

Amino Acid Sequence↗

PubMed2000

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.

Bacteria↗

PubMed1999

Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme.

A beta-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of beta-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80 degrees C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80 degrees C over 16 h, while at 90 degrees C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and mannotriose, confirming that ManA is an endo-acting beta-mannanase. Sequence comparisons with related beta-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes.

Amino Acid Sequence↗

PubMed1999

Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. strain Rt69B.1.

Three family 10 xylanase genes (xynA, xynB, and xynC) and a single family 11 xylanase gene (xynD) were identified from the extreme thermophile Caldicellulosiruptor strain Rt69B.1 through the use of consensus PCR in conjunction with sequencing and polyacrylamide gel electrophoresis. These genes appear to comprise the complete endoxylanase system of Rt69B.1. The xynA gene was found to be homologous to the xynA gene of the closely related Caldicellulosiruptor strain Rt8B.4, and primers designed previously to amplify the Rt8B.4 xynA gene could amplify homologous full-length xynA gene fragments from Rt69B.1. The complete nucleotide sequences of the Rt69B.1 xynB, xynC, and xynD genes were obtained using genomic walking PCR. The full-length xynB and xynC genes are more than 5 kb in length and encode highly modular enzymes that are the largest xylanases reported to date. XynB has an architecture similar to the family 10 xylanases from Thermoanaerobacterium saccharolyticum (XynA) and Clostridium thermocellum (XynX) and may be cell wall associated, while XynC is a bifunctional enzyme with an architecture similar to the bifunctional beta-glycanases from Caldicellulosiroptor saccharolyticus. The xynD gene encodes a two-domain family 11 xylanase that is identical in architecture to the XynB family 11 xylanase from the unrelated extreme thermophile Dictyoglomus thermophilum strain Rt46B.1. The sequence similarities between the Rt69B.1 xylanases with respect to their evolution are discussed.

Bacteria, Anaerobic↗

PubMed1998

Expression and secretion of a xylanase from the extreme thermophile, thermotoga strain FjSS3B.1, in Kluyveromyces lactis.

The yeast Kluyveromyces lactis has been developed as a host for extracellular production of thermophilic hemicellulases by employing expression vectors based on the 2 mu-like plasmid pKD1 of Kluyveromyces drosophilarium. A beta-1,4-xylanase gene (xynA) from the extreme thermophile Thermotoga sp. strain FjSS3B.1 was fused inframe with a synthetic secretion signal derived from the K. lactis killer toxin and expressed under control of the K. lactis LAC4 (beta-galactosidase) promoter. Correctly processed xylanase enzyme with full biological activity on oat spelts xylan was secreted during shake-flask cultivation of K. lactis transformants. The transcriptional activity of the LAC4 promoter dramatically affected mitotic stability of the expression vector under nonselective conditions. However, one combination of host strain and expression plasmid showed higher stability and good yield and has been employed for scaled-up production of XynA and other thermostable hemicellulases in chemostat culture. XynA secreted by K. lactis is as thermostable as the native enzyme, having a half-life of 48 h at 90 degrees C.

Amino Acid Sequence↗

PubMed1998

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 degrees 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.

Amino Acid Sequence↗

PubMed1997

Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus.

A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95,732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5'-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.

Amino Acid Sequence↗

PubMed1996

Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4.

A gene encoding a beta-mannanase (manA) has been cloned from an obligately anaerobic extreme thermophile, Caldicellulosiruptor strain Rt8B.4, which is most closely related to Caldicellulosiruptor saccharolyticus (formerly Caldocellum saccharolyticum). The gene codes for a multidomain enzyme with a C-terminal beta-mannanase domain which was amplified by the polymerase chain reaction and cloned into a temperature-inducible expression vector in Escherichia coli. Sequence comparisons have shown that the Man domain of Rt8B.4 ManA is related to a thermophilic Dictyoglomus mannanase and a mesophilic mannanase from a Bacillus species. It appears to be unrelated to the beta-mannanase domain of C. saccharolyticus, implying acquisition of the genes from unrelated sources by the two bacteria.

Amino Acid Sequence↗

PubMed1996

Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor.

A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in lambda ZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70 degrees C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.

Amino Acid Sequence↗

PubMed1995

Sequence and expression of a xylanase gene from the hyperthermophile Thermotoga sp. strain FjSS3-B.1 and characterization of the recombinant enzyme and its activity on kraft pulp.

A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp.

Amino Acid Sequence↗

PubMed1995

Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate.

A genomic library of the Dictyoglomus sp. strain Rt46B.1 was constructed in the phage vector lambda ZapII and screened for xylanase activity. A plaque expressing xylanase activity, designated B6-77, was isolated and shown to contain a genomic insert of 5.3 kb. Subcloning revealed that the xylanase activity was restricted to a internal 1,507-bp PstI-HindIII fragment which was subsequently sequenced and shown to contain a single complete open reading frame coding for a single-domain xylanase, XynA, with a putative length of 352 amino acids. Homology comparisons show that XynA is related to the family F group of xylanases. The temperature and pH optima of the recombinant enzyme were determined to be 85 degrees C and pH 6.5, respectively. However, the enzyme was active across a broad pH range, with over 50% activity between pH 5.5 and 9.5. XynA was shown to be a true endo-acting xylanase, being capable of hydrolyzing xylan to xylotriose and xylobiose, but it could not hydrolyze xylobiose to monomeric xylose. XynA was also shown to hydrolyze xylan present in Pinus radiata kraft pulp, indicating that it may be of use as an aid in pulp bleaching. The equivalent xylanase gene was also isolated from the related bacterium Dictyoglomus thermophilum, and DNA sequencing showed these genes to be identical, which, together with the 16S small-subunit rRNA gene sequencing data, indicates that Rt46B.1 and D. thermophilum are very closely related.

Amino Acid Sequence↗

PubMed1995

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.

The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.

Bacterial Proteins↗

PubMed1993

RepFIB: a basic replicon of large plasmids.

The distribution of the RepFIB replicon among a total of 20 plasmid incompatibility groups was determined using Southern blot and polymerase chain reaction analysis. The presence of the replicon was confirmed in 25 of 55 plasmids tested. The majority of plasmids carrying RepFIB are from the IncF incompatibility groups, but one plasmid from IncI and one plasmid from IncP also possess the replicon. Seven different examples of RepFIB were sequenced in the minimal replicon region to obtain 1525 bp of sequence information covering the repA gene and flanking repeat regions for comparison. An analysis of these sequences plus three sequences previously reported showed almost perfect conservation of the predicted protein sequence of RepA and of the flanking DNA repeats. DNA sequence data were analyzed using maximum parsimony techniques to describe the possible evolutionary relationships of the 10 examples of RepFIB.

Amino Acid Sequence↗

PubMed1992

The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.

Amino Acid Sequence↗