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Biomedical subjects

P L Bergquist

Publications and source records attributed to P L Bergquist.

At least 19 recordsLinked to original sources
PubMed2006

Applying fluorescence based technology to the recovery and isolation of Cryptosporidium and Giardia from industrial wastewater streams.

As increasing water shortages continue, water re-use is posing new challenges with treated wastewater becoming a significant source of non-potable water. Rapid detection strategies that target waterborne pathogens of concern to industry are gaining importance in the assessment of water quality. This study reports on the ability to recover spiked Cryptosporidium and Giardia from a variety of industrial wastewater streams of varied water quality. Incorporation of an internal quality control used commonly in finished water-enabled quantitative assessments of pathogen loads and we describe successful analysis of pre- and part-treated wastewater samples from four industrial sites. The method used combined calcium carbonate flocculation followed by flow cytometry and epifluorescence microscopy. Our focus will now aim at characterising the ambient parasites isolated from industrial wastewater with the objective of developing a suite of highly specific platform detection technologies targeted to industrial needs.

Animals↗
PubMed2004

Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 ( cbh1 ) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.

Biochemistry↗
PubMed2004

Paenibacillus isolates possess diverse dextran-degrading enzymes.

AIMS: To isolate and identify dextran-degrading organisms from sugar mill and compost samples, and to examine the diversity of the dextranolytic enzymes produced. METHODS AND RESULTS: Fifteen dextranolytic prokaryotes were purified at various temperatures from sugar-mill or compost samples, using indicator plates containing blue dextran. A 16S rRNA gene sequence analysis showed that 12 isolates purified at 40, 50 or 70 degrees C were closely aligned to Paenibacillus spp. The three isolates purified at 60 degrees C had identical 16S rDNA sequences, with highest affinity to Bacillus spp. Liquid culture of the 11 isolates purified at 40 or 50 degrees C produced dextranolytic activity in the spent media with maximal activity at 40 or 45 degrees C under the assay conditions used. Hydrolysis of blue dextran in activity gels showed that the 12 Paenibacillus isolates produced from one to five dextranolytic proteins, ranging from 70 to 120 kDa. Based on 16S rDNA sequence, growth habit in liquid culture and dextranolytic enzyme pattern, the 12 Paenibacillus-like isolates could be differentiated into six distinct groups, one of which was capable of growth at 70 degrees C. CONCLUSIONS: The Bacillales, especially the Paenibacillus, are a valuable environmental repository for dextranolytic enzymes of diverse size and potentially diverse activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Dextranolytic enzymes produced by Paenibacillus spp. are an exploitable resource for those interested in modifying the structure of dextrans.

Bacillus↗
PubMed2002

Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system.

Effective biolistic transformation of intact conidia from the filamentous fungus Trichoderma reesei was achieved using the Bio-Rad Hepta Adaptor system with seven barrels for particle launch. Transformation frequencies of up to 39 colonies per microg of circular DNA and 37 colonies per microg of linear DNA were obtained at an optimal target distance of 3 cm and a helium pressure of 1350 psi. These values are about 3.5- to 6-fold higher than transformant yields reported earlier for T. reesei using the hygromycin phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin B as a selectable marker in combination with the PDS-1000/He single barrel system. High mitotic stability of the transformants (98-100%) was demonstrated. The Hepta Adaptor device allowing bombardment of seven lots of conidia in a single plate offers clear advantage in terms of transformant numbers over the single barrel system where target cells are restricted to the center of the plate.

Cellulase↗
PubMed2002

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei.

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.

Ascomycota↗
PubMed2001

Identification of novel beta-mannan- and beta-glucan-binding modules: evidence for a superfamily of carbohydrate-binding modules.

Many glycoside hydrolases, which degrade long-chain carbohydrate polymers, possess distinct catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). On the basis of conserved protein secondary structure, we describe here the identification and experimental characterization of novel type of mannanase-associated mannan-binding module and also characterization of two CBM family 4 laminarinase-associated beta-glucan-binding modules. These modules are predicted to belong to a superfamily of CBMs which include families 4, 16, 17, 22 and a proposed new family, family 27.

Amino Acid Sequence↗
PubMed2001

Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G+C contents.

Amino Acid Sequence↗
PubMed2000

Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei.

The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei. The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T. reesei vectors. The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan. Effective transcription of xyn B in T. reesei was obtained after changing 20 codons.

Amino Acid Sequence↗
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

Rapid transformation of high cellulase-producing mutant strains of Trichoderma reesei by microprojectile bombardment.

Intact conidia of three industrially relevant strains of Trichoderma reesei were effectively transformed by particle bombardment. Transformations were carried out individually with plasmids carrying either the fungal amdS or bacterial hph gene as a selectable marker and by cotransformation with both plasmids. Transformant yields with single plasmids were up to 11 stable transformants per microg DNA at the bombardment distance of 6 cm. Mitotic stability of the transformants was 75-100% and the cotransformation efficiency averaged 92% when the first selection was performed on hygromycin B plates. The entire procedure could be completed in 1 week with the hph marker.

Amidohydrolases↗
PubMed2000

Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 A resolution.

Microorganisms employ a large array of enzymes to break down the cellulose and hemicelluloses of plant biomass. These enzymes, especially those with high thermal stability, have many uses in biotechnology. We have solved the crystal structure of a beta-1, 4-xylanase, XynB, from the extremely thermophilic bacterium Dictyoglomus thermophilum, isolate Rt46B.1. The protein crystallized from 1.6 M ammonium sulfate, 0.2 M HEPES pH 7.2 and 10% glycerol, with unit-cell parameters a = b = 91.3, c = 44.9 A and space group P4(3). The structure was solved at high resolution (1.8 A) by X-ray crystallography, using the method of isomorphous replacement with a single mercury derivative, and refined to a final R factor of 18.3% (R(free) = 22.1%). XynB has the single-domain fold typical of family 11 xylanases, comprising a jelly roll of two highly twisted beta-sheets that create a deep substrate-binding cleft. The two catalytic residues, Glu90 and Glu180, occupy this cleft. Compared with other family 11 xylanases, XynB has a greater proportion of polar surface and has a slightly extended C-terminus that, combined with the extension of beta-strand A5, gives additional hydrogen bonding and hydrophobic packing. These factors may account for the enhanced thermal stability of the enzyme.

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↗