Biomedical subjects

H Dalbøge

Publications and source records attributed to H Dalbøge.

At least 19 recordsLinked to original sources

PubMed2001

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco.

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

Amino Acid Sequence↗

PubMed1999

Crystallization and preliminary X-ray studies of beta-1, 4-galactanase from Aspergillus aculeatus.

Recombinant beta-1,4-galactanase from Aspergillus aculeatus has been crystallized and characterized by X-ray diffraction. Crystals were obtained in hanging drops by vapour-diffusion under the conditions 30% PEG 400, 0.2 M CaCl2 and 0.1 M Na HEPES buffered to pH 7.5. The crystals diffract to 2.3 A resolution and belong to one of the orthorhombic space groups I222 or I212121. The unit-cell dimensions are a = 60.42, b = 88.94 and c = 129.08 A. With one molecule in the asymmetric unit, the corresponding solvent content is approximately 48%.

Aspergillus↗

PubMed1998

Crystallization and preliminary X-ray diffraction studies of the heterogeneously glycosylated enzyme rhamnogalacturonan acetylesterase from Aspergillus aculeatus.

Well diffracting crystals of rhamnogalacturonan acetylesterase from Aspergillus aculeatus have been obtained in two polymorphic modifications despite its heterogeneous glycosylation. The best-diffracting crystals (resolution 1.55 A) are orthorhombic. The limit of the diffraction pattern of the other (trigonal) form is 2.5 A. The ability of the enzyme to crystallize appears to depend on the glycosylation of the protein sample. This aspect has been investigated by mass spectrometry, which also showed that the orthorhombic crystals have the same glycosylation as the protein sample used in the crystallization.

Acetylesterase↗

PubMed1998

Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

Base Sequence↗

PubMed1998

Using molecular techniques to identify new microbial biocatalysts.

Evolution has favoured microorganisms that produce efficient enzymes with substrate-adapted biocatalytic activities. Progress in molecular techniques, especially expression cloning, molecular screening, protein engineering and in vivo and in vitro shuffling, have paved the way for greater speed and accuracy in cloning enzyme genes from microorganisms and generating versions with improved properties. Recently, two new approaches have been added: screening directly from uncultivated microorganisms and generating additional hits by database mining using bioinformatic tools.

Amino Acid Sequence↗

PubMed1997

X-ray structure of the 154-amino-acid form of recombinant human basic fibroblast growth factor. comparison with the truncated 146-amino-acid form.

The crystal structure of the 154-amino-acid form of human basic fibroblast growth factor (hbFGF154), probably representing the intact form of hbFGF as deduced from the open reading frame of hbFGF cDNA, was determined by X-ray crystallography and refined to a crystallographic residual of 19.0% for all data between 20.0 and 2.0 A resolution. Crystals were obtained from recombinant hbFGF154 expressed in E. coli. hbFGF154 has the same overall structure as the N-terminus truncated 146-amino-acid form. The structure has a Kunitz-type fold and is built of 12 beta-strands of which six antiparallel strands form a beta-sheet barrel. In the structure it was possible to locate two additional residues at the N terminus and the last three C-terminal amino-acid residues, which seem to be disordered in all but one of the reported structures of the truncated form of hbFGF. The C-terminal amino-acid residues are part of the last beta-strand through the formation of a hydrogen bond between the main-chain amide group of Ala152 and the carbonyl O atom of Pro28. An apparent phosphate ion is bound within the basic region on the surface of the molecule and has as ligands the side chains of Asn35, Arg128 and Lys133 and two water molecules. A slightly different hydrogen-bonding pattern to the phosphate ion is observed as compared with the sulfate ions in the truncated forms [Eriksson, Cousens & Matthews (1993). Protein Sci. 2, 1274-1284; Zhang, Cousens, Barr & Sprang (1991). Proc. Natl Acad. Sci. USA, 88, 3446-3450]. One molecule of beta-mercaptoethanol forms a disulfide bridge to Cys77.

Journal Article↗

PubMed1997

Expression cloning of fungal enzyme genes; a novel approach for efficient isolation of enzyme genes of industrial relevance.

Expression cloning is a relatively new method for fast and efficient cloning of enzyme genes from fungi that are known to make complex enzyme mixtures. In contrast to traditional cloning methods that are usually dependent on knowledge of at least a partial amino acid sequence in order to synthesize appropriate DNA probes or primers, the expression cloning method solely relies on access to reliable and sensitive enzyme assays. A representative expression cDNA library is made in Saccharomyces cerevisiae form the donor strain and relevant cDNA clones are detected directly based on the encoded enzyme activity. Thus, time-consuming enzyme purification and characterization steps are avoided. The method has been applied on the characterization of extracellular enzyme genes from the filamentous fungus Aspergillus aculeatus and has resulted in the isolation of 20 different enzyme genes such as endo-glucanases, xylanases, pectinases, proteases, hemicellulases and rhamnogalacturonan-degrading enzymes. All enzymes have been expressed in Aspergillus oryzae, purified and characterized. In the present review a description of the expression cloning technique will be given as well as examples of how the technique has been used in the exploration and characterization of a commercial enzyme product that is known to consist of a complex mixture of more than 25 different enzyme activities.

Aspergillus↗

PubMed1996

Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

Amino Acid Sequence↗

PubMed1996

Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae.

A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENCI) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENCI amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 degrees C.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. Synergism between rhamnogalacturonan degrading enzymes.

A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.

Acetylesterase↗

PubMed1995

Expression cloning, purification and characterization of a beta-1,4-galactanase from Aspergillus aculeatus.

Expression cloning has been used to isolate a cDNA encoding beta-1,4-galactanase from the filamentous fungus Aspergillus aculeatus. A cDNA library was prepared from mycelia, inserted in a yeast expression vector and transformed into Saccharomyces cerevisiae. Thirteen clones secreting galactanase activity were identified from a screening of approximately 2.5 x 10(4) yeast colonies. All clones expressed transcripts of the same galactanase gene. The cDNA was re-cloned in an Aspergillus expression vector and transformed into Aspergillus oryzae. The recombinant enzyme had a molecular weight of 44,000 Da, an isoelectric point of pH 2.85, a pH optimum of pH 4.0-4.5, and a temperature optimum of 45-65 degrees C, which is similar to values obtained for a beta-1,4-galactanase purified from A. aculeatus. The enzyme degraded unsubstituted galactan to galactose and galactobiose. The deduced primary sequence of the enzyme showed no apparent homology to any known enzyme, in accordance with this being the first reported beta-1,4-galactanase cDNA. However, the deduced amino-acid sequence of a Bacillus circulans DNA sequence containing an open reading frame (ORF) with no known function, showed 36% identity and 60% similarity to the galactanase amino-acid sequence.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and expression in S. cerevisiae of two exochitinases from Trichoderma harzianum.

The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast. The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots. The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide. The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase. The substrate specificity of the recombinant enzymes expressed in S. cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus.

Two rhamnogalacturonases from the filamentous fungus Aspergillus aculeatus have been cloned and characterized. A cDNA library from A. aculeatus was constructed, and a novel rhamnogalacturonase B was isolated by expression cloning in yeast. For this purpose a new plate screening assay was developed, specific for the detection of rhamnogalacturonase activity. The rhamnogalacturonase A, known from previous reports, was shown not to be expressed in yeast in an active form. Therefore, rhamnogalacturonase A was purified, peptide sequences were obtained, and full-length cDNAs encoding the enzyme were isolated using a polymerase chain reaction-generated product as a probe. Comparison of the deduced primary structures indicates that the two rhamnogalacturonases are structurally different. This is further supported by the finding that polyclonal antibodies raised against native rhamnogalacturonase A do not cross-react with rhamnogalacturonase B. The cloned genes were transformed into Aspergillus oryzae for high level expression. The recombinant enzymes were purified and characterized, revealing significant differences in glycosylation pattern and substrate specificity as well as in pH and temperature optima and stability. Data from the hydrolysis of apple rhamnogalacturonan with the recombinant rhamnogalacturonases suggest that the two enzymes exert their action at different sites in the backbone.

Amino Acid Sequence↗

PubMed1994

NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

Amino Acid Sequence↗

PubMed1994

A novel method for efficient expression cloning of fungal enzyme genes.

We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays. A cDNA library from the fungus Humicola insolens was constructed in a S. cerevisiae/Escherichia coli shuttle vector in E. coli. Sub-pools of the library were subsequently screened for enzyme activity in S. cerevisiae. More than 130 clones were identified as positive in either an endo-beta-glucanase or an endo-xylanase assay. Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo-beta-glucanases and three types of endo-xylanases. A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A. oryzae. The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.

Amino Acid Sequence↗

PubMed1994

Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus.

A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S. cerevisiae. The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies. All clones represented transcripts of the same mannanase gene (man1). The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme. The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.

Amino Acid Sequence↗

PubMed1993

Crystallization and X-ray diffraction analysis of recombinant Coprinus cinereus peroxidase.

Crystals suitable for an X-ray diffraction investigation have been obtained of recombinant Coprinus cinereus peroxidase expressed in Aspergillus oryzae. The crystals were grown by the hanging drop method with polyethylene glycol 6000 as the precipitant. A pH range from 6.2 to 8.0 and CaCl2 or MgCl2 present at a concentration of 0.35 M were essential for the crystal growth. A metastable monoclinic modification can be obtained under certain conditions, and with variations in temperature they are transformed into a stable orthorhombic modification. With CaCl2 as the additive, the unit cell dimensions were a = 74.9 A, b = 76.8 A and c = 128.2 A. With two peroxidase molecules per asymmetric unit, the solvent content is 49% (v/v). In the diffraction pattern, the reflections Okl are systematically very weak for k = 2n + 1. Combined with an analysis of the Patterson function, this showed that the two independent molecules are related by the pseudotranslational symmetry 0.29a + 0.5b. The possible space groups are P2(1)2(1)2(1) or P2(1)22(1) because of this pseudosymmetry. The crystals diffract to a resolution of 2.9 A.

Coprinus↗

PubMed1993

Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva.

A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse-transcribed parotid gland RNA. After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E. coli. The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively. An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein. Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid. Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein. On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity.

Amino Acid Sequence↗