Lene Venke Kofod

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 NH-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. aculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.

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

Expression cloning has been used to isolate a cDNA encoding β-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 2.5×104 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°C, which is similar to values obtained for a β-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 β-1,4-galactanase cDNA. However, the deduced aminoacid 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.

Application of tailormade pectinases

Abstract Heterologous expression of pectinolytic enzymes has made it possible to tailormake pectinolytic enzyme preparations. Composition of pectinase products for existing applications can be improved, and products for new applications can be made. Apple mash treatment with galactanase, rhamnogalacturonase and rhamnogalacturonan esterase improves the cloud stability of the juice. Pectin methyl esterase is useful to gelate fruits, and the gelling effect can be enhanced by a pectin hairy region degrading enzyme combination. Rhamno-galacturonase releases soluble high molecular rhamnogalacturonans from the soy cell wall, and the use of this enzyme to produce a fibre enriched soy protein product is demonstrated. Carrot mash treatment with combinations of polygalacturonase, pectin lyase, and rhamnogalacturonase gives a puree.

Application of cloned monocomponent carbohydrases for modification of plant materials

Abstract Several plant cell wall degrading enzymes have been cloned by the expression cloning technique. These enzymes can be used to degrade isolated plant cell wall polysaccharides into oligomers or to extract poly- or oligosaccharides from insoluble and complex plant cell wall material, thereby providing soluble dietary fibre or oligosaccharides with potential beneficial physiological effects. Also the cloned enzymes can be used to control e.g. viscosity in the industrial processing of plant material. This is illustrated by the degradation of various arabinoxylans or arabinoxylan containing plant material with cloned xylanases and by the degradation of rhamnogalacturonans or rhamnogalacturonan containing plant material with cloned rhamnogalacturonases and assessory enzymes.

Crystallization and preliminary X-ray studies of rhamnogalacturonase A from Aspergillus aculeatus

Recombinant rhamnogalacturonase A from Aspergillus aculeatus has been crystallized and X-ray diffraction data has been collected. Crystals were grown by the hanging-drop vapour-diffusion technique, under the conditions 10% PEG 8000, 0.05 M KH(2)PO(4) and 0.1 M sodium acetate buffered at pH 3.5. The crystals diffract beyond 2.0 A resolution and belong to one of the orthorhombic space groups I2(1)2(1)2(1) or I222, with the unit-cell parameters a = 62.9, b = 125.4 and c = 137.0 A. There is one molecule in the asymmetric unit and a solvent content of approximately 54%. The enzyme is highly glycosylated corresponding to 5.9 kDa.