Enzymatic extractability of soybean meal proteins and carbohydrates: heat and humidity effects.

To study the incomplete enzymatic extractability of proteins and carbohydrates of thermally treated soybean meals, one unheated and three heat-treated soybean meals were produced. To obtain truly enzyme-resistant material, the meals were extracted by a repeated hydrolysis procedure using excessive concentrations of different combinations of commercial protease and carbohydrase preparations. The water extractability of protein from the different meals varied considerably (13-67%). For all soybean meals, enzymatic treatment extracted most of the original protein (89-94%). Carbohydrase preparations did not improve protein extraction. High-humidity heat treatment led to a more effective enzymatic extraction, which seemed to correlate with the extent of protein denaturation. Results with purified proteins indicated that the soybean meal matrix affects the enzymatic extraction of protein from the meals. Interactions between protein and other components (e.g., cellulose) may explain the incomplete enzymatic extractability of protein from the meals.

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.

Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis.

Non-specific monoaminopeptidase (AP; E.C. 3.4.11) and X-prolyl dipeptidyl aminopeptidase (X-PDAP; E.C. 3.4.14.5), both from Aspergillus oryzae, demonstrate strong synergism in hydrolyzing proline-containing peptides. Incubation of AP alone with the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe does not generate free amino acids. However, when AP and X-PDAP are added in combination, complete and immediate hydrolysis of all peptide bonds, other than X-Pro bonds, is observed. In the enzymatic hydrolysis of casein, soy, and gluten, degree of hydrolysis (DH) values of 54, 54, and 47% were achieved, respectively, when subtilisin (E.C. 3.4.21.62) was supplemented with AP. Addition of a third enzyme, X-PDAP, resulted in significantly higher DH values of 69, 72, and 64%, respectively, establishing the utility of this synergism in protein hydrolysis.

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.

Fermentability of an enzymatically modified solubilised potato polysaccharide (SPP).

OBJECTIVE: To study processing and fermentability in the human gastro-intestinal tract of a newly isolated, enzymatically modified, soluble, and highly concentrated (> 80% dietary fibres) solubilised potato fibre (SPP). SETTING: Gastroenterological laboratory. DESIGN, SUBJECTS AND INTERVENTIONS: Seven healthy volunteers ingested in random order on seven different days: 20 g SPP; bread made of 180 g wheat flour served with 20 g raw SPP; bread baked of 180 g wheat flour and 20 g SPP; bread made from 180 g what flour; 20 g lactulose; 20 g oat bran; and 20 g wheat bran. The hydrogen breath test was used to evaluate oro-coecal transit time (OCTT) and fermentation. RESULTS: Fermentation of SPP yielded a measurable increase in end-expiratory H2. The total incremental increase in end expiratory H2 due to SPP was unaffected of whether SPP was served alone, as the raw flour served with bread, or baked into bread. The OCTT for raw SPP was significantly delayed compared to lactulose (P = 0.01). The OCTT for SPP baked into bread was significantly delayed compared to raw SPP (P = 0.01), indicating that SPP may be used as a marker of oro-coecal transit time for as well the fluid phase as the solid phase of a meal. CONCLUSIONS: SPP is a fermentable, highly concentrated soluble fibre source. Baking SPP did not interfere with the fermentable properties. Thus, SPP may be interesting as a fibre-supplement in fibre-poor diets. The change in oro-coecal transit time for SPP, depending on the composition of the total meal, makes SPP interesting as a marker in studies of gastrointestinal transit.

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.

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.

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.

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.

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.

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.