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We report the cloning and characterization of a gene encoding a ferulic acid esterase, faeA, from Aspergillus niger and Aspergillus tubingensis. The A. niger and A. tubingensis genes have a high degree of sequence identity and contain one conserved intron. The gene product, FAEA, was overexpressed in wild-type A. tubingensis and a protease-deficient A. niger mutant. Overexpression of both genes in wild-type A. tubingensis and an A. niger protease-deficient mutant showed that the A. tubingensis gene product is more sensitive to degradation than the equivalent gene product from A. niger. FAEA from A. niger was identical to A. niger FAE-III (C. B. Faulds and G. Williamson, Microbiology 140:779-787, 1994), as assessed by molecular mass, pH and temperature optima, pI, N-terminal sequence, and activity on methyl ferulate. The faeA gene was induced by growth on wheat arabinoxylan and sugar beet pectin, and its gene product (FAEA) released ferulic acid from wheat arabinoxylan. The rate of release was enhanced by the presence of a xylanase. FAEA also hydrolyzed smaller amounts of ferulic acid from sugar beet pectin, but the rate was hardly affected by addition of an endo-pectin lyase.

Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethy1ene glycol) precipitatiodfractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 f 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 f 5 kDa. The enzyme had a PI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH,' as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg", K,,, values for L-glutamine was determined to be 0.37f0.05 mM, for chorismate 67?3 pM, and for MgCl, 0.26 -C-0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperativity of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20pM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperativity for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.Anthranilate synthase (AS; EC 4.1.3.27) catalyzes the first reaction branching from the shikimate pathway toward the biosynthesis of tryptophan occurring in bacteria, fungi and plants [I] (Fig. 1).Tryptophan is a key precursor for the indole alkaloids, among which there are several pharmacologically important compounds [2], from Catharanthus roseus, for example, the two anticancer drugs vinblastine and vincristine. The availability of tryptophan might be a limiting factor in the production of indole alkaloids. In all plants and cell cultures where AS has been examined, the enzyme was found to be strongly feedback-inhibited by tryptophan ; in accordance with this, several studies have indicated that loss of feedback regulation of AS can lead to largely unregulated accumulation of tryptophan [3 -61.AS enzymes have been purified to homogeneity and characterized from a number of microorganisms. In all mi- crobial species studied, AS is composed of two nonidentical subunits [7-91. Component I (also called a-subunit) binds the substrate chorismate and catalyzes its aromatization; component I1 @-subunit) binds the other substrate glutamine and transfers an ammonia group from glutamine to component I (chorismate). This glutamine-dependent AS reaction requires both subunit...

Cereal Chem. 75(1):51-57Hexose oxidase (EC 1.1.3.5) (HOX) was purified 51-fold from the red algae Chondrus crispus, by several chromatography methods, including hydrophobic interaction, chelating Sepharose, anion exchange, gel filtration, and chromatofocusing. Purified HOX was subjected to native PAGE and activity staining with nitroblue tetrazolium. For HOX electroeluted out of the gel and digested with endoproteinase Lys-C, the internal peptide sequence determined was: D-P-G-Y-I-V-I-D-V-N-A-G-T-(V or P)-D-K-P-D-P-X. The molecular mass, determined by gel filtration, was 126 kDa, versus 65 kDa determined by SDS-PAGE. The pI was determined to 4.64 and 4.79 as a double band on an isoelectrofocusing gel. K m was determined to 2.7 mM for D-glucose, 3.6 mM for D-galactose, 20.2 mM for cellobiose, 43.7 mM for maltose, 90.3 mM for lactose, 102 mM for xylose, and 531 mM for arabinose. The oxidation of thiol groups in gluten was determined by using Ellman's reagent: 5,5′-dithiobis (2-nitrobenzoic acid). The effect of HOX was compared to that of glucose oxidase. Both enzymes caused a dose-responsive reduction in the free thiol groups. Extensigraph measurements and baking tests confirmed that HOX caused increased dough strength and increased bread volume more efficiently than glucose oxidase used in the same dosage. MATERIALS AND METHODS ChemicalsGOX was obtained from Aspergillus niger (Grindamyl S 757, Danisco Ingredients, Denmark). Maltotriose and maltotetraose were obtained from Megazyme, Ireland. Peroxidase, odianisidine, nitroblue tetrazolium, phenazine methosulphate, and endoproteinase Lys-C were obtained from Sigma Chemical Co., St. Louis, MO. Phenyl Sepharose FF, chelating Sepharose FF, Source Q, Superdex 200, Mono P HR 5/5, isoelectrofocusing (IEF), and molecular weight standards were from Pharmacia, Sweden. The 8-16% tris-glycine gels used for electrophoresis and the precast 3-10 IEF gels were from Novex, San Diego, CA. The inorganic chemicals used were analytical grade. A Vydac C18 column was obtained from The Separation Group, CA. A Brownlee C2 column was obtained from Applied Biosystems. A Dionex PA2 column was from Dionex. Assay for HOX ActivityThe assay of Sullivan and Ikawa (1973) was scaled down to be run in microtiter plates. The absorbance was read in a microplate reader. The HOX assay method was also used for the assay of GOX. One unit is defined as the amount of enzyme that catalyzes the production of 1 µmole of H 2 O 2 per minute at 25°C, pH 6.3, and at a substrate concentration of 50 mM D-glucose. The assay was used to determine the Michaelis-Menten constant (K m ) and maximum velocity (V max ) for D-glucose, D-galactose, cellobiose, maltose, lactose, xylose, and arabinose, respectively. The data were fitted by nonlinear regression to v = V max S/(K m + S), where S is substrate concentration and K m is the concentration giving half maximum velocity. The values were determined by a curve-fitting computer program, EZ-FIT (Perella 1988). Determination of Protein and CarbohydrateThe protein concentration ...

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An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression ofaguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.