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We have identified the major endo-β-l,4-xylanase (XYN-1) in the aleurone of germinating barley grain, and show that it is expressed as a precursor of Mr 61 500 with both N- and C-terminal propeptides. XYN-1 is synthesized as an inactive enzyme in the cytoplasm, and only becomes active at a late stage of germination when the aleurone ceases to secrete hydrolases. A series of processing steps, mediated in part by aleurone cysteine endoproteases, yields a mature active enzyme of Mr 34 000. Processing and extracellular release of the mature enzyme coincide with the programmed cell death (PCD)-regulated disintegration of aleurone cells. We discuss the significance of delayed aleurone cell-wall degradation by endoxylanases in relation to the secretory capacity of the aleurone, and propose a novel role for aleurone PCD in facilitating the export of hydrolases. Chemicals/CAS: alpha-Amylase, EC 3.2.1.1; Cysteine Endopeptidases, EC 3.4.22.-; Endo-1,4-beta Xylanases, EC 3.2.1.8; Green Fluorescent Proteins, 147336-22-9; Hydrolases, EC 3.-; Indicators and Reagents; Luminescent Proteins; Plant Proteins; RNA Precursors; Starch, 9005-25-8; Xylosidases, EC 3.2.1.-

A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression is regulated at the transcriptional level. Using a β-glucuronidase (uidA) reporter strategy, D-xylose was shown to be an efficient inducer of the exlA promoter, whereas sucrose or maltodextrin were not. Upon D-xylose induction, the exlA promoter was threefold more efficient than the frequently used A. niger glucoamylase (glaA) promoter under maltodextrin induction. Detailed induction analyses demonstrated that induction was-dependent on the presence of D-xylose in the medium. Carbon-source-limited chemostat cultures with the uidA reporter strain showed that D-xylose was also a very good inducer in a fermenter, even in the presence of sucrose.

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-β-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exlA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli β- glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.

1. A study with growing chicks investigated the effects of an inclusion of an endo-xylanase preparation (LYXASAN®) to a wheat- and rye-based diet on performance and nutrient digestibility in relation to the fat source. 2. The basal diet contained 500 g wheat and 100 g rye/kg of diet. The basal diet was supplemented with either 65 g soya oil/kg or 60 g blended animal fat and 5 g soya oil/kg. 3. Endo-xylanase added to the soya oil diet did not affect weight gain, but there was a numerical improvement in food conversion efficiency which was not statistically significant. When the endo-xylanase preparation was added to the blended animal fat diet, both weight gain and food utilisation were improved by 9·5% and 6·0%, respectively (P< 0.05). 4. Digestibilities of organic matter, crude fat, crude fibre and NFE were not significantly affected by adding endo-xylanase to the soya oil diet. However, when endo-xylanase was included in the blended animal fat diet, digestibility of organic matter, crude fat, crude fibre and NFE were improved (P<0.05). The improvement in fat digestibility was the most pronounced, amounting to 9·4%. Nitrogen retention and metabolisable energy content were improved significantly by the addition of an endo-xylanase to the animal fat diet (P<0·05), by 6·6% and 6·5% respectively. 5. From the results of this study, it can be concluded that the effects on chick performance and nutrient digestibility of a dietary endo-xylanase in a wheat- and rye-based diet are influenced to a considerable degree by the type of fat in the diet.

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l-1), improved FAEA activity 24.5-fold and a yield of 1 g l-1 of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification - close to 75% - and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector. Chemicals/CAS: hemicellulose, 63100-39-0, 63100-40-3, 9034-32-6; laccase, 80498-15-3; xylan endo 1,3 beta xylosidase, 37278-89-0, 9025-55-2; Carboxylic Ester Hydrolases, EC 3.1.1.-; Endo-1,4-beta Xylanases, EC 3.2.1.8; feruloyl esterase, EC 3.1.1.73; Laccase, EC 1.10.3.2; Recombinant Proteins