Stable repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) from Aspergillus oryzae

Recombinant Aspergillus oryzae expressing a dye-decolorizing peroxidase gene (dyp) was cultivated for repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) using maltose as a carbon source. High-level rDyP activity in limitation of carbon and nitrogen sources was maintained stably for 26 cycles of repeated 1-d batches of A. oryzae pellets without any additional pH control. Cultures maintained at 4 degrees C for 20 d resumed rDyP production following a single day of incubation. One liter filtrated crude rDyP containing 4600 U rDyP decolorized 5.07 g RBBR at the apparent decolorization rate of 17.7 mg l(-1) min(-1).     

Production of lepidimoide by an endophytic fungus from polysaccharide extracted from Abelmoschus sp.: identification of the product and the organism producing it

A highly potent allelopathic factor, lepidimoide, was initially extracted from mucilage of germinated cress seeds. Polysaccharide extracted from okra (Abelmoschus esculentum Moench) is considered to have a similar structure to lepidimoide as its repeating unit. We therefore initiated the screening of enzymes capable of degrading okra polysaccharide into lepidimoide from endophytes. We discovered an endophytic fungal strain AHU9748 isolated from Coleus galeatus, which produced an oligosaccharide having similar properties to lepidimoide on thin layer chromatography. The physico-chemical data from ESI-MS, NMR spectra and other analyses also showed the purified product to be identical to lepidimoide. The strain AHU9748 was identified as a fungus belonging to the coelomycetes, closely related to the genus Colletotrichum, based on morphological characteristics and sequence analysis of the 18S rDNA and ITS region.     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Production of dye-decolorizing peroxidase (rDyP) from complex substrates by repeated-batch and fed-batch cultures of recombinant Aspergillus oryzae

We examined production levels of dye-decolorizing peroxidase (rDyP) by recombinant Aspergillus oryzae using wheat bran and rice bran powders in repeated-batch and fed-batch cultures. Similar average rDyP productivities were observed in repeated-batch cultures using wheat bran powder and rice bran powder. Average rDyP productivities in fed-batch cultures were slightly lower than those in repeated-batch cultures. The rDyP production was affected by the addition of K(2)HPO(4) in the repeated-batch and fed-batch cultures using wheat bran powder. All average rDyP productivities in this study were significantly higher than those for any other peroxidases previously reported.     

‘Lipase and protease production of dairy Penicillium sp. on milk‐protein‐based solid substrates’

The lipolytic and proteolytic activity of Penicillium camemberti PC TT033 and Penicillium roqueforti PR G3, cultured on the whey solids or simulated cheese media, were compared under several pH reaction conditions. Lipolytic activity was higher when both strains had been cultured on the whey medium than on the simulated cheese medium, whereas proteolytic activity was less influenced by the culture medium. The relationship between the reaction pH and these enzyme activities was dependent on the culture medium, which suggested that the expression level and balance of isozyme rely on the culture substrate.     

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.