Manabu Suto

Induction and catabolite repression mechanisms of cellulase in fungi

Cellulases are induced in most of fungi only when cellulose or an inducer exists. In Hypocrea jecorina and Penicillium purpurogenum, the respective inducers are sophorose and gentiobiose, which do not have beta-1,4 linkages though cellobiose, which has this linkage, is an inducer in other fungi. beta-Glucosidase, which catalyzes transglucosylation, is the key enzyme in converting cello-oligosaccharides to the inducers for cellulase induction in H. jecorina and P. purpurogenum. There are three states in the regulation of cellulase at the transcriptional level in fungi: expression at a basal level, mass secretion of cellulases induced by inducers, and glucose or catabolite repression. Expression at a basal level allows a small amount of cellulase to hydrolyze cellulose to soluble oligosaccharides or to an inducer if cellulose exists near the mycelia. Once the inducer enters the cell, it triggers full-scale transcription of the cellulase gene mediated by activator proteins and activating elements. After cellulose is degraded a large amount of glucose is liberated, which causes catabolite repression.

Endophytes as Producers of Xylanase

One hundred and sixty-nine endophytic fungi and 81 endophytic bacteria were isolated from 14 plants in total. Among them, 155 fungi (91.7%) and 52 bacteria (64%) were found to produce xylanase. The inside part of plants is a novel and good source for isolating xylanase producers in comparison with soil.

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

Abstract The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain.

Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae

A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%–56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADH1). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.

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.

Cellulase induction by cellobiose-octaacetate in Penicillium purpurogenum

Abstract Cellulase production by Penicillium purpurogenum strain P-26 was strongly induced by cellobiose-octaacetate (COA) in the presence of microcrystalline cellulose as a co-substrate. COA was solubilized during growth and reducing sugar consisting of multiple components accumulated in the culture broth. Cellulase induction was coupled with consumption of the reducing sugar. A crude protein fraction obtained from the culture supernatant could solubilize COA and the hydrolyzate stimulated cellulase formation by non-induced cells grown on glycerol. Partial purification of the culture supernatant revealed two enzymes, both of which act not only on COA to release soluble reducing sugar and acetic acid but also on other acetylesters to form acetate, indicating that COA was solubilized by acetylesterase secreted in the culture broth and the hydrolyzate, probably partially deacetylated COA, could induce cellulase.

Cellulase induction by soluble acetyl cellobioses in Penicillium purpurogenum

Abstract Cellulase in Penicillium purpurogenum strain P-26 was induced with the cellobiose-octaacetate (COA) hydrolyzate due to the crude extracellular acetyl esterase of this fungus. Of the multiple constituents of the hydrolyzate, three predominant ones were purified by silica gel chromatography. They were identified as tri-, di-, and mono- O -acetyl cellobioses with a deacetylated reducing end by mass spectrometry and 500 MHz 1 H-NMR. These acetyl cellobioses apparently induced cellulase due to their slow consumption, while reference sugars such as glucose or cellobiose did not. In particular, mono- O -acetyl cellobiose was the most effective for cellulase induction among the compounds.