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Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates

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Author: Biesebeke, R. te · Record, E. · Biezen, N. van · Heerikhuisen, M. · Franken, A. · Punt, P.J. · Hondel, C.A.M.J.J. van den
Institution: TNO Kwaliteit van Leven
Source:Applied Microbiology and Biotechnology, 1, 69, 44-50
Identifier: 238787
doi: doi:10.1007/s00253-005-1968-4
Keywords: Biology · Biotechnology · Enzyme kinetics · Enzymes · Fermentation · Growth kinetics · Hydrolysis · Microorganisms · Substrates · Yeast · Amylase · Branching mutants · Hydrolytic enzymes · Solid substrates · Mutagenesis · amylase · carrier protein · glucan 1,4 alpha glucosidase · phosphatidylglycerol · phosphatidylinositol · proteinase · fungus · gene expression · protein · allele · article · Aspergillus oryzae · enzyme activity · enzyme synthesis · fermentation · fungal biomass · fungal genetics · fungal strain · fungus growth · fungus hyphae · fungus mutant · genetic code · hydrolysis · microscopy · nonhuman · protein secretion · seed kernel · solid · sporogenesis · wheat · wild type · Amylases · Aspergillus oryzae · Biomass · Fungal Proteins · Gene Deletion · Glucan 1,4-alpha-Glucosidase · Hyphae · Morphogenesis · Mutagenesis, Insertional · Mutation · Peptide Hydrolases · Phospholipid Transfer Proteins · Spores, Fungal · Triticum · Aspergillus oryzae · Fungi · Triticum aestivum


To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L hgu) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process. © Springer-Verlag 2005.