title: The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger.
abstract: The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.
authors.affiliation: School of Biological, University of Nottingham, UK. gwallis@meristem-therapeutics.com
authors.forename: G L
authors.initials: GL
authors.lastname: Wallis
authors.forename: R J
authors.initials: RJ
authors.lastname: Swift
authors.forename: R
authors.initials: R
authors.lastname: Atterbury
authors.forename: S
authors.initials: S
authors.lastname: Trappe
authors.forename: U
authors.initials: U
authors.lastname: Rinas
authors.forename: F W
authors.initials: FW
authors.lastname: Hemming
authors.forename: M G
authors.initials: MG
authors.lastname: Wiebe
authors.forename: A P
authors.initials: AP
authors.lastname: Trinci
authors.forename: J F
authors.initials: JF
authors.lastname: Peberdy
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D009005:Monosaccharides / Q000032:analysis / Q000378:metabolism; D009154:Mutation; D011134:Polysaccharides; D011995:Recombination, Genetic
chemical_list: D003470:Culture Media; D006023:Glycoproteins; D007527:Isoenzymes; D009005:Monosaccharides; D011134:Polysaccharides; C008315:maltodextrin; D005087:Glucan 1,4-alpha-Glucosidase