The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger

The effect of ambient pH on production and glycosylation of glucoamylase (G AM) 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 p H from 4 to 5.4 during continuous cultivation of the A. niger B1 strain ins tigates 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 st rain with parental-type morphology (PS). The intracellular levels of GAM we re also lower in the LB strain. In addition, cultivation of the original pa rent BI strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold d rop in GAM production and a 5-fold drop in extracellular protein compared t o that obtained at pH 4. Glycosylation analysis of the glucoamylases purifi ed from shake-flask cultivation showed that both principal forms of GAM sec reted by the LB strain possessed enhanced galactosylation (2-fold), compare d to those of the PS. Four diagnostic methods (immunostaining, mild methano lysis, mild acid hydrolysis and beta -galactofuranosidase digestion) provid ed evidence that the majority of this galactose was of the furanoic conform ation. 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 therefor e regulates both GAM production and influences its glycosylation. (C) 2001 Elsevier Science B.V. All rights reserved.