Construction and use of chimeric a-amylases to study protein secretion in Bacillus subtilis

PhD ThesisTo investigate the influence of exoprotein charge on protein secretion, genes encoding a range of chimeric a-arnylases with altered net charge were constructed by a PCR-based technique and expressed in B. subtilis. The chimeric (x-amylases were based on ArnyL and contained specific regions from two related Bacillus oc-amylases, ArnyQ and ArnyS. The engineered changes were introduced into amyL to increase the net positive charge of the chimeric enzymes, when compared to wild type AmyL. Isoelectric focusing confirmed that chimeric cc-amylases possessed considerable positive charge and the temperature and pH optima of the most basic chimeric protein, AmyLQS50.5, were largely unaffected by the engineered changes. However, the structural stability, thermostability and the specific activity of this chimeric oc-amylase were adversely affected. In general,l ower amountso f chimeric oc-amylasews ere releasedi nto the culture supernatantsP. ulse-chasee xperimentsr evealed that the rate of processing of AmyLQS50.5 was reduced when compared to AmyL and also that the mature forms of both cc-amylasews ere subjectedt o degradationd uring or shortly after translocation, although the chimeric enzyme was affected most. When compared to AmyL, the rate of refolding of AmyLQS50.5 was reduced approximately 3- fold, maintaining this protein in a protease-sensitivec onformation for an increased period of time. Therefore, it is proposed that the extensive co- or posttranslocationald egradationo f the chimeric enzymew as a consequenceo f reduced folding kinetics on the outer surface of the cytoplasmic membrane since, when in its native conformation, AmyLQS50.5 is highly resistant to the activity of B. sublifis extracellularp roteasesT. heseo bservationsh ave important implications for the use of B. subtilis as a host for the secretion of heterologous proteins. The most positively charged chimera, AmyLQS50.5, was shown to bind significantly to cell walls isolated from B. sublifis 168, whereas AmyL and human serum albumin did not. This suggeststh at protein chargec an influence the degree to which exoproteins interact with, and bind to, the cell wall as a consequenceo f electrostatici nteractionsw ith the anionic polymers.Novo Nordisk