Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system

A series of chimeric alpha -amylase genes derived from amyL, which encodes the liquefying alpha -amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned i n Bacillus subtilis, where their ability to direct the synthesis and secret ion of active alpha -amylase was determined. Detectable alpha -amylase acti vity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wildtype AmyL and its chimeric derivatives revealed tha t, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzyme s were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologou s proteins.