Abstract
BackgroundBacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.ResultsCompared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874.ConclusionsWe demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.
Highlights
Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease
As αamylase activity was barely detected in the culture supernatant of B. subtilis harboring pHY300PLK, the indirect effects of endogenous α-amylase production were considered to be negligible under the assay conditions used in this study
In contrast to our previous report on the enzymes cellulase Egl-237 and M-protease [8], we found that the production of AmyK38 gradually decreased in the order of strains 168, MGB625, MGB723, and MGB874, and was markedly impaired as a result of the genomic deletion process between strains MGB625 and MGB723 (Figure 1)
Summary
Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Α-amylases are generally metalloenzymes that contain at least one activating and stabilizing Ca2+ ion [13], AmyK38 does not associate with Ca2+ ions; rather, its enzyme activity depends on Na+ ions and is not inhibited by chelating reagents. Due to these characteristics, AmyK38 would likely be an advantageous component of laundry and dishwashing detergents, which are alkaline solutions and typically contain bleach and chelating agents. The improvement of AmyK38 production levels is a necessary prerequisite for the commercial use of this α-amylase, which is only produced by strain KSM-K38 at markedly low levels (30 mg l-1) [12]
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