Preliminary Mechanism Of High-level α-amylase Production In Bacillus Subtilis Mediated By CcpA Gene

Α-amylase as an important industrial enzyme was widely used in food industries, textiletechnology, paper manufacturing and so on, but carbon catabolic repression which have animportant influence on the accumulation products of the Bacillus subtitles is always exiting in thefermentation processing. In order to reveal the mechanism of this kind of phenomenon, we taketwo strains of B.subtilis168and B.subtilis1.769as the research objects,analyzes the Bacillussubtitles amylase processing.on the other hand, to construct the recombinant strains isolated in toa certain extent, ease the catabolic repression by gene recombination technology, and finallythrough the Red gene knockout technology success of the CcpA and Hprk gene deletion mutant,through fermentation experiments prove that the two genes are directly related to the regulation ofCCR effect. The main research is as follows:1. Firstly, In the course of the study on the nuclease mapping,we found that transcriptioninitiates at a site8base pairs (BP) downstream of a sequence50%homologous to the promoterconsensus recognized by EσARNA polymerase. Research on EσARNA polymerase promoter cantype amylase expression further identification of Bacillus subtilis. AmyEmRNA will quicklybegan to accumulate in the stable period, But when the grape fermentation liquid of thisaccumulation was inhibited. Research on B.subtilis168and B.subtilis1.769dry preserved in ourlaboratory, hand to reveal the expression differences of alpha amylase yield and enzymeproduction time of the fermentation process, on the other hand from different carbon source forfermentation substrate differences among the two strains of alpha amylase yield reducing sugarresistance. Through two aspects of research further reveals the intrinsic factors affecting theproduction of alpha amylase by Bacillus subtilis.2. Secondly, Through the method of gene recombination, the alpha amylase gene amyE(remove promoter) of Bacillus subtitles was integrated into Paxo1plasmid and the recombinantexpression plasmid Paxo1amyE was leaded into the galactosidase gene lacZ of Bacillus subtilisstrain168.Through the transformation and selection analysis, engineering genetic strain PaEcontaining the recombinant alpha amylase gene. After adding four different carbon sources infermentation detection and plusing2%xylose induction, the recombinant strain overcame thecarbon catabolic repression causing by the glucose in a certain extent.The PaE strain could improve the yield of the alpha amylase yield.3. At last, Finally, using gene knockout technology catabolic repression regulation of Bacillussubtilis carbon on Bacillus subtilis strain168and strain1.769(CCR) transcription factor encodinggene CcpA global had targeted knockout, and control gene HprK on the regulation of metabolismlater knocked out, through the fermentation culture medium in detection of different carbon thesource of fermentation, the amylase activity was improved, which also reveals the alpha amylaseproduced by these two kinds of gene regulation.