Modified promoter
A promoter and base sequence technology, applied in the direction of cells, enzymes, biochemical equipment and methods modified by introducing foreign genetic material, can solve problems that cannot be called sufficient
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Embodiment 1
[0039] Example 1 Construction of SigE recognition promoter (sequence) to the upstream region of alkaline cellulase gene
[0040] As shown in FIG. 2 , introduction of the SigE recognition promoter sequence into the upstream region of the alkaline cellulase gene was carried out. That is, in the shuttle vector pHY300PLK Bam At the cleavage site of the HI restriction endonuclease, there will be inserted a code from Bacillus sp.( Bacillus sp.) The recombinant plasmid pHY-S237 of the DNA fragment (3.1kb) of the alkaline cellulase gene (Japanese Patent Application Publication No. 2000-210081) of KSM-S237 strain (FERM BP-7875) is used as a template, and is used in Table 1 The indicated primer sets for 237UB1 and EP1UPr prepared a 0.4 kb fragment of the upstream region of the alkaline cellulase gene (A). The primer set of EP1DNf and S237RV shown in Table 1 was also used to prepare a 2.7 kb fragment (B) of the downstream region of the alkaline cellulase gene. Then, by mixing the ob...
Embodiment 2
[0041] Example 2 Production Evaluation of Alkaline Cellulase Secretion
[0042] The recombinant plasmid pHY-S237EP1 obtained in Example 1 and the recombinant plasmid pHY-S237W as a control were introduced into the Bacillus subtilis 168 strain by the protoplast transformation method. The bacterial strain thus obtained was shake-cultured overnight at 37°C in 10 mL of LB medium, and 0.05 mL of the culture solution was inoculated into 50 mL of 2×L-maltose medium (2% tryptone, 1% yeast extract, 1 % NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, and 15 ppm tetracycline), shaking culture was carried out at 30° C. for 3 days. After the cultivation, the alkaline cellulase activity of the culture supernatant from which the bacterial cells were removed by centrifugation was measured to determine the amount of alkaline cellulase secreted and produced outside the bacterial cells by the culture. As shown in Table 2, as a result, it was found that the secreted production of alka...
Embodiment 3
[0045] Example 3 Verification of the effectiveness of the production of alkaline cellulase introduced into the upstream region of the alkaline cellulase gene with a SigE recognition promoter (sequence)
[0046]Using the plasmid pHY-S237EP1 constructed in Example 1 as a template, PCR was carried out with the primer sets of S237ppp-F2 (BamHI) and S237ppp-R2 (ALAA) shown in Table 3, and amplification containing the introduced SigE recognition promoter ( sequence) of the promoter region of the alkaline cellulase gene and a 0.6 kb DNA fragment (E) of the region encoding the secretion signal sequence. Moreover, from Bacillus sp.( Bacillus sp.) Genomic DNA extracted from the KSM-K38 strain (FERM BP-6946) was used as a template, and PCR was carried out with the primer set of K38matu-F2 (ALAA) and SP64K38-R (XbaI) shown in Table 3, and the encoding was carried out by A 1.5 kb DNA fragment (F) of alkaline cellulase (Appl. Environ. Microbiol., 67, 1744, (2001)) having the amino acid se...
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