Subtilis engineering bacterium bacillus subtilis and its construction and method for producing depilatory enzyme preparations using it
A technology of engineering bacteria and depilation enzyme, applied in the field of subtilis engineering bacteria Bacillus subtilis and its construction, and production of depilation enzyme preparations, which can solve the problems of unavoidable damage to the structure of the dermis, unsuitable for factory operation, popularization and application, complex collagenase process, etc.
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Embodiment 1
[0062] A subtilis engineering bacterium Bacillus subtilis CGMCC No.11625.
[0063] The applicant for the strains in this example has been submitted to the General Microbiology Center of the China Microbial Cultures Preservation Management Committee, located at No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, for preservation on November 6, 2015. The abbreviation of the preservation unit is CGMCC, the deposit number is: CGMCC No.11625.
[0064] The construction method of subtilis engineering bacterium Bacillus subtilis CGMCC No.11625, the method comprises the following process steps:
[0065] A. Amplification of the neutral protease gene npr
[0066] The primers were designed according to the npr sequence of the neutral protease gene of Bacillus amyloliquefaciens CGMCCNo.1.398, the donor strain in NCBI, as follows, and the upstream primer was: 5'-CCGATTACAAAAACATCAGCCGTA GGATCC CATCAGCTTAAAGAGAATCAAAC-3', where the underlined part is the BamHI restrictio...
Embodiment 2
[0100] The difference between this embodiment and embodiment 1 is:
[0101] The fermentation medium is composed of the following raw materials in unit mass percentage: wheat bran 6%; corn flour 5%; peptone 2%; NaH 2 PO 4 0.4%;K 2 HPO 4 0.04%; soybean oil 0.05%; the balance is water.
[0102] The aerated fermentation conditions are as follows: OD in the fermentation broth 600 When =0.5, add the IPTG that final concentration is 2.5mmol as inducer, when the enzyme activity in the fermented liquid descends, it is the end point of fermentation;
[0103] Described strain culture medium is made up of the raw material of following mass percent:
[0104] Peptone 5%; beef extract 5%; Nacl 5%; the balance is water; pH=6.8-7.5;
[0105] Described shaking flask seed culture medium is made up of the raw material of following mass percent:
[0106] 5% peptone; 5% beef extract; 5% Nacl; the balance is water; chloramphenicol with a concentration of 100 μg / ml is selected as the antibioti...
Embodiment 3
[0112] The difference between this embodiment and embodiment 1 is:
[0113] The fermentation medium is composed of the following raw materials per unit mass percentage: wheat bran 4%; corn flour 3.5%; peptone 1.5%; NaH 2 PO 4 0.3%;K 2 HPO 4 0.03%; soybean oil 0.03%; the balance is water.
[0114] Described strain culture medium is made up of the raw material of following mass percent:
[0115] Peptone 3%; beef extract 3%; Nacl 3%; the balance is water; pH=6.8-7.5;
[0116] Described shaking flask seed culture medium is made up of the raw material of following mass percent:
[0117] 3% peptone; 3% beef extract; 3% Nacl; the balance is water; chloramphenicol with a concentration of 55 μg / ml is selected as the antibiotic; pH=6.8-7.5.
[0118] Described seed culture medium is made up of the raw material of following mass percent:
[0119] 3% peptone; 3% beef extract; 3% Nacl; the balance is water; chloramphenicol with a concentration of 55 μg / ml is selected as the antibioti...
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