A signal peptide for optimizing the high-efficiency secretion and expression of keratinase keratinase and its application
A technology for secreting expression and keratinase, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low extracellular expression of keratinase and is difficult to meet the needs of the industry, and achieves the effect of improving secretion efficiency and facilitating industrial application.
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Embodiment 1
[0028] The preliminary screening of embodiment 1 signal peptide
[0029] (1) Synthesis of gene ker
[0030] According to the NCBI accession number JX504681 of keratinase Ker derived from Bacillus licheniformis BBE11-1, its gene sequence was searched and submitted to Shanghai Boyi Biotechnology Co., Ltd. for the whole gene synthesis of keratinase Ker.
[0031] (2) Construction of expression vector pSTOP1622-ker
[0032] Design PCR primers P1 and P2 (Table 1) according to the gene sequence of Ker, use the synthetic gene ker as a template, and use P1 and P2 as primers to perform PCR amplification to obtain the amplified product ker (signal peptide, propeptide and Nucleotide sequence of mature enzyme); PCR amplification conditions are: 98°C 5min; 98°C 20sec, 60°C 40sec, 74°C 2min, 30 cycles; 74°C, 10min; Restriction digestion, connection to vector pSTOP1622, construction of recombinant vector pSTOP1622-ker.
[0033] Table 1 Primers required for vector construction
[0034] ...
Embodiment 2
[0052] Optimization of embodiment 2 signal peptide
[0053] (1) Gene synthesis of signal peptide mutants
[0054] The preliminary screening results of signal peptides showed that signal peptides YfkN, Bpr, Mpr, PhoB, WapA, AbnA can effectively improve the secretion efficiency of keratinase Ker. According to the structural characteristics of the signal peptides, the amino acid sequences and nucleotide sequences of the above six signal peptides were partitioned, that is, N-terminal, H-segment, and C-terminal, as shown in Tables 4-9. The three regions of these six signal peptides were recombined, and a total of 216 (6×6×6) signal peptide mutants were obtained ( image 3 ). According to the gene sequences of these six signal peptides, the nucleotide sequence "5'-ACAATGGTCCAAACTAGT+the nucleotide sequence of the signal peptide mutant+GCTCAGCCGGCGAAAA-3'" was designed and synthesized by Shanghai Boyi Biotechnology Co., Ltd.
[0055] Table 4 Signal peptide YfkN
[0056]
[005...
Embodiment 3
[0073] Example 3 Verification of high secretion capacity keratinase production strain
[0074] The batch-fed fermentation method was used to perform fermentation verification on the recombinant Bacillus subtilis WB600 / pSTOP1622-24-kerds constructed in Example 2. The seed culture conditions of recombinant bacteria Bacillus subtilis WB600 / pSTOP1622-24-kerds and Bacillus subtilis WB600 / pSTOP1622-ker are as follows: LB liquid medium is used for cultivation in a 500mL Erlenmeyer flask, wherein the liquid volume of the medium is 50mL, and the cultivation temperature The temperature is 37°C, the rotational speed is 200rpm, and the incubation time is 10h. The batch-fed fermentation conditions of the recombinant bacteria are as follows: B.subtilis WB600 batch fermentation medium is used, the seeds are inoculated in a 3L automatic fermenter, the stirring speed is 400rpm, the culture temperature is 37°C, and the ventilation rate is 1.5vvm. Use ammonia water to control the pH not lower t...
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