A signal peptide mutant capable of increasing the expression of recombinant pullulanase and its application
A pullulanase and signal peptide technology, applied in the direction of peptides, enzymes, depsipeptides, etc., can solve problems such as loss of activity, and achieve the effect of increasing the amount of extracellular expression
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0019] This example illustrates the acquisition of a signal peptide mutant derived from the α-acetolactate decarboxylase gene of Bacillus brevis, and the acquisition of amino acids comprising this mutant combined with pullulanase derived from Bacillus naganoensis A method for recombining Bacillus subtilis.
[0020] 1. Construction of recombinant plasmid pMLK83-P43
[0021] According to the promoter P43 sequence annotated in Genbank, the upstream primer was designed as 5'attgctggacgcttatggac3' and the downstream primer as 5'cgggatccattcctctcttacctataat 3'. PCR reaction system 100ul: DNA template (Bacillus subtilis 1A751 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ul PrimeSTAR HS DNA polymerase 1ul, add ddH 2 0 to 100ul. PCR reaction program: 94°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1min; 72°C for 10min; store at 4°C. The PCR fragment and plasmid pMLK83 were double dige...
Embodiment 2
[0052] This example illustrates the method for producing pullulanase using the recombinant Bacillus subtilis described in Example 1.
[0053] The operation steps are as follows:
[0054] 1) Preparation of first-class species: A single colony of the genetically engineered strain of Bacillus subtilis was cultured overnight in 4 ml of LB liquid medium at 37° C. and 220 rpm on a shaker, and the obtained strain was a first-class species.
[0055] 2) Preparation of the secondary species: the primary species was inoculated in 800 ml LB liquid medium, and cultured on a shaker at 37° C. at 220 rpm until the OD600 was about 0.6 (about 4 to 5 hours).
[0056] 3) Preparation of the third-level seed: inoculate the second-level seed into an 80L LB liquid fermenter, control the temperature at 37°C, control the pH to 7.0 with citric acid and NaOH, ventilate and stir for 5-6 hours, and control the dissolved oxygen at 20-30 %, cultivate until OD600 is about 0.6 (about 5-6 hours).
[0057] 4) ...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com