Production method of L-phosphinothricin
A technology for the regeneration of glufosinate-ammonium and coenzyme, applied in the biological field, can solve the problems of only 52% reaction conversion rate, little advantage compared with chemical synthesis, low optical purity of products, etc. Overall yield and strong stereoselectivity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Example Embodiment
[0057] Example 1 Construction of genetically engineered strains
[0058] 1.1 Construction of genetically engineered bacteria expressing γ-aminobutyric acid / α-ketoglutarate aminotransferase
[0059] The transaminase genes were cloned from the genomes of E. coli K12W3110, Bacillus subtilis 168, and Bacillus magaterium YYBM1. According to the corresponding genomic DNA sequences (GenBank accession numbers are CP012868.1, CP010052.1, and CP001982.1, respectively) ) Design corresponding PCR upstream primers and downstream primers.
[0060] Primers for transaminase derived from E. coli:
[0061] EC-F sequence: 5’-CCG GAATTC ATGAGCAACAATGAATTCCATC-3’(EcoRI)
[0062] EC-R sequence: 5’-CCG CTCGAG TTAATCGCTCAGCGCATCC-3’(XholI)
[0063] Primers derived from the transaminase of Bacillus subtilis:
[0064] BS-F sequence: 5’-CCC GAGCTC ATGAGTCAAACAACAGCAAGCATCA-3’(SacI)
[0065] BS-R sequence: 5’-CCC AAGCTT TTAAGCTCGCAGGCCCGCCT-3’(HindIII)
[0066] Primers for transaminase derived from Bacillus magat...
Example Embodiment
[0104] Example 2
[0105] 2.1 Cultivation of microorganisms
[0106] The composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and fixed to volume, sterilized at 121°C for 20 minutes, set aside
[0107] The genetically engineered strain E.coli BL21(DE3) containing the transaminase gene was inoculated into 5mL LB liquid medium containing 50μg / mL kanamycin, and cultured at 37°C with shaking for 12h. Transfer to 500mL fresh LB liquid medium containing 50μg / mL Kan, and shake culture to OD at 37℃ 600 When it reaches about 0.8, add IPTG to its concentration of 0.3mM, and induce culture at 28℃ for 20h. After the cultivation, the culture solution was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, the bacterial cells were collected, and stored in an ultra-low temperature refrigerator at -70°C for later use.
[0108] 2.2 Preparation of crude enzyme solution
[0109] The bacterial cells collected after the completion of t...
Example Embodiment
[0118] Example 3
[0119] Γ-aminobutyric acid / α-ketoglutarate aminotransferase derived from E.coli K12W3110, glutamate dehydrogenase derived from E.coli K12W3110, glucose dehydrogenase derived from Bacillus megaterium DSM319, See Example 1 for strain construction. After microbial cultivation, the cells were collected and the wet cell enzyme activity was measured. Refer to Example 2 for the enzyme activity determination method. The enzyme activities of the three strains of cells were 932.5U / g wet cell, 338.3U / g wet cell, and 993.3U / g Wet cell
[0120] The solution containing PPO 100 mM, glutamic acid 5 mM, pyridoxal phosphate 1 mM, glucose 120 mM, and NADH 0.1 mM was placed in a 37° C. warm bath, and the pH of the solution was adjusted to 7.5 with 30% ammonia. Add 5g / L of engineered bacteria cells expressing γ-aminobutyric acid / α-ketoglutarate aminotransferase derived from E.coli K12W3110, and engineered bacteria expressing glutamate dehydrogenase derived from E.coli K12W3110 15g...
PUM
Property | Measurement | Unit |
---|---|---|
Pre-denatured | aaaaa | aaaaa |
Extend | aaaaa | aaaaa |
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.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap