Method for synthesizing L-theanine through enzyme process
A technology of enzymatic synthesis and theanine, applied in the field of bioengineering, to achieve the effects of good thermal stability, good activity and stability, and short reaction time
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0023] Example 1: Construction of ggt-pET20b(+) / E.coli BL21(DE3)
[0024] (1) According to the γ-glutamyl transpeptidase gene sequence of Bacillus subtilis 168 (NCBI Gene ID: 940001) on NCBI, the γ-glutamyl transpeptidase gene was obtained by chemical synthesis, and the gene was used as a template to design Primers P1, P2, P1: 5'–AT CCATGG ATAAAAAAACCGCCCAAAAGCTACGA–3’ (underlined is the enzyme cutting site NocI), P2: 5’–GGCG CTCGAG TTATTTACGTTTTAAATTAATGC-3' (the underline is the restriction site XhoI), the gene was amplified by PCR. The PCR system was 50 μl, and the PCR program was: pre-denaturation at 94°C for 4 minutes, followed by 30 cycles of "incubating at 98°C for 10 s, 55°C for 5 s, and 72°C for 120 s", and finally at 72°C for 10 min. The amplified target fragment was recovered and purified by gel and ligated with pMD18-T simple vector. After the ligation product is chemically transformed into Escherichia coli JM109, the transformation mixture is spread on an amp...
Embodiment 2
[0027] Embodiment 2: fermentation produces enzyme
[0028] (1) Fermentation culture
[0029] The genetically engineered bacteria ggt-pET20(b) / E.coli BL21(DE3) obtained in Example 1 was inoculated in LB / Amp liquid medium and then cultured at 37°C for 8-10 hours and then transferred to TB / Amp liquid medium containing 0.75% glycine, cultured at 37°C for 3 hours, then induced with 0.4mM / L IPTG (isopropylthio-β-D-galactoside), cooled to 25°C and incubated at a constant temperature for 40-48h to induce Enzymes. After the fermentation, the supernatant collected by centrifugation is the crude enzyme liquid.
[0030] (2) Enzyme activity assay
[0031] The color reaction was carried out with γ-glutamyl-p-nitroaniline (γ-GpNA) and glyceryl dipeptide (Gly-Gly) as substrates. The enzyme activity assay system is 1mL (contains a final concentration of 5mmol / L γ-glutamyl p-nitroanilide (γ-GpNA), 80mmol / L glyceryl dipeptide (Gly-Gly), 50mM borax-NaOH buffer, pH10) After adding 20 μL of appr...
Embodiment 3
[0033] Embodiment 3: the concentration of crude enzyme liquid
[0034] Slowly add thionine with a concentration of 60% relative to the mass fraction of the enzyme solution to the enzyme solution obtained in Example 2 while stirring, stir until the thionine is dissolved, and stand at 4°C for 8-10 hours to precipitate protein. The mixture was centrifuged (8000rpm, 10min) to collect the precipitate, and then reconstituted with a minimum volume of 50mM Tris-HCl buffer (pH8). According to the difference in reconstituted volume, the enzyme activity of the concentrated enzyme solution is 300-400U / ml.
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