Bacillus licheniformis for degrading ethyl carbamate and ethyl carbamate precursors
A technology of Bacillus licheniformis and ethyl carbamate, which is applied in the field of microorganisms and can solve problems such as the inability to fully reduce the content of ethyl carbamate
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Embodiment 1
[0034] Example 1: Screening of Bacillus licheniformis DX530
[0035] Separation of microorganisms from fermented grains: fresh fermented grains were mixed with sterile saline in an appropriate ratio, the mixture was diluted in gradient and spread on the MRS plate. Place the MRS plate at 37°C / 30°C for 24 hours until a single colony appears;
[0036] Isolation of citrulline-utilizing strains: the strains isolated on the MRS plate were evenly inoculated into 96 deep-well plates containing citrulline-utilizing medium, and cultured at 37°C for 24 hours.
[0037] Screening of strains with citrulline utilization ability: centrifuge and collect 200 μL of supernatant in a 96-well plate, add 300 μL of Fe-containing 3+ Ion mixed acid and 50 μL of diacetyl monoxime-thiosemicarbazide solution, diacetyl monoxime is converted into diacetyl (diacetyl) under acidic conditions, diacetyl and urea are condensed into red diazine compounds under strong acid conditions, due to The citrulline molec...
Embodiment 2
[0039] Embodiment 2: Analysis of enzyme production ability of Bacillus licheniformis DX530
[0040] Perform liquid culture on the primary screened strains in MRS medium, culture at 37°C for 24h, take 50mL of bacterial liquid at 4000rpm / min for 10min, wash twice with citric acid-sodium citrate buffer, and finally resuspend with 50mL of buffer , ice-bathed for 30 minutes, and mechanically broken for 120 seconds to obtain a crude enzyme solution.
[0041] The enzyme activity of urease and ethyl carbamate hydrolase in the crude enzyme solution was measured, and the results showed that the activity of urease was 0.23U / mL, and the activity of ethyl carbamate hydrolase was 0.27U / mL.
Embodiment 3
[0042] Example 3: Verification of key genes in the arginine deiminase pathway of Bacillus licheniformis DX530
[0043] Using the genomic DNA of Bacillus licheniformis as a template, the primers in Table 1 were used for PCR amplification. The PCR reaction system is: 1 μL of genomic DNA, 25 μL of 2×Primer star, 1 μL of upstream and downstream universal primers, and finally add double distilled water to 50 μL. The reaction conditions were as follows: pre-denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 90 seconds, a total of 34 cycles, and extension at 72°C for 10 minutes. The verification result is as figure 2 shown.
[0044] Table 1. The primers used to verify the ADI approach in the present invention
[0045]
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