Construction method of high-temperature-resistant carboxypeptidase in bacillus subtilis and application of high-temperature-resistant carboxypeptidase in low-bitterness plant oligopeptides
A technology of Bacillus subtilis and carboxypeptidase, which is applied in the field of protein processing, can solve the problems of limited application research, high cost of carboxypeptidase, low production of carboxypeptidase, etc., and achieve high broad-spectrum activity, reduced bitterness, and hydrolysis degree Enhanced effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0077] Example 1: Construction of recombinant Bacillus subtilis expressing carboxypeptidase
[0078] The specific implementation method steps are as follows:
[0079] (1) Amplification of carboxypeptidase gene
[0080] According to the deduced carboxypeptidase gene sequence of Bacillus megaterium (GenBank: CP026736.1) disclosed on NCBI, a pair of primers were designed, and the primers were designed as follows:
[0081] Upstream primer F: 5'-tgcaaaaagtgaaatcagggatgagagaagacatacagaaaattgagaa-3';
[0082] Downstream primer R: 5'-aggtgaatttcgacctctagtcagtggtgatggtgatgatgtacgtgataaatttctccatatttcttttctaga-3'.
[0083] Using the genome as a template, the carboxypeptidase gene fragment (nucleotide sequence shown in SEQ ID NO.2) was obtained by PCR technology. The PCR system was: (10 μL): 5 μL of mix enzyme, 4.4 μL of water, 0.2 μL of template, Upstream primer F 0.2 μL, downstream primer R 0.2 μL.
[0084] PCR conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95...
Embodiment 2
[0095] Example 2: Using Bacillus subtilis to express carboxypeptidases from different sources
[0096] Specific steps are as follows:
[0097] 1. Preparation of carboxypeptidase
[0098] (1) Preparation of crude carboxypeptidase enzyme solution
[0099] B.subtilis WB600 / pMA5-CPM32, B.subtilis WB600 / pMA5-CPK, B.subtilis WB600 / pMA5-CPJ, B.subtilis WB600 / pMA5-CPL prepared in Example 1 were respectively scribed at 50 μg·ml -1 On a solid LB plate containing Kan antibiotics, culture overnight in a 37°C incubator. On the second day, pick a single colony into 10 mL of LB medium and add 50 μg·ml -1 Kan antibiotics at 37°C, 150r min -1 Cultivated under conditions for 12h to prepare seed liquid;
[0100] The prepared seed solution was heated at 37 °C, 200 r min -1 Fermentation for 24h under the condition of
[0101] The above fermentation broths were respectively heated at 4 °C for 10000 r min. -1 Centrifuge for 15 min, collect the supernatant (repeated twice); prepare separately:...
Embodiment 3
[0128] Example 3: Effects of different signal peptides on the enzymatic activity of carboxypeptidase M32
[0129] Specific steps are as follows:
[0130] By adding the other three signal peptides in the Sec pathway, AprE, AmyQ (the nucleotide sequence of the signal peptide AmyQ is shown in SEQ ID NO. 6, and the nucleotide sequence of the signal peptide AprE is shown in SEQ ID NO. 7), NprB (The nucleotide sequence is shown in SEQ ID NO. 8), and the addition method is as follows Figure 9 shown, the specific steps are:
[0131] (1) Redesign primers according to the signal peptide sequence disclosed on NCBI, and the primer design is shown in Table 4:
[0132] Table 4 Signal peptide primers
[0133]
[0134] Note: Underline indicates homology arm, bold indicates 6×His tag, italics indicates signal peptide
[0135] (2) Using the Bacillus megaterium genome preserved in the laboratory as a template, the nucleotide sequences of the signal peptides including the signal peptide A...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More