A mutant of Bacillus licheniformis lysozyme and its application in preservation of rainbow trout
A technology of Bacillus licheniformis and lysozyme, applied in the application, enzyme, hydrolase and other directions, can solve the problem of low specific enzyme activity, and achieve the effects of simple preparation method, good application prospect, strong killing and inhibition effect
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Embodiment 1
[0017] Example 1 Construction of lysozyme mutant plasmids pPIC9K-AmyL-lysozyme-M1, pPIC9K-AmyL-lysozyme-M2 and pPIC9K-AmyL-lysozyme-M3
[0018] In order to further improve the specific enzyme activity of lysozyme derived from Bacillus licheniformis, it is necessary to carry out molecular modification of lysozyme to develop a new type of lysozyme that is more suitable for modern production needs. The hydrophobic short peptide was linked to the C-terminus of egg white lysozyme through recombinant technology, and it was found that the modified lysozyme could significantly improve the bactericidal activity against Escherichia coli. The hydrophobic short peptides Val-Leu-Ile-Ala-Pro, Val-Ala-Leu-leu-Phe or Val-Leu-Leu-Ala-Ile-Ile-Phe were selected for screening and C-terminal fusion was performed, and the effect on Bacillus licheniformis Lysozyme undergoes hydrophobic modification to enhance its antibacterial effect.
[0019] The original Bacillus licheniformis lysozyme gene (see ...
Embodiment 2
[0026] Example 2 Construction of wild-type and mutant lysozyme Pichia recombinant strains
[0027] The constructed lysozyme mutant plasmid and the original wild-type plasmid were linearized with Kpn2I endonuclease, and the recovered fragment was electrotransformed into Pichia pastoris GS115, and coated with MD (1.34% YNB, 2% glucose, 4× 10 -5 % Biotin, 1.5% agar) plates, cultured at 30°C for 48h. Pick a single colony from the plate for colony PCR verification. Select the correct positive clones and inoculate them in YPD (1% yeast extract, 2% peptone, 2% glucose) medium containing 5 mL of 0.25 mg / mL G418, culture at 30 °C for 48 h, and store the culture at the final concentration of 20% glycerol, frozen in -80 ℃ refrigerator, thus obtained wild-type lysozyme Pichia expression strain GS115 (AmyL-lysozyme) and lysozyme mutant Pichia expression strain GS115 (AmyL-lysozyme- M1), GS115 (AmyL-lysozyme-M2) and GS115 (AmyL-lysozyme-M3).
Embodiment 3
[0028] Example 3 Induced fermentation of lysozyme recombinant strain
[0029]Streak the expressing bacteria of lysozyme wild type and mutants frozen in -80°C refrigerator in Example 2 on the YPD plate containing 0.25 mg / mLG418, and culture at 30°C for 48h. Select a single clone colony and inoculate it into a test tube containing 5 mL of YPD medium containing 0.25 mg / mL G418, culture it at 200 rpm at 30°C for 24 hours, and inoculate it into a test tube containing 100 μg / mL ampicillin, 50 μg / mL kanamycin in 30 mL BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 4×10 -5 %Biotin, 1% glycerol (V / V), 1% potassium phosphate buffer (pH6.0)) in a 250mL shake flask, cultured at 200 rpm at 30°C for 24 h, and then inoculated to Contain 30 mL of BMMY containing 1% methanol ((1% yeast extract, 2% peptone, 1.34% YNB, 4×10 -5 % Biotin, 1% Methanol (V / V), 1% Potassium Phosphate Buffer (pH 6.0)) culture medium in 250mL shake flasks, cultured at 30°C and 200rpm for 7 days. Add 0.5% methanol ev...
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