Keratinase mutants with thermostability and catalytic activity improved
A kind of keratinase mutation, keratinase technology, applied in the directions of hydrolase, botanical equipment and method, biochemical equipment and method, etc., can solve the problem of low extracellular catalytic activity and the like
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Embodiment 1
[0024] Example 1: Construction of keratinase site-directed mutants
[0025] (1) Selection of keratinase P1C2T2 mutation site
[0026] Based on the keratinase mutant P1C2T2 in the previous patent (CN201410276309.3) of this experiment, five site-directed mutants were constructed.
[0027] In the present invention, the crystal structure (PDB ID: 3TI7) of subtilisin BprV with the highest degree of similarity is used as a template, and the three-dimensional simulation structure of keratinase P1C2T2 mature protein is constructed by Modeller V9.11 software ( figure 1 ). Through amino acid primary sequence alignment, it was found that the similarity between subtilisin BprV and keratinase P1C2T2 reached 47%. Although the model obtained by using Modeller software can largely meet the requirements of rational design, further optimization is still needed. The invention adopts NAMD molecular dynamics software to carry out molecular dynamics calculation on the initially constructed model ...
Embodiment 2
[0035] Example 2: Expression and purification of keratinase mutants
[0036]Pick and transfer the positive single clone of Escherichia coli BL21(DE3) into the expression host and grow in LB liquid medium (containing 80 μg / ml ampicana antibiotic) for 8-10 hours, and transfer the seed fermentation broth to TB liquid culture according to 5% inoculation amount base (containing 80 μg / ml ampicana antibiotic); Escherichia coli was cultured on a shaker at 37°C for 2 hours until OD 600 = about 1.2, add 0.2mM final concentration of IPTG to induce cells to express keratinase extracellularly, and continue to culture and ferment on a shaker at 20°C for 48h, then centrifuge the fermentation broth at 4°C and 8000rpm for 15min to remove bacteria, and collect the centrifuged fermentation. The supernatant was analyzed by SDS-PAGE. It was found that the extracellular protein band was single, and the molecular weight of the site-directed mutant was close to the 46kDa of P1C2T2 ( figure 2 ), an...
Embodiment 3
[0038] Embodiment 3: enzyme activity analysis method
[0039] 1) Enzyme activity assay method
[0040] The enzymatic activity of keratinase was determined by Folin-phenol reagent chromogenic method. Under certain conditions, keratinase hydrolyzes keratin substrates to release tyrosine. Folin-phenol reagent can be reduced by phenolic compounds under alkaline conditions to turn blue (a mixture of molybdenum blue and tungsten blue), while the keratin substrate is hydrolyzed to release free tyrosine as phenolic substances, and color development occurs The reaction can be within a certain range. The depth of the color is proportional to the release of tyrosine, so the colorimetry can be performed at a wavelength of 660nm to calculate the enzyme activity. Definition of enzyme activity unit: Under the above conditions, with the unreacted sample as a blank control, the amount of enzyme per milliliter that catalyzes the decomposition of keratin to generate 1 μg of tyrosine per minute...
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