Method for preparing gold nanoparticles by using keratinase and application thereof
A technology of gold nanoparticles and keratinase, which is applied in the field of biological preparation of gold nanoparticles, can solve the problems of uneven size distribution of nanoparticles, high requirements, high reaction energy consumption, etc., to solve the limitation of preparation equipment, reduce pollution, and simplify detection The effect of steps
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Embodiment 1
[0022] Enzyme production fermentation optimization and high-efficiency expression of embodiment 1 keratinase
[0023] B. subtilis WB600 was selected as the host bacterium, and the keratinase gene kerBv was recombined to obtain the recombinant bacterium B. subtilis WB600 / pMA5-kerBv. Enzyme production experiments in shake flasks and 5L fermenters.
[0024] (1) Carbon source experiment
[0025] According to the enzyme activity measured by the enzyme solution taken out at 48 hours, among the different carbon sources selected, the enzyme activity produced by the bacteria with glycerol as the carbon source was higher, and the glycerol content in the medium was 2%. At present, other studies have shown that the addition of glucose in the medium can inhibit the enzyme production of the strain, which is consistent with the results of this experiment.
[0026] (2) Optimization of carbon source concentration
[0027] After confirming that glycerol was the best carbon source, different ...
Embodiment 2
[0050] Embodiment 2 keratinase prepares gold nanoparticles
[0051] Through the bioreduction method, the enzyme solution fermented by the strain is used as a reducing agent to reduce the chloroauric acid to an atomic state, and obtain spherical and dispersed gold nanoparticles. The specific process is as follows:
[0052] The keratinase enzyme liquid obtained by fermentation on the tank is sterilized by filtration with a 0.22 μm microporous membrane for use. The biosynthesis process of nano-gold was gradually optimized by controlling three factors in the synthesis reaction, namely the concentration of chloroauric acid (0.5, 1, 1.5, 2, 2.5, 5 mM; figure 1 ), synthetic reaction time (1, 2, 3, 4, 5, 6h, etc.; figure 2 ), and the amount of enzyme added (700, 875, 1050, 1225, 1400, 1575, 1750U; image 3 ). The biosynthesis reaction was carried out in a 10 mL centrifuge tube with a reaction volume of 8 mL. The reaction container was wrapped with tin foil, and the reaction was s...
Embodiment 3
[0054] The characterization of embodiment 3 gold nanoparticles
[0055] The above-mentioned solutions with obvious absorption peaks were measured with a particle size analyzer, and the results of the experimental group were as follows: Figure 4 , it can be seen from the figure that the particle size of the obtained nano-gold solution is about 75nm, and the particle size measured by DLS will be higher than its true value. This phenomenon has also been reported many times in other studies. However, after the oscillation time exceeds 5 hours, a large amount of gold nanoparticles will aggregate and produce visible precipitation; when the oscillation reaction is less than 5 hours, there is no obvious absorption peak at around 550nm, indicating that the particles have not yet formed in large quantities.
[0056] Figure 5 As the particle size measurement results of the gold nanoparticles synthesized by the control group, it can be seen that the particle size of the control group i...
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