Magnetic chitosan microsphere for enzyme immobilization as well as preparation method and application thereof
A technology of chitosan microspheres and enzyme immobilization, which is applied in the direction of immobilization on/in organic carriers, can solve the problems of samples having to be diluted, large loss of carrier recovery, etc., and achieve easy separation and recovery and recovery of enzyme activity. The effect of high rate and good mechanical properties
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Embodiment 1
[0049] (1) Weigh 0.5g chitosan powder in a 100mL three-necked bottle, add 30mL mass fraction of 5% acetic acid solution, stir at room temperature, after the chitosan is fully dissolved, add 30mL liquid paraffin, and stir for 10 minutes;
[0050] (2) Warm up the product of step (1) to 50°C, add 2-3 drops of emulsifier Span80 dropwise, and emulsify for 10 minutes to form fine chitosan droplets;
[0051] (3) Add 1.0 mL of formaldehyde solution to the obtained product of step (2), and stir and react for 1.5 hours;
[0052] (4) Warm up the product of step (3) to 70°C, add dropwise 5-6 drops of NaOH solution with a mass fraction of 10%, keep the pH of the solution alkaline, and slowly add 2.0mL of epoxy chloride Propane, reacted for 5 hours;
[0053] (5) The gain of step (4) is filtered, washed with distilled water, then washed with sherwood oil, absolute ethanol, and water successively until the pH value of the washing liquid reaches neutrality, and chitosan microspheres (NCTS) are ...
Embodiment 2
[0097] (1) Accurately weigh 10 mg of modified chitosan microsphere carrier (NCTS-EDA), and separate after swelling with 10 mL of phosphate buffer solution (PBS, pH=7) for 24 hours;
[0098](2) Add 10 mL of 5% glutaraldehyde solution to the obtained product of step (1), shake and activate at 25° C. for 5 hours and filter, and wash off excess glutaraldehyde with distilled water;
[0099] (3) Add 2.0 mg of cellulase enzyme solution to the obtained product of step (2), shake and fix at 30° C. for 4 hours, filter, and wash the filter residue repeatedly with phosphate buffer to obtain immobilized enzyme. The immobilized enzymes were stored in the refrigerator (4°C) for later use.
[0100] The method for measuring cellulase activity and the method for measuring the enzyme activity of the immobilized enzyme obtained in Example 2 will be described below.
[0101] Determination of free enzyme activity: Take an appropriate amount of cellulase and dissolve it in HAc-NaAc buffer to prepar...
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