A kind of bentonite single-layer exfoliation nanosheet assembly enzyme and preparation method thereof
A bentonite and nanosheet technology, applied in the field of bioengineering, can solve the problems of low load, weak force, large volume, etc., and achieve the effects of enhancing enzyme catalytic activity, improving catalytic stability, and reducing shedding problems.
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Embodiment 1
[0033] Weigh 4.5 g of NaCl and dissolve it in 500 mL of acetate buffer solution at pH 4.0, add 5 g of sodium bentonite to make a bentonite suspension with a concentration of 1%. At room temperature, place the suspension in a sonicator for sonication (frequency 40KHz), and at the same time turn on the stirrer to stir at 3000rpm, and stop 20min after every 1h of sonication. After continuous treatment for 4 hours, centrifuge at 3000 rpm for 10 minutes to discard the precipitate, add pH 4.0 acetate buffer to dilute to obtain 1 mg / mL bentonite exfoliated nanosheet colloidal solution. Take 100 mL of the colloidal solution, add 100 mg of bovine pancreatic lipase and mix, and shake the mixture at 25° C. and 200 rpm for 20 h. After the end, the mixture was centrifuged at 15000 rpm for 10 min to obtain a precipitate. The precipitate was washed several times with deionized water until neutral, and then freeze-dried for 12 hours to obtain a solid immobilized lipase. The supernatant and ...
Embodiment 2
[0036] Weigh Na 2 SO 4 3 g was dissolved in 500 mL of acetate buffer solution at pH 4.0, and 5 g of sodium bentonite was added to make a bentonite suspension with a concentration of 1%. At room temperature, place the suspension in a sonicator for sonication (frequency: 50KHz), and at the same time turn on the stirrer to stir at 2500rpm, and stop for 20min after every 1h of sonication. After continuous treatment for 3 hours, centrifuge at 3000 rpm for 10 minutes to discard the precipitate, add pH 4.0 acetate buffer to dilute to obtain 2 mg / mL bentonite exfoliated nanosheet colloidal solution. Take 100 mL of this solution, add 400 mg of lipase YLL (Yarrawia lipolytica lipase), mix and shake at 25°C and 200 rpm for 24 hours. After the end, the mixture was centrifuged at 15000 rpm for 10 min to obtain a precipitate. The precipitate was washed several times with deionized water until neutral, and then freeze-dried for 12 hours to obtain a solid immobilized lipase. The supernata...
Embodiment 3
[0038] Weigh 4.5g of NaCl and dissolve it in 500mL of pH6.0 phosphate buffer solution, add 2.5g of calcium-based bentonite to make a bentonite suspension with a concentration of 0.5%. At room temperature, place the suspension in a sonicator for sonication (60KHz frequency), and at the same time turn on the stirrer to stir at 3000rpm, and stop 20min after every 1h of sonication. After continuous treatment for 5 h, centrifuge at 3000 rpm for 10 min to discard the precipitate, add pH 6.0 phosphate buffer to dilute to obtain 0.5 mg / mL bentonite exfoliated nanosheet colloidal solution. Take 100 mL of this solution, add 180 mg of trypsin, and shake at 25° C. and 200 rpm for 24 h. After the end, the reaction solution was centrifuged at 15000 rpm for 10 min to obtain a precipitate. The precipitate was washed several times with deionized water until neutral, and the immobilized trypsin solid was obtained after freeze-drying for 12 hours. The supernatant and washing liquid were combin...
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