Imprinted lipase and application thereof
A lipase and imprinting technology, applied in the directions of enzymes, hydrolases, enzymes, etc., can solve the problems of difficult separation, low optical purity of L-lactic acid, and high impurity content, and achieve high optical purity, simple operation, and high enzyme activity. Effect
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[0062] Example 1
[0063] I. Preparation of molecular blotting crosslinked enzyme
[0064] The 100 mg Antarctic Pseudoma yeast fat enzyme B was dissolved in 10 ml of phosphate buffer (pH 7.0, 10 mM), and 500 mg high-optically purity L-lactic acid was added as a blot template, mixed under 4 ° C, 400 rpm, mixed 20 min, then add Liquid nitrogen or in an ultra-low temperature refrigerator is frozen overnight, followed by freezing the vacuum freeze drying machine. 50 ml of isopropanol reagent was added to lyophilized enzyme powder to obtain isopropanol solution, where isopropyl alcohol reagent serve as a precipitated enzyme protein molecule, while simultaneously removing the printing template as detergent. The SMCC of 0.05 g / 100 mL (volume meter of isopropyl alcohol) was then crosslinked as a crosslinking agent. After mixing 20 min under 400 rpm, centrifuged at 4 ° C, 6000 rpm. The supernatant was discarded, 50 ml of isopropanol reagent was added to the precipitate, 4 ° C, 8000 rpm c...
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[0073] Example 2
[0074] I. Preparation of molecular blotting crosslinked enzyme
[0075] The 200 mg Antarctic Pseudoma yeast fat enzyme B was dissolved in 10 ml of phosphate buffer (pH 7.0, 10 mM), and 300 mg of high-optically purity L-lactic acid was added as a blot template, mixed under 4 ° C, 300 rpm, mixed 25 min, then joined Liquid nitrogen or in an ultra-low temperature refrigerator is frozen overnight, followed by freezing the vacuum freeze drying machine. 80 ml of isopropanol reagent was added to lyophilized enzyme powder to obtain an isopropanol solution, of which isopropanol reagent serves as a precipitated enzyme protein molecule as a detergent. The SMCC of 0.07 g / 100 ml (volume meter of isopropyl alcohol) was added as a crosslinking agent. After mixing 25 min under 300 rpm, centrifuged at 4 ° C, 6000 rpm. The supernatant was discarded, and 80 ml of isopropanol reagent was added again in the precipitate, 4 ° C, 8000 rpm centrifugation was washed 3 times. Finally, th...
Example Embodiment
[0084] Example 3
[0085] I. Preparation of molecular blotting crosslinked enzyme
[0086] Weigh 400mg of porcine pancreatic lipase was dissolved in 10ml phosphate buffer solution (pH 7.0, 10) was added 900mg L- lactic acid with high optical purity as western blot template at 4 ℃, 35min at 500rpm mixing conditions, followed by addition of liquid nitrogen or into the ultra-low temperature refrigerator freezer overnight and then transferred to a vacuum freeze dryer and lyophilized. Was added to the enzyme powder after lyophilization 90mL isopropanol, to give isopropanol enzyme solution, wherein the precipitation of isopropanol as a precipitant reagent enzyme protein molecule, while removing the imprinting template as a detergent. The enzyme solution was added again isopropanol 0.09g / 100ml (isopropanol in volume) as a crosslinking agent SMCC crosslinking. After 35min at 500rpm mixing conditions, at 4 ℃, centrifuged at 6000rpm 20min under conditions. The supernatant was discarded, 9...
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