Carrier-free immobilized Rhizopus oryzae lipase and its preparation method and application in the production of biodiesel
A carrier-free immobilization technology of Rhizopus oryzae lipase, which is applied in biofuels, multi-enzyme systems, fermentation, etc., can solve the problems of poor efficiency and reusability, and achieve increased reuse rate, good effect, The effect of the simple preparation process
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Embodiment 1
[0044] Preparation of carrier-free immobilized Rhizopus oryzae lipase: extract the total RNA of Rhizopusoryzae IFO 4697, use the total RNA as a template to reverse transcribe to obtain cDNA, design primers to amplify the gene of lipase ROL on the cDNA, and then use EcoRI and KpnI two The restriction site connects the ROL gene to the Pichia pastoris expression vector pPICZα, and electrotransforms it into Pichia pastoris X33 for intracellular recombinant expression. On the culture medium containing olive oil, the colonies confirmed to express the lipase were picked and fermented for enzyme production. The Pichia pastoris fermentation broth containing lipase is collected, and then the fermentation broth is freeze-dried. Purify the fermentation broth of Pichia pastoris X33 expressing lipase, collect the solution containing lipase after purification and freeze-dry, dissolve 100mg of lipase freeze-dried powder in 2mL of phosphate buffer, centrifuge for 10mim, and take 10 mg bovine ...
Embodiment 2
[0047] Preparation of carrier-free immobilized Rhizopus oryzae lipase: obtain the fermentation broth of Pichia pastoris X33 expressing lipase according to the method of Example 1, collect and purify the solution containing lipase and freeze-dry, and dissolve 60 mg of lipase freeze-dried powder In 1.5mL of phosphate buffer, centrifuge for 10mim, take the supernatant and add 6mg of bovine serum albumin (BSA); add 2mL of ethylene glycol dimethyl ether dropwise at 4°C to precipitate the lipase, and let it stand After 30min, a solution containing lipase precipitation was formed; in the prepared solution, 30% (v / v) formaldehyde solution was added dropwise to form a mixed solution, and then the mixed solution was put into a 37° C. shaker and incubated at 200 rpm. Add 1 mL of acetone to the mixed solution, centrifuge for 10 min, discard the supernatant, and wash the precipitate three times with pre-cooled acetone to obtain lipase cross-linked enzyme aggregates.
[0048] Biodiesel prep...
Embodiment 3
[0050] Preparation of carrier-free immobilized Rhizopus oryzae lipase: obtain the fermentation broth of Pichia pastoris X33 expressing lipase according to the method of Example 1, collect and purify the solution containing lipase and freeze-dry, and dissolve 50 mg of lipase freeze-dried powder In 1mL of phosphate buffer, centrifuge for 10mim, take the supernatant and add 5mg of bovine serum albumin (BSA); add 1mL of ethylene glycol dimethyl ether dropwise at 4°C to precipitate the lipase, and let it stand for 30min Finally, a solution containing lipase precipitation is formed; in the prepared solution, 30% (v / v) glutaraldehyde solution is added dropwise to form a mixed solution, and then the mixed solution is put into a 37° C. shaker and incubated at 200 rpm, Add 1 mL of acetone to this mixed solution, centrifuge for 10 min, discard the supernatant, and wash the precipitate three times with pre-cooled acetone to obtain lipase cross-linked enzyme aggregates.
[0051] Preparatio...
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