Carrier-free immobilized rhizopus oryzae lipase as well as preparation method thereof and application thereof in producing biodiesel

Abstract

The invention provides carrier-free immobilized rhizopus oryzae lipase as well as a preparation method thereof and an application thereof. The preparation method comprises the following steps: (1) charging a protein cross-linking additive into a buffer solution containing rhizopus oryzae lipase to form a first solution, wherein the protein cross-linking additive is serum albumin; (2) charging glycol dimethyl ether into the first solution to precipitate the rhizopus oryzae lipase, and standing the solution to obtain a second solution containing the rhizopus oryzae lipase; (3) charging a cross-linking agent into the second solution to form a mixed solution, then incubating the mixed solution, wherein the cross-linking agent is selected from acetic anhydride, formaldehyde and glutaraldehyde; (4) precipitating the mixed solution incubated in the step (3), to obtain a cross-linked enzyme aggregation. The carrier-free immobilized rhizopus oryzae lipase is high in catalytic oil transesterification reaction and is still high in activity after being reused for multiple times.

Description

technical field

[0001] The invention relates to carrier-free immobilized lipase and its preparation method and application, in particular to carrier-free immobilized Rhizopus oryzae lipase and its preparation method and application for biodiesel production. Background technique

[0002] Lipase (Lipase, EC3.1.1.3), that is, triacylglycerol acyl hydrolase, is a class of enzymes that can not only catalyze the hydrolysis of lipids but also carry out transesterification reactions. Lipase widely exists in many animals, plants and microorganisms, especially in microorganisms. At present, 65 genera of microorganisms including bacteria, actinomycetes, yeast and filamentous fungi are known to produce lipase. Most lipases can be used to catalyze the hydrolysis of oils, but only some lipases can be used to catalyze the transesterification of oils. Lipase hydrolyzes oil to generate free fatty acids and glycerol, monoglycerides or diglycerides, and its transesterification reaction is to...

Examples

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, lipase freeze-dried powder 60mg Dissolve in 1.5mL of phosphate buffer, centrifuge at 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, statically After leaving for 30 minutes, a solution containing lipase precipitation was formed; 30% (v / v) formaldehyde solution was added dropwise to the prepared solution to form a mixed solution, and then the mixed solution was incubated at 200 rpm in a shaker at 37° C. 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.

[0048] Biodiesel preparatio...

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, lipase freeze-dried powder 50mg Dissolve 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 After 30min, a solution containing lipase precipitation was formed; 30% (v / v) glutaraldehyde solution was added dropwise to the prepared solution to form a mixed solution, and then the mixed solution was incubated at 200rpm in a shaker at 37°C 1 mL of acetone was added to the mixed solution, centrifuged for 10 min, the supernatant was discarded, and the precipitate was washed three times with pre-cooled acetone to obtain lipase cross-linked enzyme aggregates.

[0051] Preparation of ...

technical field

[0001] The invention relates to carrier-free immobilized lipase and its preparation method and application, in particular to carrier-free immobilized Rhizopus oryzae lipase and its preparation method and application for biodiesel production. Background technique

[0002] Lipase (Lipase, EC3.1.1.3), that is, triacylglycerol acyl hydrolase, is a class of enzymes that can not only catalyze the hydrolysis of lipids but also carry out transesterification reactions. Lipase widely exists in many animals, plants and microorganisms, especially in microorganisms. At present, 65 genera of microorganisms including bacteria, actinomycetes, yeast and filamentous fungi are known to produce lipase. Most lipases can be used to catalyze the hydrolysis of oils, but only some lipases can be used to catalyze the transesterification of oils. Lipase hydrolyzes oil to generate free fatty acids and glycerol, monoglycerides or diglycerides, and its transesterification reaction is to...

Examples

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, lipase freeze-dried powder 60mg Dissolve in 1.5mL of phosphate buffer, centrifuge at 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, statically After leaving for 30 minutes, a solution containing lipase precipitation was formed; 30% (v / v) formaldehyde solution was added dropwise to the prepared solution to form a mixed solution, and then the mixed solution was incubated at 200 rpm in a shaker at 37° C. 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.

[0048] Biodiesel preparatio...

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, lipase freeze-dried powder 50mg Dissolve 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 After 30min, a solution containing lipase precipitation was formed; 30% (v / v) glutaraldehyde solution was added dropwise to the prepared solution to form a mixed solution, and then the mixed solution was incubated at 200rpm in a shaker at 37°C 1 mL of acetone was added to the mixed solution, centrifuged for 10 min, the supernatant was discarded, and the precipitate was washed three times with pre-cooled acetone to obtain lipase cross-linked enzyme aggregates.

[0051] Preparation of ...

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