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Method for preparing high-activity recombination lipoidase

A technology of lipase and specific activity, which is applied in the field of preparation of recombinant lipase, can solve problems such as difficulty in soluble expression and difficulty in large-scale production

Inactive Publication Date: 2010-03-31
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

However, the soluble expression of the enzyme is very difficult and difficult to produce on a large scale
[0007] In addition, although there are examples of successful renaturation of some recombinant proteins using renaturation technology, there is no report on the successful renaturation of lipase by renaturation technology.

Method used

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  • Method for preparing high-activity recombination lipoidase
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preparation example Construction

[0039] One of the keys of the preparation method of the highly active recombinant lipase of the present invention lies in lipase inclusion body denaturation-renaturation technology, which mainly includes suitable denaturation conditions and optimized renaturation conditions for inclusion bodies.

[0040] In a preferred embodiment, the denaturation condition is preferably 8M urea rather than 6M guanidine hydrochloride; the refolding method is preferably a dilution refolding method; the composition of the refolding solution includes a redox pair: 0.5mM GSSG+1mM GSH; pH5.0-pH9 .0 buffer system; the buffer pair is preferably Tris-HCl and phosphate buffer; the refolding temperature is preferably low temperature, such as 4°C; the refolding time is preferably 20h-60h.

[0041]Concentration and / or purification can also be carried out for the recombinant lipase obtained after renaturation.

[0042] A preferred concentration method is anion resin adsorption; preferred anion resins inclu...

Embodiment 1

[0053] Induced Expression of Recombinant Bacteria Producing Lipase and SDS-PAGE Protein Electrophoresis Identification

[0054] I. Engineering bacteria

[0055] Escherichia coli BL21(DE3) (purchased from East China University of Science and Technology; see Jian-He Xu et al., Journal of Molecular Catalysis B: Enzymatic 47 (2007) 105-110) with the expression plasmid of the recombinant lipase gene was inoculated in the Ampicillin (100 μg / ml) solid LB medium growth. The engineered bacterium expresses lipase in the form of inclusion body, and the lipase comes from Serratia marcescens ECU1010 with the preservation number CGMCC 1219 (patent application 200410067046.1).

[0056] II. Fermentation expression

[0057] Pick a single colony of engineering bacteria and put it in 5ml LB (containing 100 μg / ml ampicillin) liquid medium, shake the bacteria overnight at 37°C; Bacteria to OD600nm 0.3-0.5, induced by IPTG, 3-5 hours after induction, centrifuged to collect the bacteria.

[...

Embodiment 2

[0062] Washing and denaturation of inclusion bodies

[0063] I. Denaturation and washing of inclusion bodies: Escherichia coli expressing lipase inclusion bodies was lysed, centrifuged, and the precipitate was collected. The identification by SDS-PAGE electrophoresis showed that the precipitate was the expression part of lipase inclusion body. The inclusion bodies were collected, suspended with 0.5% Triton-X 100, washed, centrifuged, and fully washed with Tris-HCl buffer without Triton-X 100.

[0064] II. After centrifuging the inclusion bodies thoroughly washed with 0.5% Triton-X 100, dissolve the lipase inclusion bodies separately in 8M, 6M or 4M urea according to the ratio of 20mg / ml (wet inclusion body / denatured solution), 6M or 3M guanidine hydrochloride, or 1% or 0.25% SDS; the results are shown in Table 1.

[0065] Table 1 Solubility of lipase inclusion bodies under different denaturing conditions

[0066]

[0067] After fully dissolving, centrifuge and take the s...

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Abstract

The invention discloses a method for preparing high-activity recombination lipoidase. The method comprises the following steps: (a) mixing lipase inclusion body and 3-10M urea aqueous solution so as to dissolve the inclusion body to obtain modified solution containing modified lipase; (b) diluting and renaturing the modified solution of the step (a) in renaturation solution with low ionic strength to obtain the renaturation solution containing active lipase; and (c) separating and purifying the renaturation solution of the step (b) to obtain lipase with specific activity being more than or equal to 10000U / g, or concentrating the renaturation solution of the step (b) to obtain concentration solution of the lipase with the specific activity being more than or equal to 10000U / g. The method of the invention can effectively obtain high-activity and high-purity recombination lipoidase.

Description

technical field [0001] The invention relates to a preparation method of recombinant lipase, in particular to a method for preparing high-activity and high-purity recombinant lipase through denaturation and renaturation of inclusion body. Background technique [0002] NSAIDs such as the common ketoprofen and naproxen are an important class of drugs. Since both ketoprofen and naproxen are chiral compounds, the pharmacological activities of their different enantiomers are different. Dextro-ketoprofen, or known as (S)-ketoprofen, has a very wide application in the pharmaceutical industry; and levo-ketoprofen, or known as (R)-ketoprofen Fen can be added to toothpaste to prevent and treat osteoporosis. The (S)-(+)-enantiomer of Naproxen is 28 times more active than its (R)-(-)-enantiomer. In order to improve drug efficacy, reduce drug side effects, expand drug safety range, reduce complications and correctly evaluate drugs, it is necessary to prepare optically pure single enant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20
Inventor 李素霞庞怀宇许建和
Owner EAST CHINA UNIV OF SCI & TECH
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