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Activity remodeling method for changing lipase catalytic activity

A technology of catalytic activity and lipase, applied in the field of enzyme engineering, can solve the problems of lipase losing ester synthesis ability, lipase not showing esterification activity, etc.

Active Publication Date: 2016-06-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The present invention adopts the method of in vitro refolding to process the lipase, obtains the lipase with the ability to catalyze ester synthesis, and solves the problems that many lipases do not show esterification activity and lipase loses the ability of ester synthesis after heterologous expression of engineering bacteria problems, laid the foundation for broadening the industrial application of lipase

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  • Activity remodeling method for changing lipase catalytic activity
  • Activity remodeling method for changing lipase catalytic activity
  • Activity remodeling method for changing lipase catalytic activity

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1: Extraction of Membrane Environment Complex WTS

[0071] TritonX-100, CHAPS, Tween80, deoxycholic acid and other surfactants were used to extract membrane environment complexes and membrane-bound proteins from the cultured Rhizopus sinensis, and then passed through the Bio-BeadsSM-2 adsorbent of American bio-rad company. Adsorb the surfactant in the extract to precipitate the membrane-bound protein, and the supernatant obtained after high-speed centrifugation is the membrane environment complex. The specific method is as follows:

[0072] Rhizopus sinensis CCTCCM201021 was cultured on potato-dextrose agar medium at 30°C for 3 days, and the spores were washed with sterile water. 200μl of spore suspension was inoculated into a shake flask (250mL Erlenmeyer flask) containing 20mL of fermentation medium, at 30°C, 150r min -1 Conditioned for 72h. The concentration of spore suspension is about 10 7 pcs ml -1 . After the bacterial cell culture, the mycelium was...

Embodiment 2

[0074] Example 2: Active Remodeling Method

[0075] Perform in vitro refolding by any of the following methods:

[0076] Protocol A: Adding Membrane Environment Complexes During Refolding

[0077] (1) Protein denaturation: take lipase protein solution or suspension, slowly add 0.5g of ground urea powder per milliliter of solution under low temperature shaking, stir magnetically at 4°C for 2-4h, take out 100μL every half hour for hydrolysis activity Determination, complete loss of activity means that the tertiary structure is completely opened to form a peptide chain, and the protein is completely denatured. Denatured protein 12000r·min -1 Centrifuge for 10 minutes, take the supernatant for concentration determination, and ensure that the protein concentration is not higher than 2.5 mg·mL -1 , to ensure that the protein can be completely refolded. (2) Protein renaturation: Add excess prepared membrane environment complex at a volume ratio of 1:1, stir magnetically for 5-10 ...

Embodiment 3

[0083] Example 3: Escherichia coli heterologous expression of Rhizopus chinensis mature peptide lipase MP 1 Restoration of vitality

[0084]Primers were designed according to the whole genome sequence of Rhizopus sinorifica lipase, and the mature peptide lipase gene A06672 of Rhizopus sinensis was amplified by PCR (http: / / genome.jgi.doe.gov / Rhich1 / Rhich1.home. html) Connection expression vector 6xHis-MBP 3 -TEV-pET15 (constructed by inserting the MBP fusion protein gene fragment (J.Am.Chem.Soc.2012,134,375–385) with a 6xHis tag at the N-terminal of pQEH6MBP and containing a TEV protease cleavage site into pET15), Transformation of E.coliBL21trxB(DE3), IPTG induction to obtain fusion protein MBP (His)8 -MP 1 , HisTrap was purified and digested with TEV protease, and then purified by HisTrap and DEAE column to obtain pure enzyme MP 1 .

[0085] According to the scheme B and scheme A of Example 2, add the membrane environment complex during denaturation and add the membrane ...

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Abstract

The invention discloses an activity remolding method for changing lipase catalytic activity, and belongs to the technical field of enzyme engineering. Remolded lipase with high esterification activity is obtained by adding different adjusting factors in the lipase in-vitro remolding process, and therefore the requirement for lipase non-aqueous biocatalysis is met. The method comprises the steps that various microorganism lipases comprising the engineered escherichia coli heterogenous expression lipase and the commodity lipase with no esterification activity or low esterification activity are subjected to 8M urea denaturating treatment, a membrane environment complex WTS extracted from rhizopus chinensis cells is added in the renaturation process, the activity remolded lipase with the significantly-improved esterification activity can be obtained, and meanwhile the common hydrolytic activity of the lipase is also changed. The lipase is more suitable for industrial application due to recovery of esterification activity.

Description

technical field [0001] The invention relates to an activity remodeling method for changing the catalytic activity of lipase, which belongs to the technical field of enzyme engineering. Background technique [0002] Lipase (EC3.1.1.3), triacylglyceride hydrolase, is a biocatalyst widely present in animals, plants and various microorganisms. It was first discovered in 1901. Its main catalytic function is that it can - Lipid interfacial hydrolysis of long-chain triacylglycerides. The reason why lipase has attracted much attention in biocatalysis is that, in addition to its ability to catalyze the hydrolysis of ester bonds in compounds, lipase can also catalyze reverse esterification and transesterification reactions in non-aqueous phases, so it can be used in biological The field of chemical engineering has received extensive attention. The ability of lipases to perform very specific chemical transformations (biotransformations) has made them increasingly popular in the food,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20
CPCC12N9/20C12Y301/01003
Inventor 王栋徐岩张璋
Owner JIANGNAN UNIV
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