CN115725542A - Cholesterol esterase activity aggregate and application thereof - Google Patents
Cholesterol esterase activity aggregate and application thereof Download PDFInfo
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- CN115725542A CN115725542A CN202211172547.0A CN202211172547A CN115725542A CN 115725542 A CN115725542 A CN 115725542A CN 202211172547 A CN202211172547 A CN 202211172547A CN 115725542 A CN115725542 A CN 115725542A
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a cholesterol esterase activity aggregate and application thereof. According to the invention, the cholesterol esterase and the SpyTag short peptide tag are recombined into fusion protein, and the fusion protein is converted into micron-sized active aggregates which are easy to precipitate at a specific pH and temperature, so that the recombined cholesterol esterase can be subjected to self-precipitation, and is rapidly separated and purified through centrifugation to prepare the cholesterol esterase active aggregates by a one-step method. The purification method is simple and time-saving in operation, large in treatment capacity and small in equipment limitation, and the cholesterol esterase activity aggregate obtained by the method provided by the invention is convenient to store, stable in enzyme activity and has the advantages of high efficiency, single product and reusability.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a cholesterol esterase activity aggregate formed by inducing recombinant cholesterol esterase by Spy-Tag and application thereof.
Background
Cholesterol esterase (EC 3.1.1.1.3 cholesterol esterase, che), also known as sterol esterase, catalyzes the hydrolysis of cholesterol esters in aqueous phase to convert them to cholesterol and fatty acids, and also performs esterification and transesterification reactions in the presence of organic solvents, which are widely found in mammals and microorganisms. CHE derived from mammals belongs to intracellular enzymes, and is mainly present in cells of the pancreas, blood, liver, kidney, and the like. In 1976 UWAJIMA et al first reported that 1 strain of CHE-producing Pseudomonas fluorescens was isolated, and then more CHEs of microbial origin were found, such as Streptomyces, saccharomyces, staphylococcus aureus, etc. The CHE has wide application in the fields of food, diagnostic reagent, sewage treatment, pulping and papermaking industry, fabric degreasing and the like.
At present, for the purification and separation of cholesterol esterase, the purification cost of preparing enzyme by the traditional method accounts for about 70 percent of the total cost, and the cost is higher in the production process. In addition, the harsh conditions of industrial application increase the instability and recycling difficulty of the free enzyme, and shorten the industrial life of the enzyme. In recent years, purification tags including His tags are commonly used in laboratories to purify enzymes, and are common tags based on affinity, which can basically achieve a purity of about 90% or higher of the target enzyme content after operations such as nickel column purification, and the like, and the method is widely used. However, the use of nickel columns also makes enzyme purification too expensive. Therefore, the development of low-cost immobilization methods is an important research area for the application of cholesterol esterase.
Disclosure of Invention
In view of the defects of the prior art, the first object of the present invention is to provide a cholesterol esterase activity aggregate which has the advantages of high efficiency, good stability, single product and reusability, and can be widely used in hydrolysis application of ester.
In order to achieve the above technical objects, the present inventors have combined with the research experience of cholesterol esterase for many years, and formed a cholesterol esterase active aggregate by fusing a new short peptide tag gene, and the obtained enzyme active aggregate can rapidly separate a target enzyme from impurities by simple centrifugation, and has enzyme activity, and can be directly used without denaturation and renaturation as compared with inclusion bodies formed by induction, thereby greatly reducing the cost of purifying the enzyme.
Specifically, the technical scheme of the invention is as follows: a cholesterol esterase activity aggregate comprises cholesterol esterase and a self-aggregation short peptide, wherein the self-aggregation short peptide is a SpyTag short peptide.
Further preferably, said cholesterol esterase activity aggregate further comprises a linker peptide, said linker peptide being located between said cholesterol esterase and said self-aggregating short peptide.
The cholesterol esterase activity aggregate has a microstructure in the form of round particles under a scanning electron microscope, and the particle size of the cholesterol esterase activity aggregate is 500-800 nm.
Further preferably, the amino acid sequence of the cholesterol esterase is shown as SEQ ID NO. 1; the amino acid sequence of the SpyTag short peptide is shown in SEQ ID NO. 2.
It is a second object of the present invention to provide a nucleic acid molecule encoding the above-mentioned cholesterol esterase activity aggregate, and to provide a recombinant vector comprising the nucleic acid molecule.
The third purpose of the invention is to provide an engineering bacterium, which comprises the recombinant vector. Further preferably, the engineering bacterium is escherichia coli.
The fourth purpose of the invention is to provide a preparation method of the cholesterol esterase activity aggregate, which adopts the engineering bacteria to ferment to obtain the cholesterol esterase activity aggregate.
More specifically, the preparation method of the cholesterol esterase activity aggregate comprises the following steps:
(1) Introducing a gene fragment of cholesterol esterase into a nucleotide sequence of a SpyTag short peptide, combining SpyTag with a cholesterol esterase gene fragment through a linker (EAAAKEAAAKEAAAK), and inserting the SpyTag and the cholesterol esterase gene fragment into a pET-22b (+) plasmid vector to construct a pET-His-Q75NT4-SpyTag expression vector;
(2) Introducing the pET-His-Q75NT4-SpyTag plasmid obtained in the step (1) into escherichia coli for expression to prepare engineering bacteria;
(3) Activating the engineering bacteria obtained in the step (2) in an LB culture medium, performing amplification culture in a TB culture medium, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression to obtain a culture solution;
(4) Centrifuging the culture solution obtained in the step (3) at 9000-11000rpm at room temperature for 10-20min to obtain a first precipitate and obtain thalli;
(5) According to the thalli: the buffer solution is (20-30): adding the thalli obtained in the step (4) into PBS buffer solution according to the volume ratio of 1, and oscillating by using an oscillator to enable the thalli to be uniformly suspended in the buffer solution for ultrasonic disruption;
(6) And (4) centrifuging the material obtained in the step (5) at the temperature of 3.8-4.2 ℃ and the rpm of 11000-13000 for 10-20min to obtain a second precipitate, namely the cholesterol esterase activity aggregate.
In a preferred embodiment of the invention, the nucleotide sequence of the gene fragment of the cholesterol esterase is shown as SEQ ID No. 3.
In a preferred embodiment of the invention, the nucleotide sequence of the Spytag short peptide is shown in SEQ ID NO. 4.
Further preferably, the Escherichia coli is E.coil BL21 (DE 3).
Further preferably, the activation of step (3) is culturing to OD 600 ＝0.5-0.8。
Further preferably, the temperature of the scale-up culture in the step (3) is 37 ℃, the shaking culture is carried out at 200rpm for 3-4h, and the inoculation amount is 1: 100.
Further preferably, the conditions of the ultrasonication are: the power is 300w, each time of ultrasound is 2s, the interval is 4s, the circulation is 99 times, and the steps are repeated twice.
The cholesterol esterase activity aggregate protein designed by the invention has simple expression conditions, convenient purification and low production cost, thereby being suitable for industrial production. The invention provides a new strategy for the separation and immobilization of recombinant protein, and has wide application prospect in the fields of biological separation and biological macromolecule immobilization. The cholesterol esterase activity aggregate can be widely used in an ester hydrolysis system as a novel immobilized enzyme. Accordingly, a fifth object of the present invention is to provide a use of the above-mentioned cholesterol esterase activity aggregate in preparation of fucoxanthin by enzymolysis of fucoxanthin.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, the cholesterol esterase and the Spytag short peptide tag are recombined into the fusion protein, and the fusion protein is changed into a micron-sized aggregate easy to precipitate at a specific pH and temperature, so that the recombined cholesterol esterase can be subjected to self-precipitation to form an active aggregate, and is quickly separated and purified by centrifugation.
(2) The recombinant cholesterol esterase activity aggregate prepared by the invention has the advantages of convenient storage, stable enzyme activity, high efficiency, single product, reusability and the like, and the cholesterol esterase activity aggregate still has 92.5 percent of initial activity after being recycled for 3 times, and also keeps 60.1 percent of initial activity after being recycled for 12 times. In addition, the preparation method has the advantages of simple route and low production cost, and is suitable for industrial production.
(3) The invention selects the cholesterol esterase active aggregate as the tool enzyme to prepare the fucoxanthin, greatly reduces the dosage of enzyme preparations, improves the efficiency of preparing the fucoxanthin, enlarges the application range of the enzyme and ensures that the enzyme has wide application in the fields of chemistry and biomedicine.
Drawings
FIG. 1: expression vector plasmid His-pET-Q75NT4-SpyTag map.
FIG. 2: SDS-PAGE electropherogram analysis of protein Q75NT4, wherein lane M: marker; lane 1: the Q75NT4 recombinant strain is crushed and centrifuged to precipitate (Q75 NT4 cholesterol esterase theoretical molecular weight (42.919 kDa)); lane 2: and (3) crushing and centrifuging the Q75NT4 recombinant bacteria to obtain a supernatant.
FIG. 3: a profile analysis profile of cholesterol esterase activity aggregates; wherein (a) SEM analysis (5000 ×); (b) SEM analysis (10000 ×, after milling); (c) DSL analysis.
FIG. 4: graph for analysis of the reuse efficiency of cholesterol esterase activity aggregates.
FIG. 5: liquid chromatogram of fucoxanthin prepared by catalysis of cholesterol esterase activity aggregate.
Detailed Description
The following examples will further illustrate the present invention with reference to the accompanying drawings. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: preparation of cholesteryl esterase activity aggregates
(1) Introducing a gene fragment of cholesterol esterase into a nucleotide sequence of a SpyTag short peptide, combining a linker (EAAAKEAAAKEAAAK) + SpyTag with a cholesterol esterase gene fragment, and inserting the combined fragment into a pET-22b (+) plasmid vector to construct a pET-His-Q75NT4-SpyTag expression vector shown in figure 1. Wherein, the amino acid sequence of the cholesterol esterase is shown as SEQ ID NO.1, the nucleotide sequence of the cholesterol esterase is shown as SEQ ID NO.3, the amino acid sequence of the Spy-tag short peptide is shown as SEQ ID NO.2, and the nucleotide sequence of the Spytag short peptide is shown as SEQ ID NO. 4.
(2) Transferring the pET-His-Q75NT4-SpyTag obtained in the step (1) into Escherichia coli E.coil BL21 (DE 3) to prepare engineering bacteria;
(3) Inoculating the glycerol strain of the engineering bacteria obtained in the step (2) into LB liquid containing ampicillin (Amp) (100 mg/L) according to the volume ratio of 1: 100 to culture for 12h to OD 600 The value is 0.6, inoculating into TB medium at an inoculum size of 1: 100, placing in a shaker at 37 deg.C, culturing at 200r/min for 3-4h, and adding IPTGInducing and culturing (the concentration is 100 mmol/L) for 24h to obtain a culture solution;
(4) Centrifuging the culture solution obtained in the step (3) at 10000rpm for 20min at room temperature to obtain a first precipitate, adding PBS according to the volume ratio of the culture solution to the PBS of 20: 1, and carrying out heavy suspension washing for 3 times to obtain thalli;
(5) According to a 25:1 (thallus: buffer) adding the thallus obtained in the step (4) into PBS buffer, oscillating by using an oscillator to enable the thallus to be uniformly suspended in the buffer for ultrasonic disruption, wherein the conditions of ultrasonic disruption are as follows: the power is 300w, each time of ultrasound is 2s, the interval is 4s, the circulation is 99 times, and the steps are repeated twice;
(6) Centrifuging the material obtained in step (5) at 4 deg.C and 12000rpm for 20min to obtain a second precipitate, which is recombinant cholesteryl esterase activity aggregate, and obtaining the material shown in lane 1 of FIG. 2, wherein the yield of cholesteryl esterase activity aggregate is 59.8%, as shown in Table 1.
TABLE 1 yield of cholesterol esterase active aggregates
Note: a: mass of the analyte in 1L of the medium, b: based on the mass of the analyte in 1g of the cells.
Example 2: morphology analysis of cholesterol esterase activity aggregates
As shown in FIG. 3, the microstructure of the recombinant cholesterol esterase activity aggregate prepared in the embodiment is round particles under a transmission electron microscope, and the particle size is 500-800 nm.
Example 3: enzyme activity assay of cholesterol esterase activity aggregates
The cholesterol esterase activity aggregate prepared in the example was resuspended in PBS buffer, and the enzyme activity was measured by determining whether the cholesterol esterase activity aggregate could catalyze the hydrolysis of the substrate cholesterol oleate. The results show that the cholesterol esterase activity aggrecanase can catalyze the hydrolysis of cholesterol oleate, has cholesterol esterase activity, and has 82.8 percent of activity compared with that of free enzyme.
Example 4: determination of Cholesterol esterase Activity aggregate reuse Rate
The above-mentioned repeated utilization rate of the cholesterol esterase activity aggregate was measured as follows: adding various reagents according to steps under the optimal reaction condition by using the cholesterol esterase activity aggregate and cholesterol oleate, measuring the enzyme activity, and adding a new cholesterol oleate substrate after continuously centrifuging and washing to carry out the next round of experiment. The enzyme activity of the first round of reaction is 100 percent, the relative enzyme activity of each round of reaction is calculated, 12 rounds are repeated in total, and the repeated utilization rate of the cholesterol esterase activity aggregate is calculated according to the enzyme activity. The reuse efficiency of cholesterol esterase activity aggregates is shown in fig. 4.
Example 5: efficient fucoxanthin preparation method by cholesterol esterase activity aggregate method
Adding fucoxanthin and sodium taurocholate into a conical flask according to the proportion of 1:5, taking the cholesterol esterase active aggregate as a tool enzyme, adding PBS buffer solution, reacting for 1h under the condition of 37 ℃ water bath, and calculating the conversion rate of the cholesterol esterase active aggregate to fucoxanthin to 87.33%.
Example 6: efficient fucoxanthin preparation method by cholesterol esterase activity aggregate method
Adding fucoxanthin and sodium taurocholate into a conical flask according to the proportion of 1.
Example 7: efficient fucoxanthin preparation method by cholesterol esterase activity aggregate method
Adding fucoxanthin and sodium taurocholate into a conical flask according to the proportion of 1:5, adding the cholesterol esterase activity aggregate as a tool enzyme into PBS buffer solution, reacting for 3h under the condition of 37 ℃ water bath, and calculating the conversion rate of the cholesterol esterase activity aggregate to fucoxanthin to be 89.26%.
Example 8: efficient fucoxanthin preparation method by cholesterol esterase activity aggregate method
Adding fucoxanthin and sodium taurocholate into a conical flask according to the proportion of 1.
Example 8: refining fucoxanthol
Fucoxanthin prepared by the method is refined by preparative chromatography to obtain fucoxanthin monomer, and the purity of the fucoxanthin monomer is 98.7% by liquid chromatography analysis, and the chromatogram is shown in figure 5.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (10)
1. A cholesterol esterase activity aggregate comprises cholesterol esterase and a self-aggregation short peptide, wherein the self-aggregation short peptide is a SpyTag short peptide.
2. The cholesterol esterase activity aggregate according to claim 1, further comprising a linker peptide, wherein the linker peptide is located between the cholesterol esterase and the self-aggregating short peptide.
3. The cholesterol esterase activity aggregate according to claim 1, wherein the amino acid sequence of the cholesterol esterase is shown in SEQ ID No. 1.
4. The cholesterol esterase activity aggregate according to claim 1, wherein the amino acid sequence of the SpyTag short peptide is shown in SEQ ID No. 2.
5. A nucleic acid molecule encoding the cholesterol esterase active aggregate of any of claims 1-4.
6. A recombinant vector comprising the nucleic acid molecule of claim 5.
7. An engineered bacterium comprising the recombinant vector of claim 6.
8. The engineered bacterium of claim 7, wherein the engineered bacterium is Escherichia coli.
9. A method for preparing cholesterol esterase activity aggregate, which comprises fermenting the engineering bacteria of claim 6 or 7 to obtain the cholesterol esterase activity aggregate.
10. Use of a cholesterol esterase active aggregate according to any of claims 1-4 for the enzymatic hydrolysis of fucoxanthin to produce fucoxanthin alcohol.
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Citations (3)
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|---|---|---|---|---|
| CN106591345A (en) * | 2016-12-26 | 2017-04-26 | 华侨大学 | Method for separation and purification and immobilization integration of recombinant double enzyme |
| CN107058273A (en) * | 2017-05-05 | 2017-08-18 | 西北工业大学 | A kind of visible protein based on heme-binding domain expresses the application of fusion tag |
| WO2022119926A1 (en) * | 2020-12-01 | 2022-06-09 | President And Fellows Of Harvard College | Engineered probiotic compositions and uses thereof |
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| CN106591345A (en) * | 2016-12-26 | 2017-04-26 | 华侨大学 | Method for separation and purification and immobilization integration of recombinant double enzyme |
| CN107058273A (en) * | 2017-05-05 | 2017-08-18 | 西北工业大学 | A kind of visible protein based on heme-binding domain expresses the application of fusion tag |
| WO2022119926A1 (en) * | 2020-12-01 | 2022-06-09 | President And Fellows Of Harvard College | Engineered probiotic compositions and uses thereof |
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| ZHANGLIN LIN等: "Facile expression and purification of active human growth hormone in E. coli by a cleavable self-aggregating tag scheme", PROTEIN EXPRESSION AND PURIFICATION, vol. 188, 31 December 2021 (2021-12-31), pages 105974 * |
| 任楠楠等: "微生物甾醇酯酶的研究进展", 中国酿造, vol. 36, no. 06, 31 December 2017 (2017-12-31), pages 9 - 13 * |
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