Recombinant lipase capable of realizing efficient froth flotation and application of recombinant lipase

Abstract

The invention provides recombinant lipase capable of realizing efficient froth flotation as well as a construction method and application of the recombinant lipase, and belongs to the technical field of bioengineering. The preparation method comprises the following steps: connecting lipase (Lipase) with elastin-like protein (ELP), so as to obtain the recombinant lipase Lipase-ELP; the amino acid sequence of the recombinant lipase is as shown in SEQ ID NO: 1, and the nucleotide sequence for coding the recombinant lipase is as shown in SEQ ID NO: 2. The elastin-like ELP in the recombinant lipase not only can increase the thermal stability of Lipase, but also can increase the surface hydrophobicity of Lipase, so that the foaming ability and foam stability of the recombinant lipase are improved, the recombinant lipase can be well adsorbed on a gas-liquid interface, and the foam flotation efficiency of the recombinant lipase is improved; the recombinant lipase has good application in froth flotation separation and purification.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant lipase capable of efficient foam floatation and application thereof. Background Art

[0002] Lipase is a class of enzymes that can catalyze the hydrolysis of fatty acid glycerides (i.e., oils and fats) to produce glycerol and fatty acids. Its main role in nature is to promote the digestion and absorption of fats, and to play a role in the energy metabolism process in organisms. In the field of biotechnology, lipase is used to synthesize various useful chemicals, such as drugs, spices and bioactive molecules, and is used in many industrial fields such as food, beverages, oils and fats, detergents, feed, textiles, leather, fine chemicals, medicines, cosmetics, papermaking and environmental management. In addition, lipase can catalyze the transesterification reaction of vegetable oils or animal fats with alcohols such as methanol to produce biodiesel.

[0003] The isolation and purification of lipases may result in high dilution, low yield, and reduced activity, and lipases are structurally unstable due to their heat sensitivity and sensitivity to extreme pH values. The structural integrity of the enzyme is key to its biological activity, and downstream processing requires high activity and high yield purification. Therefore, it is necessary to improve the activity and purity of lipases.

[0004] Foam separation technology is mainly based on the principle of surface adsorption. It injects bubbles into the solution and uses the difference in surface activity between the components in the solution to make substances with strong surface activity adsorbed on the gas-liquid interface. As the bubbles rise, they separate from the main liquid phase to form a foam layer, and finally separate and enrich the solutes adsorbed on the bubble surface. Foam separation technology has the advantages of mild operating conditions, no use of organic solvents, short time consumption, simple device structure, and low cost. It can not only increase enzyme production without significantly reducing enzyme activity, but also achieve the enrichment of low-concentration substances, which is conducive to subsequent experiments.

[0005] The hydrophobicity of proteins is one of the important factors affecting foam stability and flotation efficiency. Hydrophobic proteins are more likely to bind to bubbles and are therefore more likely to be carried by bubbles and separated during froth flotation. The hydrophobic surface is conducive to the adsorption of proteins at the gas-liquid interface and the stabilization of foam, thereby improving the efficiency of froth flotation. Therefore, it is necessary to develop a recombinant lipase that can be used for efficient froth flotation. Summary of the invention

[0006] In view of some deficiencies in the prior art, the present invention provides a recombinant lipase capable of efficient foam flotation, a construction method and an application thereof; the present invention connects lipase (Lipase) with elastin-like protein ELP to obtain the recombinant lipase Lipase-ELP; the amino acid sequence of the recombinant lipase is shown in SEQ ID NO:1, and the nucleotide sequence encoding the recombinant lipase is shown in SEQ ID NO:2; the elastin-like protein ELP in the recombinant lipase can not only increase the thermal stability of Lipase, but also increase the surface hydrophobicity of Lipase, thereby improving the foaming ability and foam stability of the recombinant lipase, making it better adsorbed on the gas-liquid interface, thereby improving the efficiency of foam flotation of the recombinant lipase; the recombinant lipase has a good application in foam flotation separation and purification.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0008] The present invention first provides a recombinant lipase, denoted as Lipase-ELP, which is obtained by connecting lipase Lipase with elastin-like protein ELP; the elastin-like protein ELP is a pentapeptide sequence (Val-Pro-Gly-Val-Gly)n, where n is repeated for 20-100.

[0009] Preferably, the amino acid sequence of the lipase Lipase is shown as SEQ ID NO:3, and the nucleotide sequence encoding the lipase Lipase is shown as SEQ ID NO:4.

[0010] Preferably, the amino acid sequence of the recombinant lipase is as shown in SEQ ID NO:1.

[0011] Preferably, n is 50.

[0012] The present invention also provides a nucleic acid encoding the above recombinant lipase, and its sequence is shown in SEQ ID NO:2.

[0013] The present invention also provides a recombinant expression plasmid, which comprises the nucleic acid encoding the recombinant lipase shown in SEQ ID NO:2.

[0014] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains the recombinant expression plasmid.

[0015] The present invention also provides a method for preparing recombinant lipase, which comprises fermenting and culturing the recombinant bacteria to obtain the recombinant lipase.

[0016] The present invention also provides the use of the recombinant lipase in foam flotation purification and separation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention constructs a recombinant lipase Lipase-ELP by gene modification technology, and the recombinant lipase is obtained by connecting lipase Lipase with elastin-like ELP; the elastin-like ELP is a pentapeptide sequence (Val-Pro-Gly-Val-Gly) n, and n is repeated for 20-100. Wherein, Val, Pro are highly hydrophobic amino acids; Gly is also a hydrophobic amino acid. Hydrophobicity is the main force for the aggregation of protein monomers, and oligomerization can enhance the interaction between protein monomers and fix N-terminal, C-terminal and high-flexibility loop, thereby improving the thermal stability of protein. In addition, ELP not only forms hydrogen bonds with water molecules, but also can be combined with other ELP molecules. According to online and experimental analysis, as the ELP sequence repeat number (n) increases, the more recombinant enzyme hydrophobic amino acids, the longer the ELP sequence repeat number (n), the more hydrogen bonds. The increase of hydrogen bonds, especially the increase of hydrogen bonds between the main chain and the main chain, significantly increases the thermal stability of the fusion enzyme. In Lipase-ELP, ELP can increase the thermal stability and storage stability of Lipase.

[0019] The present invention fuses ELP tags with lipase Lipase to express, and hydrophobicity is beneficial to the adsorption of lipase Lipase at gas-liquid interface, and as the ELP sequence repeat number (n) increases, the more recombinant enzyme hydrophobic amino acids. Heat treatment can increase the exposure of hydrophobic amino acids inside the lipase Lipase molecule, resulting in an increase in surface hydrophobicity, thereby improving the foaming ability and foam stability of protein. In addition, using ELP and lipase Lipase to express, not only can increase the thermal stability of Lipase, but also result in an increase in surface hydrophobicity, thereby improving the foaming ability and foam stability of lipase Lipase, can be preferably adsorbed on the gas-liquid interface, and then improve the efficiency of the foam flotation of lipase Lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the construction of recombinant plasmid pET28a(+)-Lipase-ELP50.

[0021] Figure 2The figures show SDS and Ni-NTA purification of recombinant plasmids pET28a(+)-Lipase (A) and pET28a(+)-Lipase-ELP50 (B); in the figure, lane Ctrl is the total protein of BL21(DE3) containing empty plasmid induced with 0.4 mM IPTG for 16 h; lanes Lys, Sup, and Pel are the total protein, supernatant, and precipitate of BL21(DE3) containing pET28a(+)-Lipase and ET28a(+)-Lipase-ELP50 induced with 0.4 mM IPTG for 16, respectively; lanes 150, 250, and 450 are the effluents after elution with 150, 250, and 450 mM imidazole, respectively.

[0022] Figure 3 This is a graph showing the thermal stability of Lipase-ELP50 and Lipase; wherein, in the figure, A is the thermal stability of Lipase-ELP50 and Lipase enzymes within 1 hour at different temperatures (40-90°C); B is the residual activity of Lipase-ELP50 and Lipase enzymes after heating at 70°C for 1 hour; C is the residual activity of Lipase-ELP50 and Lipase enzymes after heating at 90°C for 1 hour (C).

[0023] Figure 4 The pH stability (A) and storage stability (B) of Lipase-ELP50 and Lipase.

[0024] Figure 5 A1 and B1 are online analysis diagrams of the hydrophobicity of Lipase-ELP50 (A1) and Lipase (B1); A2 and B2 are fluorescence emission spectra of different concentrations of Lipase-ELP50 (A2) and Lipase (B2) systems and diagrams showing changes in fluorescence intensity with recombinant protein concentration.

[0025] Figure 6 Graph showing the effects of initial protein concentration (a), liquid inlet volume (b), temperature (c) and gas flow rate (d) on the Lipase-ELP foam separation efficiency. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following examples, if specific conditions are not specified, they are all carried out according to conventional known conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially. Unless otherwise specified, the present invention adopts the prior art in this field.

[0027] The following examples are described with the repetition number of elastin-like protein ELP being 50, but the repetition number can be any value between 20 and 100. pET28a(+) plasmid was purchased from Sangon Biotech (Shanghai) Engineering Co., Ltd.; PUC18 plasmid was purchased from Sangon Biotech (Shanghai) Engineering Co., Ltd.

[0028] The culture medium formula used is:

[0029] Solid LB medium: agar powder 15 g / L, tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.4;

[0030] Liquid LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.4;

[0031] In the following examples, SEQ ID NO: 1:

[0032] MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTAR MGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS(VPGVG)n, n=20-100

[0033] SEQ ID NO:2:

[0034] ATGAACTTTCCCCGCGCTTCCCGCCTGATGCAGGCCGCCGTTCTCGGCGGGCTGATGGCCGTGTCGGCCGCCGCCACCGCCCAGACCAACCCCTACGCCCGCGGCCCGAACCCGACAGCCGCCTCACTCGAAGCCAGCGCCGGCCCGTTCACCGTGCGCTCGTTCACCGTGAGCCGCCCGAGCGGCTACGGCGCCGGCACCGTGTACTACCCCACCAACGCCGGCGGCACCGTGGGCGCCATCGCCATCGTGCCGGGCTACACCGCGCGCCAGTCGAGCATCAAATGGTGGGGCCCGCGCCTGGCCTCGCACGGCTTCGTGGTCATCACCATCGACACCAACTCCACGCTCGACCAGCCGTCCAGCCGCTCGTCGCAGCAGATGGCCGCGCTGCGCCAGGTGGCCTCGCTCAACGGCACCAGCAGCAGCCCGATCTACGGCAAGGTCGACACCGCCCGCATGGGCGTGATGGGCTGGTCGATGGGCGGTGGCGGCTCGCTGATCTCGGCGGCCAACAACCCGTCGCTGAAAGCCGCGGCGCCGCAGGCCCCGTGGGACAGCTCGACCAACTTCTCGTCGGTCACCGTGCCCACGCTGATCTTCGCCTGCGAGAACGACAGCATCGCCCCGGTCAACTCGTCCGCCCTGCCGATCTACGACAGCATGTCGCGCAATGCGAAGCAGTTCCTCGAGATCAACGGTGGCTCGCACTCCTGCGCCAACAGCGGCAACAGCAACCAGGCGCTGATCGGCAAGAAGGGCGTGGCCTGGATGAAGCGCTTCATGGACAACGACACGCGCTACTCCACCTTCGCCTGCGAGAACCCGAACAGCACCCGCGTGTCGGACTTCCGCACCGCGAACTGCAGC(GTAC CGGGTGTGGGT)n, n = 20 - 100

[0035] Amino acid sequence of ELP SEQ ID NO:5:

[0036] (VPGVG)n, n = 20 - 100

[0037] The nucleotide sequence of ELP is SEQ ID NO:6:

[0038] (GTACCGGGTGTGGGT)n,n=20-100

[0039] Since the number of repetitions cannot be expressed in the sequence listing, the repetitive sequence is not expressed.

[0040] Example 1: Construction of recombinant expression plasmid pET28a(+)LIPASE-ELP

[0041] (1) GenScript Company of Nanjing City synthesized the nucleotide sequence of lipase Lipase (Ideonella sakaiensis, accession number GAP38373.1), added BamHI and EcoRI restriction sites to the 5' and 3' ends of the Lipase sequence, respectively, to obtain the Lipase nucleotide sequence with BamHI and EcoRI restriction sites, and then constructed the Lipase nucleotide sequence with BamHI and EcoRI restriction sites into the pET28a(+) plasmid, which was named pET28a(+)-Lipase.

[0042] The nucleotide sequence of ELP synthesized by Nanjing GenScript Company, where n is selected as 50, is recorded as ELP50. ELP50 has an EcoR I restriction site at 5' and an Xho I restriction site at 3', and then it is constructed on the PUC18 plasmid, recorded as PUC18-ELP50 plasmid.

[0043] (2) EcoR I and Xho I were used to double-digest pET 28a(+)-Lipase and PUC18-ELP50 plasmids to construct the recombinant plasmid pET28a(+)-Lipase-ELP50( Figure 1 ), wherein the double enzyme digestion system is:

[0044] EcoR I: 1 μL;

[0045] Xho I: 1 μL;

[0046] 10×Buffer: 2 μL;

[0047] PUC18--ELP50(pET28a(+)-Lipase): 5μL;

[0048] Sterile water: make up to 20 μL.

[0049] The double enzyme digestion system was added to a centrifuge tube and mixed evenly, and then digested at 37°C for 2-3h. After the digestion, the ELP50 gene fragment and the linearized pET28a(+)-Lipase were recovered according to the method of a conventional gel recovery kit, and then the ELP50 gene fragment and pET28a(+)-Lipase obtained by gel recovery were connected by T4 ligase (Takara Biotechnology (Dalian) Co., Ltd.). The connection system is:

[0050] 10×T4 Ligase buffer: 2μL;

[0051] pET28a(+)-Lipase: Depends on the recovery concentration;

[0052] ELP50: Depends on the recovery concentration;

[0053] T4 DNA Ligase (10U / μL): 1μL;

[0054] Sterile water: make up to 20 μL.

[0055] The above ligation system was reacted in an incubator at 16° C. for 18 h. After the reaction, the recombinant plasmid pET28a(+)-Lipase-ELP50 was obtained.

[0056] (3) Take out the competent E. coli BL21 (DE3) from the -80°C freezer, place it on ice to thaw, add 5 μL of the recombinant plasmids pET28a(+)-Lipase-ELP50 and pET28a(+)-Lipase respectively, and gently tap the bottom of the EP tube to mix. Place it on ice for 30 minutes, then place it in a 42°C water bath for 45 seconds, and quickly place it on ice for two minutes.

[0057] After standing, add 700 μL of preheated LB medium in the clean bench, mix well, and shake at 37°C, 200 rpm for 2 hours to revive the cells. Collect the cells by centrifugation at 10,000 rpm for one minute at room temperature, discard 600 μL of supernatant, mix the remaining supernatant with the cells, spread on LB solid medium containing kanamycin, and culture in a 37°C constant temperature incubator overnight to obtain recombinant E. coli expressing Lipase-ELP50 and Lipase, respectively.

[0058] Example 2: Expanding the culture of Escherichia coli expressing Lipase-ELP and Lipase

[0059] (1) The freshly transformed recombinant E. coli was transferred to a solid LB medium containing 50 μg / mL kanamycin and then cultured overnight at 37°C. After the culture was completed, a single colony in the medium was transferred to 5 mL of liquid LB medium containing 50 μg / mL kanamycin and then cultured overnight at 37°C and 200 rpm in an orbital shaker. After the culture was completed, 3 mL of the recombinant E. coli was inoculated into 300 mL of liquid LB medium containing 50 μg / mL kanamycin and grown at 37°C and 200 rpm for about 3 h until the OD600 of the bacteria reached between 0.4 and 0.6.

[0060] (2) After the cultivation in step (1), the liquid LB culture medium was placed on ice for 20 minutes, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added thereto at a final concentration of 0.4 mM, and the culture was shaken at 180 rpm at 37°C for 16-20 h (overnight) to induce Lipase-ELP50 expression. After the induction, the liquid culture medium was centrifuged at 3000 rpm and 4°C for 20 minutes, and the supernatant was discarded to obtain cell pellets cultured at different temperatures, which were stored at -80°C for later use.

[0061] (3) The cell pellets obtained in step (2) were thawed and resuspended in 10 mL of Tris-HCl (50 mM, pH 8.0), and then centrifuged at 3000 rpm and 4°C for 20 minutes, the supernatant was discarded, and the cells were collected and washed twice with 50 mM Tris-HCl (pH 8.0).

[0062] The cells were resuspended in 20 mL Tris-HCl buffer containing 1 mM PMSF (200 μL), and ultrasonic cell disruptors were used for 30 minutes on ice, followed by 6 seconds of alternating ultrasonic treatment and intermittent cooling for 6 seconds to obtain a solution containing cell precipitates (total protein). The total protein was centrifuged at 13,000 rpm for 30 minutes at 4 ° C to obtain a supernatant and a precipitate. The supernatant was transferred to a new EP tube, and the precipitate was resuspended with 50 mM Tris-HCl, pH 8.0. The total protein, supernatant, and precipitate were subjected to SDS-PAGE to analyze the expression and solubility of Lipase-ELP50 and Lipase.

[0063] from Figure 2 It can be seen that under the induction condition of 37°C, a band was added at the target size of 27 and 47 kDa in the total protein and supernatant of Lipase and Lipase-ELP50, respectively, which indicates that the induction expression of Lipase and Lipase-ELP50 was successful, and most of the Lipase-ELP50 and Lipase proteins were in the supernatant and were soluble proteins ( Figure 2 A).

[0064] The amino acid sequence of Lipase-ELP50 is shown in SEQ ID NO:1, and the nucleotide sequence encoding Lipase-ELP50 is shown in SEQ ID NO:2.

[0065] Example 3: Purification of Lipase and Lipase-ELP50 recombinant proteins using Ni-NTA nickel column

[0066] The supernatant containing Lipase-ELP50 and Lipase was purified by Ni-NTA column. The specific steps are as follows:

[0067] Add 10 mL of nickel-removing buffer to the column, rinse twice until the column changes from blue to white, rinse the column twice with 10 mL of deionized water, and then slowly add 10 mL of NiSO4 buffer to the column, cover the upper and lower lids, and incubate with rotation in a chromatography cabinet at 4°C for 30 min.

[0068] After the incubation, open the bottom cover to allow excess NiSO4 to flow out, then rinse once with 10 mL of deionized water and 10 mL of PBS, then add 4 mL of protein sample and rotate and incubate in a 4°C chromatography cabinet for 2 hours. After the incubation, open the bottom cover to allow protein samples that are not bound to the column to flow out, and rinse once again with 15 mL of PBS buffer. Then elute with 2 mL of 150 mM, 250 mM, and 450 mM imidazole buffers, respectively.

[0069] Prepare protein loading samples and determine the purification results of Lipase and Lipase-ELP50 by SDS-PAGE. Figure 2 As shown in the figure, a band of about 27 and 47 kDa appeared at 450 mM, which is consistent with the theoretical molecular weight of the target protein, indicating that the recombinant protein Lipase and Lipase-ELP50 ( Figure 2 B).

[0070] Example 4: Detection of recombinant lipase activity

[0071] Dilute the p-nitrophenol (p-NP) standard solution with anhydrous ethanol solution to an appropriate gradient, add 5 mL of 0.5 mol / L trichloroacetic acid to mix, and then add 15 mL of 0.5 mol / L NaOH to adjust the pH until the pH is consistent with that before adding acid. Measure the absorbance respectively and draw an absorbance-concentration standard curve.

[0072] Take 4mL of p-nitrophenol palmitate (p-NPP) substrate solution, preheat at 37℃ for 5 minutes, add 1mL of Lipase-ELP50 or Lipase enzyme solution separated and purified by Ni-NTA nickel column, react for 10 minutes, immediately add 5mL of 0.5mol / L trichloroacetic acid and mix well, stand for 5 minutes to terminate the reaction, then add 15mL of 0.5mol / L NaOH to adjust the pH value until the pH value is consistent with that before the reaction (replace 1mL of enzyme solution with 1mL of deionized water, and other conditions remain unchanged to obtain a blank), and measure the absorbance at 410nm. Calculate the concentration of p-nitrophenol generated according to the standard curve, and then calculate the enzyme activity.

[0073] The unit of lipase activity is defined as: under certain conditions, the amount of enzyme that releases 1 μmol of p-nitrophenol per minute is defined as 1 lipase activity unit (U).

[0074] The calculation formula is: X = cV / tV',

[0075] Where X is lipase activity (U / ml), c is p-nitrophenol concentration (μmol / L), V is the final volume of the reaction solution after acid-base adjustment (1 mL), V' is the amount of enzyme solution (1 mL), and t is the action time (min).

[0076] The specific activities of Lipase-ELP50 and Lipase were detected to be 41.09 and 43.12 U / mg, respectively. The results showed that the ELP label had no significant effect on the enzyme activity.

[0077] Example 5: Investigation of thermal, pH and storage stability of recombinant lipase

[0078] In this example, the thermal stability, pH stability and storage stability of Lipase-ELP50 were investigated to evaluate the stability of the recombinant lipase, as shown below.

[0079] (1) Investigation of thermal stability:

[0080] Lipase and Lipase-ELP50 were incubated at 20-90°C for 1 h, respectively. After the incubation, the relative enzyme activities were determined according to the method described in Example 3. The results were as follows: Figure 3 As shown in A.

[0081] As can be seen from the figure, with the increase of temperature, the enzyme activities of Lipase and Lipase-ELP50 both showed a trend of first increasing and then decreasing. The optimum temperatures of Lipase-ELP50 and Lipase were 40℃ and 30℃, respectively. Under 70℃ conditions, the enzyme activity of Lipase-ELP50 dropped to about 50% after 30min treatment (with the purified enzyme activity as 100%), while Lipase-ELP50 still retained 64% of its enzyme activity after 1h treatment ( Figure 3 B); when the treatment temperature was 90℃ and the treatment time was 20min, the enzyme activity of Lipase-ELP50 was 55%, while Lipase only had 28% enzyme activity; after 1h of treatment, Lipase was almost completely inactivated, while Lipase-ELP50 still maintained 21% enzyme activity ( Figure 3 C).

[0082] It can be seen that under the same temperature conditions, Lipase-ELP50 has better thermal stability than Lipase.

[0083] (2) Investigation of pH stability:

[0084] In this step, Lipase and Lipase-ELP50 were placed in different pH buffers (pH 3.0-9.0) and placed in a 37°C water bath for 60 min, and then the pH buffer was removed by dialysis. After dialysis, the residual enzyme activity was detected. The test results are as follows: Figure 4 As shown in A.

[0085] As can be seen from the figure, both Lipase and Lipase-ELP50 showed maximum activity at pH 6.5, that is, the optimal pH of both was 6.5. At pH 6.5.0-9.0, Lipase-ELP50 maintained more than 80% activity, while Lipase only maintained more than 70% activity. This shows that Lipase-ELP50 has higher pH stability than Lipase. Therefore, the addition of ELP tag can improve the pH tolerance of the enzyme.

[0086] (3) Investigation of storage stability:

[0087] In this step, the purified Lipase and Lipase-ELP50 were stored at 4°C and 40°C for 28 days, and the residual enzyme activity was detected every 4 days to investigate the storage stability of Lipase-ELP50 and Lipase. The results are as follows: Figure 4 shown.

[0088] As can be seen from the figure, with the extension of storage time, the activities of Lipase and Lipase-ELP50 both showed a downward trend, but the downward trend of Lipase was more obvious; the stability of these two enzymes at 4°C was higher than that at 40°C, indicating that the high temperature resistance of both was weak, but under these two temperature conditions, the stability of Lipase-ELP50 was higher than that of Lipase. In addition, Lipase-ELP50 still had 88% activity after 28 days at 4°C, while Lipase had only 76% activity; Lipase-ELP50 still had 83% activity after 28 days at 40°C, while Lipase had only 70% activity, indicating that the storage stability of Lipase-ELP50 was better than that of Lipase.

[0089] Example 6: Determination of surface hydrophobicity of recombinant lipase

[0090] In this example, the surface hydrophobicity of Lipase-ELP50 was investigated using Expasy's Prot Scale (https: / / web.expasy.org / protscale / ). The results showed that the addition of ELPs tags can significantly enhance the hydrophobicity of recombinant lipase, and the more repeated the ELPs tag sequence is, the stronger the hydrophobicity of the recombinant lipase is ( Figure 5 A1, 5B1).

[0091] This example also uses a fluorescent probe method to study the effect of temperature on the hydrophobicity of the recombinase surface. The investigation principle is as follows:

[0092] 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) is a commonly used fluorescent probe for studying protein conformation. It has a very low fluorescence quantum yield in aqueous solution. After binding to the hydrophobic residues of protein molecules through non-covalent bonds, the fluorescence spectrum will blue-shift as the non-polarity of the region increases, and the fluorescence intensity will increase significantly, so it can be used to measure the surface hydrophobicity of proteins.

[0093] The specific steps are:

[0094] Using 1 mg / mL enzyme sample as the mother solution, add fluorescent probe ANS solution (8.0 mmol / L) to the protein solution to a final concentration of 0.4 mmol / L. After thorough mixing, balance at room temperature in the dark for 1 hour. Take 300 μL of the balanced solution in a quartz fluorescence micro-cuvette, and measure the fluorescence emission spectrum of the protein solution in the range of 420-580 nm under the conditions of excitation wavelength of 380 nm, slit width of 5.0 nm, and scanning speed of 800 nm / min. Repeat the measurement 3 times for each concentration, and establish a linear relationship between fluorescence intensity and substrate concentration based on the concentration gradient method to determine the surface hydrophobicity. The results are as follows: Figure 5 shown.

[0095] As can be seen from the figure, under an excitation wavelength of 380nm, the maximum emission wavelength of ANS alone is around 520nm, and the fluorescence intensity value is extremely small, at 5.05; after adding Lipase-ELP50 and Lipase, the maximum emission wavelength blue-shifts, and the fluorescence intensity increases significantly. The maximum emission wavelengths of Lipase / ANS and Lipase-ELP50 / ANS blue-shift to around 470nm and 480nm, respectively; as the concentrations of Lipase-ELP50 and Lipase increase, the fluorescence intensity at the maximum emission wavelength gradually increases, and the fluorescence intensity at the maximum emission wavelength is plotted against the concentrations of Lipase-ELP50 and Lipase, and the slope of the resulting straight line is the surface hydrophobicity index S0 of the recombinant enzyme.

[0096] As shown in the figure, the S0 of Lipase and Lipase-ELP50 are 366.12 and 879.33, respectively. Figure 5 A2, B2). This indicates that Lipase-ELP50 has a large number of hydrophobic areas on its surface compared to Lipase, and its surface hydrophobicity is higher. This is because the ELPs tag of the present invention is composed of a (VPGVG) pentapeptide repeat sequence, Val and Pro are highly hydrophobic amino acids; Gly is also a hydrophobic amino acid, which enhances the hydrophobicity of Lipase-ELP50 and is beneficial to the subsequent separation efficiency of the recombinant enzyme foam flotation.

[0097] Example 7: Foam flotation separation of recombinant lipase

[0098] According to the method described in Example 2, recombinant lipase is expressed, impurities are removed by centrifugation, and ultrasonic crushing is performed. The crushed supernatant is stored in a 4°C refrigerator for subsequent use. The supernatant is diluted to the required concentration using Tris-HCl. The column is repeatedly rinsed with tap water and deionized water, and the device is connected according to the principle of bottom-in and top-out. Open the valve of the gas cylinder and adjust the flowmeter to the required value at the same time. After the value stabilizes, the tube on the flowmeter can be connected to the device. Recover the foam at the outlet above the column. Defoam the foam, record its volume, and measure the concentration and enzyme activity.

[0099] This example studies the effects of four factors, namely, recombinant lipase concentration (0.2-1.0 mg / mL), liquid volume (75-250 mL), air flow rate (50-250 mL / min) and temperature (25-45°C) on separation efficiency. The foam separation efficiency of the recombinant enzyme was evaluated by using enrichment ratio (Ep), recovery rate (Rp), fusion enzyme enrichment ratio (Ee) and enzyme activity recovery rate (Re). Figure 6 shown.

[0100]

[0101] In the formula, C f is the protein concentration of the defoaming solution, mg / mL; C0 is the initial protein concentration, mg / mL; V f is the volume of defoaming solution, mL; V0 is the amount of liquid inlet, mL; A f is the specific enzyme activity of the defoaming solution, U / mg; A0 is the initial specific enzyme activity, U / mg.

[0102] from Figure 6 a It can be seen that with the increase of initial protein concentration, the E p and E e Significantly decreased, R p and R e The increase in initial protein concentration reduces the equilibrium surface tension of the crude enzyme lysate, and the adsorption capacity of the surfactant on the bubble surface is stronger, resulting in R p and R e Based on the comprehensive results, the initial protein concentration of Lipase-ELP50 for foam separation was determined to be 0.4 mg / mL.

[0103] from Figure 6 b It can be seen that as the amount of liquid increased, the E p and E e Reduce, R p This is because the residence time of bubbles in the foam phase is shortened, and the foam cannot be fully discharged in a short time, resulting in an increase in the liquid holdup at the tower mouth, E p and E e The increase in liquid volume prolongs the time that bubbles stay in the liquid phase, which is more conducive to the full adsorption of surfactant on the bubble surface. p After comprehensive consideration, the foam separation liquid inlet volume was determined to be 100 mL.

[0104] from Figure 6 c It can be seen that as the temperature increases from 25℃ to 45℃, the E p and E e Increase, R p and R e When the temperature is low, the viscosity of the solution is high, a liquid film with greater viscoelasticity is formed, and the foam contains a higher amount of liquid, so the enrichment ratio is low and the recovery rate is high. When the temperature is high, the viscosity of the solution decreases, the resistance of the interstitial liquid flowing from top to bottom in the foam layer is small, and the drainage rate is accelerated. p Increase, R p High temperature is not conducive to the stability of Lipase-ELP50, and the enzyme activity is reduced. e Lower. Increase the temperature R p The downward trend is Re This indicates that high temperature is more conducive to the foam separation of Lipase-ELP50 than other surfactants in the system. In summary, 30℃ was selected as the appropriate temperature for foam separation.

[0105] from Figure 6 d It can be seen that with the increase of gas flow rate, the E of foam separation Lipase-ELP50 p and E e Significantly decreased, R p and R e When the gas flow rate is low, the flow rate of the bubbles is small, the residence time in the liquid phase is long, and the adsorption mass transfer at the gas-liquid interface can be fully carried out; the foam layer has a long drainage time, which makes the interstitial liquid carry less liquid, E p and E e When the gas flow rate is increased, the bubble flow rate from bottom to top is accelerated, the bubble stays in the liquid layer for a short time, and the surfactant is not fully combined at the gas-liquid interface, making E p and E e The gas flow rate increases, the number of bubbles generated per unit time increases, the downward flow resistance of the interstitial liquid increases, and the liquid content of the foam layer is higher, R p and R e Based on the above considerations, the gas flow rate for foam separation is determined to be 150 mL / min.

[0106] In summary, the optimal foam separation conditions were: liquid volume 100 mL, initial protein concentration 0.4 mg / mL, gas flow rate 150 mL / min, and temperature 30 °C.

[0107] Lipase-ELP50 and Lipase were separated and purified in the optimal foam separation system, and the purification results are shown in Table 1.

[0108] Table 1. Purification efficiency of Lipase-ELP50 and Lipase foam separation

[0109]

[0110]

[0111] As can be seen from Table 1, the Ep, Rp, Ee, and Re of Lipase-ELP50 are 2.52, 86.24%, 4.02, and 93.67%, respectively. Those of Lipase are 1.85, 62.32%, 1.52, and 55.12%, respectively. There is almost no activity in the residual liquid of Lipase-ELP50 after foam separation, while 44.88% activity is still left in the residual liquid after Lipase separation. This shows that in foam flotation separation and purification, Lipase-ELP obtains a higher recovery rate and a high enrichment ratio than Lipase, indicating that the fusion ELPs tag can significantly increase the efficiency of lipase foam flotation.

[0112] In summary, the present invention connects lipase (Lipase) with elastin-like protein ELP to obtain the recombinant lipase Lipase-ELP; the ELPs are composed of hydrophobic amino acids, and as the number of ELP sequence repetitions (n) increases, the recombinant enzyme has more hydrophobic amino acids; the elastin-like protein ELP in the recombinant lipase can not only increase the thermal stability of Lipase, but also lead to an increase in the hydrophobicity of the Lipase surface, thereby improving the foaming ability and foam stability of the recombinant lipase, so that it is better adsorbed on the gas-liquid interface, thereby improving the efficiency of foam flotation of the recombinant lipase.

[0113] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A recombinant lipase, characterized in that The recombinant lipase is obtained by connecting lipase Lipase with elastin-like protein ELP; the elastin-like protein ELP is a pentapeptide sequence (Val-Pro-Gly-Val-Gly) n, where n is repeated for 20-100.

2. The recombinant lipase according to claim 1, characterized in that The amino acid sequence of the lipase Lipase is shown in SEQ ID NO:3, and the nucleotide sequence encoding the lipase Lipase is shown in SEQ ID NO:

4.

3. The recombinant lipase according to claim 1, characterized in that The amino acid sequence of the recombinant lipase is shown in SEQ ID NO:

1.

4. The recombinant lipase according to claim 1, characterized in that The n is 50.

5. A nucleic acid encoding the recombinant lipase according to any one of claims 1 to 4, the sequence of which is shown in SEQ ID NO:

2.

6. A recombinant expression plasmid, characterized in that: The recombinant expression plasmid comprises the nucleic acid encoding the recombinant lipase shown in SEQ ID NO: 2 according to claim 5.

7. A recombinant bacterium, characterized in that: The recombinant bacteria contains the recombinant expression plasmid according to claim 6.

8. A method for preparing recombinant lipase, characterized in that: The method comprises fermenting and culturing the recombinant bacteria according to claim 7 to obtain the recombinant lipase.

9. Use of the recombinant lipase according to claim 1 in foam flotation purification and separation.

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