A lipase with high stability and its application

Abstract

The present invention provides a lipase with high stability and its application, belonging to the field of enzyme technology and genetic engineering technology. The lipase has an amino acid sequence shown in SEQ ID No.1, or replaces the leucine at position 305 of the SEQ ID No.1 sequence with arginine; the lipase is used to produce fatty acid derivatives, and the production method comprises the following steps: S1, mixing oil and ethanol, adding the lipase, and reacting in a reactor; S2, centrifuging the reaction solution, collecting the upper oil phase; S3, performing a membrane treatment on the collected upper oil phase; S4, removing ethanol from the oil phase; S5, distilling to obtain fatty acid derivatives. The lipase of the present invention can catalyze the conversion of oil into fatty acid derivatives under relatively high temperature conditions, and the enzyme activity does not show a significant decline for a long time.

Description

Technical Field

[0001] The invention relates to the technical fields of enzyme technology and genetic engineering, in particular to a lipase with high stability and application thereof. Background Art

[0002] Lipase is an enzyme that can decompose oil into fatty acids, monoglycerides or diglycerides, and glycerol. Lipase is widely used in the food industry, paper industry, energy industry, and cosmetics industry due to its wide source, environmental friendliness, safety, non-toxicity, and wide range of effects. It has important application value in the production of biodiesel.

[0003] Many food processing and biodiesel production processes are carried out at high reaction temperatures, but lipase is sensitive to heat and is easily inactivated and denatured under high temperature conditions, which causes its catalytic efficiency to drop rapidly, making it difficult to cope with the harsh conditions in the production process. Therefore, a lipase with high activity and high thermal stability is needed to achieve efficient catalytic reactions.

[0004] CN112266906A provides a thermostable acidic lipase LIP and its gene and application. The lipase encoded by the patent has high enzyme activity under acidic and neutral conditions, with an optimum temperature of 55°C, an optimum pH of 4.5, and good thermal stability under 70°C. However, the activity of the lipase provided by the patent decreases rapidly with increasing temperature. Therefore, in addition to high temperature resistance, the stability of the lipase at high temperature also needs to be modified. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a lipase with high stability and its application. The lipase of the present invention can catalyze the conversion of oil into fatty acid derivatives as shown in formula (I) under high temperature conditions, and the enzyme activity does not show obvious decline for a long time.

[0006] The technical solution of the present invention is as follows:

[0007] A lipase with high stability, the lipase has an amino acid sequence shown in SEQ ID No.1.

[0008] The present invention also provides another lipase with high stability, wherein the lipase has an amino acid sequence as shown in SEQ ID No. 1, and the leucine at position 305 of the SEQ ID No. 1 sequence is replaced by arginine.

[0009] The present invention also provides the use of the two lipases with high stability, wherein the lipases are used to produce fatty acid derivatives as shown in formula (I):

[0010]

[0011] Among them, R is a fatty acid backbone with different carbon chain lengths.

[0012] Furthermore, the production method of the fatty acid derivative represented by formula (I) comprises the following steps:

[0013] S1, mixing oil and ethanol, adding the lipase, and reacting in a reactor;

[0014] S2, centrifuging the reaction solution and collecting the upper oil phase;

[0015] S3, performing membrane treatment on the collected upper oil phase;

[0016] S4, removing ethanol from the oil phase;

[0017] S5. Distill to obtain fatty acid derivatives.

[0018] Preferably, the oil described in step S1 includes but is not limited to coconut oil, sunflower seed oil, jojoba seed oil, rosehip oil, grape seed oil, almond oil, white meadowfoam seed oil, macadamia oil, olive oil, nut seed oil, camellia seed oil, prune oil, sea buckthorn seed oil, peony seed oil, hazelnut oil, rice germ oil, wheat germ oil, borage seed oil, safflower seed oil, echium seed oil, bilberry seed oil, baobab seed oil, palm fruit oil, Chilean hazel oil, African wild mango oil, white lupine oil, and olive shell oil.

[0019] Preferably, in step S1, the mass ratio of the oil to ethanol is 1:0.01-100; the mass of the lipase is 0.1%-30% of the mass of the oil.

[0020] Preferably, the reaction temperature in step S1 is 20-80° C., and the reaction time is 1-48 h.

[0021] Preferably, the membrane treatment in step S3 is filtering using a ceramic membrane, an ultrafiltration membrane or a nanofiltration membrane.

[0022] Preferably, the method for removing ethanol from the oil phase in step S4 includes rotary evaporation, flash evaporation, molecular distillation or rectification.

[0023] The beneficial technical effects of the present invention are:

[0024] 1. The lipase of the present invention has the characteristics of high stability and can react at relatively high temperature. When used to produce fatty acid derivatives as shown in formula (I), the obtained product has the characteristics of low acid value and peroxide value.

[0025] 2. The lipase of the present invention can react under higher reaction conditions, thereby shortening the reaction time of the esterification reaction and improving the reaction efficiency. In addition, the lipase can remain stable for a long time at high temperature, thereby improving the yield of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the determination of the optimal reaction temperature of lipase;

[0027] Figure 2 The results of lipase thermal tolerance test;

[0028] Figure 3 This is the result of molecular dynamics simulation of lipase;

[0029] Figure 4 The results of enzyme stability test after modification. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1: Screening of thermotolerant lipase-producing strains

[0032] A1. Prepare a selective culture medium for lipase screening, the composition of the culture medium is:

[0033] K 2 HPO 4 1.5 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 0.5 g / L, FeSO 4 1 mg / L, macadamia oil 15g / L, agar 15g / L, add 0.5g / L rhodamine B after autoclaving.

[0034] A2. After activating the samples collected from high temperature environments (such as hot springs, saunas, and near volcanoes) in Guangzhou and other places, evenly spread them on solid plates of selective culture medium, and culture them at a constant temperature of 60°C for 48 hours. Based on the size of the hydrolysis circle on the plate, strains with lipase activity were preliminarily screened out.

[0035] A3. Rescreening: The strains obtained from the initial screening were inoculated into the fermentation medium for cultivation. The enzyme activity of each fermentation broth was determined by measuring the absorbance value of p-nitrophenol (p-NP) produced by the hydrolysis of p-nitrophenyl palmitate (p-NPP) at 410 nm, thereby selecting the strain with the highest lipase activity.

[0036] The formula of the fermentation medium is: glucose 5g / L, peptone 2g / L, urea 6g / L, K 2 HPO 4 1.5 g / L, (NH 4 ) 2 SO 4 1 g / L, MgSO 4 0.5 g / L, macadamia oil 100g / L.

[0037] The fermentation culture method is: select a single colony from a solid plate into a liquid culture medium, and ferment and culture at 45°C to prepare a seed solution. Then, the seed solution is transferred to a fermentation medium and fermented at 45°C.

[0038] The formula of the liquid culture medium is: K 2 HPO 4 1.5 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 0.5 g / L, FeSO 4 1mg / L, macadamia oil 15g / L.

[0039] The strain obtained by screening was identified by the 16S method, and after comparison, the obtained strain was Geobacillus. The whole genome of the strain obtained by screening was further sequenced by Nanopore sequencing, and the amino acid sequence of the lipase therein was obtained as shown in SEQ ID NO.1.

[0040] Example 2: Construction of strain expressing high stability lipase

[0041] B1. In order to realize the application of the lipase obtained in Example 1, the lipase was selected to be expressed intracellularly in an Escherichia coli host. The transmembrane region of the protein was analyzed using a protein transmembrane analysis tool to obtain that the protein was secreted. Therefore, the first 30 amino acids (transmembrane region) of the lipase were removed and optimized for the codon preference of Escherichia coli, and synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthesized nucleotide sequence is shown in SEQ ID NO.2.

[0042] B2. The lipase encoding gene was amplified using primers 1 and 2, and the plasmid pET28a was digested with NcoI and EcoRI. The two fragments were purified and connected using the Gibson seamless cloning kit, and then transformed into E. coli DH5α competent cells. The positive clones were screened using LB solid plates containing 50 μg / mL kanamycin, and the correctness of the gene cloning was further identified by sequencing. The correct plasmid obtained was named pJJL-24-06-1.

[0043] The sequence of primer 1 is:

[0044] ctttaagaaggagatataccATGCTGCGCGCGAACGACGCTCC;

[0045] The sequence of primer 2 is:

[0046] gcttgtcgacggagctcgaattcTCAAGGTTGTAAGCTCGCCAG.

[0047] B3. Transform pJJL-24-06-1 into E. coli BL21 (DE3) competent cells. Use LB solid plates containing 50 μg / mL kanamycin to screen positive clones, and verify the correct transformant obtained by PCR and name it JJL-24-06-1.

[0048] Example 3: Obtaining highly stable lipase

[0049] C1. Inoculate a single colony of the engineered bacteria JJL-24-06-1 into 10 mL of LB medium (add kanamycin at a final concentration of 50 μg / mL), and culture at 37° C. and 220 rpm for 12-16 h until mid-logarithmic phase to obtain seed solution.

[0050] C2. Transfer 2 mL of seed solution to a 500 mL shake flask containing 100 mL of LB medium (with kanamycin added at a final concentration of 50 μg / mL) and culture at 37°C and 220 rpm until OD 600 Reach 0.6-0.8.

[0051] C3. Add IPTG at a final concentration of 1 mM and continue culturing for 8 h.

[0052] C4. Collect the bacterial precipitate by centrifugation, wash it twice with 50 mM PBS solution, and resuspend it to OD 600 is 50.

[0053] C5. High-pressure homogenization to break up the resuspended bacteria at a pressure of about 800 bar for three times.

[0054] C6. Centrifuge and collect the supernatant for subsequent reactions.

[0055] Example 4: Determination of the optimal reaction temperature of lipase

[0056] The method for determining the enzyme activity is as follows: lipase hydrolyzes p-nitrophenol palmitate p-NPP to release p-nitrophenol, which is yellow-green in color under alkaline conditions. The enzyme activity of lipase is calculated by measuring the absorbance value at a wavelength of 410 nm.

[0057] The enzyme activity is defined as: 1 mg of lipase hydrolyzes p-nitrophenol palmitate for 1 min to release 1 μmol of p-nitrophenol, which is 1 enzyme activity unit U, μmol / mg·min.

[0058] The lipase activity was measured at 20-80℃. Figure 1 As shown, it can be seen that within the range of 20-70°C, as the reaction temperature increases, the lipase activity gradually increases, and when the reaction temperature is 70°C, the highest enzyme activity is 351 U. Then, as the reaction temperature increases, the lipase activity decreases rapidly.

[0059] Example 5: Determination of thermal tolerance of lipase

[0060] The thermal tolerance of lipase was measured at 50℃, 55℃, 60℃, 65℃ and 70℃ respectively. The specific test method is:

[0061] The lipase obtained in Example 3 was placed at 50°C, 55°C, 60°C, 65°C, and 70°C for 12 hours, and samples were taken every 3 hours to measure the lipase activity. Figure 2 As shown, it can be seen that at different storage temperatures, the lipase activity decreases to varying degrees as the storage time increases. When the storage temperature is not higher than 60°C, the lipase activity can be retained by more than 85% after 12 hours. When the storage temperature reaches 70°C, the lipase activity still remains at 60% after 12 hours. Therefore, lipase has good heat tolerance.

[0062] Example 6: Modification of thermal tolerance of lipase

[0063] Molecular dynamics simulation was used to simulate the unstable region of the lipase shown in SEQ ID NO.1. Figure 3 As shown in Figure 2, the leucine at position 305 (L277) has the characteristic of poor stability. Therefore, the leucine was mutated to arginine, and the stability of the mutated enzyme was measured. The thermal stability of the original enzyme and the mutated enzyme was measured at 70°C. The results are shown in Figure 2. Figure 4As shown. From the results, it can be seen that after being placed at 70℃ for 12h, the modified enzyme can still maintain more than 80% of its activity, while the activity of the original enzyme is only 60%. The stability advantage of the modified enzyme is obvious.

[0064] Example 7: Yield of enzymatic esterification of fat before transformation

[0065] The method of producing the fatty acid derivatives as shown in formula (I) by lipase catalysis comprises the following steps:

[0066] Macadamia oil was used as a substrate and ethanol as an acyl donor. The lipase prepared in Example 3 was added to react. The addition ratio of macadamia oil, ethanol and lipase was 1:1:0.2. The reaction temperature was 60°C. The reaction was carried out in a continuously stirred reactor for 12 hours. The yield of fatty acid derivatives was determined by gas chromatography, and the yield was 52.1%.

[0067] Example 8: Esterification reaction process of modified lipase

[0068] The method of producing the fatty acid derivatives as shown in formula (I) by lipase catalysis comprises the following steps:

[0069] S1. Using macadamia oil as a substrate and ethanol as an acyl donor, the lipase prepared in Example 6 was added to react. The mass ratio of macadamia oil, ethanol and lipase added was 1:1:0.2. The reaction temperature was 60°C. The reaction was carried out in a continuously stirred reactor for 12 hours. The yield of crude fatty acid derivatives was determined by gas chromatography, and the yield was 72.1%.

[0070] S2, centrifuging the obtained reaction product at 8000g and 25°C, and taking the upper oil phase;

[0071] S3, filtering the obtained oil phase using an IRIS 3042 membrane;

[0072] S4, using a rotary evaporator at 45°C and a vacuum degree of 0.09 MPa to remove ethanol contained in the oil phase;

[0073] S5. Using a distillation tower, obtain the fatty acid derivative represented by formula (I).

[0074] The acid value and peroxide value of the obtained fatty acid derivative were measured. The acid value of the obtained product was 0.211 mgKOH / g, and the peroxide value was 0.12 g / 100 g.

[0075] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A lipase, characterized in that The amino acid sequence of the lipase is that the leucine at position 305 of the SEQ ID No. 1 sequence is replaced by arginine.

2. Use of the lipase according to claim 1, characterized in that: The lipase is used to produce a fatty acid derivative as shown in formula (I): Among them, R is a fatty acid backbone with different carbon chain lengths.

3. The use according to claim 2, characterized in that: The production method of the fatty acid derivative represented by formula (I) comprises the following steps: S1, mixing oil and ethanol, adding the lipase, and reacting in a reactor; S2, centrifuging the reaction solution and collecting the upper oil phase; S3, performing membrane treatment on the collected upper oil phase; S4, removing ethanol from the oil phase; S5. Distill to obtain fatty acid derivatives.

4. The use according to claim 3, characterized in that: The oils and fats described in step S1 include but are not limited to coconut oil, sunflower seed oil, jojoba seed oil, rosehip oil, grape seed oil, almond oil, white meadowfoam seed oil, macadamia oil, olive oil, nut seed oil, camellia seed oil, prune oil, sea buckthorn seed oil, peony seed oil, hazelnut oil, rice germ oil, wheat germ oil, borage seed oil, safflower seed oil, echium seed oil, bilberry seed oil, baobab seed oil, palm fruit oil, Chilean hazelnut oil, African wild mango oil, white lupine oil, and olive shell oil.

5. The use according to claim 3, characterized in that: In step S1, the mass ratio of the oil to ethanol is 1:0.01-100; the mass of the lipase is 0.1%-30% of the mass of the oil.

6. The use according to claim 3, characterized in that: The reaction temperature in step S1 is 20-80° C. and the reaction time is 1-48 h.

7. The use according to claim 3, characterized in that: The membrane treatment in step S3 is filtering using a ceramic membrane, an ultrafiltration membrane or a nanofiltration membrane.

8. The use according to claim 3, characterized in that: The method for removing ethanol from the oil phase in step S4 includes rotary evaporation, flash evaporation, molecular distillation or rectification.

Images