Recombinant high-temperature-resistant lipase PFHL, recombinant strain for expressing enzyme and application of recombinant high-temperature-resistant lipase PFHL
By optimizing and expressing recombinant high-temperature lipase PFHL for lipase genes, the inactivation problem of existing lipase under high temperature and alkaline conditions is solved, and the adaptability and catalytic activity of higher temperature and pH ranges is achieved. It is suitable for Various industrial applications.
Patent Information
- Application Number
- CN202411210110.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-13
AI Technical Summary
Existing lipases are prone to inactivation under high temperature or alkaline conditions, and lack thermal stability and acid-base tolerance, which limits their industrial applications, and have poor hydrolysis specificity for long-chain fatty acids, making it difficult to meet the needs of industrial production. .
By codon optimization of the lipase gene, specific primers are designed for amplification, recombinant plasmids are constructed and expressed in E. coli, recombinant high-temperature lipase PFHL is obtained. The optimal catalytic temperature of this enzyme is 60℃, between 50 and 80 It maintains 80% activity within the range of °C, maintains 70% activity within the range of pH 7.0 to 10.0, and has a preference specificity for medium and long-chain fatty acid esters.
It realizes the high temperature and alkalinity stability of lipase, expands its applicable temperature and pH range, improves catalytic activity and specificity, and is suitable for industrial fields such as oil and fat modification, ester synthesis, drug synthesis and papermaking, reducing production cost.
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Figure CN119120421A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant high temperature resistant lipase PFHL, a recombinant strain expressing the enzyme and applications thereof. Background Art
[0002] Lipase (EC 3.1.1.3) is a triacylglycerol hydrolase, and its main function is to catalyze the hydrolysis of long-chain triglycerides into diglycerides, monoglycerides, glycerol and fatty acids at the oil-water interface. In addition to catalyzing the hydrolysis of glyceride compounds, lipase can also be used to catalyze ester synthesis, transesterification, synthesis of biosurfactants, peptide synthesis, polymer synthesis and drug synthesis. It has the characteristics of catalytic diversity and substrate specificity, and is widely used in many fields such as grain and oil production, food industry, papermaking industry, daily chemical industry, agricultural chemical industry, detergent industry and drug synthesis.
[0003] Lipase ranks third in sales among enzyme preparations, second only to protease and amylase. Currently, lipases used in industrial applications are mainly derived from yeast and bacteria, with the optimum temperature and pH range of 35-45°C and 6.5-8.0, which are suitable for medium-low temperature and neutral application environments. However, there are some common problems with medium-low temperature lipases in industrial applications, such as easy inactivation under high temperature or alkaline conditions, which to a certain extent limits the industrial application of lipases. Regarding the hydrolysis specificity of fatty acid chain length, the vast majority of lipases reported so far preferentially recognize short-chain fatty acids, and only a few can catalyze reactions of long-chain fatty acids. In addition, in the actual production process, there are still problems such as low activity, poor thermal stability, and low specificity. Moreover, with the continuous development of the industrial application of microbial lipase, the market has put forward higher requirements on the properties and indicators of lipase preparations, such as different optimal temperatures and pH values, higher temperature stability and acid-base tolerance, ideal catalytic activity and reaction specificity, tolerance to unconventional solvents (organic solvents, ionic liquids, etc.), etc. Therefore, there is still an urgent need to explore and characterize new resources of lipase preparations with excellent performance so that they can better meet industrial needs. Summary of the invention
[0004] In view of some deficiencies in the prior art, the present invention provides a recombinant high-temperature resistant lipase PFHL, a recombinant strain expressing the enzyme and an application thereof; the present invention performs codon optimization on the lipase gene, then designs primers for a large number of specific amplification of the lipase based on the optimized gene, constructs a recombinant strain expressing the enzyme, induces expression, and obtains the recombinant high-temperature resistant lipase PFHL; the optimum catalytic reaction temperature of the recombinant high-temperature resistant lipase PFHL is 60°C, and the reaction can still maintain 80% of the activity at a temperature range of 50-80°C, and the reaction can still maintain 70% of the activity at a reaction pH of pH 7.0-10.0; the lipase also has a preference for medium- and long-chain fatty acid ester substrates, and different medium- and long-chain fatty acids produced by hydrolyzing glyceride with lipase have different functions, and the lipase has good practicality.
[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:
[0006] The present invention first provides a recombinant thermostable lipase PFHL, the amino acid sequence of the recombinant thermostable lipase PFHL is shown as SEQ ID NO.2.
[0007] The present invention also provides a gene Pfhl encoding the above-mentioned recombinant thermostable lipase PFHL, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0008] The present invention also provides a specific primer pair for amplifying the above-mentioned Pfhl, wherein the primer pair is:
[0009] ccgcgcggcagccatATGAACATGGATGCCAGCAC(SEQ ID NO:3);
[0010] ggtgctcgagttaCAGGCCTTTGCTTTTCAGGC (SEQ ID NO: 4).
[0011] The present invention also provides a recombinant plasmid, which carries the nucleotide fragment of the gene Pfhl.
[0012] The present invention also provides a recombinant strain, wherein the recombinant strain is introduced into a host bacterium; preferably, the host bacterium includes Escherichia coli.
[0013] The present invention also provides the application of the recombinant high temperature resistant lipase PFHL in oil modification, ester synthesis, drug synthesis and papermaking.
[0014] Preferably, when the recombinant thermostable lipase PFHL is used, the reaction temperature is 50-80° C. and the reaction pH is 7.0-10.0.
[0015] An enzyme preparation for producing low-calorie oils and fats, comprising the above-mentioned recombinant thermostable lipase PFHL.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The existing natural lipases from microorganisms only have an optimum reaction temperature of 35 to 45°C. The present invention uses genetic engineering technology to obtain a recombinant high-temperature resistant lipase PFHL, the optimum catalytic reaction temperature of which is 60°C, and the reaction can still maintain 80% activity at a temperature range of 50 to 80°C, and can still maintain 70% activity at a reaction pH of 7.0 to 10.0. The recombinant high-temperature resistant lipase PFHL of the present invention has outstanding advantages such as high temperature resistance and a wide pH range, and will have huge market prospects in various industrial fields, and is of great significance for supplementing the types of high-temperature resistant lipases used in industrial production, and can better meet production needs.
[0018] The recombinant thermostable lipase PFHL prepared by the present invention has good stability under high temperature conditions. The residual enzyme activity of the recombinant thermostable lipase PFHL of the present invention is maintained at more than 70% within 2 hours of incubation at 50-70°C, and the residual enzyme activity can be maintained at more than 85% within 2 hours of incubation at the optimum reaction temperature of 60°C, and has good temperature stability. This characteristic can be widely used in industrial fields such as oil modification, biodiesel, detergents and papermaking to solve the problem of temperature limiting enzyme catalytic reactions in practical applications and reduce industrial application costs. For example, in some ester synthesis processes, such as the catalytic synthesis of some plant sterol esters, palmitic acid esters, vitamin esters and coumaric acid monoterpene esters, the esterification reaction needs to be carried out at a relatively high temperature to obtain its maximum conversion rate; lipase is also required as a catalyst in the production of environmentally friendly bio-based plasticizers, and the general reaction temperature is 50-60°C; in addition, in the papermaking industry, the pulping temperature and flotation temperature of lipase used for waste paper deinking are generally 50-65°C, so lipase is also required to have heat-resistant properties; and during the processing of lipase, enzyme activity loss will occur during high-temperature granulation, drying and other processes, and the activity loss rate of some lipases with poor thermal stability is as high as 80% or more. At the same time, the enzyme preparation is also required to have sufficient stability during transportation, storage and other processes. Therefore, the recombinant high-temperature resistant lipase of the present invention can effectively reduce industrial production costs and still maintain strong stability at high temperatures.
[0019] The recombinant thermostable lipase PFHL prepared by the present invention also has a preference for medium-chain fatty acid ester substrates. Different medium-chain fatty acids produced by lipase hydrolysis of glycerides have different functions. Medium-chain fatty acids can be widely used in medicine, health edible oils and aquaculture; long-chain saturated fatty acids such as palmitic acid and stearic acid are mainly used to produce fatty acid salts as emulsifiers; long-chain unsaturated fatty acids have a wide range of physiological activities in metabolism and are used for health care, etc. Therefore, the lipase prepared by the present invention also has good application value in many fields such as food, medicine, health care and feed industry.
[0020] In the technical scheme of the present invention, the Escherichia coli expression strain used has a high level of recombinant protein expression ability, a high expression level, mild fermentation enzyme production conditions, a wide source of culture medium, a low fermentation production cost, and the characteristics of sustainable development, which has a good promoting effect on actual production applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The figure is the genetic map of the pET-28a(+)-Pfhl plasmid used in the present invention.
[0022] Figure 2 The present invention is an SDS-PAGE gel diagram of the expression product of Pfhl of the present invention; wherein lane M shows protein standard molecular weight markers, which are 180kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, and 15kDa from top to bottom; lane 1 shows the supernatant of Escherichia coli BL21(DE3) / pET-28a(+) cell disruption after induction; lane 2 shows the supernatant of Escherichia coli BL21(DE3) / pET-28a(+)-Pfhl cell disruption after induction; lane 3 shows the precipitate of Escherichia coli BL21(DE3) / pET-28a(+)-Pfhl cell disruption after induction; lanes 4 and 5 show the lipase PFHL protein purified by Ni-NTA after induction.
[0023] Figure 3 This is a curve diagram of the relative enzyme activity of the recombinant thermostable lipase PFHL and temperature.
[0024] Figure 4 This is the thermal stability curve of the recombinant thermostable lipase PFHL.
[0025] Figure 5 This is a curve diagram of the relative enzyme activity of the recombinant thermostable lipase PFHL and pH.
[0026] Figure 6This is a diagram of the fatty acid chain length specificity of the recombinant thermostable lipase PFHL (p-NPC2, p-NPC4, p-NPC8, p-NPC12, p-NPC16, p-NPC18). DETAILED DESCRIPTION
[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are followed. The reagents or instruments used are not indicated by the manufacturer and are conventional products that can be purchased on the market.
[0028] The primer synthesis and sequencing work used in the following examples were completed by Suzhou Jinweizhi Biotechnology Co., Ltd. The protein expression vector used was pET-28a(+) (purchased from Wuhan Miaoling Biotechnology Co., Ltd.), and the Escherichia coli BL21 and DH5α used were purchased from Shanghai Shenggong Biotechnology.
[0029] Example 1: Cloning of lipase gene and construction of recombinant plasmid
[0030] The lipase gene from Pseudomonas fluorescens was codon-optimized according to the preference of Escherichia coli to obtain a lipase gene Pfhl fragment with nucleotides as shown in SEQ ID NO: 1. The GC content of the lipase gene Pfhl was 50.23% and the codon adaptability index was 0.70.
[0031] The lipase gene Pfhl was submitted to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis of the target gene fragment, and then the synthesized gene fragment was used as a template to amplify the target gene by PCR using a pair of forward and reverse primers (SEQ ID NO: 3 and SEQ ID NO: 4). The procedure was performed for 28-35 cycles. After the PCR reaction was completed, 45 μL of the reaction product was taken for 1% agarose gel electrophoresis, and the PCR results were observed by comparing with DNAMaker. The pET-28a (+) empty plasmid was subjected to reverse PCR with primers, and the obtained linearized vector and the purified target fragment were seamlessly cloned to obtain a recombinant plasmid carrying the nucleotide fragment of the gene Pfhl. The recombinant plasmid was as follows: Figure 1 The PCR reaction system is shown in Table 1, and the PCR amplification program settings are shown in Table 2.
[0032] Table 1. PCR reaction system
[0033] Reagents volume 2×Phanta Flash Master Mix 25μL Upstream primer 2.5μL Downstream primer 2.5μL DNA template 1μL <![CDATA[ddH2O]]> 19μL
[0034] Table 2. PCR amplification program
[0035] Cycle steps temperature time Pre-denaturation 98℃ 30sec transsexual 98℃ 10sec annealing Tm 5sec extend 72℃ 4-5sec / kb Complete extension 72℃ 1min
[0036] SEQ ID NO.1:
[0037] atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccatATGAACATGGATGCCAGCACCCAGTATCCGATTGTGCTGGTGCATGGCCTGTTTGGCTTTGATACCATTGCCGGTTATCCGTATTTTTTTGATATTAAAGAGGCGCTGGAATATGCAGGTGCACGTGTGTTTGCAGTTAATATTCCGGCAGTGAATGGTAATGAAGAACGTGGTGAAAAACTGCTGGAACAGGTGAATCGCATTCTGCAGGAAACCGGTGCCGCCCGCGTTAATCTGATTGGCCATAGCCAGGGCCCGCTGGCCGCCCGTTATGTGGCTGCACTGTATCCGGAAAAAGTTGCCAGCGTTACCAGTGTGAGTTGTCCGAATCATGGTAGCGAAATTGCAGATCAGCTGCATAAAGCCCTGACCCCGGGTGAACTGCCGGAAGCCTTTGCCCTGGCACTGCTGAGCGCCGTGGGTACCTTTATTAGCCTGATTAGTGGTCATCCGAAAAATCCGCAGGATCCGCAGGCAGCCTTTGAAAGTCTGACCAGTGCCGGTCTGGCCGAATTTAATCGCAAATATCCGCAGGGCGTTCCGAAAGTGTGGGGCGGTGAAGGTAATGAAATTGAAAATGGTGTTTACTACTACAGCTGGAGCGGCATTCTGCAGAATTTTCAGACCCCGCAGATTCTGGATCCGACCCATATTAATTGCATTGTGCTGAGCAAACTGTTTTATAAAGAAAAAGACGCAAACGATGGCCTGGTTGGCCGCTATAGTAGTCATCTGGGCAAAGTTATTCGCAGTGATTATCCGATGGATCATTTTGATGCCGTGAATCAGATGGCCGGCATTACCAGCTGGAATGTGAGCCCGGTGAGTCTGTATGTGGAACATGCCGTTCGCCTGAAAAGCAAAGGCCTGtaa;
[0038] SEQ ID NO.2:
[0039] MGSSHHHHHHSSGLVPRGSHMNMDASTQYPIVLVHGLFGFDTIAGYPYFFDIKEALEYAGARVFAVNIPAVNGNEERGEKLLEQVNRILQETGAARVNLIGHSQGPLAARYVAALYPEKVASVTSVSCPNHGSEIADQLHKALTPGELPEAFALA LLSAVGTFISLISGHPKNPQDPQAAFESLTSAGLAEFNRKYPQGVPKVWGGEGNEIENGVYYYSWSGILQNFQTPQILDPTHINCIVLSKLFYKEkDaNDGLVGRYSSHLGKVIRSDYPMDHFDAVNQMAGITSWNVSPVSLYVEHAVRLKSKGL;
[0040] SEQ ID NO:3:
[0041] ccgcgcggcagccatATGAACATGGATGCCAGCAC;
[0042] SEQ ID NO:4:
[0043] ggtgctcgagttaCAGGCCTTTGCTTTTCAGGC;
[0044] Example 2: Inducible expression of recombinant thermostable lipase PFHL
[0045] The recombinant plasmid obtained in Example 1 was transformed into Escherichia coli for heterologous expression, and the specific steps were as follows:
[0046] Take 10 μL of recombinant plasmid and add it to 100 μL of competent cell E. coli DH5α centrifuge tube, put it in ice bath for 30 minutes, quickly heat shock to 42℃ for 1 minute, and immediately put it on ice for 2 minutes. Then add fresh LB medium, recover and culture for 1 hour, centrifuge at 5000rpm for 5 minutes, discard 900 μL of supernatant, and spread the remaining bacteria on LB solid plate containing 50 μg / mL kanamycin, and place it in 37℃ incubator to culture, and obtain positive clones.
[0047] The positive clones were inoculated into LB test tube medium containing 50 μg / mL kanamycin, cultured at 37°C in a shaking incubator, plasmids were extracted, and the band size was compared with the standard marker band by gel electrophoresis and sequenced. The strain with the correct pET-28a(+)-Pfhl was added with an equal volume of 20% glycerol and stored at -80°C. The correct plasmid was transformed into E.coli BL21(DE3) competent cells to construct an expression strain, and E.coli BL21(DE3)(pET-28a(+)-Pfhl) was obtained, and expression was induced.
[0048] The fresh bacterial suspension of E. coli BL21 (DE3) (pET-28a (+) -Pfhl) with correct sequencing results was transferred to a 50 mL LB medium flask containing 50 mg / mL Kan at a ratio of 1% (v / v) and cultured at 37°C until OD 600nm The final concentration of isopropylthiogalactoside (IPTG) was 0.6-0.8, and the final concentration of 0.5mmol / L was added for induction, and the culture was induced at 16°C and 100rpm for 24h. Then the bacteria were collected by centrifugation, and the bacterial precipitate was resuspended and washed twice with 20mM PBS buffer at pH8.0, and the cells were ultrasonically broken in an ice water bath (total time was about 30min, working for 3s, and intervals of 5s). When the bacterial solution changed from a viscous state to a transparent state, it was ready, and it was centrifuged at 10000rpm for 10min. The supernatant and precipitate were boiled with electrophoresis buffer and then verified by SDS-PAGE gel electrophoresis for protein expression. After expression, the recombinant thermostable lipase PFHL was obtained.
[0049] from Figure 2 It can be seen that compared with the protein of the E. coli BL21 (DE3) (pET-28a (+)) empty vector in lane 1, there is an obvious protein band at 33.8 kDa in lanes 2 and 3, indicating that E. coli BL21 (DE3)
[0050] (pET-28a(+)-Pfhl) successfully expressed the recombinant lipase protein PFHL under the induction of IPTG. The molecular weight of the protein PFHL is 31.7kDa, but because it carries a 6×His tag and a tag cleavage site, the molecular weight of the recombinant protein is predicted to be 33.8kDa, which is consistent with the SDS-PAGE result.
[0051] Example 3: Purification of recombinant lipase PFHL
[0052] The recombinant thermostable lipase PFHL induced and expressed in Example 2 has His tags at both ends and can be expressed using Ni 2+ -NTA affinity chromatography column was used for purification, and the specific steps are as follows:
[0053] First, wash the column with 5 mL of deionized water, and then wash the column with 3 mL of Binding Buffer to balance the column bed filling. The supernatant obtained by centrifugation, that is, the crude enzyme solution, is repeatedly loaded into the column to bind the protein to the resin as much as possible, and then the column is washed with 3 mL of 20 mM imidazole concentration Washing Buffer to wash away most of the impurities that cannot bind to the resin. The target protein PFHL is eluted with 5 mL of 500 mM imidazole concentration Elution Buffer, and the eluate is collected in a 1.5 mL EP tube, which is the pure enzyme solution.
[0054] The concentration of the purified recombinant thermostable lipase PFHL was determined by BCA protein concentration determination. The purified recombinant thermostable lipase PFHL was diluted to the corresponding multiple and subjected to color development reaction. After the color development was stable, the OD was determined. 595nm The protein concentration of the recombinant thermostable lipase PFHL can be obtained according to the standard curve.
[0055] Depend on Figure 2 It can be seen that the recombinant protein has been successfully induced to express in the expression strain, and after Ni 2+ -NTA affinity chromatography column purification can obtain a pure single band (such as Figure 2 4 in the swimming lane), indicating that the target protein was successfully purified, and the protein concentration of the diluted pure enzyme was determined to be 1.06 mg / mL.
[0056] Example 4: Enzymatic properties of recombinant thermostable lipase PFH
[0057] In this example, p-nitrophenol palmitate was used as a reaction substrate, and the activity of the purified recombinant thermostable lipase PFHL at different temperatures and pH values was determined by the p-NP method to investigate the enzymatic properties of the purified recombinant thermostable lipase PFHL.
[0058] (1) Effects of different temperatures on the activity of recombinant thermostable lipase PFHL:
[0059] Using p-nitrophenol palmitate as substrate, add purified diluted enzyme solution to Tris-HCl buffer at pH 8.0, set up 3 parallels, and then put them into water baths at 20-70℃ for reaction. The highest enzyme activity was set as 100%, and the relative enzyme activity under other conditions was calculated. The results are as follows: Figure 3 As shown. And determine its thermal stability, that is, half-life, place equal volumes of enzyme solution in water baths at different temperatures for insulation, take samples every 2 hours to determine the enzyme activity, set the highest enzyme activity as 100%, and calculate the relative enzyme activity under other conditions. The results are shown in Figure 4 shown.
[0060] from Figure 3 It can be seen that within the temperature range of 20-90°C, the activity of the recombinant thermostable lipase PFHL showed a trend of first increasing and then decreasing. Among them, the optimum reaction temperature of the recombinant thermostable lipase PFHL was 60°C, and the relative enzyme activity was the highest, which was set to 100%. At 50-80°C, the relative enzyme activity was maintained at more than 80%; while at temperatures below 40°C, the relative enzyme activity decreased with decreasing temperature. This shows that the recombinant thermostable lipase PFHL has a stronger tolerance to high temperatures and is more suitable for industrial applications; but it is more sensitive to low temperatures. In a low temperature environment, the enzyme activity of the recombinant thermostable lipase PFHL will be greatly inhibited.
[0061] from Figure 4 It can be seen that the residual enzyme activity of the recombinant thermostable lipase PFHL was maintained above 70% within 2 h of incubation at 50-70°C, and the half-life was 2.3 h at the optimal reaction temperature of 60°C, showing good temperature stability.
[0062] (2) Effect of different pH on the activity of recombinant thermostable lipase PFHL:
[0063] In order to determine the effect of different pH on the lipase, 50mmol / L PBS buffer with different pH values, such as pH 6.0-8.0, Tris-HCl buffer with pH 8.0-9.0, and Gly-NaOH buffer with pH 9.0-11.0, were prepared as reaction systems, and p-nitrophenol palmitate was used as substrate. The reactions were carried out in a 37℃ water bath, and the highest enzyme activity was set as 100%. The relative enzyme activities under other conditions were calculated. The results are shown in Figure 2. Figure 5 shown.
[0064] from Figure 5 It can be seen that in the pH range of 6.0-11.0, the lipase activity shows a trend of first increasing and then decreasing. Among them, the optimal reaction pH of lipase PFHL is 7.0, and the relative enzyme activity is the highest, which is set to 100%; and it has higher enzyme activity in the alkaline reaction environment of pH 7.0-10.0, and its relative enzyme activity is maintained above 70%, but the enzyme activity decreases rapidly under acidic conditions, and is only half of its enzyme activity in an alkaline environment.
[0065] In summary, it is shown that the optimal reaction temperature of the recombinant lipase PFHL is 60°C, which is a high-temperature lipase, and it still has good enzyme activity at 50-80°C, with a certain temperature tolerance. Its optimal reaction pH is 7.0, and the enzyme activity is stable within pH7.0-10.0, and it has a certain alkaline resistance. Therefore, this enzyme is a heat-resistant and alkali-resistant lipase, and it will have a good application prospect in catalyzing reactions under high temperature and alkaline environments.
[0066] Example 5: Study on fatty acid specificity of recombinant lipase PFHL
[0067] In this example, p-nitrophenol fatty acid esters with different alkyl carbon chain lengths (p-NPC2, p-NPC4, p-NPC8, p-NPC12, p-NPC16, p-NPC18) were used as substrates to investigate the fatty acid chain length specificity of lipase PFHL at pH 7.0 and temperature 60°C. Figure 6 It can be seen that lipase PFHL has the best specificity for medium-chain fatty acid ester p-NPC12, and its relative enzyme activity decreases with the decrease of fatty acid carbon chain length. For short-chain fatty acid esters p-NPC2 and p-NPC4, the specificity is low, and the hydrolysis activity is 86.32% and 68.10% lower than that of p-NPC12; the specificity for long-chain fatty acid ester p-NPC16 is also good, and the hydrolysis activity is only 9.21% lower than that of p-NPC12. Therefore, it can be seen that lipase PFHL has good specificity for medium- and long-chain fatty acid esters, especially the highest hydrolysis activity for p-NPC12 and p-NPC16.
[0068] In summary, the present invention performs codon optimization on the lipase gene, and then designs primers for a large number of specific amplification of lipase based on the optimized gene, and constructs a recombinant strain expressing the enzyme, induces expression, and obtains a recombinant thermostable lipase PFHL; the optimal catalytic reaction temperature of the recombinant thermostable lipase PFHL is 60°C, and the reaction can still maintain 80% activity at a temperature range of 50 to 80°C, and the reaction can still maintain 70% activity at a reaction pH of pH 7.0 to 10.0; the lipase also has a preference for medium- and long-chain fatty acid ester substrates, and different medium- and long-chain fatty acids produced by lipase hydrolysis of glycerides have different functions, and have good practicality. The recombinant thermostable lipase PFHL can be used to meet various high-heat industrial applications, such as in health food, drug synthesis, oil modification, biodiesel, detergents, and papermaking industries.
[0069] 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 thermostable lipase PFHL, characterized in that: The amino acid sequence of the recombinant thermostable lipase PFHL is shown in SEQ ID NO.
2.
2. The gene Pfhl encoding the recombinant thermostable lipase PFHL according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
3. The specific primer pair for amplifying Pfhl according to claim 2 is characterized in that, The primer pairs are: ccgcgcggcagccatATGAACATGGATGCCAGCAC(SEQ ID NO:3); ggtgctcgagttaCAGGCCTTTGCTTTTCAGGC (SEQ ID NO: 4).
4. A recombinant plasmid, characterized in that: The recombinant plasmid carries the nucleotide fragment of the gene Pfhl according to claim 2.
5. A recombinant strain, characterized in that: The recombinant strain is obtained by introducing the recombinant plasmid according to claim 4 into the host bacteria.
6. The recombinant strain according to claim 5, characterized in that The host bacteria include Escherichia coli.
7. Use of the recombinant thermostable lipase PFHL according to claim 1 in oil modification, ester synthesis, drug synthesis and papermaking.
8. The use according to claim 7, characterized in that: When the recombinant thermostable lipase PFHL is used, the reaction temperature condition is 50-80°C and the reaction pH condition is 7.0-10.
0.
9. An enzyme preparation, characterized in that The enzyme preparation comprises the recombinant thermostable lipase PFHL according to claim 1.