RML lipase chimera and its application

Abstract

The present invention discloses an RML lipase chimera and its application. The amino acid sequence of the RML lipase chimera is shown in SEQ ID NO. 2. The RML lipase chimera has both lipase and phospholipase activity, and its hydrolysis activity on phospholipid substrates is increased by 161 times compared to wild-type RML lipase, better meeting the enzyme performance requirements in phospholipid processing. It can be used for oil degumming, phospholipid modification, and lysophospholipid production, and has applications in the fields of biology, food, medicine, beauty, agriculture, and industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and in particular relates to a Rhizomucor miehei lipase (RML) chimera with high phospholipid hydrolysis activity and application thereof. Background Art

[0002] Methods for oil degumming include hydration degumming, acid refining degumming, adsorption degumming, thermal polymerization degumming, and chemical reagent degumming. Compared to other degumming methods, enzymatic degumming significantly reduces chemical consumption and produces virtually no wastewater, offering potential advantages in environmental protection and quality. Typically, an aqueous solution of an enzyme preparation, such as phospholipase, is added to an oil-water mixture. This mixture is then stirred through a high-shear agitator to form an emulsion. The enzyme reacts with the phospholipids in the crude oil at a specific temperature, removing the fatty acids at the first or second position. This allows the gum to hydrate and aggregate, and finally be removed through a high-speed centrifuge. Degumming is a crucial step in vegetable oil refining, and its effectiveness directly impacts refining efficiency and product quality. Incomplete degumming increases the decolorization burden, impacting both oil quality and the economic benefits of refining.

[0003] When it comes to the development of enzymes for degumming, research has largely focused on phospholipases. Enzymatic degumming technologies that use novel phospholipases to remove phospholipids from oils and fats are also gaining increasing attention. However, experiments have revealed that lipases also possess phospholipases' hydrolytic activity. Therefore, highly active phospholipid hydrolysis substrates can be obtained by screening or modifying lipases. Currently, Novozymes has successfully developed a lipase chimera, Lecitase Ultra, for enzymatic degumming in oil and fat processing. Lecitase Ultra, released by Novozymes, is the only commercially available lipase for vegetable oil degumming. However, there are relatively few reported chimeras. Domestic development of degumming enzymes, particularly lipase-based degumming enzymes, is still in its infancy.

[0004] Lysophospholipids can be used as emulsifying dispersants and emulsifiers in food processing and have certain antibacterial effects that are superior to those of ordinary phospholipids. They have been approved as natural food additives in many countries. Lysophospholipids, when added to skincare cosmetics, can regulate and improve skin respiration and smooth wrinkles. Lysophospholipids also have unique physiological functions in the pharmaceutical field. Currently, lysophospholipids are generally obtained by treating them with enzymes containing phospholipase A1 or A2. However, there are few reports on the modification of lipases to obtain higher phospholipase activity.

[0005] Protein engineering is an effective technique for improving the catalytic properties of bioenzymes, such as increasing thermal stability, enhancing catalytic efficiency, and modifying substrate selectivity. This technology has been applied to create enzymes with both lipase and phospholipase hydrolysis activities, which have promising applications in phospholipid modification. These enzymes can reduce the glyceride content in raw materials while increasing the content of lysophospholipid products. However, such bioenzyme preparations are currently lacking. Summary of the Invention

[0006] Based on this, one of the objectives of the present invention is to provide a RML lipase chimera, wherein the lipase chimera has both lipase activity and phospholipase hydrolysis activity.

[0007] The specific technical solutions for achieving the above-mentioned invention objectives include the following:

[0008] An RML lipase chimera, the amino acid sequence of the RML lipase chimera is shown in SEQ ID NO.2.

[0009] The present invention also provides a gene encoding the above-mentioned RML lipase chimera, whose nucleotide sequence is shown in SEQ ID NO.3, or its nucleotide sequence is the reverse sequence of SEQ ID NO.3.

[0010] The present invention also provides the use of the RML lipase chimera or the coding gene of the RML lipase chimera in oil degumming.

[0011] The present invention also provides the use of the RML lipase chimera or the coding gene of the RML lipase chimera in the preparation of lysophospholipids.

[0012] The present invention also provides a recombinant expression vector into which the coding gene of the RML lipase chimera is inserted.

[0013] The present invention also provides a recombinant engineering bacterium transformed with the above recombinant expression vector.

[0014] In some embodiments, the host strain of the recombinant engineered bacteria is Pichia pastoris.

[0015] The present invention also provides the use of the recombinant expression vector or the recombinant engineering bacteria in oil degumming.

[0016] The present invention also provides the use of the above-mentioned recombinant expression vector or the above-mentioned recombinant engineering bacteria in the preparation of lysophospholipids.

[0017] The present invention also provides a method for oil degumming, which utilizes the RML lipase chimera to catalyze a reaction on an oil-water mixture.

[0018] The present invention also provides a method for preparing lysophospholipids, which utilizes the RML lipase chimera to catalyze the conversion of phospholipid substrates into lysophospholipids.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] In the present invention, a special polypeptide gene is fused with the Rhizomucor miehei lipase RML encoding gene to obtain an RML lipase chimera. The RML lipase chimera has lipase activity and phospholipase activity at the same time. Compared with the wild-type RML lipase, the hydrolysis activity of the phospholipid substrate is increased by 161 times, which is more in line with the requirements for enzyme performance in phospholipid processing. It can be used for oil degumming and production of hemolysin, and is applied to biology, food, medicine, beauty, agriculture, industry and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an electrophoresis detection diagram of the purified RML lipase chimera in Example 1 of the present invention.

[0022] Figure 2 This is the result of the effect of pH on the enzyme activity of the RML lipase chimera in Example 4 of the present invention.

[0023] Figure 3 This is the result of the effect of temperature on the enzyme activity of the RML lipase chimera in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0026] Rhizomucor miehei lipase (RML) is a mesophilic lipase with an optimum temperature for triglyceride hydrolysis at 45°C and an optimum pH of 8.0. It exhibits the highest catalytic efficiency at these optimal conditions. To enhance the application of RML, current RML chimeras are being designed primarily to improve its thermal stability and methanol tolerance. For example, Li et al. introduced single-point mutations and disulfide bonds into RML through various computational methods. In a mutant library consisting of 36 chimeras, 24 chimeras showed higher thermal stability. The half-life of the best chimera at 70°C increased by 12.5 times, and the catalytic efficiency was 39% higher than that of the wild type. Sanches et al. formed a cross-linked polymer between PAA and aldehyde-dextran to immobilize the lipase RML, forming a "nanoencapsulation" structure. The stability of the RML immobilized by this method increased by 439 times, and it also had better recycling rate and thermal stability. Tian et al. used a semi-rational design method to modify the α-helix of RML by N-glycosylation. The enzyme activity of the optimal chimera obtained was 66.81 times that of the wild type, and the methanol tolerance was also significantly improved. The yield of biodiesel produced using the chimera was increased to 90.46%. The inventors of the present invention have modified RML lipase to have high phospholipase activity, which can be used for oil degumming and production of lysophospholipids, and is applied in the fields of biology, food, medicine, beauty, agriculture, industry and so on.

[0027] In one aspect of the present invention, a chimera of RML lipase (whose amino acid sequence is shown in SEQ ID NO.1) (whose amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3) is provided, wherein the chimera has high phospholipase activity.

[0028] Amino acid sequence of RML lipase (SEQ ID NO. 1):

[0029] VPIKRQSNSTVDSLPPLIPSRTSAPSSSPSTTDPEAPAMSRNGPLPSDVETKYGM

[0030] ALNATSYPDSVVQAMSIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATW

[0031] DCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLT

[0032] FVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSL

[0033] GGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRT

[0034] VNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFT

[0035] SVLDHLSYFGINTGLCTHHHHHH

[0036] Amino acid sequence of the RML lipase chimera (SEQ ID NO.2):

[0037] VPIKRQSNSTVDSLPPLIPSRTSAPSSSPSTTDPEAPAMSRNGPLPSDVETKYGM

[0038] ALNATSYPDSVVQAMSIDGGIRAATSQEINELTYYTTLSANSYCRTVIPGATW

[0039] DCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLT

[0040] FVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSL

[0041] GGATALLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRT

[0042] VNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFT

[0043] SVLDHLSYFQNTESCNHHHHHH

[0044] Nucleotide sequence encoding RML lipase chimera (SEQ ID NO. 3): GTGCCAATCAACAGCACGGTGGATAGTCTGCCACCCCTCATCCCCTCTCGAACCTCGGCACCTTCATCATCACCAAGCACAACCGACCCTGAAGCTCCAGCCATGAGTCGCAATGGACCGCTGCCCTCGGATGTAGAGACTAAATATGGCATGGCTTTGAATGCTACTTCCTATCCGGATTCTGTGGTCCAAGCAATGAGTATTGATGGAGGTATAAGAGCCGCAACCTCACAGGAGATCAATGAATTGACTTATTACACCACATTATCTGCCAACTCATACTGCCGTACTGTCATTCCCGGAGCTACCTGGGACTGTATACATTGTGATGCAACTGAGGACCTGAAAATTATCAAGACTTGGTCCACCTTGATTTATGATACAAATGCAATGGTGGC ACGTGGTGACTCCGAAAAAACTATCTATATTGTCTTCAGAGGTTCATCATCGATCAGAAACTGGATTGCTGATTTAACCTTTGTGCCAGTATCATATCCTCCAGTCAGTGGTACAAAAGTACACAAGGGATTCTTGGACAGTTACGGAGAAGTGCAAAATGAGCTTGTTGCTACTGTTCTTGACCAGTTCAAGCAATATCCCTCTTA CAAGGTGGCTGTTACAGGTCACTCATTAGGTGGTGCTACTGCTTTGCTTTGCGCCCTGGATCTGTATCAAAGAGAAGAAGGACTGTCATCCTCTAACTTGTTCCTTTACACTCAAGGTCAACCACGTGTAGGTGACCCTGCCTTTGCCAACTACGTTGTTTCCACCGGTATTCCTTACAGGAGGACTGTCAATGAAAGAGATATAGT TCCTCATCTTCCACCTGCAGCTTTTGGTTTTTTGCACGCTGGTGAGGAGTATTGGATTACTGACAATTCTCCAGAGACTGTTCAGGTCTGTACATCTGATCTGGAAACCTGATTGTTCTAACTCTATTGTTCCTTCACAAGTGTTCTTGACCATCTGTCTTACTTTCAAAATACTGAATCATGTAACCATCATCATCATCATCAT

[0045] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] Example 1 Construction of RML lipase chimera expression vector

[0047] The polypeptide (whose nucleotide sequence is SEQ ID NO. 4) was fused with the nucleotide gene sequence (mother copy) of RML lipase using fusion PCR technology to obtain a chimeric expression vector.

[0048] The nucleotide sequence of the polypeptide (SEQ ID NO.4):

[0049] CAAAATACTGAATCATGTAAC

[0050] The specific steps include:

[0051] (1) Design 3 pairs of primers for the peptide sequence (as shown in Table 1)

[0052] Table 1 Primer list

[0053]

[0054] (2) Using the lipase plasmid pPICZαA-RML (stored at South China University of Technology) as a template and the primer pairs shown in Table 1 as primers, fusion PCR amplification was performed. The PCR amplification reaction system is shown in Table 2.

[0055] Table 2 Reaction system

[0056]

[0057]

[0058] The PCR reaction program was as follows: pre-denaturation at 98°C for 3 min; 30 cycles of denaturation at 98°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 3 min; and extension at 72°C for 5 min.

[0059] (3) PCR products were tested by 1% agarose gel electrophoresis. After the PCR products were confirmed, DpnI was added to remove the methylated original template strand. The enzyme digestion system was: 8 μL PCR product, 1 μL buffer, 1 μL enzyme solution, and the enzyme digestion reaction conditions were 37°C for 1 hour.

[0060] (4) The enzyme-digested product was transformed into Escherichia coli Top10. After overnight culture at 37°C, a single clone was picked and cultured in LB liquid culture medium. The plasmid was extracted and sequenced to determine whether the target polypeptide had been correctly fused with the parent gene.

[0061] In this example, three lipase chimera vectors were designed and successfully constructed (i.e., expression vectors containing the RML lipase chimera encoding gene; hereinafter, the vector constructed by amplification with primer pair 1 is named RML lipase chimera 1, the vector constructed by amplification with primer pair 2 is named RML lipase chimera 2, and the vector constructed by amplification with primer pair 3 is named RML lipase chimera 3).

[0062] Example 2 Preparation and purification of RML lipase chimeric enzyme protein

[0063] The following steps are involved:

[0064] 1. The three expression vectors containing the lipase chimera encoding genes constructed in Example 1 were transformed into the genome of Pichia pastoris X-33 using electroporation to obtain genetically engineered bacteria.

[0065] 2. Inoculate the genetically engineered bacteria into the YPG first-level seed culture medium for expansion. When the OD value reaches 1.6-2, transfer it to the second-level YPG seed culture medium and culture for 12-16 hours.

[0066] 3. Inoculate the seed liquid into the fermentation tank culture medium at a ratio of 1:10 for high-density fermentation. Induce expression when the wet weight of the bacteria reaches 150-180g / L. The inducer is methanol. Harvest the bacteria after 72-108h of induction. Centrifuge the bacterial liquid at 10000rpm for 20min and collect the supernatant, which is the crude enzyme liquid.

[0067] 4. The crude enzyme solution of RML lipase chimera was concentrated and desalted with 20 mM pH 7.4 PBS buffer, loaded onto an anion exchange chromatography column (Q FF, GE Healthcare) at a flow rate of 2 mL / min, and then eluted with 20 mM pH 7.4 PBS buffer (containing 300 mM NaCl) to obtain purified RML lipase chimera.

[0068] 5. To ensure protein stability, the target protein was salted and then washed with 20 mM PBS buffer at pH 7.4. After the above steps, the RML lipase chimera with a purity of more than 90% was obtained. The SDS-PAGE test results were as follows: Figure 1 As shown, lanes 1-3 are RML lipase chimeras 1-3, respectively. Figure 1 It can be seen that the RML lipase chimera achieved good purification effect, and the protein molecular weights were approximately 32.0 kDa, 31.7 kDa, and 31.5 kDa, respectively.

[0069] 6. Target protein concentration determination: Mix 20 μL of the protein solution to be tested with 200 μL of Bradford reagent and react at room temperature for 5 minutes before measuring the A 595, combined with the standard curve, the protein concentrations of the RML lipase chimera were calculated to be 1.25 mg / mL, 1.30 mg / mL, and 1.17 mg / mL, respectively.

[0070] Example 3 Lipase and phospholipase activity determination of RML lipase chimera

[0071] The enzyme activity was determined by alkaline titration method.

[0072] Definition of enzyme activity: Under certain reaction conditions, the amount of enzyme required to catalyze the hydrolysis of substrate to produce 1 μmol fatty acid per minute is defined as one enzyme activity unit, expressed as U, i.e. 1U.

[0073] The enzyme activity was calculated by the following formula:

[0074]

[0075] Where: X is the specific enzyme activity, U / mg; V1: the volume of sodium hydroxide consumed by the experimental group, mL; V0: the volume of sodium hydroxide consumed by the control group, mL; t: reaction time, min; c: the protein concentration of the reaction enzyme solution, mg / mL; v: the volume of enzyme solution added to the reaction, mL.

[0076] The specific method is:

[0077] 1. In a 50 mL stoppered Erlenmeyer flask, add 4 mL of soybean lecithin emulsion (phospholipase activity assay substrate) or olive oil emulsion (lipase activity assay substrate) and 5 mL of buffer and preheat in a constant temperature water bath shaker for 5 min. Add 1 mL of RML wild-type lipase or RML lipase chimera pure enzyme solution to the experimental group and 1 mL of the corresponding inactivated enzyme solution to the control group. After reacting at 200 rpm for 5 min, add 15 mL of 95% ethanol to terminate the reaction.

[0078] 2. After the reaction is completed, add 2 drops of 1% phenolphthalein solution and titrate with 0.05 mol / L NaOH standard solution. Calculate the volume of NaOH consumed and then calculate the phospholipase or lipase activity units.

[0079] This example measured the phospholipase and lipase specific activities of wild-type RML lipase and three RML lipase chimeras. The results are shown in Table 3.

[0080] Table 3 Results of enzyme activity determination of wild-type and chimeric RML lipase

[0081]

[0082] As shown in Table 3, the lipase and phospholipase activities of the three RML lipase chimeras constructed in the present invention were all improved compared to the wild-type RML lipase. The lipase activities were 4.7, 3.4, and 2.6 times that of the wild-type, respectively, and the phospholipase activities were 161, 81, and 91 times that of the wild-type, respectively. Among them, RML lipase chimera 1 had the highest phospholipase specific enzyme activity of 69.52 U / mg and the highest lipase specific enzyme activity of 62.15 U / mg. This chimera is the optimal chimera of the present invention. Its amino acid sequence is shown in SEQ ID NO. 2, and the nucleotide sequence encoding this lipase chimera is shown in SEQ ID NO. 3.

[0083] Example 4 Determination of the Optimal Reaction pH of RML Lipase Chimera

[0084] This example determined the optimal reaction pH of the best chimera of the present invention, namely RML lipase chimera 1. Specifically, the following steps were included:

[0085] 1. Phospholipase activity was determined at 40°C using soybean lecithin emulsion as substrate in 20 mM reaction buffers with pH values ​​of 3.0, 4.0, 5.0, 6.0, and 7.0 (pH 3.0-5.0 citric acid-disodium hydrogen phosphate buffer, pH 6.0-7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer). Each experiment was repeated three times.

[0086] 2. Draw a graph with pH as the horizontal axis and enzyme activity as the vertical axis.

[0087] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the optimal reaction pH of the RML lipase chimera of the present invention is 4.0, compared with the optimal pH of 8.0 of the wild-type RML lipase (since the phospholipase activity of the wild-type is approximately 0, it is difficult to measure its optimal pH, so there is no wild-type curve in the figure), and an obvious acid shift occurs, indicating that the RML lipase chimera of the present invention is an acidic lipase suitable for use in oil degumming.

[0088] Example 5 Determination of the Optimal Reaction Temperature of RML Lipase Chimera

[0089] This example determined the optimal reaction temperature of the best chimera of the present invention, namely RML lipase chimera 1. Specifically, the following steps were included:

[0090] 1. Phospholipase activity was measured using soybean lecithin emulsion as substrate in 20 mM reaction buffer (citric acid-disodium hydrogen phosphate buffer) at pH 5.0 at 30°C, 35°C, 40°C, 45°C, and 50°C. Each experiment was repeated three times.

[0091] 2. Draw a graph with temperature as the horizontal axis and enzyme activity as the vertical axis.

[0092] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that the optimal reaction temperature of the RML lipase chimera of the present invention is 40°C, which is not much different from the optimal temperature of RML lipase (45°C) (since the wild-type phospholipase activity is approximately 0, it is difficult to measure its optimal temperature in this example, so there is no wild-type curve in the figure), indicating that the RML lipase chimera of the present invention can achieve good catalytic effect at medium temperature.

[0093] Example 6 Effect of RML lipase chimera on oil degumming

[0094] The following steps are involved:

[0095] 1. Weigh 300 g of crude soybean oil, heat it to 70°C in a water bath, add 0.18 mL of 45% citric acid, and homogenize at 10,000 rpm for 1 min.

[0096] 2. Stir at 70°C for 25 minutes, then cool to 40°C. Add a certain amount of 4% NaOH solution to adjust the system pH to 4. Add 3% water and 20,000 U / kg enzyme solution. Homogenize at 10,000 rpm for 1 minute, and then react at 500 rpm for 5 hours.

[0097] 3. After the reaction is complete, heat to 90°C and inactivate the enzyme for 10 minutes. Centrifuge at 10,000 rpm for 10 minutes, collect the upper oil phase, and determine the phosphorus content. The phosphorus content in oils and fats should be analyzed according to the molybdenum blue colorimetric method in GB 5537-1985.

[0098] The experimental results are shown in Table 4.

[0099] Table 4 Effects of RML lipase chimera and wild type on soybean crude oil degumming

[0100]

[0101] As can be seen from Table 4, when the RML lipase chimera of the present invention was used to degummed soybean oil, compared with the wild-type RML lipase, under the same reaction time and reaction conditions, the wild-type could only reduce the phosphorus content in the crude soybean oil from 197.0 mg / kg to 165.3 mg / kg, while the RML lipase chimera of the present invention reduced the phosphorus content in the crude soybean oil from 197.0 mg / kg to 7.1 mg / kg, meeting the requirement of phospholipid content below 10 mg / kg, and can achieve a good degumming effect on crude soybean oil, indicating that it has application value and prospects.

[0102] Example 7 Preparation of lysophospholipids using RML lipase chimera

[0103] Using 10% phosphatidylcholine as the substrate, 0.07% CaCl2 and an appropriate amount of enzyme solution were added, mixed evenly, and reacted at 40°C for 12 hours. The conversion rate of lysophospholipids was then determined by high performance liquid chromatography.

[0104] The conversion rate of lysophospholipids was determined by high-performance liquid chromatography. Sample preparation method: 40 μL of sample was mixed with 960 μL of methanol, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was filtered through a 0.22 μm filter membrane to prepare the high-performance liquid chromatography sample.

[0105] The detector was an ELSD detector, the chromatographic column was a Symmetry C18 column (4.6 mm × 150 mm, 5 mm, Wasters, USA), the mobile phase was acetonitrile: methanol: water (40:50:10, v / v / v), the mobile phase flow rate was 1.0 mL / min, the column temperature was 25°C, the injection volume was 10 μL; the N2 flow rate was 2 L / min, and the drift tube temperature was 80°C.

[0106] The experimental results are shown in Table 5.

[0107] Table 5 Conversion efficiency of RML lipase chimera and wild type to lysophospholipids

[0108]

[0109] As can be seen from Table 5, the conversion rate of lysophospholipids prepared using wild-type RML lipase is only 17%, while the conversion rate of lysophospholipids produced by the RML lipase chimera of the present invention can reach 74.9%, which is a significant improvement.

[0110] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0111] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A RML lipase chimera, characterized in that The amino acid sequence of the RML lipase chimera is shown in SEQ ID NO.

2.

2. The gene encoding the RML lipase chimera according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

4. Use of the RML lipase chimera according to claim 1, or the gene encoding the RML lipase chimera according to claim 2 or 3, in oil degumming.

5. Use of the RML lipase chimera according to claim 1, or the gene encoding the RML lipase chimera according to claim 2 or 3, in the preparation of lysophospholipids.

6. A recombinant expression vector into which the gene encoding the RML lipase chimera according to claim 2 or 3 is inserted.

7. A recombinant engineered bacterium into which the recombinant expression vector according to claim 6 is introduced.

8. Use of the recombinant expression vector according to claim 6 or the recombinant engineered bacteria according to claim 7 in oil degumming or preparation of lysophospholipids.

9. A method for degumming oils and fats, characterized in that: The RML lipase chimera according to claim 1 is used to catalyze the reaction of an oil-water mixture.

10. A method for preparing lysophospholipids, characterized in that: The RML lipase chimera according to claim 1 is used to catalyze the conversion of phospholipid substrates into lysophospholipids.

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