A lipase gene, a recombinant expression vector, a recombinant expression strain, a lipase, and a preparation method and use thereof

Abstract

The present invention discloses a lipase gene, a recombinant expression vector, a recombinant expression strain, a lipase, and a preparation method and use thereof, and belongs to the field of genetic engineering technology. The present invention provides an optimized MAS1 lipase, which overcomes the problem that the wild-type MAS1 lipase is almost unable to be actively expressed in the cytoplasm by optimizing the amino acid sequence of the wild-type MAS1 lipase. The optimized MAS1 lipase of the present invention can be directly expressed in the cytoplasm in an efficient, soluble, and active manner, is easy to crystallize, and has significantly improved yield and enzyme activity and good uniformity. The present invention simplifies the production process, reduces production costs, and provides a highly advantageous technical solution for obtaining high-yield, high-purity, and high-activity MAS1 lipase, which has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a lipase gene, a recombinant expression vector, a recombinant expression strain, a lipase, and a preparation method and use thereof. Background Art

[0002] Lipases are important industrial enzymes widely used in food processing, detergents, biodiesel production, and pharmaceuticals. MAS1, a lipase from Streptomyces sp. strain W007, belongs to the bacterial lipase subfamily I.7 and exhibits excellent thermal stability, which makes it potentially useful in high-temperature reaction conditions.

[0003] To meet the high demand for lipases in industrial applications, genetic engineering is often used to produce them through heterologous expression using recombinant microorganisms (such as E. coli). However, the expression of wild-type MAS1 lipase in E. coli faces many challenges:

[0004] Limitations of Cytoplasmic Expression: When wild-type MAS1 is expressed in the E. coli cytoplasm using conventional cytoplasmic expression vectors (such as pET21b or other similar vectors), the expression product primarily exists as inclusion bodies. Inclusion bodies are biologically inactive protein aggregates that require complex refolding steps to obtain active enzyme. This not only increases production cost and difficulty, but also often results in low refolding efficiency, resulting in extremely low yields of active lipase.

[0005] Therefore, developing a strategy to efficiently express MAS1 lipase in Escherichia coli with high yield, good purity, and ease of production is of great significance for promoting its industrial application. Summary of the Invention

[0006] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a lipase gene, a recombinant expression vector, a recombinant expression strain, a lipase and a preparation method and use thereof.

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

[0008] The present invention provides a lipase gene, the nucleotide sequence of the lipase gene is shown as SEQ ID No. 2.

[0009] The present invention also provides a recombinant expression vector comprising the lipase gene.

[0010] Furthermore, the expression vector does not contain a signal peptide.

[0011] Furthermore, the expression vector not containing a signal peptide is pET21b.

[0012] Furthermore, the expression vector is an expression vector comprising a signal peptide.

[0013] Furthermore, the expression vector containing the signal peptide is pET22b.

[0014] The present invention also provides a recombinant expression strain, which comprises the above-mentioned recombinant expression vector.

[0015] Furthermore, the strain is Escherichia coli.

[0016] Furthermore, the Escherichia coli is BL21 (DE3).

[0017] The present invention also provides a lipase, the amino acid sequence of the lipase is shown as SEQ ID No. 1.

[0018] Furthermore, the method comprises the following steps:

[0019] (1) constructing the above-mentioned recombinant expression vector;

[0020] (2) The recombinant expression vector obtained in step (1) is used to transform the host strain to obtain a recombinant expression strain, and the expression is induced.

[0021] The present invention also provides the application of the lipase in ester hydrolysis and ester synthesis.

[0022] Furthermore, the ester hydrolysis is achieved through a forward reaction under the action of lipase, and the ester synthesis is achieved through a reverse reaction under the action of lipase.

[0023] Furthermore, the applications include the synthesis and hydrolysis of ester compounds in fine chemicals; the resolution of chiral drugs in the pharmaceutical industry; the transesterification reaction of biodiesel; the modification of oils and fats and the synthesis of flavor substances in the food industry; and the catalytic degreasing in the washing industry.

[0024] The present invention has achieved the following beneficial effects:

[0025] The present invention provides an optimized MAS1 lipase. By optimizing the amino acid sequence of the wild-type MAS1 lipase, this overcomes the problem of the wild-type MAS1 lipase being virtually inactive in the cytoplasm. The optimized MAS1 lipase can be efficiently, soluble, and actively expressed directly in the cytoplasm, is easily crystallized, and exhibits significantly improved yield and enzyme activity with good homogeneity. The present invention simplifies the production process and reduces production costs, providing a highly advantageous technical solution for obtaining high-yield, high-purity, and highly active MAS1 lipase, promising broad application prospects.

[0026] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0027] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 SDS-PAGE results of the optimized MAS1 lipase expressed in pET21b and purified. BI: pre-induction sample; AI: post-induction sample; L: whole cell suspension sample; S: supernatant sample after lysis; P: pellet sample after lysis; FT: Ni column flow-through sample; 13-30: all samples eluted with an imidazole linear gradient. Arrows indicate single protein bands corresponding to the optimized MAS1 lipase.

[0029] Figure 2 The SDS-PAGE results of wild-type MAS1 lipase expressed in pET21b. BI: sample before induction; AI: sample after induction; L: whole cell suspension; S: supernatant after cell lysis; P: pellet after cell lysis. The arrow indicates wild-type MAS1 lipase.

[0030] Figure 3 This is the SDS-PAGE result of the optimized MAS1 lipase after purification on a desalting column. Before: sample before desalting; 10 / 11 / 12: sample collected on the desalting column; Storage: sample finally stored after purification on the desalting column.

[0031] Figure 4 Photo of protein crystals of MAS1 lipase optimized for the present invention. Figure 4 A and Figure 4 B are photos of protein crystals formed by the optimized MAS1 lipase in different crystallization droplets.

[0032] Figure 5This is the SDS-PAGE result of the optimized MAS1 lipase expressed in pET22b and purified. BI: pre-induction sample; AI: post-induction sample; L: whole cell suspension sample; S: supernatant sample after lysis; P: pellet sample after lysis; FT: Ni column flow-through sample; 30 mM: 30 mM imidazole equilibrated sample; 13 / 17 / 21: all samples eluted using a linear imidazole gradient; arrows indicate two protein bands, corresponding to the optimized MAS1 with the signal peptide completely removed and the optimized MAS1 precursor with the signal peptide not completely removed, respectively.

[0033] Figure 6 The SDS-PAGE results of wild-type MAS1 lipase expressed and purified in pET22b. BI: pre-induction sample; AI: post-induction sample; L: whole cell suspension sample; S: supernatant sample after cell lysis; P: pellet sample after cell lysis; FT: Ni column flow-through sample; 12 / 15 / 17 / 21 / 22 / 24: samples eluted using an imidazole linear gradient. The arrows indicate two protein bands, corresponding to wild-type MAS1 with the signal peptide completely removed and the wild-type MAS1 precursor with the signal peptide not completely removed, respectively.

[0034] Figure 7 This is the enzyme activity test result of the MAS1 lipase optimized in the present invention. DETAILED DESCRIPTION

[0035] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0036] The optimized MAS1 lipase of the present invention is obtained on the basis of wild-type MAS1 lipase.

[0037] Wild-type MAS1 lipase is derived from Streptomyces sp. strain W007 (NCBI Reference Sequence: WP_007448656.1). This enzyme is a thermostable lipase belonging to the bacterial lipase subfamily I.7. Its catalytic triad consists of S109, D200, and H232.

[0038] The following experiments without temperature indication are for normal temperature reactions, where normal temperature is room temperature, which is 25±5℃.

[0039] Example 1: Preparation of the optimized MAS1 lipase of the present invention

[0040] The present invention optimizes the amino acid sequence of wild-type MAS1 lipase. The main purpose of the optimization is to further enhance its structural stability based on the analysis of MAS1's spatial structure, making it easier to correctly fold in the basic environment of the E. coli cytoplasm, thereby achieving soluble active expression.

[0041] The amino acid sequence of the optimized MAS1 lipase of the present invention (SEQ ID No. 1) is as follows:

[0042] >Optimized MAS1 (266 aa)

[0043] MATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSVDNWLVLAPYLVNRGYCVFSLDYGQLPGVPFFHLGGPIEKSAQQLRDFVDKVLAATGAPKVDLVGHSQGGMMPRYYLKFLGGADKVHALV GIAPNNHGTTLDGLTRLLPYFPGVEDFIDANTPGLADQVAGSPFITKLNAGGDTVPGVRYTVIATRYDQVVTPYTTQFLTGPNVRNVLLQDLCPLDYSDHVAIGTTDRIAFHEVANALDPARATPTTCASVIG

[0044] The nucleotide sequence of the optimized MAS1 lipase of the present invention (SEQ ID No. 2) is as follows:

[0045] >Optimized MAS1 (798 bp) (Due to codon degeneracy, the nucleotide sequence may be diverse, but the encoded amino acid sequence remains consistent with SEQ ID No. 1)

[0046] ATGGCTACCGCTACCGCTGCTACCCAGCTGCTGAAGCTACCTCTCGTGGTTGGAACGATTACTCTTGTAAACCATCTGCTGCTCACCCACGTCCAGTTGTTCTGGTTCACGGTACCTTCGGTAACTCTGTTGATAACTGGCTGGTTCTGGCTCCATACCTGGTTAACCGTGGTTACTGCGTTTTCTCCTGGACTACG GTCAGCTGCCAGGTGTTCCATTCTTCCACGGTCTGGGCCCAATCGAAAAATCTGCTCAGCAGCTGCGTGACTTCGTTGATAAAGTTCTGGCTGCTACTGGTGCTCCAAAAGTTGACCTGGTTGGTCACTCTCAGGGTGGTATGATGCCACGTTACTACCTGAAATTCCTGGGTGGTGCTGATAAAGTTCACGCACTGGTT GGTATCGCTCCAAACAACCACGGCACCACCCTGGACGGCCTGACCCGTCTGCTGCCATACTTCCCAGGTGTTGAAGATTTCATCGATGCAAACACTCCTGGTCTGGCTGACCAGGTTGCTGGTTCTCCATTCATCACCAACTGAACGCAGGTGGCGACACCGTTCCAGGTGTGCGTTACACCGTTATCGCTACCCGTT ACGATCAGGTTGTTACCCCATACACCACCCAGTTCCTGACTGGTCCAAACGTTCGTAACGTTCTGCTGCAGGATCTGTGTCCACTGGACTATTCTGACCACGTTGCTATCGGTACCACCGACCGTATCGCTTTCCACGAAGTTGCAAACGCTCTGGACCCAGCACGTGCAACCCCAACCACCTGTGCTTCTGTTATCGGT

[0047] The preparation method of the optimized MAS1 lipase of the present invention is as follows:

[0048] 1. Cytoplasmic expression strategy

[0049] (1) The nucleotide sequence encoding the optimized MAS1 lipase (SEQ ID No. 2) was cloned into a conventional Escherichia coli expression vector pET21b without a signal peptide (the vector pET21b itself carries the coding for a C-terminal histidine tag).

[0050] (2) The expression vector containing the sequence shown in SEQ ID No. 2 obtained in step (1) is transformed into the Escherichia coli expression host BL21 (DE3) for induced expression. After the induced expression is completed, the bacteria are collected and lysed. The supernatant of the lysate (containing the optimized MAS1 lipase) can be directly used for catalysis and application; or the supernatant of the lysate can be further purified to obtain a pure product of the optimized MAS1 lipase.

[0051] The purification step utilizes affinity chromatography: the lysate supernatant is loaded onto a Ni ion affinity chromatography column in the presence of 10-50 mM imidazole, and impurities are washed with a solution containing 10-50 mM imidazole. After impurities are removed, the optimized MAS1 lipase is specifically eluted from the affinity chromatography column using a solution containing 150-250 mM imidazole, and the eluted sample is collected to obtain the purified optimized MAS1 lipase.

[0052] 2. Periplasmic expression strategy

[0053] (1) The nucleotide sequence encoding the optimized MAS1 lipase (SEQ ID No. 2) was cloned into the Escherichia coli expression vector pET22b encoding the pelB signal peptide (amino acid sequence: MKYLLPTAAAGLLLLAAQPAMA).

[0054] (2) The expression vector containing the sequence shown in SEQ ID No. 2 obtained in step (1) is transformed into the Escherichia coli expression host BL21 (DE3) for induced expression. After the induced expression is completed, the bacteria are collected and lysed. The supernatant of the lysate (containing the optimized MAS1 lipase) can be directly used for catalysis and application; or the supernatant of the lysate can be further purified to obtain a pure product of the optimized MAS1 lipase.

[0055] The purification step is the same affinity chromatography method as in the "cytoplasmic expression strategy".

[0056] The following is the preparation method of the control sample of the present invention.

[0057] Comparative Example 1: Preparation of wild-type MAS1 lipase

[0058] The amino acid sequence of wild-type MAS1 lipase (SEQ ID No. 3) is as follows:

[0059] >Wild-type MAS1 lipase (266 aa)

[0060] MATATAATPAAEATSRGWNDYSCKPSAAHPRPVVLVHGTFGNSIDNWLVLAPYLVNRGYCVFSLDYGQLPGVPFFHGGLGPIDKSAEQLDVFVDKVLDATGAPKADLVGHSQGGMMPNYYLKFLGGADKVNALV GIAPDNHGTTLLGLTKLLPFFPGVEKFISDNTPGLADQVAGSPFITKLTAGGDTVPGVRYTVIATKYDQVVTPYRTQYLDGPNVRNVLLQDLCPVDLSEHVAIGTIDRIAFHEVANALDPARATPTTCASVIG

[0061] The nucleotide sequence of wild-type MAS1 lipase (SEQ ID No. 4) is as follows:

[0062] >Wild-type MAS1 (798 bp) (Due to codon degeneracy, the nucleotide sequence may be diverse, but the encoded amino acid sequence remains consistent with SEQ ID No. 3)

[0063] ATGGCCACCGCCACCGCCGCCACCCCGGCCGCCGAAGCCACCTCTCGTGGTTGGAATGATTATTCTTGTAAACCGTCTGCCGCCCATCCGCGTCCGGTTGTTCTGGTTCATGGCACCTTTGGTAATTCTATTGATAATTGGCTGGTTCTGGCCCCGTATCTGGTTAATCGTGGTTATTGTGTTTTTTCCTGGATTATG GTCAGCTGCCGGGTGTTCCGTTTTTTCATGGTCTGGGTCCGATTGATAAATCTGCCGAACAGCTGGATGTTTTTGTTGATAAAGTTCTGGATGCCACCGGTGCCCCGAAAGCCGACCTGGTTGGTCATTCTCAAGGTGGTATGATGCCGAATTATTATCTGAAATTTCTGGGTGGTGCCGATAAAGTTAATGCCCTGGTT GGTATTGCCCCGGATAATCATGGCACCACCCTGCTGGGTCTGACCAAACTGCTGCCGTTTTTTCCGGGTGTTGAAAAATTTATTTCTGATAATACCCCGGGTCTGGCCGACCAAGTTGCCGGTTCTCCGTTTATTACCAAACTGACCGCCGGTGGTGATACCGTTCCGGGTGTTCGTTATACCGTTATTGCCACCAAAT ATGACCAAGTGTTACCCCGTATCGTACTCAGTATCTGGATGGTCCGAATGTTCGTAATGTTCTGCTGCAAGACCTGTGTCCGGTTGACCTGTCTGAACATGTTGCCATTGGCACCATTGACCGTATTGCCTTTCATGAAGTTGCCAATGCCCTGGACCCGGCCCGTGCCACCCCGACCACCTGTGCCTCTGTTATTGGT

[0064] The preparation method of the optimized MAS1 lipase in Example 1 was referred to, except that the nucleotide sequence (SEQ ID No. 2) in step (1) of the two expression strategies was replaced with the nucleotide sequence (SEQ ID No. 4), thereby obtaining the wild-type MAS1 lipase.

[0065] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0066] Experimental Example 1: Comparison of the effects of lipases obtained by cytoplasmic expression strategy

[0067] 1. The expression products obtained by the cytoplasmic expression strategy in Example 1 and Comparative Example 1 (i.e., the optimized MAS1 lipase and the wild-type MAS1 lipase) were analyzed respectively:

[0068] The results of SDS-PAGE detection were as follows Figure 1-2 shown.

[0069] The optimized MAS1 lipase of the present invention expressed by pET21b ( Figure 1 ) is mainly present in the supernatant of cell lysates in a soluble form, and the formation of inclusion bodies is almost invisible ( Figure 1 Lane labeled p). MAS1 lipase purified by affinity chromatography appeared as a single, clear protein band on SDS-PAGE, indicating high purity and homogeneity, and no problem of incomplete signal peptide cleavage.

[0070] Wild-type MAS1 lipase expressed in pET21b ( Figure 2 ) exists mainly in the cytoplasm in the form of insoluble inclusion bodies ( Figure 2 The precipitate of the cell lysate showed a clear MAS1 band, while soluble MAS1 was almost undetectable in the supernatant, indicating that active enzyme could hardly be obtained.

[0071] If you want to remove imidazole or other solutes introduced during affinity chromatography purification, you can continue to use dialysis or desalting and other classic methods to replace the solvent to obtain the optimized MAS1 lipase under the target solution conditions. The optimized MAS1 lipase of the present invention was replaced with the buffer solvent after the desalting column. Figure 3 As shown, it is still in the form of a single strip.

[0072] 2. Protein purity yield: The optimized MAS1 lipase of the present invention showed significantly increased expression, reaching approximately 100 mg of pure protein per liter of culture after affinity chromatography purification (accounting for losses during the purification process, the actual expression level should be much higher than 100 mg per liter of culture). In contrast, the wild-type MAS1 lipase showed no distinct bands in the cell lysate supernatant, and no active lipase protein could be obtained.

[0073] 3. Protein crystallization: The purity and homogeneity of the MAS1 lipase optimized by the present invention are high. Figure 4 ) found that its high-quality single crystals are very easy to obtain, which laid the foundation for further structural biology research and indirectly proved its high uniformity.

[0074] 4. Enzymatic Activity: The optimized MAS1 lipase of the present invention can be expressed and purified normally in the cytoplasm; however, wild-type MAS1 lipase cannot be expressed in the cytoplasm. Using the same cytoplasmic expression method, it is not possible to simultaneously produce both the optimized and wild-type MAS1 lipases. Although the enzymatic activity of the optimized MAS1 lipase can be measured, direct comparison of their activity is not possible.

[0075] Experimental Example 2: Comparison of the effects of lipases obtained by periplasmic expression strategy

[0076] 1. The expression products obtained by the periplasmic expression strategy of Example 1 and Comparative Example 1 (i.e., the optimized MAS1 lipase and the wild-type MAS1 lipase) were analyzed respectively:

[0077] The results of SDS-PAGE detection were as follows Figure 5-6 shown.

[0078] The pET22b vector contains a PelB signal peptide that directs the protein into the periplasm and exists in an active form.

[0079] SDS-PAGE ( Figure 5 ) Detection of the optimized MAS1 lipase after affinity chromatography purification revealed two protein bands, corresponding to the mature optimized MAS1 with the signal peptide completely removed and the optimized MAS1 precursor with the signal peptide not completely removed. This indicates that even with the optimized sequence, incomplete cleavage of the signal peptide can occur when the signal peptide is used for periplasmic expression. The protein yield of the optimized MAS1 after periplasmic expression and purification was approximately 58 mg / L. While this yield was lower than the cytoplasmic pure protein yield (100 mg / L), it was still significantly higher than the periplasmic pure protein yield of wild-type MAS1 (20 mg / L), representing nearly three times that of the wild-type.

[0080] Soluble active wild-type MAS1 lipase can be obtained through periplasmic expression, but the yield of pure protein is low, about 20 mg / L culture. SDS-PAGE detection ( Figure 6 ) After purification of wild-type MAS1 lipase, two adjacent protein bands were observed, corresponding to the mature wild-type MAS1 with complete signal peptide removal and the wild-type MAS1 precursor with incomplete signal peptide removal. This suggests that incomplete signal peptide cleavage results in heterogeneous wild-type MAS1 lipase product.

[0081] The periplasmic expression strategy presents two major issues: 1. Low expression efficiency: Due to the relatively limited space in the E. coli periplasm, protein storage capacity is limited, resulting in lower yields of active MAS1 protein obtained via this approach compared to cytoplasmic expression strategies. 2. Product heterogeneity: After directing the protein into the periplasm, the signal peptide should theoretically be cleaved by a signal peptidase. However, in practice, signal peptide cleavage is often incomplete, resulting in the purified MAS1 lipase existing in two forms: a mature form without the signal peptide, and a form still attached to the signal peptide. This results in MAS1 appearing as two protein bands during purity testing using SDS-PAGE gel electrophoresis, increasing the difficulty of subsequent purification and affecting product uniformity and batch stability.

[0082] 2. Enzymatic Activity: Both the optimized MAS1 lipase and wild-type MAS1 lipase are actively expressed in the periplasm and can be purified, allowing for a comparison of their enzymatic activities. The hydrolysis activity of the optimized MAS1 lipase and wild-type MAS1 was determined using p-nitrophenol octanoate as the substrate.

[0083] The p-nitrophenol octanoate substrate was dissolved in isopropanol to a 10 mmol / L stock solution, which was then stored at 4°C in the dark until use. To a 1 mL reaction system, 100 μL of the substrate stock solution was added, and the volume was made up to 900 μL with phosphate buffered saline (PBS) (pH 7.4). The catalytic reaction was initiated by the addition of 100 μL of lipase solution, resulting in a final lipase concentration of 0.0005 mg / mL. The reaction was carried out at room temperature. 100 μL of the reaction solution was sampled 1, 2, and 3 minutes after the start of the reaction and immediately terminated by the addition of 20 μL of 10% (w / v) sodium dodecyl sulfate (SDS) solution. The product of lipase-catalyzed substrate hydrolysis, p-nitrophenol, exhibits an absorbance peak at 405 nm. A standard curve was established by measuring the absorbance of a series of p-nitrophenol standards at varying concentrations using a microplate reader. The amount of the product generated by lipase hydrolysis was then calibrated.

[0084] The protein expression level of the optimized MAS1 lipase in the periplasm of the present invention is higher than that of the wild-type MAS1 lipase, and the activity of the optimized MAS1 lipase per unit protein concentration is also higher than that of the wild-type MAS1 ( Figure 7 ).

[0085] In summary, the present invention provides an optimized MAS1 lipase. By optimizing the amino acid sequence of the wild-type MAS1 lipase, this overcomes the problem of the wild-type being virtually inactive in the cytoplasm. The optimized MAS1 lipase of the present invention can be efficiently, soluble, and actively expressed directly in the cytoplasm, is easily crystallized, and exhibits significantly improved yield and enzyme activity with good homogeneity. The present invention simplifies the production process and reduces production costs, providing a highly advantageous technical solution for obtaining high-yield, high-purity, and highly active MAS1 lipase, promising broad application prospects.

Claims

1. A lipase gene, characterized in that The nucleotide sequence of the lipase gene is shown in SEQ ID No.

2.

2. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the lipase gene according to claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The expression vector does not contain a signal peptide.

4. The recombinant expression vector according to claim 3, characterized in that The expression vector not containing a signal peptide is pET21b.

5. The recombinant expression vector according to claim 2, characterized in that The expression vector is an expression vector comprising a signal peptide.

6. The recombinant expression vector according to claim 5, characterized in that The expression vector containing the signal peptide is pET22b.

7. A recombinant expression strain, characterized in that: The recombinant expression strain comprises the recombinant expression vector according to any one of claims 2 to 6.

8. A lipase, characterized in that The amino acid sequence of the lipase is shown in SEQ ID No.

1.

9. The method for preparing the lipase according to claim 8, characterized in that: The method comprises the following steps: (1) constructing the recombinant expression vector according to any one of claims 2 to 6; (2) The recombinant expression vector obtained in step (1) is used to transform the host strain to obtain a recombinant expression strain, and the expression is induced.

10. Use of the lipase according to claim 8 in ester hydrolysis.

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