A phospholipase A1 and its integrated secretory expression method in Bacillus subtilis and its application

Abstract

The present invention relates to the technical field of recombinant enzymes. The present invention provides a phospholipase A1 and a method and application of its integrated secretory expression in Bacillus subtilis. The amino acid sequence of the phospholipase A1 is shown in SEQ ID NO.1. The present invention constructs various signal peptides and expression vectors combined from the Bacillus subtilis homologous protein signal peptide library, and combines a high-throughput screening method to screen out signal peptides that can improve PLA1 secretion, thereby optimizing the secretion of PLA1. Compared with the original signal peptide, the secretion amount of PLA1 is increased by 10 times, and the enzyme activity is increased by 10 times, which is of great significance for the reduction of PLA1 production cost and further large-scale application. In addition, the phospholipase A1 enzyme preparation prepared by the present invention can reduce the phosphorus content of rice oil to 70-75ppm at a temperature of 55°C and acidic conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant enzymes, and in particular to a phospholipase A1 and a method and application of integrating and secreting expression thereof in Bacillus subtilis. Background Art

[0002] Phospholipases are a type of lipase that can catalyze the hydrolysis of glycerophospholipids. According to the different action sites of the enzyme and phospholipids, they are divided into phospholipase A1, phospholipase A2, phospholipase C and phospholipase D. Among them, PLA1 can catalyze the hydrolysis of the first ester acyl bond of phospholipids to generate lysophospholipids and fatty acids. Lysophospholipids have strong hydrophilicity and can remove phospholipids through hydration, so they can be used for vegetable oil degumming. In addition, phospholipases can be widely used in oil refining, phospholipid modification, food, medical and agricultural pesticides, etc., and are important industrial enzyme preparations. Due to the wide range of industrial uses of phospholipase A1, research on it is becoming more and more extensive. Phospholipase A1 is widely present in animals, plants and microorganisms. Microorganisms are one of its important sources. Most of them are secreted and expressed, which is easy to mass produce and has become the main source of industrial enzymes. Common phospholipase-producing microorganisms include Pseudomonas alcaligenes, Vibrio harveyi, Serratia marcescens, Streptomyces, Aspergillus oryzae, Thermomyces lanuginosus and Fusarium oxysporum, but wild strains have low yields, long culture times, and are difficult to separate and purify, and there are certain hidden dangers in food safety. With the increasing demand for product safety, bioenzyme preparations play an increasingly important role in major fields such as food processing, biomedicine, biochemical engineering, and enzyme-added detergents. Enzymes from microorganisms must meet the requirements of biosafety of enzyme-producing microorganisms, culture medium, simple culture methods, and short culture time before they can be used for mass production. As can be seen from the above, most of the microorganisms that produce phospholipase have problems such as pathogenicity (Pseudomonas, Vibrio), difficulty in cultivation, and long fermentation time (fungi). As a GRAS (generally regarded as safe) strain, Bacillus subtilis has a wide range of applications in the field of bioenzyme preparations due to its low fermentation cost and excellent protein secretion level (up to 20-25g / L enzyme). At present, 60% of the hosts of commercial enzymes come from Bacillus.

[0003] The presence of phospholipids in vegetable oils will bring many problems to oil refining. Therefore, removing phospholipids from vegetable oils is a key step in oil refining (this step is called degumming in oil processing). However, incomplete degumming will reduce the refining yield of oils and increase the burden of decolorization and deodorization in the later stage. Commonly used degumming methods mainly include hydration degumming and acid degumming. Although they are easy to operate, the degumming effect is not ideal. They are only suitable for processing crude oil with good quality. In addition, a large amount of wastewater will be generated during the degumming process, which increases the wastewater treatment load and treatment costs, and is not conducive to clean production and environmental protection. Compared with traditional degumming methods, enzymatic degumming has the characteristics of mild reaction conditions, no damage to oil structure, less oil loss, economy, and environmental protection. The phospholipase preparation Lecitase developed by Novozymes has been commercially applied since 2000. Enzymatic degumming is favored because of its mild action conditions, no pollution, and low cost. The use of phospholipase for vegetable oil degumming is the development trend of modern oil refining.

[0004] At present, most of the phospholipases used in China rely on imports, such as Novozymes lipase / phospholipase Lecitase Novo, Lecitase 10L and Lecitase Ultra, which have an absolute market share. In order to change the current situation of phospholipases relying on imports and obtain strains with excellent performance for industrial production of phospholipases, PLA1 (PLA1) gene modification and optimization were carried out to construct Bacillus subtilis genetically engineered bacteria with high efficiency in producing PLA1, and the recombinant PLA1 produced by the engineered strain was used for application research on soybean oil and rice oil degumming.

[0005] Phospholipase A from Thermomyces lanuginosus has good performance in many aspects. It is heat-resistant and can tolerate a temperature of 55°C. It is suitable for use at medium temperature (40-55°C) and can be stored for a long time at 4°C. It has a wide pH range and the optimum pH is 7.5. In addition, phospholipase A1 is well expressed in Bacillus subtilis and can reach a level of 1-2g / L after high-density fermentation, which has good industrial application prospects. At present, the optimization of heterologous and efficient secretory expression of phospholipase A1 is mainly reflected in the optimization of phospholipase A1 gene and its secretion optimization. For the gene optimization of phospholipase A, it is mainly focused on the construction of mutants and the optimization of gene codon preference. For the secretion of phospholipase, it is mainly focused on the optimization of promoter and signal peptide, the removal of rare codons in mRNA, the optimization of secondary structure, the addition of mRNA stablizer, the optimization of ribosomes and the optimization of high-density fermentation process.

[0006] Patent literature on the improvement of the properties of natural phospholipases is gradually increasing. Especially after the three-dimensional structure of lipases is analyzed, it is feasible to obtain corresponding property improvements through semi-rational mutations at corresponding sites, such as obtaining mutants with improved alkaline tolerance, heat resistance, and both lipase activity and phospholipase activity. However, although the properties of the screened mutants are optimized, the comprehensive performance still cannot meet the requirements of industrial applications, such as increased secretion difficulty and prolonged culture time, which ultimately leads to increased production costs. In order to reduce the actual use cost and further improve the industrial application value of phospholipase A, people have tried to express the phospholipase gene A (pla1) from Thermomyces lanuginosus in Bacillus subtilis by heterologous secretion, optimize the gene codon, the promoter of the expression vector, the signal peptide, and mutate the gene, and optimize its fermentation conditions.

[0007] In order to increase the protein expression of phospholipase A in Bacillus subtilis, the focus is mainly on the improvement of promoter, mRNA secondary structure and high-density fermentation process. However, too strong promoters and excess exogenous proteins accumulate in cells, forming inclusion bodies or blocking secretion channels, which leads to poor bacterial growth and a decrease in the amount of secreted proteins. There is a high risk. At present, there is no report on the secretion expression of phospholipase A in Bacillus subtilis and the practical application of the recombinant phospholipase A produced; the high-density fermentation process has been improved many times and has certain development potential, but due to the maturity of the process, it is not easy to gain a greater advantage in the industry. It is a common technology to optimize the secretion of heterologous proteins by changing signal peptides. Many researchers have tried to improve the secretion of target proteins by screening signal peptides. For example, Kool, a researcher, fused dbi0038 from Bacillus licheniformis with the fused protease BNP to obtain the best secretion expression. Compared with the original signal peptide, the secretion effect was increased to 7 times the original. This shows that not only heterologous proteins with poor secretion expression, but also homologous proteins with good secretion expression can achieve secretion optimization of the protein by replacing the signal peptide.

[0008] The signal peptide of phospholipase A1 was expressed in Bacillus subtilis, and no phospholipase activity was detected in the fermentation supernatant. As people's understanding of the types of secretory proteins of Bacillus deepens, they begin to try to screen signal peptides with secretion optimization properties for specific proteins on a large scale.

[0009] However, the signal peptides that are currently used more frequently are mainly the signal peptides corresponding to several common high-level secretory proteins such as NprB, NprE and AmyQ. Different signal peptides have different effects on the expression of different proteins, so these signal peptides may not necessarily achieve the ideal optimization of the secretory expression of PLA1.

[0010] According to the different types of vectors used, Bacillus subtilis can express foreign genes in two ways: free plasmid expression and chromosome integration expression. The foreign genes expressed by chromosome integration can maintain good stability in the host, while the plasmid vectors of free expression are usually unstable and cause plasmid loss, and usually require antibiotics to maintain. However, antibiotics are prohibited in food and drug production, so Bacillus subtilis integration expression is a safer and more reliable way to express genetically engineered bacteria.

[0011] In terms of application effect, Clausen compared the degumming characteristics of Lecitase Novo from Fusarium oxysporum and Lecitase 10L from Thermomyces lanuginosus. The results showed that the former preferentially hydrolyzed the phospholipids in the oil phase and could hydrolyze almost all the phospholipids into lysophospholipids under the condition of sufficient reaction time; while the latter preferentially hydrolyzed the phospholipids in the water phase, but the degumming effect was not as good as Lecitase Novo. Li Qiusheng et al. used Lecitase Novo to degummed soybean oil and rapeseed oil, and the phosphorus content was reduced to less than 10 mg / kg after 5 hours of reaction. Lecitase Ultra is a fusion of the lipase gene of Thermomyces lanuginose and the lipase gene of Fusarium oxysporum, expressed in Aspergillus oryzae and obtained by fermentation. The enzyme inherits the stability of the former and the specificity of the latter. As an upgrade of Lecitase Novo, Lecitase Ultra has stronger thermal stability and pH stability. Studies have found that Lecitase Ultra has the dual characteristics of phospholipase and lipase. When the temperature is higher than 40°C, it exhibits phospholipase activity and can hydrolyze phospholipids; when the temperature is lower than 40°C, it mainly exhibits lipase activity and can hydrolyze triglycerides. Yang et al. used Lecitase Ultra for degumming soybean oil and rapeseed oil and successfully reduced the phosphorus content to below 10 mg / kg. With the continuous maturity of technology, Lecitase Ultra has been used for degumming of various vegetable oils such as soybean oil, rapeseed oil, rice bran oil, sunflower oil, and tea seed oil, with good results and wide applicability. However, the price of Novozymes' Lecitase series lipases remains at a high level, which restricts the further large-scale use of phospholipases. Summary of the invention

[0012] The present invention aims to provide a phospholipase A1 and a method and application of integrating secretory expression thereof in Bacillus subtilis, a signal peptide for efficient secretory expression in Bacillus subtilis, and integrating one to two phospholipase A1 expression units into the same integration site of the Bacillus subtilis chromosome to increase the expression amount of phospholipase A1, obtain the recombinant phospholipase A1 and use it in rice bran oil degumming.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides a phospholipase A1, the amino acid sequence of the phospholipase A1 is shown as SEQ ID NO.1.

[0015] The invention also provides application of the phospholipase A1 in oil processing.

[0016] Preferably, the oil processing process is oil degumming.

[0017] The present invention also provides a gene encoding the phospholipase A1, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0018] The invention also provides an expression vector containing the gene, wherein the expression vector is a plasmid containing a promoter, a signal peptide and a phospholipase A1 gene.

[0019] Preferably, the promoter is an overlapping promoter P43 derived from Bacillus subtilis or a promoter of the glycerol-3-phosphate dehydrogenase gene glpD derived from Bacillus subtilis;

[0020] The amino acid sequence of the signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4;

[0021] The plasmid is an integration plasmid pMLK83 and its derivative plasmids, or pAXO1 and its derivative plasmids.

[0022] The present invention also provides the use of the expression vector in constructing a recombinant strain expressing phospholipase A1.

[0023] The present invention also provides a recombinant strain expressing phospholipase A1, wherein the strain contains the expression vector.

[0024] Preferably, the strain is Bacillus subtilis 1A751, Bacillus subtilis WB600 or Bacillus subtilis WB800.

[0025] Preferably, the deposited name of the strain is Bacillus subtilis WB600 / p43-spAl-lipA, in Latin Bacillus subtilis WB600 / p43-spAl-lipA, the deposit date is June 17, 2024, the depository unit is China Center for Type Culture Collection, and the deposit number is CCTCC NO: M20241238.

[0026] The present invention provides a phospholipase A1 and a method and application for integrating secretory expression in Bacillus subtilis. The amino acid sequence of the phospholipase A1 is shown in SEQ ID NO.1. The present invention constructs various signal peptides and expression vectors combined from the Bacillus subtilis homologous protein signal peptide library, and screens out signal peptides that can improve PLA1 secretion by combining a high-throughput screening method, thereby optimizing the secretion of PLA1. Compared with the original signal peptide, the secretion amount of PLA1 is increased by 10 times, and the enzyme activity is increased by 10 times, which is of great significance for the reduction of PLA1 production cost and further large-scale application. In addition, the phospholipase A1 enzyme preparation prepared by the present invention can reduce the phosphorus content of rice oil to 70-75ppm at a temperature of 55°C and acidic conditions.

[0027] The invention synthesizes the phospholipase A1 gene of Thermomyces lanuginosus, and the synthesized coding gene is more in line with the gene coding preference of Bacillus subtilis for efficient expression, and the phospholipase A1 gene is modified according to the gene consistency; the Bacillus subtilis expression plasmid of the phospholipase A1 of Thermomyces lanuginosus is constructed, the signal peptide for secretory expression of phospholipase A1 is optimized, the transformation of Bacillus subtilis and the screening of high-yield strains of recombinant phospholipase A1, and the application of the generated recombinant phospholipase A. The invention provides an amino acid sequence of a mutant phospholipase A1 after gene mutation and optimized based on the codon preference of Bacillus subtilis, and the amino acid sequence is shown in SEQ ID NO.1. In addition, a nucleotide sequence of a mutant phospholipase A1 is also provided, and the nucleotide sequence is shown in SEQ ID NO.2. The invention also provides an expression vector combining various signal peptides with phospholipase A1 from a Bacillus subtilis homologous protein signal peptide library, and a signal peptide that can improve the secretion of phospholipase A1 is screened out in combination with a high-throughput screening method, thereby optimizing the secretion of phospholipase A1. The amino acid sequence of the signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence of the signal peptide is provided, wherein the nucleotide encodes the signal peptide for optimizing the efficient secretory expression of phospholipase A. The nucleotide sequence is shown in SEQ ID NO.4.

[0028] Compared with the prior art, it has the following beneficial effects:

[0029] 1. Compared with wild-type phospholipase A1, the mutant enzyme obtained by site-directed mutagenesis has higher specific activity and heat resistance, and is more suitable for application requirements;

[0030] 2. The signal peptide screened and obtained by the present invention realizes the efficient secretory expression of phospholipase A. Compared with the original signal peptide, the secretion amount of phospholipase A is increased by 10 times, which is of great significance for the reduction of phospholipase A production cost and further large-scale application;

[0031] 3. The method for screening and obtaining the optimized secretion signal peptide of phospholipase A1 of the present invention will not cause the blockage of inclusion bodies or secretion channels, thereby affecting the downstream fermentation, relative to the optimization of transcription efficiency;

[0032] 4. The present invention utilizes a food-safe expression system to produce phospholipase A1. The exogenous genes contained in the integrated expression strain can be stably propagated and efficiently expressed. The product meets food requirements and has no antibacterial activity.

[0033] Collection Instructions

[0034] The deposited name of the strain is Bacillus subtilis WB600 / p43-spAl-lipA, in Latin Bacillussubtilis WB600 / p43-spAl-lipA, the deposit date is June 17, 2024, the depository unit is China Center for Type Culture Collection, the deposit number is CCTCCNO: M 20241238, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Construction process for the engineered strain of Bacillus subtilis WB600 / p43-spA1-lipA;

[0036] Figure 2 Comparison of the sedimentation effect of phospholipase A1 and commercial enzyme Lecitase Ultra in rice oil degumming (the left picture shows sedimentation for 2 hours, and the right picture shows sedimentation for 3 hours); ①~③ are phospholipase A1, ④ is commercial enzyme Lecitase Ultra, and ⑤~⑥ are domestic commercial phospholipase A1;

[0037] Figure 3 This is a comparison of the sedimentation effects of phospholipase A1 and commercial enzyme Lecitase Ultra in soybean oil degumming, among which ① is the commercial enzyme Lecitase Ultra, ②~④ are the phospholipase A1 of this study, and ⑤~⑥ are domestic commercial phospholipase A1. DETAILED DESCRIPTION

[0038] The present invention provides a phospholipase A1, the amino acid sequence of the phospholipase A1 is shown in SEQ ID NO.1:

[0039] ARPVRRAVPQDLLDQFELFSQYSAAAYCAANNHAPVGSDVTCSENVCPEVDAADATFLYSFEDSGLGDVTGLLALDNTNKLIVLSFRGTRSVENWIANLNADLTEISDICSGCEGHDGFVTSWRSVADTIREQVQNAV NEHPDYRVVFTGHSLGGALATIAAAALRGNGYNIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRDWGYSHSSPEYWVTSGNDVPVTANDITVVEGIDSTDGNNQGNIPDIPSHLWYFGPISECD.

[0040] The invention also provides application of the phospholipase A1 in oil processing.

[0041] In the present invention, the oil processing process is preferably oil degumming.

[0042] The present invention also provides a gene encoding the phospholipase A1, the nucleotide sequence of the gene is shown in SEQ ID NO.2:

[0043] .

[0044] The invention also provides an expression vector containing the gene, wherein the expression vector is a plasmid containing a promoter, a signal peptide and a phospholipase A1 gene.

[0045] In the present invention, the promoter is preferably an overlapping promoter P43 derived from Bacillus subtilis or a promoter of the glycerol-3-phosphate dehydrogenase gene glpD derived from Bacillus subtilis;

[0046] The amino acid sequence of the signal peptide is shown in SEQ ID NO.3:

[0047] MRIQKRRTHVENILRILLPPIMILSLILTPTPPIHA;

[0048] The nucleotide sequence is shown in SEQ ID NO.4:

[0049] atgagaatacagaaaagacgaacacacgtcgaaaacattctccgtattcttttgcccccaattatgatacttagc ctaatcctcccaacaccacccattcatgca;

[0050] The plasmid is preferably an integration plasmid pMLK83 and its derivative plasmids, or pAXO1 and its derivative plasmids.

[0051] The present invention also provides the use of the expression vector in constructing a recombinant strain expressing phospholipase A1.

[0052] The present invention also provides a recombinant strain expressing phospholipase A1, wherein the strain contains the expression vector.

[0053] In the present invention, the strain is preferably Bacillus subtilis 1A751, Bacillus subtilis WB600 or Bacillus subtilis WB800.

[0054] In the present invention, the deposited name of the strain is Bacillus subtilis WB600 / p43-spAl-lipA, the Latin name is Bacillus subtilis WB600 / p43-spAl-lipA, the deposit date is June 17, 2024, the depository unit is China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20241238.

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Example 1 Screening of signal peptides for efficient secretory expression of phospholipase A

[0057] Referring to the method of (Christian Degering et al, 2010, TaKaRaInstruction), expression vectors combining various signal peptides with phospholipase A were constructed from the Bacillus subtilis homologous protein signal peptide library, and signal peptides that improve the secretion effect of phospholipase A to varying degrees were obtained from clones in combination with a high-throughput screening method, specifically comprising the following steps:

[0058] 1. Mutation and synthesis of the phospholipase A1 gene of Thermomyces lanuginosus. According to the phospholipase A1 gene sequence of Thermomyces lanuginosus in GenBank EU004196, after sequence consistency comparison, the phospholipase A1 was mutated into a triple mutant S105T / A116N / V133D, and the gene lipA1 was synthesized according to the codon preference of Bacillus subtilis; after adding 6×his tag to the end of the phospholipase A gene, the restriction sites at both ends of the phospholipase A gene were designed as NdeI and HindⅢ, and the phospholipase A full gene DNA fragment containing the restriction sites was obtained by gene synthesis technology.

[0059] 2. Construction of pBE-S-aprE-lipA1 plasmid. The lipA1 gene and pBE-S plasmid were treated with NdeI and HindⅢ, and the DNA fragment containing the target band was separated and recovered by agarose gel electrophoresis. The recovered DNA fragment was connected to the pBE-S plasmid using T4 ligase to construct the pBE-S-aprE-lipA1 recombinant plasmid. The ligation product was introduced into E.coli DH5α by chemical transformation and coated on an LBA plate containing 100μg / mL ampicillin for screening. The correct single colony was verified by sequencing and saved as the E.coli DH5α / pBE-S-aprE-lipA1 strain. The strain was inoculated into 5mL LBA liquid culture medium and cultured at 37℃ and 220rpm for 8h, and then the pBE-S-aprE-lipA1 plasmid was extracted.

[0060] 3. Construct pHYp43-aprE-lipA1. Using the plasmid pBE-S-aprE-lipA1 in step 2 as a template, use the specific primers lipA1-F: ggtaccattataggtcttaagcaaaaggagagggacg (SEQ ID NO.5) and lipA1-R: gcagatctgtgtacgaagcttaatggtgatggtgatgg (SEQ ID NO.6) to perform PCR amplification to obtain the DNA fragment from Afl II to lipA gene. At the same time, design primers pHYp43-F: cgtacacagatctgcaggtcga (SEQ ID NO.7) and pHYp43-R: acctataatggtaccgctatcacttta (SEQ ID NO.8) to amplify the pHYp43 fragment by PCR. After the two PCR products were recovered by gel, the recovered products were purified by In- The pHYp43-aprE-lipA1 recombinant plasmid was constructed by ligation according to the instructions of HD Cloning Kit.

[0061] 4. Optimization and screening of secretion signal peptides.

[0062] (1) According to the instructions of the B. subtilis Secretory Protein Expression System Kit (TaKaRa, cas: #3380), the SP mix library containing 173 signal peptides was inserted into the pHYp43-aprE-lipA1 plasmid to construct the pHYp43-SP mix-lipA1 recombinant plasmid. The ligation product was introduced into E. coli DH5α by chemical transformation and coated on LBA plates containing 100 μg / mL ampicillin for screening.

[0063] (2) Extraction of pHYp43-SP mix-lipA1 plasmid. All colonies on the LBA plate were washed with 1 mL of sterile water and added to 10 mL of LBA liquid culture medium. The culture was cultured at 37°C and 220 rpm for 12 h to extract the bacterial liquid plasmid pHYp43-SP mix-lipA1.

[0064] (3) Preparation of Bacillus subtilis WB600 competent cells. Inoculate Bacillus subtilis WB600 on an LB plate, culture at 37°C for 24 hours, pick a single colony and inoculate it in GM I solution, and culture it overnight at 30°C and 125rpm with shaking for 16 hours. Take 500μL and transfer it to 5mL GM I, and culture it at 37°C and 220rpm for 3.5 hours. Take another 250μL of the culture solution from the previous step and transfer it to 5mL GMII, and culture it at 37°C and 220rpm for 90 minutes. Finally, centrifuge at 5000rpm for 10 minutes to collect the cells. Gently suspend the cells with 10% of the original culture volume of the supernatant. The suspended cells are the Bacillus subtilis WB600 competent cells. 10×SPsalt solution: K 2 HPO 4 15%, K 2 HPO 4 6%, ammonium sulfate 2%, magnesium sulfate 0.2%, sodium citrate 1%. Prepare 10% yeast powder, 1% hydrolyzed casein, 25mM MgCl 2 , 0.25% tryptophan, 0.1 M CaCl 2 20% glucose solution, 10% yeast extract, 1% hydrolyzed casein, 25mM MgCl 2 Sterilization is required at 115°C for 20 minutes. 0.25% tryptophan, 0.1M CaCl 2Filter and sterilize with a 0.22μm filter membrane. 0.25% tryptophan needs to be stored at -20℃, and the rest should be stored at 4℃. GM I solution: 1mL 10×SP salt solution, 0.25mL 20% glucose, 0.2mL 1% hydrolyzed casein, 0.1mL 10% yeast juice, 0.2mL 0.25% tryptophan, add water to make up to 10mL. GMII solution: 1mL 10×SP salt solution, 0.25mL 20% glucose, 0.04mL 1% hydrolyzed casein, 0.05mL 10% yeast juice, 0.2mL 0.25% tryptophan, 0.05mL 0.1MCaCl 2 , 1 mL 25 mM MgCl 2 , add water to make up to 10mL.

[0065] (4) Chemical transformation of Bacillus subtilis. 500 μL of Bacillus subtilis WB600 competent cells were mixed with 1 μg of pHYp43-SP mix-lipA1 plasmid and cultured at 37°C, 200 rpm for 120 min with shaking. The culture was then spread on an LBTC plate containing 20 μg / mL tetracycline and cultured at 37°C overnight.

[0066] (5) Cultivate Bacillus subtilis WB600 / pHYp43-SP mix-lipA1. Inoculate the single colonies on the LBTC plate into 5 mL of LBTC liquid culture medium and culture at 37°C, 220 rpm for 8 h as seed solution. Inoculate the seed solution into 100 mL of fermentation medium containing 20 μg / mL tetracycline and culture at 37°C, 220 rpm for 48 h. Fermentation medium: 3% glucose, 2% peptone, 0.1% potassium dihydrogen phosphate, 0.2% potassium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate, and 0.01% calcium chloride.

[0067] (6) Select the secretion-optimized strain. Centrifuge the fermentation broth at 10,000 rpm for 2 min, and use the pNPPC substrate method to measure the enzyme activity of the supernatant. The strain corresponding to the fermentation broth supernatant with the highest enzyme activity is the secretion-optimized strain.

[0068] (7) Extract the plasmid of the secretion optimized strain. Take 10 mL of the fermentation broth of the secretion optimized strain, centrifuge at 10,000 rpm for 2 min, discard the supernatant and retain the bacteria. Use 500 μL of solution I containing 100 mg / mL lysozyme to resuspend the bacteria, oscillate at 37°C and 220 rpm for 30 min, and then extract the plasmid according to the instructions of the plasmid extraction kit. Sequence the extracted plasmid to determine whether the signal peptide has been modified. The sequencing primer is lipA-F: ggtaccattataggtcttaagcaaaaggagagggacg (SEQ ID NO.5).

[0069] (8) Confirming the signal peptide of the secretion-optimized strain. The signal peptide that can improve the secretion effect of phospholipase A was screened through the above steps and named spA1. The amino acid sequence is MRIQKRRTHVENILRILL PPIMILSLILPTPPIHA (SEQ ID NO.3), and the nucleotide sequence encoding the amino acid sequence is atgagaatacagaaaagacgaacacacgtcgaaaacattctccgtattcttttgcccccaattatgatacttagcct aatcctcccaacaccacccattcatgca (SEQ ID NO.4)

[0070] Example 2 Optimization effect of the signal peptide obtained in Example 1 on secretory expression

[0071] The NPPC substrate method was used to determine the enzymatic activity of the signal peptide obtained in Example 1, the currently used signal peptide, and the wild-type signal peptide on the secreted phospholipase A1. The bacterial solution was cultured on a shaker, and the enzyme activity was measured according to the following operation:

[0072] NPPC substrate method for measuring phospholipase C enzyme activity: Based on the fact that phospholipase A can hydrolyze the glycerol phospholipid structural analogue p-nitrophenolphosphocholine (p-NPPC) to produce a colored group (p-nitrophenol), which has a maximum absorption peak at a wavelength of 410nm, p-NPPC can be used as a reaction substrate, and the absorbance of the reaction solution can be measured at a wavelength of 410nm. The amount of p-nitrophenol produced by PLA hydrolyzing p-NPPC can be calculated by substituting it into the standard curve, thereby quantitatively detecting the activity of phospholipase C.

[0073] Enzyme reaction system composition: 0.25mol / L Tris-HCl (pH 7.2), 0.1mmol / L ZnCl 2 , 60g / 100mL sorbitol, 10mmol / L p-NPPC, add 200μL fermentation enzyme solution to the reaction system and react at 37℃ for 30min, and immediately measure the absorbance at 410nm wavelength with a spectrophotometer. Definition of enzyme activity unit: Under the conditions of pH 7.2 and 37℃, the amount of enzyme required to hydrolyze p-NPPC to produce 1nmol of p-nitrophenol per minute is 1 enzyme activity unit (U).

[0074] Table 1 Comparison of expression levels of different signal peptides PLA1

[0075]

[0076] As can be seen from Table 1, the signal peptide spA1 obtained in the present invention can significantly improve the secretory expression of PLA compared with the wild-type signal peptide and the signal peptide currently used.

[0077] Example 3 Integrated secretory expression of phospholipase A1

[0078] 1. Construct pMLK83-p43-spA1-lipA. A fragment containing the p43 promoter to the lipA gene was amplified from the pHYp43-spA1-lipA plasmid by PCR, and two restriction sites, SalI and BamHI, were added at both ends of the fragment. The primers were pMLK83-F (sequence: ACGCGTCGACAACGCTTTGCCCAAGC) (SEQ ID NO.11) and pMLK83-R (sequence: GGCCGGATCCCAGATCTGTGTACGAAGCTT) (SEQ ID NO.12). The integration vector pMLK83 plasmid was double-digested with BamHI and SalI. The amplified fragment and the pMLK83 plasmid after restriction digestion were separated by agarose gel electrophoresis, and the gel fragment containing the target band was recovered. The recovered fragment was connected to the pMLK83 plasmid using T4 ligase, and the ligation product was introduced into E.coli DH5α competent cells by chemical transformation. Through screening and identification, the correct clones were selected for liquid culture, and the pMLK83-p43-spA1-lipA1 plasmid was extracted.

[0079] 2. Construction of Bacillus subtilis engineered bacteria WB600 / p43-spA1-lipA. The successfully constructed pMLK83-p43-spA1-lipA1 plasmid was introduced into Bacillus subtilis WB600 by chemical transformation, and the transformed cells were spread on LBK plates containing 20μg / mL kanamycin. The grown colonies were selected and inoculated in parallel into two 1% starch LBK plates containing 20μg / mL kanamycin, and cultured at 37°C for 16h. Among them, one plate was used for color development reaction with Gram's iodine solution. By observation, colonies that did not form a hydrolysis circle were selected, indicating that the lipA1 gene had been successfully integrated into the α-amylase gene site of the Bacillus subtilis chromosome genome through homologous double exchange, thereby constructing the Bacillus subtilis engineered bacteria WB600 / p43-spA1-lipA.

[0080] 3. Cultivate the engineered Bacillus subtilis WB600 / p43-spA1-lipA. Select a single colony that has not formed a hydrolysis circle and inoculate it into 5mL LB liquid culture medium, and culture it at 37°C, 220rpm for 8h as seed liquid. Subsequently, inoculate the seed liquid into 100mL fermentation medium and culture it under the same conditions for 48h. Collect the fermentation supernatant by centrifugation, and use the pNPPC substrate method to determine the enzyme activity to further confirm the successful integration and expression of the lipA1 gene in Bacillus subtilis WB600.

[0081] Example 4 Preparation of recombinant phospholipase A1 by large-scale fermentation of Bacillus subtilis engineered strain WB600 / p43-spA1-lipA

[0082] The culture medium of the Bacillus subtilis engineered bacteria WB600 / p43-spA1-lipA is a common LB liquid culture medium, without the need for inducers and antibiotics. The operation steps are as follows:

[0083] (1) Preparation of primary strain: A single colony of the genetically engineered strain of Bacillus subtilis was cultured in 4 mL of LB liquid medium at 37° C. and 220 rpm overnight to obtain the primary strain;

[0084] (2) Preparation of secondary seed: The primary seed was inoculated into 800 mL of LB liquid medium and cultured at 37°C and 220 rpm until the OD600 reached about 0.6 (culture for 5 h);

[0085] (3) Preparation of the third-level seed: The second-level seed was inoculated into an 80 LLB liquid fermentation tank and cultured at 37°C with citric acid and NaOH to pH 7.0, with ventilation and stirring, and the dissolved oxygen was controlled at 25%. 600 0.6 (cultured for 6 h);

[0086] (4) Production tank fermentation: The third-grade seed was inoculated into a 3-ton fermentation tank, LB liquid culture medium, 37°C, ventilation and stirring, dissolved oxygen controlled at 25%, pH 7 controlled with citric acid and NaOH, cultured for 26 hours, centrifuged at 10,000 rpm for sterilization, and the supernatant was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 7500 Da to obtain a concentrated stock solution of phospholipase A1.

[0087] Example 5 Application of recombinant phospholipase A1 in degumming of vegetable oil (rice oil)

[0088] According to the method of using commercial lipase (Lecitase Ultra), the suitable conditions for enzymatic degumming of phospholipase A1 were determined: the enzyme preparation was diluted 100 times, the pH was 6.0, the enzyme addition amount was 100U / kg (80ppm), the total water addition amount was 3%, the homogenization condition was 10,000rpm, 1min, and then the degumming was carried out on a shaking table at 55℃ and 100rpm for 4h. Under these conditions, the reaction was allowed to stand after standing, and the oil impurities were well settled, the phosphorus content of the oil layer was reduced to 74ppm, and the removal rate of phospholipids in the degummed oil was as high as more than 80%, indicating that the recombinant enzyme phospholipase PLA1 obtained by integrating Bacillus subtilis expression has good application prospects in degumming.

[0089] in conclusion

[0090] The present invention integrates the phospholipase A1 expression element into the chromosome of Bacillus subtilis for the first time, takes the bacillus subtilis as the strain, and produces the phospholipase A1 by fermentation using the conventional production process.

[0091] The advantages of the present invention are: the present invention utilizes a food-safe expression system to produce phospholipase A1, the exogenous gene contained in the integrated expression strain can be stably propagated and efficiently expressed, the product meets food requirements, and the product has no antibacterial activity

[0092] As can be seen from the above embodiments, the present invention provides a phospholipase A1 and a method and application for its integrated secretory expression in Bacillus subtilis, wherein the amino acid sequence of the phospholipase A1 is shown in SEQ ID NO.1. The present invention constructs various signal peptides and expression vectors combined from the Bacillus subtilis homologous protein signal peptide library, and screens out signal peptides that can improve PLA1 secretion by combining a high-throughput screening method, thereby optimizing the secretion of PLA1. Compared with the original signal peptide, the secretion amount of PLA1 is increased by 10 times, and the enzyme activity is increased by 10 times, which is of great significance for the reduction of PLA1 production cost and further large-scale application. In addition, the phospholipase A1 enzyme preparation prepared by the present invention can reduce the phosphorus content of rice oil to 70-75ppm at a temperature of 55°C and under acidic conditions.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an expression vector containing a phospholipase A1 gene in constructing a recombinant strain expressing phospholipase A1, characterized in that: The recombinant strain contains the expression vector; The expression vector is a plasmid containing a promoter, a signal peptide and a phospholipase A1 gene; The promoter is an overlapping promoter P43 derived from Bacillus subtilis or a promoter of the glycerol-3-phosphate dehydrogenase gene glpD derived from Bacillus subtilis; The amino acid sequence of the signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; The plasmid is an integration plasmid pMLK83 and its derivative plasmids, or pAXO1 and its derivative plasmids; The nucleotide sequence of the phospholipase A1 gene is shown in SEQ ID NO.2; The type of the recombinant strain is Bacillus subtilis WB600.

2. A recombinant strain expressing phospholipase A1, characterized in that: The strain contains the expression vector according to claim 1, and the type of the recombinant strain is Bacillus subtilis WB600.

3. The strain according to claim 2, characterized in that The deposited name of the strain is Bacillus subtilis WB600 / p43-spAl-lipA, which is Bacillus subtilis WB600 / p43-spAl-lipA, preservation date: June 17, 2024, preservation unit: China Center for Type Culture Collection, preservation number: CCTCC NO: M 20241238.

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