Method for remarkably improving soluble expression of horse-derived acylase in pichia pastoris

Abstract

The invention belongs to the technical field of biology, and discloses a method for improving soluble expression of horse-derived acylase in pichia pastoris. On the premise of not changing the types of amino acids, by optimizing the N-terminal nucleic acid sequence of horse-derived acylase, a method capable of remarkably improving the soluble expression of the pig acylase is accidentally found, and meanwhile, the enzyme activity of the pig acylase is remarkably improved by 4.41 times. The method disclosed by the invention is simple to operate and obvious in effect, and the market competitiveness is improved for large-scale production and practical application of the horse-derived acylase.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving the soluble expression of horse acylase in Pichia pastoris. Background Art

[0002] Acylases are a class of enzymes that catalyze esterification reactions in organisms. They catalyze the combination of acids and alcohols into esters. Acylases are usually key enzymes in various organisms and participate in a variety of biochemical processes, such as fatty acid metabolism, biosynthesis, and degradation. Our team has confirmed that horse-derived acylase has the activity to synthesize lauroylglycine. In order to improve its protein synthesis efficiency in industrial production, it is necessary to find a suitable protein expression system for it.

[0003] The Pichia pastoris expression system has obvious advantages in expressing foreign proteins, such as no endotoxin effects, efficient secretory expression, fast growth rate, and effective post-translational protein modification. The yeast expression system contains a unique and powerful alcohol oxidase gene promoter, which can strictly regulate the expression of foreign genes with methanol, and the expression level is high; at the same time, the system also has the advantages of simple operation, stable properties, less expression background protein, and easy production process amplification. In recent years, many recombinant proteins have been expressed efficiently in Pichia pastoris, reaching the gram level, but there are still many proteins with low expression levels, which limits their industrial use.

[0004] The soluble expression level of horseradish acylase directly expressed in Pichia pastoris is low, mostly in the form of inclusion bodies, which seriously limits its industrial production. Summary of the invention

[0005] In order to improve the soluble expression level of horse acylase in Pichia pastoris, the inventors tried to optimize the strain culture and induction conditions, add fusion tags, optimize the expression strain and other strategies, but failed to achieve a significant increase in the soluble expression of horse acylase. During the research process, the inventors accidentally discovered that the change of the N-terminal nucleic acid sequence of horse acylase unexpectedly significantly improved the soluble expression of the protein and significantly improved its enzyme activity. It is against this background that the purpose of the present invention is to provide a Pichia pastoris expression system that significantly improves the soluble expression of horse acylase.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: The present invention provides a gene encoding horse-derived acylase, wherein the nucleotide sequence of 1 to 10 amino acids at the N-terminus of the gene encoding horse-derived acylase is replaced with ATGGCGTCTAAGGGGCGAGAGGATGAACAT.

[0007] Preferably, the amino acid sequence of the horse acylase is as shown in SEQ ID NO. 1. Preferably, the gene encoding the horse acylase is based on the nucleotide sequence shown in SEQ ID NO. 2, with the nucleotide sequence encoding 1 to 10 amino acids at the N-terminus replaced with ATGGCGTCTAAGGGGCGAGAGGATGAACAT.

[0008] More preferably, the nucleotide sequence is as shown in SEQ ID NO.3.

[0009] The invention contains the recombinant expression vector encoding the horse source acylase gene.

[0010] Preferably, the vector framework thereof is pPIC9K.

[0011] The present invention further provides a recombinant bacterium encoding the horse acylase gene or the recombinant expression vector.

[0012] Preferably, it is Pichia pastoris.

[0013] The invention provides a method for expressing or preparing horse acylase, which produces horse acylase by culturing the recombinant bacteria.

[0014] Optionally, the method further comprises the step of isolating the produced horse acylase.

[0015] Preferably, the method for isolating the produced horse acylase is to centrifuge the recombinant bacteria after induced expression and collect the supernatant to obtain the horse acylase.

[0016] The significant beneficial effect achieved by the present invention is that an optimized Pichia pastoris expression system is successfully constructed, which can significantly improve the soluble expression level of horse acylase and increase its enzyme activity by 4.41 times. Specifically, by optimizing the nucleotide sequence of 1 to 10 amino acids at the N-terminus of horse acylase, the soluble expression of horse acylase in Pichia pastoris is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 .Comparison of HAcy and HAcy-M protein expression. DETAILED DESCRIPTION

[0018] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, to facilitate understanding of the technical solution of the present invention, and is not used to limit the present invention.

[0019] The culture medium used in the examples is: YPD liquid medium (1 L): Dissolve 50 g YPD solid in 1 L distilled water, adjust the pH to 6.5, sterilize at 121°C for 20 min, and cool for later use.

[0020] 10×YNB (1 L): 134 g yeast nitrogen source solid was dissolved in 1 L distilled water and sterilized by filtration using a 0.22 μm filter.

[0021] 500× Biotin (50 mL): Dissolve 10 mg of biotin solid in 50 mL of distilled water, filter sterilize with a 0.22 μm filter, and store at 4°C.

[0022] BMGY liquid culture medium (1 L): add 10 g yeast extract and 20 g peptone to 879 mL distilled water, sterilize at 121°C for 20 min, and after cooling, add 100 mL 10×YNB, 1 mL 500×biotin, and 20 mL 50% glycerol in a clean bench.

[0023] BMMY liquid culture medium (1 L): add 10 g yeast extract and 20 g peptone to 894 mL distilled water, sterilize at 121 °C for 20 min, and add 100 mL 10×YNB, 1 mL 500×biotin, and 5 mL methanol in a clean bench after cooling.

[0024] Example 1: Construction and expression of Pichia pastoris recombinant expression strain of horse acylase According to the horse-derived acylase gene HAcy included in NCBI (GenBank: XP_001492888.2, the encoded amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2), the horse-derived acylase gene was artificially synthesized, and PCR amplification was performed using the gene as a template, and EcoRI restriction sites and NotI restriction sites were added at both ends to complete the construction of the gene expression frame.

[0025] The horse-derived acylase gene fragment was amplified by specific primers upstream primer F: 5'-GAATTCATGGCCAGCAAGGGTCGCG-3' and downstream primer R: 5'-GCGGCCGCTCAGCTGTCACTGGGCAGG -3', and the pPIC9K vector was double-digested with EcoR I and Not I. After the acylase gene fragment and pPIC9K plasmid were double-digested with EcoR I and Not I, the acylase gene was ligated with the double-digested pPIC9K vector using T4 DNA ligase to construct the recombinant plasmid pPIC9K-Acy. pPIC9K-HAcy was transformed into E.coil DH5α and the plasmid was extracted.

[0026] The plasmid was linearized with restriction endonuclease SacI and electroporated into GS115. A 100 μg / ml YPD resistance plate was used for primary screening, and a 800 μg / ml YPD resistance plate was used for secondary screening.

[0027] The positive transformants were inoculated into BMGY liquid medium and cultured at 30°C until the OD600 value of the bacterial solution reached 2-6. Centrifuge at 5000rpm for 5min at room temperature, remove the supernatant, and collect the bacteria. Subsequently, the bacteria were resuspended in BMMY liquid medium until the OD600 value was about 1.0, and continued to be cultured in a shaking incubator at 30°C and 220rpm. Pure methanol was added to the culture medium every 24 hours to adjust the final concentration to 0.5%-1.0%. After methanol induction for 96 hours, centrifuge at 8000rpm for 10min, collect the filtered supernatant, and obtain horse acylase HAcy.

[0028] Example 2: Construction and expression of an optimized expression strain for the N-terminus of acylase The inventors accidentally discovered that after optimizing the codons of the first 10 amino acids at the N-terminus of horse acylase (the optimized nucleotide sequence is shown in SEQ ID NO: 3), an unexpected effect was obtained. The specific construction method is as follows: The recombinant plasmid pPIC9K-Acy constructed in Example 1 was used as a template for whole plasmid PCR amplification. The amount of each component added to the PCR system (total volume 50 μL): PrimerStar 25 μL, ddH2O 20 μL, primer F (5'-AATTCATGGCGTCTAAGGGGCGAGAGGATGAACATCCATCCGTGACGCTC-3') and primer R (5'-GAGCGTCACGGATGGATGTTCATCCTCTCGCCCCTTAGACGCCATGAATT-3') 1.5 μL each, template 2 uL.

[0029] The PCR amplification program was as follows: 98℃ pre-denaturation for 2min; enter the cycle (98℃ denaturation for 10s; 56℃ annealing for 15s; 72℃ extension for 70s), a total of 25 cycles, and 72℃ final extension for 5min. After the PCR reaction, 1μL of DpnI was added and digested at 37℃ for 30min. Then it was transformed into E.coil DH5α and the plasmid was extracted. The recombinant plasmid pPIC9K-HAcy-M with optimized N-terminus was obtained.

[0030] According to the method shown in Example 1, the N-terminus-optimized horse acylase HAcy-M was obtained.

[0031] Example 3 Identification of the expression results of HAcy and HAcy-M proteins The horse acylase enzyme solution obtained in Example 1 and Example 2 was concentrated 5 times using an ultrafiltration tube, and 20 ul of 2X loading + 20 ul of the supernatant and the precipitate suspension were taken respectively, and boiled in a water bath for 10 min. After cooling, 20 ul of the sample was taken for electrophoresis. The electrophoresis results are shown in FIG. Figure 1 As shown, the protein size is 46 kDa.

[0032] from Figure 1 It can be seen that the soluble expression of HAcy-M is significantly increased compared with HAcy.

[0033] The present invention systematically analyzed and found that the free energy of the mRNA of the first 10 amino acids at the N-terminus of HAcy-M is -4.28, while that of HAcy-M is -6.59 (the free energy prediction is performed using RNAFOLD software). It is speculated that the soluble protein expression is related to the free energy, and the greater the free energy prediction value, the higher the soluble protein expression.

[0034] Example 4 Comparison of Enzyme Activities of Horse Acylase HAcy and HAcy-M Obtain the bacterial OD using Example 1 and Example 2 600 =25 The enzyme solution obtained was used to measure the activity of lauroylglycine synthesis. Synthesis reaction system (10 mL): 360 mg of lauric acid, 5 ml of saturated glycine solution, 5 ml of enzyme solution. Reaction at 37°C for 14 h.

[0035] Analysis conditions: Chromatographic column: C18, 4.6mm×250mm, 5μm; Detection wavelength: ultraviolet (UV) = 200nm; Mobile phase: 85% acetonitrile and 15% 0.05% trifluoroacetic acid aqueous solution; Column temperature: 30°C; Flow rate: 1mL / min; Detection time: 15min.

[0036] After the reaction, the concentration of lauroyl glycine in HAcy-M was 5.41 times that of HAcy, indicating that the enzyme activity of HAcy-M was increased by 4.41 times compared with HAcy.

Claims

1. A gene encoding horse acylase, characterized in that: The nucleotide sequence of 1 to 10 amino acids at the N-terminus of the gene encoding horse acylase is replaced with ATGGCGTCTAAGGGGCGAGAGGATGAACAT.

2. The gene encoding horse acylase according to claim 1, characterized in that The amino acid sequence of the horse source acylase is shown in SEQ ID NO.1; Preferably, the gene encoding horse acylase is based on the nucleotide sequence shown in SEQ ID NO.2, with the nucleotide sequence encoding 1 to 10 amino acids at the N-terminus replaced with ATGGCGTCTAAGGGGCGAGAGGATGAACAT.

3. The gene encoding horse acylase according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

3.

4. A recombinant expression vector comprising the gene encoding the horse acylase according to any one of claims 1 to 3.

5. The recombinant expression vector according to claim 4, characterized in that Its vector framework is pPIC9K.

6. A recombinant bacterium comprising the gene encoding the horse acylase according to any one of claims 1 to 3, or the recombinant expression vector according to claim 4 or 5.

7. The recombinant bacterium according to claim 6, characterized in that It is Pichia pastoris.

8. A method for expressing or preparing horse acylase, comprising culturing the recombinant bacterium according to claim 4 or 5 to produce horse acylase.

9. The method according to claim 8, characterized in that The method also includes the step of isolating the produced horse acylase.

10. The method according to claim 9, characterized in that The step of separating the produced horse-derived acylase is to centrifuge the recombinant bacteria after induced expression and collect the supernatant to obtain the horse-derived acylase.

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