A method for increasing the production of lysozyme in European oysters

Abstract

The present invention discloses a method for increasing the yield of European oyster lysozyme, and belongs to the field of bioengineering. The present invention screens and obtains European oyster-derived lysozyme with relatively high enzyme activity from 12 different sources of lysozyme gene sequences from vertebrates, mollusks, poultry and insects, and realizes the active expression of the enzyme in Pichia pastoris. The present invention also fuses short peptides and protein tags of different amino acids to the N-terminus of OElyz. When the Sumo tag is fused, the extracellular enzyme activity of recombinant OElyz reaches 382.6U / mL at the shake flask level, and this yield level can meet the application requirements of lysozyme in the fields of medicine, biotechnology and food.

Description

Technical Field

[0001] The invention relates to a method for improving the yield of European oyster lysozyme, and belongs to the field of bioengineering. Background Art

[0002] Lysozymes (Lyzs) are cell wall hydrolases that can break the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposing the insoluble mucopolysaccharide in the cell wall into soluble glycopeptides. Due to its bactericidal activity, Lyzs has important application value in the fields of medicine, biotechnology, and food. For example, Lyzs has antibacterial, antiviral and tissue recovery functions. Lysozyme lozenges are often used in medicine to treat oral ulcers and acute and chronic pharyngitis. Lyzs has the characteristic of specifically hydrolyzing cell walls. Lysozymes are often used in the biotechnology field to prepare protoplasts, extract and manufacture enzymes, nucleic acids and active peptides. Lyzs can kill intestinal putrefactive cocci and promote the proliferation of bifidobacteria in the infant intestine. It is a good additive for infant food and beverages. According to the differences in amino acid sequence and source, Lyzs can be divided into: C-type lysozyme (chicken lysozyme), G-type lysozyme (goose lysozyme), I-type lysozyme (invertebrate lysozyme) and P-type lysozyme (bacteriophage lysozyme).

[0003] At present, egg lysozyme is the main source of commercial lysozyme, and its extraction method is crystallization. The isoelectric point of egg lysozyme is 10.7. Adding 5% NaCl salt solution to it can change the pH value of the solution, so as to use the principle of protein isoelectric point precipitation to crystallize and precipitate egg lysozyme to achieve the purpose of separation and purification. Although this method is simple to operate, it is too time-consuming. It usually takes a week to obtain high-purity lysozyme; the recovery rate is low, only 60-80% can be recovered; the raw materials are limited, and the removal of impurities during the crystallization process is incomplete.

[0004] Using genetic engineering technology to produce lysozyme heterologously can fundamentally solve the problem of limited raw materials for lysozyme. It has been reported that human lysozyme genes were transferred into animals such as goats, cows and mice, and lysozyme proteins were obtained in milk through mammary gland secretion, but this method is cumbersome to operate and the purification work is relatively complicated. The microbial method of producing lysozyme is both convenient and fast, and does not require the consumption of a large amount of raw materials. It only needs to provide sufficient nutrients for the microorganisms so that the lysozyme genes can be expressed in host cells to form proteins. Lysozyme with high purity can be obtained through simple separation and purification. Since lysozyme molecules often contain disulfide bonds, their expression in prokaryotic hosts such as Escherichia coli is not ideal. Eukaryotic cells with a more complete expression system are a better choice for producing lysozyme. Pichia pastoris GS115 has the advantages of fast growth, simple genetic manipulation, high secretion efficiency, and glycosylation modification of exogenous proteins. It is a commonly used eukaryotic expression system for achieving high-level secretory expression of recombinant enzymes. For example, Masuda et al. used Pichia pastoris cells to heterologously express egg lysozyme, with a yield of 400 μg / mL; Ercan et al. used yeast as a host strain to express human lysozyme, with a yield of 187 U / mL.

[0005] Currently, egg lysozyme and human lysozyme have been heterologously expressed in yeast cells, but there are still problems with low activity and low yield, which cannot meet the large demand for lysozyme in industry. Therefore, screening for highly active and thermally stable lysozyme target genes and achieving their efficient expression in Pichia pastoris is an urgent problem to be solved. Summary of the invention

[0006] The present invention provides an oyster-derived lysozyme having an amino acid sequence as shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the lysozyme.

[0008] In one embodiment, the gene has the nucleotide sequence shown in SEQ ID NO.2.

[0009] The present invention also provides recombinant Pichia pastoris expressing the lysozyme.

[0010] In one embodiment, the Pichia pastoris uses Pichia pastoris GS115 as a host.

[0011] In one embodiment, the recombinant Pichia pastoris uses pPIC9k as an expression vector.

[0012] In one embodiment, the N-terminus of the lysozyme (before the first amino acid) is fused with a short peptide H6, K6, R6, D6, E6, N6, Q6, or a protein tag AP2, Flag, HL 28, Sumo or Strep.

[0013] The present invention also provides a method for increasing the expression of lysozyme, wherein the method comprises fusing a short peptide H6, K6, R6, D6, E6, N6, Q6, or a protein tag AP2, Flag, HL 28 , Sumo or Strep.

[0014] In one embodiment, the amino acid sequences of the short peptides H6, K6, R6, D6, E6, N6, and Q6 are HHHHHH, KKKKKK, RRRRRR, DDDDDD, EEEEEE, NNNNNN, and QQQQQQ, respectively; the amino acid sequences of AP2, Flag, and HL 28 The amino acid sequences of Sumo or Strep are shown in SEQ ID NOs. 14 to 18, respectively.

[0015] The present invention also provides a method for producing oyster-derived lysozyme, which comprises culturing the recombinant Pichia pastoris in a culture medium for a period of time and collecting the lysozyme in the cell culture fluid.

[0016] In one embodiment, the culture medium is BMMY medium.

[0017] In one embodiment, the culturing is carried out at 28-30° C. and 150-200 rpm for at least 96 h.

[0018] The present invention also claims the use of the lysozyme, the Pichia pastoris or the method in producing a product containing lysozyme.

[0019] The present invention also claims to protect the use of the lysozyme in cell lysis.

[0020] Beneficial effects:

[0021] The present invention screened lysozyme gene sequences of 12 different sources of vertebrates, mollusks, poultry and insects to obtain European oyster-derived lysozyme (OElyz) with relatively high enzyme activity and good thermal stability, and achieved active expression of the enzyme in Pichia pastoris;

[0022] The present invention also fuses different amino acid short peptides and protein tags to the N-terminus of OElyz. When the Sumo tag is fused, the extracellular enzyme activity of the recombinant OElyz reaches 382.6 U / mL at the shake flask level. This production level can meet the application requirements of lysozyme in the fields of medicine, biotechnology, and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The expression and activity analysis of lysozymes from different sources in fermentation broth. (A) SDS-PAGE analysis of the expression of lysozymes from different sources. M: standard protein; 1: pPIC9k; 2: ADAlyz; 3: ADlyz; 4: BTlyz; 5: CGlyz; 6: DMlyz; 7: HS1lyz; 8: HS2lyz; 9: LLlyz; 10: MLlyz; 11: OElyz; 12: OMlyz; 13: SSlyz. (B) Activity analysis of lysozymes from different sources.

[0024] Figure 2 Expression and activity analysis of recombinant enzymes with different amino acid peptides and tags fused to the N-terminus of OElyz. (A) SDS-PAGE analysis of the expression of recombinant OElyz with different amino acid peptides and tags fused to the N-terminus in fermentation broth. M: standard protein; 1: OElyz; 2: pPIC9k; 3: D6-OElyz; 4: E6-OElyz; 5: H6-OElyz; 6: K6-OElyz; 7: N6-OElyz; 8: Q6-OElyz; 9: R6-OElyz; 10: AP2-OElyz; 11: Flag-OElyz; 12: HL 28 -OElyz; 13:Sumo-OElyz; 14:Strep-OElyz. (B) Activity analysis of recombinant OElyz with short peptides of different amino acids and tags fused to the N-terminus in fermentation broth. DETAILED DESCRIPTION

[0025] (I) Strains and vectors

[0026] The Pichia pastoris expression vector pPIC9K and strains P. pastoris GS115, E. coli JM109 and Micrococcus luteus were purchased from Novagen.

[0027] (II) Enzymes and other biochemical reagents

[0028] Restriction enzyme Sal I and DNA purification kit were purchased from Thermo Fisher Scientific (China) Co., Ltd. Plasmid extraction kit, G418 antibiotic, ampicillin, kanamycin, yeast nitrogen base (YNB) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; tryptone and yeast extract were purchased from OXOID, UK, and the remaining reagents were domestic analytical grade.

[0029] (III) Culture medium

[0030] LB medium (g / L): 5.0% yeast powder, 10.0% peptone, 10.0% NaCl, pH 7.0. Solid medium contains 20.0g / L agar. When screening E. coli clones or liquid culture, add ampicillin or kanamycin to the medium at a final concentration of 100μg / mL as needed.

[0031] YPD medium (g / L): 10.0% yeast powder, 20.0% peptone, 20.0% glucose. Solid medium contains 20.0g / L agar. G418 at a final concentration of 4mg / mL is added to the solid medium as needed to screen the number of copies of the target gene of the recombinant.

[0032] MD medium (g / L): glucose 20.0, yeast nitrogen base (YNB) 13.4, biotin 4×10 -4 , agar 20.0.

[0033] BMGY medium (g / L): yeast powder 10.0, peptone 20.0, yeast nitrogen base (YNB) 13.4, glycerol 10 mL, biotin 4×10 -4 , 100 mM potassium phosphate buffer, pH 6.0.

[0034] BMMY medium (g / L): yeast powder 10.0, peptone 20.0, yeast nitrogen base (YNB) 13.4, methanol 10mL, biotin 4×10 -4 , 100 mM potassium phosphate buffer, pH 6.0.

[0035] Escherichia coli and Micrococcus luteus were cultured in LB liquid or solid medium (with corresponding antibiotics added as needed) at 37°C for 12 h, with a liquid culture speed of 200 rpm. Pichia pastoris was cultured in YPD liquid or solid medium at 30°C for 24 h, with a liquid culture speed of 200 rpm.

[0036] The molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook, or according to the kits and product instructions.

[0037] Lysozyme activity detection method: Micrococcus luteus (M.luteus) was used as the standard substrate for detecting lysozyme activity. The specific method was as follows: 500 μL of lysozyme fermentation broth and 2.5 mL of substrate (suspended in 0.2 M pH = 6.2 phosphate buffer, OD 450 The OD was recorded every 10 seconds for 1 minute at 37°C. 450 Reading. One unit of enzyme activity (U) is defined as OD per minute under the assay conditions. 450The amount of enzyme required to decrease by 0.001.

[0038] Example 1 Construction of recombinant expression strains of lysozymes from different sources

[0039] The amino acid sequences of lysozymes from mosquito Anopheles dirus A, sturgeon Acipenser dabryanus, cattle Bostaurus, oyster Crassostrea gigas, fruit fly Drosophila melanogaster, human Homo sapiens-1, human Homo sapiens-2, black pheasant Lophura leucomelanos, clam Meretrix lusoria, European oyster Ostreaedulis, rainbow trout Oncorhynchus mykiss, and copper pheasant Syrmaticus soemmerringii reported by NCBI were used as the reference sequences. The 100 sequences of the 247 genes (SEQ ID NOs. ACC77967.1, AXH73012.1, AAC37312.1, Q6L6Q6.1, NP_476828.1, AAA59536.1, AAA36188.1, AAB20837.1, P86383.1, Q6L6Q5.1, CAA42084.1 and AAB31830.1) were submitted to an online tool (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) for signal peptide prediction, and the functional sequences that did not contain signal peptides were optimized according to the codon preference of Pichia pastoris. The optimized gene sequences (shown in SEQ ID NOs. 3 to 11, SEQ ID NO. 1, and SEQ ID NOs. 12 to 13, respectively) were synthesized by General Biosystems (Anhui) Co., Ltd. and ligated to the EcoRI and NotR of the expression vector pPIC9K. I restriction site, and obtained recombinant plasmids pPIC9K-adalyz, pPIC9K-adlyz, pPIC9K-btlyz, pPIC9K-cglyz, pPIC9K-dmlyz, pPIC9K-hs1lyz, pPIC9K-hs2lyz, pPIC9K-lllyz, pPIC9K-mllyz, pPIC9K-oelyz, pPIC9K-omlyz and pPIC9K-sslyz. The above recombinant plasmids were linearized at 37°C for 2h according to the following reaction system.

[0040] Linearized system:

[0041]

[0042] The linearized fragments were recovered and purified, and the purified linear fragments were transformed into P. pastoris GS115 by electroporation. The bacterial suspension was spread on MD medium and cultured at 30°C until single colonies appeared. The single colonies were picked to contain 4 mg / mL G418-resistant YPD plates were used to screen high-copy transformants of the target gene and named them P.pastoris-pPIC9K-adalyz, P.pastoris-pPIC9K-adlyz, P.pastoris-pPIC9K-btlyz, P.pastoris-pPIC9K-cglyz, P.pastoris-pPIC9K-dmlyz, P.pastoris-pPIC9K-hs1lyz, P.pastoris-pPIC9K-hs2lyz, P.pastoris-pPIC9K-lllyz, P.pastoris-pPIC9K-mllyz, P.pastoris-pPIC9K-oelyz, P.pastoris-pPIC9K-omlyz and P.pastoris-pPIC9K-sslyz.

[0043] Example 2 Shake flask fermentation of recombinant strains

[0044] The recombinants P.pastoris-pPIC9K-adalyz, P.pastoris-pPIC9K-adlyz, P.pastoris-pPIC9K-btlyz, P.pastoris-pPIC9K-cglyz, P.pastoris-pPIC9K-dmlyz, P.pastoris-pPIC9K-hs1lyz, P.pastoris-pPIC9K-hs2lyz, P.pastoris-pPIC9K-lllyz, P.pastoris-pPIC9K-mllyz, P.pastoris-pPIC9K-oelyz, P.pastoris-pPIC9K-omlyz and P.pastoris-pPIC9K-sslyz constructed in Example 1 were streaked on YPD solid plates and cultured in a constant temperature incubator at 30°C until single colonies grew. The single colonies were inoculated into 250 mL shake flasks containing 50 mL YPD liquid medium and cultured overnight at 30°C and 200 rpm to obtain seed cultures. The seed cultures were transferred to 250 mL shake flasks containing 50 mL BMGY medium at a 10% (v / v) inoculation rate and cultured at 30°C and 200 rpm until OD 600When the cell density reached 6, all the cells were collected and washed three times with 0.9% NaCl, then transferred to a 250 mL shake flask containing 50 mL BMMY medium and cultured at 30°C and 200 rpm for 96 h, supplemented with methanol at a final concentration of 1% (v / v) every 24 h.

[0045] The shake flask fermentation broth was collected for 96 hours, and the target protein of the fermentation broth was analyzed by SDS-PAGE, and the enzyme activity in the fermentation broth was detected. Figure 1 As shown in A, recombinant enzymes ADAlyz (14.4 kDa), ADlyz (15.9 kDa), BTlyz (15.4 kDa), CGlyz (14.0 kDa), DMlyz (14.6 kDa), HS1lyz (15.6 kDa), HS2lyz (15.7 kDa), LLlyz (15.2 kDa), MLlyz (14.3 kDa), OElyz (13.8 kDa), OMlyz (15.2 kDa) and SSlyz (15.2 kDa) were successfully expressed and secreted outside the cells. The target band size was consistent with the theoretical molecular weight of the protein, among which BTlyz had the highest expression level. The lysozyme activity in the fermentation broth was analyzed, and the results were as follows: Figure 1 As shown in B, OElyz had the highest activity, with the enzyme activity in the fermentation broth reaching 40U / mL.

[0046] Example 3 Construction of recombinant expression strains with N-terminal fusion of short peptides with different amino acids and tags

[0047] Amino acid short peptides with different charges and different protein tags can usually improve the yield and secretion efficiency of exogenous recombinant proteins. 28 The amino acid sequences of Sumo, Sumo and Strep were optimized according to the codon preference of Pichia pastoris (as shown in Table 1), and the ends of the optimized sequences were fused with the European oyster lysozyme gene sequence oelyz in Example 1, respectively, and handed over to General Biosystems (Anhui) Co., Ltd. for synthesis and ligated between the EcoRI and Not I restriction sites of the expression vector pPIC9K to obtain recombinant plasmids pPIC9K-h6-oelyz, pPIC9K-k6-oelyz, pPIC9K-r6-oelyz, pPIC9K-d6-oelyz, pPIC9K-e6-oelyz, pPIC9K-n6-oelyz, pPIC9K-q6-oelyz, pPIC9K-ap2-oelyz, pPIC9K-flag-oelyz, pPIC9K-hl 28The above recombinant plasmids were linearized by incubation at 37°C for 2 h according to the following reaction system.

[0048] Linearized system:

[0049]

[0050] Table 1 Short peptides and sequences

[0051]

[0052] The linearized fragments were recovered and purified, and the purified linear fragments were transformed into P. pastoris GS115 by electroporation. The bacterial suspension was spread on MD medium and cultured at 30°C until single colonies appeared. The single colonies were picked to contain 4 mg / mL G418-resistant YPD plates were used to screen high-copy transformants of the target gene and named P.pastoris-pPIC9K-h6-oelyz, P.pastoris-pPIC9K-k6-oelyz, P.pastoris-pPIC9K-r6-oelyz, P.pastoris-pPIC9K-d6-oelyz, P.pastoris-pPIC9K-e6-oelyz, P.pastoris-pPIC9K-n6-oelyz, P.pastoris-pPIC9K-q6-oelyz, P.pastoris-pPIC9K-ap2-oelyz, P.pastoris-pPIC9K-flag-oelyz, and P.pastoris-pPIC9K-hl 28 -oelyz, P.pastoris-pPIC9K-sumo-oelyz and P.pastoris-pPIC9K-strep-oelyz.

[0053] Example 4 Shake flask fermentation of recombinant strains

[0054] The recombinants P. pastoris-pPIC9K-h6-oelyz, P. pastoris-pPIC9K-k6-oelyz, P. pastoris-pPIC9K-r6-oelyz, P. pastoris-pPIC9K-d6-oelyz, P. pastoris-pPIC9K-e6-oelyz, P. pastoris-pPIC9K-n6-oelyz, P. pastoris-pPIC9K-q6-oelyz, P. pastoris-pPIC9K-ap2-oelyz, P. pastoris-pPIC9K-flag-oelyz, P. pastoris-pPIC9K-hl constructed in Example 3 were used. 28 -oelyz, P.pastoris-pPIC9K-sumo-oelyz and P.pastoris-pPIC9K-strep-oelyz were streaked on YPD solid plates and placed in a 30°C constant temperature incubator to grow single colonies. The single colonies were inoculated into 250 mL shake flasks containing 50 mL YPD liquid culture medium and cultured overnight at 30°C and 200 rpm to obtain seed cultures. The seed cultures were transferred to 250 mL shake flasks containing 50 mL BMGY culture medium at a 10% (v / v) inoculation amount and cultured at 30°C and 200 rpm until OD 600 When the cell density reached 6, all the cells were collected and washed three times with 0.9% NaCl, then transferred to a 250 mL shake flask containing 50 mL BMMY medium and cultured at 30°C and 200 rpm for 96 h, supplemented with methanol at a final concentration of 1% (v / v) every 24 h.

[0055] The shake flask fermentation broth was collected for 96 hours, and the target protein of the fermentation broth was analyzed by SDS-PAGE, and the enzyme activity in the fermentation broth was detected. Figure 2 As shown in A, the recombinant enzymes H6-OElyz (14.7 kDa), K6-OElyz (14.6 kDa), R6-OElyz (14.8 kDa), D6-OElyz (14.5 kDa), E6-OElyz (14.6 kDa), N6-OElyz (14.5 kDa), Q6-OElyz (14.6 kDa), AP2-OElyz (15.4 kDa), Flag-OElyz (14.8 kDa), HL 28-OElyz (17.1 kDa), Sumo-OElyz (25.1 kDa) and Strep-OElyz (15.2 kDa) were successfully expressed and secreted outside the cells. The target band size was consistent with the theoretical molecular weight of the protein. The short peptide E6 and protein tags AP2 and HL 28 Sumo can significantly increase the production and secretion of OElyz in Pichia pastoris. The production of recombinant OElyz in the fermentation broth was analyzed. Figure 2 As shown in B, E6-OElyz, AP2-OElyz, HL 28 The lysozyme activities of -OElyz and Sumo-OElyz were 302.7U / mL, 90.4U / mL, 317.3U / mL and 382.6U / mL, respectively, which were 7.6, 2.3, 7.9 and 9.6 times that of OElyz, among which Sumo-OElyz had the highest yield.

[0056] Example 5 Study on the properties of lysozyme

[0057] According to the method of Example 4, the recombinant strains P. pastoris-pPIC9K-oelyz, P. pastoris-pPIC9K-e6-oelyz, P. pastoris-pPIC9K-ap2-oelyz, P. pastoris-pPIC9K-hl 28 -oelyz and P. pastoris-pPIC9K-sumo-oelyz were fermented, and the shake flask fermentation broth was collected at 96h and kept at 70℃ for 15min to determine the residual activity of lysozyme. The results showed that OElyz, E6-OElyz, AP2-OElyz, HL 28 -OElyz and Sumo-OElyz still had 37.6U / mL, 298.4U / mL, 85.1U / mL, 313.7U / mL and 376.0U / mL enzyme activities, with no obvious loss of enzyme activity compared to before heat treatment, indicating that OElyz has good thermal stability.

[0058] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A lysozyme fusion protein, characterized in that A short peptide EEEEEE is fused to the N-terminus of lysozyme; the amino acid sequence of the lysozyme is shown in SEQ ID NO.

1.

2. A recombinant microorganism, characterized in that Express the lysozyme fusion protein according to claim 1.

3. The recombinant microorganism according to claim 2, characterized in that Pichia pastoris was used as the host.

4. The recombinant microorganism according to claim 3, characterized in that pPIC9k was used as the expression vector.

5. A method for increasing the expression level of lysozyme, characterized in that: A short peptide EEEEEE is fused to the N-terminus of the lysozyme amino acid sequence shown in SEQ ID NO.

1.

6. A method for producing oyster-derived lysozyme, characterized in that: The recombinant microorganism according to any one of claims 2 to 4 is cultured in a culture medium for a period of time, and lysozyme in the cell culture fluid is collected.

7. Use of the lysozyme fusion protein according to claim 1, the recombinant microorganism according to any one of claims 2 to 4, and the method according to any one of claims 5 to 6 in producing a product containing lysozyme.

8. Use of the lysozyme fusion protein according to claim 1 in bacterial cell lysis.

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