WO2022242033A1 - 一种生产猪肌红蛋白的基因工程菌及其发酵与纯化 - Google Patents

一种生产猪肌红蛋白的基因工程菌及其发酵与纯化 Download PDF

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WO2022242033A1
WO2022242033A1 PCT/CN2021/126637 CN2021126637W WO2022242033A1 WO 2022242033 A1 WO2022242033 A1 WO 2022242033A1 CN 2021126637 W CN2021126637 W CN 2021126637W WO 2022242033 A1 WO2022242033 A1 WO 2022242033A1
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myoglobin
fermentation
porcine
seq
porcine myoglobin
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French (fr)
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赵鑫锐
张博涵
周景文
堵国成
李江华
陈坚
余飞
鲁伟
钱源
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江苏东汇生物科技有限公司
江南大学
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Priority claimed from CN202110560189.XA external-priority patent/CN113265346B/zh
Priority claimed from CN202110558529.5A external-priority patent/CN113150120B/zh
Priority claimed from CN202110561197.6A external-priority patent/CN113186147B/zh
Application filed by 江苏东汇生物科技有限公司, 江南大学 filed Critical 江苏东汇生物科技有限公司
Priority to US17/743,716 priority Critical patent/US11639515B2/en
Publication of WO2022242033A1 publication Critical patent/WO2022242033A1/zh

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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention relates to a genetically engineered bacterium for producing porcine myoglobin and its fermentation and purification, belonging to the technical field of genetic engineering.
  • Myoglobin is a heme-containing globular protein that is structurally similar to the alpha subunit of hemoglobin and stores oxygen. It can also be used in iron supplements, disease diagnosis and other fields. Myoglobin is closely related to the color of meat. Different states of myoglobin cause different changes in the color of meat. There are generally three forms of myoglobin, oxy-Myoglobin (oxy-Mb), deoxygenated myoglobin The ratio between myoglobin (deoxy-Myoglobin, deoxy-Mb) and met-myoglobin (met-Mb, met-Mb) makes the meat appear in different colors. In recent years, with the rise of artificial meat products, in order to simulate the real meat color, myoglobin will be added to artificial meat products.
  • porcine myoglobin there are two main ways to produce porcine myoglobin.
  • porcine hemoglobin Compared with traditional chemical methods, microbial synthesis has many advantages, such as reduced environmental pollution, stable product quality, relatively simple downstream extraction, and greater possibility of low-cost production.
  • the synthesis of porcine hemoglobin by microorganisms needs to solve two problems: first, to determine the suitable medium for the fermentation of recombinant porcine myoglobin, which has never been expressed in genetically engineered strains before; second, to determine Fermentation strategies to increase porcine myoglobin production.
  • the purification of protein must first solve the problem of fermentation broth concentration.
  • the fermentation broth contains antifoaming agents, and antifoaming agents with high concentrations will cause irreversible dissolution reactions on most ultrafiltration membranes, resulting in the inability of the target protein To achieve the purpose of concentration.
  • the problem of protein degradation must be solved. Porcine myoglobin is easy to degrade, and the problem of industrialized large-scale purification and production.
  • the present invention successfully expresses porcine myoglobin in Pichia pastoris through the screening of promoter and signal peptide, and The yield can reach 46.15mg/L under shake flask conditions. And further through the optimization of fermentation conditions, the yield of porcine myoglobin was improved.
  • the fermented liquid involved in the present invention contains antifoaming agent, and antifoaming agent with higher concentration will cause irreversible dissolution reaction to most ultrafiltration membranes, resulting in the leakage of the target protein and making it impossible to concentrate. Therefore, the ultrafiltration cup is not suitable for the concentration of porcine myoglobin.
  • salting out can achieve the purpose of concentrating the fermented liquid, it is not suitable for processing a large amount of fermented liquid because it will cause certain losses during the salting out process.
  • this patent tries three food-grade purification methods without affinity tags. It can be mainly divided into the following three types: salting-out-desalting-gel filtration chromatography (AKTA), salting-out-desalting-DEAE anion exchange chromatography (AKTA), ultrafiltration concentration-Q anion exchange chromatography (gravity column).
  • AKTA salting-out-desalting-gel filtration chromatography
  • AKTA salting-out-desalting-DEAE anion exchange chromatography
  • ultrafiltration concentration-Q anion exchange chromatography gravity column
  • the AKTA protein purifier has low processing capacity and low purification efficiency, so it is not suitable for large-scale industrial production.
  • the use of gravity columns can increase the purification throughput, reduce the purification time, and avoid the degradation of porcine myoglobin.
  • Ultrafiltration concentration-Q anion exchange chromatography (gravity column) has the advantages of simple operation, low cost and high purification rate, and can be used in large-scale industrial production.
  • the first object of the present invention is to provide a genetically engineered bacterium, which uses Pichia pastoris as a host and expresses porcine myoglobin in Pichia pastoris, and the NCBI Reference Sequence of said porcine myoglobin is NP_999401 .1.
  • nucleotide sequence of the gene encoding porcine myoglobin is shown in SEQ ID NO:1.
  • the porcine myoglobin gene is expressed through an expression vector containing a GAP promoter or a G1 promoter.
  • nucleotide sequence encoding the G1 promoter is shown in SEQ ID NO:12.
  • the signal peptide of the expression vector is ⁇ -factor (nucleotide sequence as shown in SEQ ID NO: 1), ⁇ -amalyse (nucleotide sequence as shown in SEQ ID NO: 2) , Glucoamylase (nucleotide sequence as shown in SEQ ID NO:3), Inulinase (nucleotide sequence as shown in SEQ ID NO:4), Invertase (nucleotide sequence as shown in SEQ ID NO:5), Lysozyme (nucleotide sequence as shown in SEQ ID NO:6), Killer protein (nucleotide sequence as shown in SEQ ID NO:7), Serum albumin (nucleotide sequence as shown in SEQ ID NO:8), sp23 (The nucleotide sequence is shown in SEQ ID NO:9), nsB (The nucleotide sequence is shown in SEQ ID NO:10) or pre-Ost1- ⁇ factor (The
  • Pichia pastoris X33 or Pichia pastoris KM71 is used as the host.
  • the second object of the present invention is to provide a method for fermenting and producing porcine myoglobin, said method fermenting genetically engineered bacteria expressing porcine myoglobin in a system containing heme, said system using glycerol or glucose as carbon source.
  • the medium in the heme-containing system is YPD medium, YPG medium or BMGY medium.
  • the fermentation system contains 20-40 mg/L heme.
  • the carbon source in the fermentation system is 10-20g/L glycerol, 10-20g/L glucose or 10-20g/L sorbitol, and the nitrogen source is 15-25g/L peptone, 15-20g/L 25g/L corn steep liquor, 15-25g/L beef extract, 15-25g/L diammonium hydrogen phosphate or 15-25g/L ammonium sulfate.
  • glycerol is added to the fermentation system.
  • the fermenter contains 10-30 g of peptone, 5-15 g of glycerin, 5-15 g of yeast extract and 1 ⁇ 10 -4 to 5 ⁇ 10 -4 g of biotin.
  • the third object of the present invention is to provide a method for isolating and purifying porcine myoglobin from a microbial fermentation broth, said method is to use any method as described in (a) to (c) to separate and purify porcine myoglobin from a fermentation broth protein:
  • the fermented liquid is obtained by fermenting and producing the genetically engineered bacteria, or by using the method.
  • the porcine myoglobin is concentrated by adding ammonium sulfate.
  • ammonium sulfate powder is slowly added to the fermentation supernatant and stirred until the concentration of ammonium sulfate reaches 50-60% saturation, left standing at 1-4°C for 2h; centrifuged at 5000-10000g for 25-35min at 4°C Collect the supernatant; add ammonium sulfate powder to the supernatant until the concentration of ammonium sulfate reaches 60-70%, and let it stand overnight at 1-4°C; centrifuge at 5000-10000g for 25-35min to collect the precipitate; use 10mM, pH 9.20 Tris-HCl The buffer reconstitutes the pellet to obtain a concentrate.
  • the concentrated solution obtained is loaded into a desalting column, and the Tris-HCl buffer solution with a pH of 10-15 mM and pH 9.0-10.0 is used for equilibration and washing.
  • desalting and by detecting the conductivity, collecting the elution peak before the conductivity change, and collecting the desalted sample; loading the obtained desalted sample into an anion exchange column, buffering with 10-15mM, pH 9.0-10.0 Tris-HCl
  • the solution was equilibrated, and then 1-2M NaCl buffer solution was used for gradient elution, and the second elution peak was collected by detecting UV 280nm to obtain purified porcine myoglobin.
  • the anion exchange chromatography fillers are DEAE-Sepharose and Q Beads 6FF respectively.
  • the obtained concentrated solution is loaded into a desalting column, and the desalting column is carried out with 10-15 mM Tris-HCl buffer solution with a pH of 9.0-10.0. Equilibrium and elution, and by detecting the conductivity, collecting the elution peak before the conductivity change, and collecting the desalted sample; loading the obtained desalted sample into the gel filtration chromatography column, using 10-15mM, pH 9.0- Equilibrate with 10.0 Tris-HCl buffer, then elute with 10-15mM, pH 9.0-10.0 Tris-HCl buffer, and collect the elution peak by detecting UV280nm to obtain purified porcine myoglobin.
  • the gel filtration chromatography filler is Superdex.
  • the obtained concentrated solution is further concentrated using a membrane bag, and the obtained concentrated supernatant is loaded onto an anion exchange column, and the concentration is 10-15mM, pH 9.0-10.0 Equilibrate with Tris-HCl buffer solution, then use 1-2M NaCl buffer solution for gradient elution, and collect the eluate with an elution concentration of 20% NaCl, which is pure myoglobin.
  • the membrane bag is: Vivaflow 200 tangential flow filtration membrane bag.
  • buffer B solution needs to be used for elution, and the salinity of buffer B is 10%-90%.
  • a membrane packet is used to truncate myoglobin with a molecular weight cut off greater than 10 kDa.
  • the packing material of the desalting column is Sephadex G-25.
  • the membrane material of the concentrated membrane cassette is polypropylene.
  • the obtained pure porcine myoglobin can be subjected to heme extraction and optical detection.
  • the fourth object of the present invention is to provide the application of the genetically engineered bacteria in the preparation of products containing myoglobin or myoglobin derivatives.
  • the fifth object of the present invention is to provide the fermentation method for improving the production of porcine myoglobin by recombinant Pichia pastoris, or the application of the method for producing porcine myoglobin in the production of porcine myoglobin or its derivative products.
  • the present invention realizes the high-efficiency synthesis and production of porcine myoglobin in Pichia pastoris, and solves the previous problem that porcine myoglobin cannot be expressed by using microbial hosts.
  • the high-efficiency expression of porcine myoglobin in Pichia pastoris can be realized through the selection of an appropriate expression system.
  • the yield of porcine myoglobin can reach 46.15 mg/L, which lays the foundation for the application of porcine myoglobin in artificial meat and other food processing fields.
  • porcine myoglobin Further optimize the conditions of dissolved oxygen and hemoglobin concentration during the fermentation process, thereby enhancing the growth of the bacteria and the ability to secrete and synthesize porcine myoglobin, and realize the production of porcine myoglobin at the fermenter level in Pichia pastoris efficient expression in .
  • the yield of porcine myoglobin can reach 285.42mg/L, which lays the foundation for the application of porcine myoglobin in food processing fields such as artificial meat.
  • Figure 1 shows the fermentation of recombinant bacteria P.pastoris X33- ⁇ GAP-Mb, P.pastoris GS115- ⁇ GAP-Mb, P.pastoris KM71- ⁇ GAP-Mb, P.pastoris SMD1168- ⁇ GAP-Mb to produce porcine myoglobin Glue map and yield map.
  • Fig. 2 is the gel map and yield map of pig myoglobin produced by fermentation of recombinant bacteria with different signal peptides.
  • Fig. 3 is a comparison gel map and yield map of pig myoglobin produced by fermentation of recombinant bacteria P. pastoris X33-G1-Mb and P. pastoris X33- ⁇ GAP-Mb containing G1 promoter.
  • Fig. 4 is a graph showing the optimization results of shake flask horizontal fermentation medium, optimum carbon source and optimum nitrogen source.
  • Fig. 5 is a graph showing the optimization results of shake flask horizontal fermentation conditions (temperature, added concentration of heme).
  • Figure 6 is a graph showing the optimization results of dissolved oxygen at the level of the fermenter.
  • Fig. 7 is a graph showing the optimization results of hemoglobin feeding concentration at the level of the fermenter.
  • Fig. 8 is a comparison result of fermenter horizontal culture of recombinant Pichia pastoris respectively containing GAP and G1 promoters.
  • Fig. 9 is the electrophoresis diagram of different ammonium sulfate salting out concentrated proteins in Example 3.
  • Fig. 10 is the electrophoresis diagram of protein purified by desalting-gel filtration chromatography and desalting-DEAE anion exchange chromatography.
  • Fig. 11 is an electrophoresis diagram of protein purified by ultrafiltration concentration-Q anion exchange chromatography.
  • Figure 12 is a comparison chart of the purity and purification rate of pig myoglobin purified by different methods.
  • Figure 13 is the extraction and analysis diagram of porcine myoglobin tracking heme.
  • Determination of bacterial cell OD Take 1 mL of bacterial liquid in a centrifuge tube, absorb the corresponding bacterial liquid, dilute the corresponding multiple with sterile water, and measure it in an ultraviolet spectrophotometer. This step is repeated three times.
  • YPD medium Each liter of YPD medium contains 20g of peptone, 20g of glucose and 10g of yeast extract; 20g of agar powder is added per liter of solid medium.
  • YPG medium Each liter of YPD medium contains 20g of peptone, 20g of glucose and 10g of glycerol; 20g of agar powder is added per liter of solid medium.
  • BMGY medium Each liter of BMGY medium contains 20g of peptone, 10g of glycerol, 10g of yeast extract, and 4 ⁇ 10 -4 g of biotin.
  • composition of the fermentation medium in the fermenter BMGY medium plus a final concentration of 40mg/L heme.
  • Shake flask fermentation medium composition BMGY medium plus a final concentration of 40mg/L heme.
  • Example 1 Construction and protein expression of Pichia pastoris recombinant bacteria expressing porcine myoglobin
  • the porcine myoglobin gene (nucleotide sequence shown in SEQ ID NO: 1) was connected to the multiple cloning site of the integrated expression vector pGAPZ ⁇ A to construct the recombinant plasmid pGAPZ ⁇ A-Mb.
  • the constructed recombinant plasmid pGAPZ ⁇ A-Mb was transformed into Escherichia coli DH5 ⁇ , the transformation solution was spread on the LB plate containing 20 ⁇ g/mL Zeocin, and a single clone was grown at 37°C, and the single clone was subjected to colony PCR and After sequencing verification, the plasmids were extracted from the correct positive clones, and the extracted plasmids were transformed into Pichia pastoris X33, Pichia pastoris GS115, Pichia pastoris KM71, and Pichia pastoris SMD1168 by electroporation to construct recombinant bacteria P. pastoris X33- ⁇ GAP-Mb, P. pastoris GS115- ⁇ GAP-Mb, P. pastoris KM71- ⁇ GAP-Mb, P. pastoris SMD1168- ⁇ GAP-Mb.
  • the expression of porcine myoglobin in the fermentation supernatant was detected by SDS-PAGE and Bradford protein concentration assay kit.
  • Example 2 Transformation of Pichia pastoris recombinant bacteria expressing porcine myoglobin and protein expression
  • Synthetic signal peptide ⁇ -amalyse (nucleotide sequence shown in SEQ ID NO: 2), Glucoamylase (nucleotide sequence shown in SEQ ID NO: 3), Inulinase (nucleotide sequence shown in SEQ ID NO: 4 shown), Invertase (nucleotide sequence shown in SEQ ID NO: 5), Lysozyme (nucleotide sequence shown in SEQ ID NO: 6), Killer protein (nucleotide sequence shown in SEQ ID NO: 7 ), Serum albumin-HSA (nucleotide sequence as shown in SEQ ID NO: 8), sp23 (nucleotide sequence as shown in SEQ ID NO: 9), nsB (nucleotide sequence as shown in SEQ ID NO: 10 Shown) and pre-Ost1-alpha factor (nucleotide sequence shown in SEQ ID NO: 11), and they were respectively inserted into the plasmid
  • 1Construction of pGAPZ ⁇ A- ⁇ -amalyse GAP-Mb plasmid use pGAPZ ⁇ A-Mb plasmid as a template, and use ⁇ -amalyse-F1 and ⁇ -amalyse-R primers for the first round of amplification to obtain the first round of PCR products; then The first-round PCR product was used as a template, ⁇ -amalyse-F2 and ⁇ -amalyse-R primers were used for the second-round amplification, and the PCR product was recovered. The correct pGAPZ ⁇ A- ⁇ -amalyse-Mb plasmid was verified by DNA sequencing.
  • 8Construction of pGAPZ ⁇ A-sp23 GAP-Mb plasmid For specific construction steps, refer to the above plasmid construction method.
  • the primers for the first round of PCR are sp23-F1 and sp23-R, and the primers for the second round of PCR are sp23-F2 and sp23-R , construct the plasmid.
  • 10Construction of pGAPZ ⁇ A-pre-Ost1 GAP-Mb plasmid For specific construction steps, refer to the above plasmid construction method.
  • the primers for the first round of PCR are pre-Ost1-F1 and pre-Ost1-R, and the primers for the second round of PCR are pre-Ost1-F1 and pre-Ost1-R.
  • -Ost1-F2 and pre-Ost1-R the plasmids were constructed.
  • the plasmid is extracted from the correct positive clone, and the extracted plasmid is transformed into Pichia pastoris X33 strain by electroporation, and respectively constructed Obtain recombinant bacteria P.pastoris X33- ⁇ -amalyse GAP-Mb, P.pastoris X33-Glucoamylase GAP-Mb, P.pastoris X33-Inulinase GAP-Mb, P.pastoris X33-Invertase GAP-Mb, P.pastoris X33- Killer protein GAP-Mb, P. pastoris X33-HSA GAP-Mb, P. pastoris X33-sp23 GAP-Mb, P. pastoris X33-nsB GAP-Mb, P. pastoris X33-pre-Ost1 GAP-Mb.
  • the expression of porcine myoglobin in the fermentation supernatant was detected by SDS-PAGE and Bradford protein concentration assay kit.
  • lanes 1 to 9 represent P. pastoris X33-SP23 GAP-Mb, X33-HSA GAP-Mb, X33- ⁇ -amylase GAP-Mb, X33-nsB GAP-Mb, X33 -Inulinase GAP-Mb, X33-Invertase GAP-Mb, X33-Glucoamylase GAP-Mb, X33-Killer protein GAP-Mb, X33-Lysozyme GAP-Mb; the lanes in the right gel are P.pastoris X33-pre- Ost1-Mb, X33-HSA GAP-Mb.
  • P. pastoris X33- ⁇ GAP-Mb, X33-SP23 GAP-Mb, X33-HSA GAP-Mb, X33-pre-Ost1-Mb can secrete porcine myoglobin.
  • the yields were 21.90, 12.38, 8.84, and 19.01 mg/L, respectively.
  • G1 promoter sequence (the nucleotide sequence is shown in SEQ ID NO: 12), use the plasmid pGAPZ ⁇ -A-Mb as a template, and replace the GAP promoter with the G1 promoter to obtain a recombinant plasmid by enzyme digestion and enzyme ligation pG1-Mb.
  • the expression of porcine myoglobin in the fermentation supernatant was detected by SDS-PAGE and Bradford protein concentration assay kit.
  • the recombinant bacteria constructed using the G1 promoter can express porcine myoglobin, and the protein production is increased to 46.15 mg/L compared with 21.90 mg/mL of the recombinant bacteria containing the GAP promoter.
  • Embodiment 3 Shake flask horizontal fermentation medium and optimization of fermentation conditions
  • Preparation of primary seed liquid Streak the strain Pichia pastoris X33- ⁇ GAP-Mb obtained in Example 1 stored at -80°C on a plate, pick a single colony and inoculate it in 50 mL of YPD medium containing 5 mL of In the bacterial shaker tube, 30°C, 220rpm, shaker culture for 16-18h is the first-class seed solution.
  • the secondary seed solution was inoculated into a 250mL shake flask containing 49mL of fermentation medium (respectively YPG, BMGY, YPD medium) with an inoculum size of 2% (2mL/100mL), and the fermentation medium contained 10g/L of glycerol and heme with a final concentration of 20mg/L, and fermented at 30°C and 220rpm for at least 60h.
  • fermentation medium respectively YPG, BMGY, YPD medium
  • the fermentation medium contained 10g/L of glycerol and heme with a final concentration of 20mg/L, and fermented at 30°C and 220rpm for at least 60h.
  • the secondary seed liquid was inoculated with 2% (2mL/100mL) inoculation amount into 49mL fermentation medium (each liter of medium contained 20g peptone, 10g yeast extract, 4 ⁇ 10 -4 g biotin, the final concentration was 20mg/L heme), 10g/L glycerol, 10g/L glucose, and 10g/L sorbitol were added to the fermentation medium in addition, and the fermentation was at least 60h.
  • the secondary seed solution was inoculated with 2% (2mL/100mL) inoculation amount into 49mL fermentation medium (10g glycerol, 10g yeast extract, 4 ⁇ 10 -4 g biotin per liter of medium, the final concentration was 20mg/L heme) in a 250mL shake flask, add 20g/L peptone, 20g/L corn steep liquor, 20g/L beef extract, 20g/L diammonium hydrogen phosphate, 20g/L sulfuric acid in the fermentation medium Ammonium, fermented for at least 60h.
  • 49mL fermentation medium (10g glycerol, 10g yeast extract, 4 ⁇ 10 -4 g biotin per liter of medium, the final concentration was 20mg/L heme) in a 250mL shake flask, add 20g/L peptone, 20g/L corn steep liquor, 20g/L beef extract, 20g/L diammonium hydrogen phosphate, 20g/L sulfuric acid in the fermentation medium Am
  • Secondary seed liquid is inoculated in the 250mL shaking flask that contains 49mL fermentation medium (fermentation medium is BMGY) with 2% inoculum size, and the glycerol that contains 10g/L and the blood red that final concentration is 20mg/L in the fermentation medium Vegetarian, ferment for at least 60 hours.
  • fermentation medium is BMGY
  • BMGY fermentation medium
  • glycerol that contains 10g/L and the blood red that final concentration is 20mg/L in the fermentation medium Vegetarian, ferment for at least 60 hours.
  • the secondary seed solution was inoculated into a 250mL shake flask containing 49mL fermentation medium (the fermentation medium was BMGY) with an inoculum size of 2% (2mL/100mL).
  • the fermentation medium contained a final concentration of 5, 10, 20 , 40mg/L heme, fermented for at least 60h. Results As shown in Figure 5B, when the final concentration of heme in the culture medium was 40 mg/L, the yield of porcine myoglobin was higher than that under other conditions.
  • Embodiment 4 produce porcine myoglobin (fermenter level) under different dissolved oxygen conditions
  • Preparation of primary seed liquid Streak the strain Pichia pastoris X33- ⁇ GAP-Mb obtained in Example 1 stored at -80°C on a plate, pick a single colony and inoculate it in 50 mL of YPD medium containing 5 mL of In the bacterial shaker tube, 30°C, 220rpm, shaker culture for 16-18h is the first-class seed solution.
  • Fermentation conditions Inoculate the secondary seed liquid into a 5L fermenter containing 1.8L fermentation medium (the fermentation medium is BMGY) with an inoculum size of 10% (10mL/100mL).
  • the fermentation medium contains 10g/L glycerol and hemoglobin at a final concentration of 20 mg/L.
  • the pH is controlled around 5.50.
  • Example 5 Add different concentrations of heme to produce porcine myoglobin
  • Preparation of primary seed liquid Streak the strains Pichia pastoris X33- ⁇ GAP-Mb and Pichia pastoris X33- ⁇ G1-Mb stored in Example 1 stored at -80°C on the plate, and pick a single colony for inoculation Cultivate in a 50mL sterile shaking tube containing 5mL YPD medium at 30°C and 220rpm on a shaking table for 16-18h to obtain the primary seed solution.
  • Fermentation conditions Inoculate the seed liquid with 10% inoculum size into a 5L fermenter containing 1.8L fermentation medium.
  • the fermentation medium contains 10g/L glycerol and heme with a final concentration of 20mg/L. Fermentation was carried out according to the 30% DO-Stat fermentation strategy, the ventilation volume was 1.5VVM, and the stirring speed was 200-800rpm. After about 12 hours of fermentation, the glycerin was exhausted, and at this time, feeding was started.
  • the feeding speed is mainly controlled automatically by DO and stirring. When DO>30%, start to add 50% (W/V) glycerin. , the stirring speed was increased (initial rotation speed was 200rpm, maximum rotation speed was 800rpm).
  • heme with final concentrations of 50mg/L, 100mg/L, 150mg/L and 200mg/L was added at the same time as glycerin, and the fermentation was carried out for 84 hours.
  • the seed solution of Pichia pastoris X33- ⁇ G1-Mb was prepared according to the above steps and fermented. The results are shown in Figure 8.
  • the detection results of porcine myoglobin production corresponding to the two strains of P.pastoris X33-G1-Mb and P.pastoris X33-GAP-Mb are shown in the figure, and the production of porcine myoglobin corresponding to the two strains of P.pastoris X33-GAP-Mb
  • the yield of pig myoglobin corresponding to P.pastoris X33-G1-Mb strain was 285.42mg/L.
  • Example 6 Separation and Purification of Porcine Myoglobin in Fermentation Broth by Salting Out-Desalting-Anion Exchange Chromatography
  • Salting out of fermentation broth Precipitate in one step, slowly add ammonium sulfate powder to the fermentation supernatant and stir until the concentration of ammonium sulfate reaches 30%, 40%, 50%, 60%, 70%, 80% ammonium sulfate saturation Temperature, let stand at 4°C for 2h; 4°C, centrifuge at 10,000g for 30min to collect the precipitate, redissolve the precipitate with 10mM Tris-HCl buffer, pH 9.20, and verify the analysis by SDS-PAGE.
  • Figure 9 shows the results of salting out, wherein lanes 1-6 correspond to 30% ammonium sulfate, 40% ammonium sulfate, 50% ammonium sulfate, 60% ammonium sulfate, 70% ammonium sulfate, and 80% ammonium sulfate.
  • the ammonium sulfate concentration was 60%, a large amount of pig myoglobin was salted out, and when the ammonium sulfate concentration was 80%, the pig myoglobin was no longer concentrated. Therefore, two concentrations of ammonium sulfate, 50% and 70%, were selected for two-stage salting-out.
  • Two-stage ammonium sulfate precipitation (salting out): Slowly add ammonium sulfate powder to the fermentation supernatant and stir until the concentration of ammonium sulfate reaches 50% saturation, let stand at 4°C for 2h; centrifuge at 10,000g for 30min at 4°C to collect the supernatant ; Add ammonium sulfate powder to the supernatant until the concentration of ammonium sulfate reaches 70%, and let it stand at 4°C overnight; centrifuge at 10000g for 30min to collect the precipitate; redissolve the precipitate with 10mM, pH 9.20 Tris-HCl buffer;
  • Anion exchange chromatography load the desalted sample obtained in step 2 onto an anion exchange column DEAE-Sepharose, equilibrate with 10mM Tris-HCl buffer solution with pH 9.20, and then perform gradient elution with 1M NaCl buffer solution, And collect the second elution peak by detecting UV 280nm.
  • step 2 and step 3 were verified and analyzed by SDS-PAGE. The results are shown in Figure 2. Lane 1 is the result of desalting, and lane 3 is the result of purification. The purity is 81.60%, and the recovery rate of purification is 48.25%.
  • Example 7 Separation and purification of porcine myoglobin in fermentation broth by salting-out-desalting-gel filtration chromatography
  • step 3 The samples obtained in step 3 were respectively verified and analyzed by SDS-PAGE, and the results are shown in the No. 2 swimming lane of FIG. 10 , the purity was 100%, and the purification recovery rate was 14.84%.
  • Example 8 Separation and purification of porcine myoglobin in fermentation broth by concentration-anion exchange chromatography
  • Sample preparation adjust the pH of the fermented broth after concentration (according to the two-stage ammonium sulfate precipitation step described in Example 6) to 9.20 with Tris base, and filter the fermented broth with a 0.45 ⁇ m filter membrane. Afterwards, the fermentation broth was connected to the Vivaflow 200 membrane bag reflux system, and the myoglobin with a molecular weight greater than 10kDa was intercepted and concentrated.
  • the specific operation steps of membrane bag backflow are as follows: Connect the Vivaflow 200 membrane bag according to the instructions to form a backflow system. Then carry out ultrafiltration and concentration under the action of a peristaltic pump to obtain an ultrafiltration concentrate.
  • Q anion exchange column packing balance Use 5 times the column volume of buffer A solution to balance the Q Beads 6FF anion packing.
  • Sample loading add a sample of 1 times the column volume, and the sample will flow down spontaneously due to gravity. After the liquid was exhausted, add buffer A solution to balance the sample.
  • lane 1 in Figure 12A corresponds to the purification result, with a purity of 88.04%, and the highest purification rate can reach 66.05%
  • Figure 12B corresponds to the gradient elution
  • Lane 2 corresponds to 20% buffer B eluate
  • lanes 1 and 3-9 correspond to 10% buffer B (10% salt concentration) and 30%-90% buffer B, respectively.
  • Embodiment 9 Heme extraction and optical detection

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Abstract

本发明提供了一种生产猪肌红蛋白的基因工程菌及其发酵与纯化方法。本发明通过在毕赤酵母中整合了猪肌红蛋白的基因，实现了猪肌红蛋白的高效分泌与表达。在此基础上，对重组毕赤酵母进行培养基和培养条件的优化，可显著提升猪肌红蛋白的产量，使得发酵罐条件下产量可达到285.42mg/L。并通过在发酵液中创造性地分步添加不同浓度的硫酸铵，最终浓缩得到的肌红蛋白纯度达88.04％，纯化率可达66.05％。本发明从猪肌红蛋白的合成、发酵到纯化等各个步骤实现了猪肌红蛋白的高效表达及高度纯化，为工业化生产猪肌红蛋白提供了广阔的前景。

Description

一种生产猪肌红蛋白的基因工程菌及其发酵与纯化 技术领域
本发明涉及一种生产猪肌红蛋白的基因工程菌及其发酵与纯化，属于基因工程技术领域。
背景技术
肌红蛋白是一种含血红素的球状蛋白，它在结构上与血红蛋白的α亚基相似，可储存氧气。也可用于铁补充剂、疾病诊断等领域。肌红蛋白与肉类的颜色息息相关，不同状态的肌红蛋白使得肉类的颜色发生不同的变化，肌红蛋白一般有三种存在形式，氧化肌红蛋白(oxy-Myoglobin，oxy-Mb)，脱氧肌红蛋白(deoxy-Myoglobin，deoxy-Mb)和高铁肌红蛋白(met-Mb，met-Mb)，这三者之间的比例使得肉以不同的颜色进行呈现。近年来，随着人造肉制品的兴起，为了模拟出真实的肉色，会在人造肉的产品中添加肌红蛋白。
目前，猪肌红蛋白的生产主要有两种途径。第一，传统化学提取法。从心脏组织中提取猪肌红蛋白，这种方法存在高成本、长周期、得率低、工艺复杂以及副产物多不利于分离等问题，难以应用于大规模工业化生产；第二，利用微生物细胞工厂异源合成猪肌红蛋白。该方法目前还未有报道，因此很有必要开展关于猪肌红蛋白食品级宿主表达系统的研究。
微生物合成法和传统化学法相比，有着很多的优势，比如减少环境污染、产品质量稳定、下游提取相对简单，且实现低成本成产的可能性更大。利用微生物法合成猪血红蛋白需要解决两个方面的难题：第一，确定重组猪肌红蛋白发酵合适的培养基，在此之前猪肌红蛋白从未在基因工程菌株中进行表达；第二，确定发酵策略，从而提高猪肌红蛋白的产量。
此外，目前从发酵液中分离纯化蛋白通常有亲和层析、离子交换层析以及凝胶过滤层析，且大多依托于AKTA蛋白纯化仪。在大多报道中，最常用的是亲和层析中的His标签纯化，使目的蛋白与镍离子结合，后用咪唑洗脱，从而进行纯化。然而，用于酶制剂的纯化标签不推荐用于食品加工，因为组氨酸有转化成组胺的风险，从而引发人体的过敏反应，因此不能采用His标签对食品级的猪肌红蛋白进行纯化。
蛋白的纯化，首先得解决发酵液浓缩的问题。在利用毕赤酵母重组菌发酵生产猪肌红蛋白的时候，发酵液中含有消泡剂，而浓度较高的消泡剂会对大多数的超滤膜造成不可逆的溶解反应，导致目的蛋白无法达到浓缩的目的。其次得解决蛋白降解的问题，猪肌红蛋白容易降解，以及工业化大规模纯化生产的问题。
发明内容
为了解决目前猪肌红蛋白生产困难、还没有合适有效、简便的方法制备猪肌红蛋白的问题，本发明通过启动子、信号肽的筛选在毕赤酵母中成功表达了猪肌红蛋白，并使得在摇瓶条件下产量可达到46.15mg/L。并进一步通过发酵条件的优化，提升了猪肌红蛋白的产量。
针对发酵液浓缩的问题，本发明涉及的发酵液含有消泡剂，浓度较高的消泡剂会对大多数的超滤膜造成不可逆的溶解反应，导致目的蛋白渗漏从而无法进行浓缩。因此超滤杯不适合用于猪肌红蛋白的浓缩。盐析虽然能够达到浓缩发酵液的目的，但是由于盐析的过程中会造成一定的损失，因此不适合处理大量的发酵液。此外，尝试了使用Vivaflow 200超滤膜包对猪肌红蛋白进行浓缩，该方法能够快速浓缩发酵液，并且有效避免蛋白的损失。为了对猪肌红蛋白进行高效纯化，本专利尝试了三种没有亲和标签的食品级纯化方法。主要可以分为以下三种：盐析-脱盐-凝胶过滤层析(AKTA)，盐析-脱盐-DEAE阴离子交换层析(AKTA)，超滤浓缩-Q阴离子交换层析(重力柱)。但是AKTA蛋白纯化仪的处理量低、纯化效率低，因此不适合用于大规模的工业生产。重力柱的使用，可以提高纯化处理量，减少纯化时间，避免猪肌红蛋白的降解。超滤浓缩-Q阴离子交换层析(重力柱)具有操作简便，成本低，纯化率高等优点，可用于大规模工业生产。
本发明的第一个目的是提供一种基因工程菌，所述基因工程菌以毕赤酵母为宿主，在毕赤酵母中表达 猪肌红蛋白，所述猪肌红蛋白的NCBI Reference Sequence为NP_999401.1。
在一种实施方式中，编码猪肌红蛋白的基因的核苷酸序列如SEQ ID NO:1所示。
在一种实施方式中，所述猪肌红蛋白基因通过含有GAP启动子或G1启动子的表达载体表达。
在一种实施方式中，编码所述G1启动子的核苷酸序列如SEQ ID NO:12所示。
在一种实施方式中，所述表达载体的信号肽为α-factor(核苷酸序列如SEQ ID NO:1所示)、α-amalyse(核苷酸序列如SEQ ID NO:2所示)、Glucoamylase(核苷酸序列如SEQ ID NO:3所示)、Inulinase(核苷酸序列如SEQ ID NO:4所示)、Invertase(核苷酸序列如SEQ ID NO:5所示)、Lysozyme(核苷酸序列如SEQ ID NO:6所示)、Killer protein(核苷酸序列如SEQ ID NO:7所示)、Serum albumin(核苷酸序列如SEQ ID NO:8所示)、sp23(核苷酸序列如SEQ ID NO:9所示)、nsB(核苷酸序列如SEQ ID NO:10所示)或pre-Ost1-αfactor(核苷酸序列如SEQ ID NO:11所示)。
在一种实施方式中，以毕赤酵母X33或毕赤酵母KM71为宿主。
本发明的第二个目的是提供一种发酵生产猪肌红蛋白的方法，所述方法将表达猪肌红蛋白的基因工程菌在含有血红素的体系中发酵，所述体系以甘油或葡萄糖为碳源。
在一种实施方式中，所述含有血红素的体系中的培养基为YPD培养基、YPG培养基或BMGY培养基。
在一种实施方式中，在摇瓶发酵条件下，将所述基因工程菌种子液培养至OD 600＝6～10，以1～5mL/100mL的接种量接种至发酵体系中，在25～35℃，pH＝4.0～7.0，150～300rpm下发酵至少60h。
在一种实施方式中，发酵体系中含有20～40mg/L的血红素。
在一种实施方式中，发酵体系中的碳源为10～20g/L的甘油、10～20g/L葡萄糖或10～20g/L山梨醇，氮源为15～25g/L的蛋白胨、15～25g/L玉米浆、15～25g/L牛肉膏、15～25g/L磷酸氢二铵或15～25g/L硫酸铵。
在一种实施方式中，在发酵罐发酵条件下，将所述基因工程菌种子液培养至OD 600＝8～10，按反应体系体积的5％～10％的量接种至发酵体系中，在25～35℃，pH＝4.0～7.0，通气量为1.0～2.0VVM，DO控制在20％～30％发酵至少70h。
在一种实施方式中，当DO不低于30％，向发酵体系中添加50～150mg/L的血红素。
在一种实施方式中，当DO不低于30％，向发酵体系中添加甘油。
在一种实施方式中，发酵罐中含有10～30g蛋白胨、5～15g甘油、5～15g酵母提取物和1×10 -4～5×10 -4g生物素。
本发明的第三个目的是提供从微生物发酵液中分离纯化猪肌红蛋白的方法，所述方法是利用如(a)～(c)任一所述方法从发酵液中分离纯化猪肌红蛋白：
(a)盐析-脱盐-阴离子交换法；
(b)盐析-脱盐-凝胶过滤层析法；
(c)浓缩-阴离子交换法；
所述发酵液为利用所述基因工程菌发酵生产得到，或是利用所述方法得到的。
在一种实施方式中，所述猪肌红蛋白通过添加硫酸铵进行浓缩。
在一种实施方式中，向发酵上清中缓慢加入硫酸铵粉末并搅拌至硫酸铵浓度达到50～60％饱和度，于1～4℃静置2h；4℃，5000～10000g离心25～35min收集上清；向上清中加入硫酸铵粉末，至硫酸铵浓度达到60～70％，于1～4℃静置过夜；5000～10000g离心25～35min收集沉淀；用10mM、pH 9.20的Tris-HCl缓冲液将沉淀复溶得到浓缩液。
在一种实施方式中，当采用盐析-脱盐-阴离子交换法时，将得到的浓缩液上样到脱盐柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡以及洗脱，并通过检测电导率，收集电导率变化前的洗脱峰，收集得到脱盐样品；将得到的脱盐样品上样到阴离子交换柱中，用10～15mM，pH 9.0～10.0的Tris- HCl缓冲液进行平衡，再用1～2M NaCl缓冲溶液进行梯度洗脱，并通过检测UV 280nm收集第二个洗脱峰，得到纯化后的猪肌红蛋白。
在一种实施方式中，阴离子交换层析填料分别为DEAE-Sepharose、Q Beads 6FF。
在一种实施方式中，当采用盐析-脱盐-凝胶过滤层析法时，将得到的浓缩液上样到脱盐柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡以及洗脱，并通过检测电导率，收集电导率变化前的洗脱峰，收集得到脱盐样品；将得到的脱盐样品上样到凝胶过滤层析柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡，再用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行洗脱，并通过检测UV280nm收集洗脱峰，得到纯化后的猪肌红蛋白。
在一种实施方式中，凝胶过滤层析的填料为Superdex。
在一种实施方式中，当采用浓缩-阴离子交换法时，将得到的浓缩液利用膜包进一步浓缩，将得到的浓缩上清上样到阴离子交换柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡，再用1～2M NaCl缓冲溶液进行梯度洗脱，收集洗脱浓度为20％NaCl的洗脱液，即为肌红蛋白纯品。
在一种实施方式中，所述膜包为：Vivaflow 200切向流过滤膜包。
在一种实施方式中，需用buffer B溶液进行洗脱，buffer B的盐度为10％～90％。
在一种实施方式中，利用膜包截取分子量大于10kDa的肌红蛋白。
在一种实施方式中，脱盐柱的填料为Sephadex G-25。
在一种实施方式中，所述的浓缩膜包的膜材料为聚丙烯。
在一种实施方式中，所得猪肌红蛋白纯品可进行血红素的提取，以及光学检测。
本发明的第四个目的是提供所述的基因工程菌在制备含肌红蛋白或肌红蛋白衍生物的产品中的应用。
本发明的第五个目的是提供所述提高重组毕赤酵母产猪肌红蛋白的发酵方法，或生产猪肌红蛋白的方法在生产猪肌红蛋白或其衍生产品方面的应用。
有益效果：
(1)本发明实现了猪肌红蛋白在毕赤酵母中的高效合成与生产，解决了之前猪肌红蛋白无法应用微生物宿主进行表达的问题。通过合适的表达系统的选择，可实现猪肌红蛋白在毕赤酵母中的高效表达。在摇瓶水平，所得猪肌红蛋白产量可达46.15mg/L，该结果为猪肌红蛋白在人造肉等食品加工领域的应用奠定了基础。
(2)进一步通过对发酵过程中溶氧条件及血红素流加浓度进行优化，从而增强菌体的生长和分泌合成猪肌红蛋白的能力，实现了发酵罐水平猪肌红蛋白在毕赤酵母中的高效表达。在发酵罐水平，所得猪肌红蛋白产量可达285.42mg/L，该结果为猪肌红蛋白在人造肉等食品加工领域的应用奠定了基础。
(3)通过在发酵液中创造性地分步添加不同浓度的硫酸铵，对表达猪肌红蛋白的重组毕赤酵母的发酵液进行浓缩，并对其进一步超滤浓缩，并对浓缩液进行纯化得到的肌红蛋白纯度达88.04％，纯化率可达66.05％，实现了从发酵液中纯化猪肌红蛋白，解决了之前猪肌红蛋白发酵液难以纯化的问题，并实现了猪肌红蛋白发酵液的高效纯化。
附图说明
图1为重组菌P.pastoris X33-α GAP-Mb、P.pastoris GS115-α GAP-Mb、P.pastoris KM71-α GAP-Mb、P.pastoris SMD1168-α GAP-Mb发酵生产猪肌红蛋白的胶图及产量图。
图2为不同信号肽的重组菌发酵生产猪肌红蛋白的胶图及产量图。
图3为含有G1启动子重组菌P.pastoris X33-G1-Mb、P.pastoris X33-α GAP-Mb发酵生产猪肌红蛋白的对比胶图及产量图。
图4为摇瓶水平发酵培养基、最适碳源、最适氮源优化结果图。
图5为摇瓶水平发酵条件(温度、血红素添加浓度)优化结果图。
图6为发酵罐水平溶氧优化结果图。
图7为发酵罐水平血红素流加浓度优化结果图。
图8为分别含有GAP和G1启动子的重组毕赤酵母的发酵罐水平培养对比结果图。
图9为实施例3中不同硫酸铵盐析浓缩蛋白电泳图。
图10为脱盐-凝胶过滤层析、脱盐-DEAE阴离子交换层析法纯化蛋白电泳图。
图11为超滤浓缩-Q阴离子交换层析法纯化蛋白电泳图。
图12为不同方法纯化猪肌红蛋白的纯度与纯化率对比图。
图13为猪肌红蛋白追踪血红素的提取与分析图。
具体实施方式
菌体OD的测定：取1mL菌液于离心管中，吸取相应的菌液，用无菌水稀释相应的倍数，于紫外分光光度计中进行测量，此步骤重复三次。
蛋白含量的测定：使用碧云天生物技术研究所研发的Bradford蛋白浓度测定试剂盒进行检测，具体操作步骤参参见试剂盒使用说明。
YPD培养基：每升YPD培养基中含有20g蛋白胨，20g葡萄糖和10g酵母提取物；固体培养基每升加入20g琼脂粉。
YPG培养基：每升YPD培养基中含有20g蛋白胨，20g葡萄糖和10g甘油；固体培养基每升加入20g琼脂粉。
BMGY培养基：每升BMGY培养基中含有20g蛋白胨，10g甘油，10g酵母提取物，4×10 -4g生物素。
发酵罐发酵培养基组成：BMGY培养基外加终浓度为40mg/L血红素。
摇瓶发酵培养基组成：BMGY培养基外加终浓度为40mg/L血红素。
蛋白含量的测定：使用碧云天生物技术研究所研发的Bradford蛋白浓度测定试剂盒进行检测，具体操作步骤参看试剂盒使用说明。
表1 实施例中所使用的引物
Figure PCTCN2021126637-appb-000001
Figure PCTCN2021126637-appb-000002
实施例1：表达猪肌红蛋白毕赤酵母重组菌的构建及蛋白表达
将猪肌红蛋白基因(核苷酸序列如SEQ ID NO:1所示)，连接至整合型表达载体pGAPZα A的多克隆位点，构建得到重组质粒pGAPZα A-Mb。
将构建得到的重组质粒pGAPZα A-Mb，转化至大肠杆菌DH5α中，将转化液涂布于含有20μg/mL Zeocin的LB平板上，在37℃至长出单克隆，将单克隆经菌落PCR和测序验证后，从正确的阳性克隆中提取质粒，将提取的质粒通过电转的方法分别转化至毕赤酵母X33、毕赤酵母GS115、毕赤酵母KM71、毕赤酵母SMD1168中，分别构建得到重组菌P.pastoris X33-α GAP-Mb、P.pastoris GS115-α GAP-Mb、P.pastoris KM71-α GAP-Mb、P.pastoris SMD1168-α GAP-Mb。
将构建得到的毕赤酵母重组菌株分别发酵生产蛋白：将OD 600＝6～8的毕赤酵母种子液接种至含有终浓度为20mg/L血红素的48mL YPD培养基中，接种量为2％(2mL/100mL)，在30℃，220rpm下发酵至少60h。应用SDS-PAGE与Bradford蛋白浓度测定试剂盒检测发酵上清中猪肌红蛋白的表达情况。
如图1所示，利用重组菌P.pastoris X33-α GAP-Mb(产量为24.25mg/L)、P.pastoris KM71-α GAP-Mb(产量为20.01mg/L)能够实现猪血红蛋白的表达，而P.pastoris GS115-α GAP-Mb、P.pastoris SMD1168-α GAP-Mb无法实现猪肌红蛋白的表达。其中，泳道1～4分别对应了P.pastoris X33-α GAP-Mb、P.pastoris GS115-α GAP-Mb、P.pastoris SMD1168-α GAP-Mb、P.pastoris KM71-α GAP-Mb。
实施例2：表达猪肌红蛋白毕赤酵母重组菌的改造及蛋白的表达
(1)信号肽的改造
合成信号肽α-amalyse(核苷酸序列如SEQ ID NO:2所示)、Glucoamylase(核苷酸序列如SEQ ID NO:3所示)、Inulinase(核苷酸序列如SEQ ID NO:4所示)、Invertase(核苷酸序列如SEQ ID NO:5所示)、Lysozyme(核苷酸序列如SEQ ID NO:6所示)、Killer protein(核苷酸序列如SEQ ID NO:7所示)、Serum albumin-HSA(核苷酸序列如SEQ ID NO:8所示)、sp23(核苷酸序列如SEQ ID NO:9所示)、nsB(核苷酸序列如SEQ ID NO:10所示)和pre-Ost1-alpha factor(核苷酸序列如SEQ ID NO:11所示)，并将其分别插入质粒pGAPZα A-Mb中，以替代原始的α-factor信号序列。
①pGAPZα A-α-amalyse GAP-Mb质粒的构建：以pGAPZα A-Mb质粒为模板，以α-amalyse-F1和α-amalyse-R引物进行第一轮扩增，得到第一轮PCR产物；然后将第一轮PCR产物用作模板，将α-amalyse-F2和α-amalyse-R引物用于第二轮扩增，回收PCR产物。通过DNA测序验证得到正确的pGAPZα A-α-amalyse-Mb质粒。
②pGAPZα A-Glucoamalyse GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为Glu-F1和Glu-R，第二轮PCR的引物为Glu-F2和Glu-R，构建得到质粒。
③pGAPZα A-Inu GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为Inu-F1和Inu-R，第二轮PCR的引物为Inu-F2和Inu-R，构建得到质粒。
④pGAPZα A-Invertase GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为Invert-F1和Invert-R，第二轮PCR的引物为Invert-F2和Invert-R，构建得到质粒。
⑤pGAPZα A-Lysoenzyme GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为Lyso-F1和Lyso-R，第二轮PCR的引物为Lyso-F2和Lys-R，构建得到质粒。
⑥pGAPZα A-Killer protein GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为Killer-F1和Killer-R，第二轮PCR的引物为Killer-F2和Killer protein-R，构建得到质粒。
⑦pGAPZα A-HSA GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为HSA-F1和HSA-R，第二轮PCR的引物为HSA-F2和HSA-R，构建得到质粒。
⑧pGAPZα A-sp23 GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为sp23-F1和sp23-R，第二轮PCR的引物为sp23-F2和sp23-R，构建得到质粒。
⑨pGAPZα A-nsB GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为nsB-F1和nsB-R，第二轮PCR的引物为nsB-F2和nsB-R，构建得到质粒。
⑩pGAPZα A-pre-Ost1 GAP-Mb质粒的构建：具体构建步骤参见上述质粒的构建方法，第一轮PCR的引物为pre-Ost1-F1和pre-Ost1-R，第二轮PCR的引物为pre-Ost1-F2和pre-Ost1-R，构建得到质粒。
将构建得到的重组质粒pGAPZα A-α-amalyse GAP-Mb、pGAPZαA-Glucoamylase GAP-Mb、pGAPZαA-Inulinase GAP-Mb、pGAPZαA-Invertase GAP-Mb、pGAPZαA-Lysozyme GAP-Mb、pGAPZαA-Killer protein GAP-Mb、pGAPZαA-HSA GAP-Mb、pGAPZαA-sp23 GAP-Mb、pGAPZαA-nsB GAP-Mb、pGAPZαA-pre-Ost1 GAP-Mb，分别转化至大肠杆菌DH5α中，将转化液涂布于含有Zeocin的LB平板上，在37℃至长出单克隆，将单克隆经菌落PCR和测序验证后，从正确的阳性克隆中提取质粒，将提取的质粒通过电转的方法转化至毕赤酵母X33菌株，分别构建得到重组菌P.pastoris X33-α-amalyse GAP-Mb、P.pastoris X33-Glucoamylase GAP-Mb、P.pastoris X33-Inulinase GAP-Mb、P.pastoris X33-Invertase GAP-Mb、P.pastoris X33-Killer protein GAP-Mb、P.pastoris X33-HSA GAP-Mb、P.pastoris X33-sp23 GAP-Mb、P.pastoris X33-nsB GAP-Mb、P.pastoris X33-pre-Ost1 GAP-Mb。
将构建得到的毕赤酵母重组菌株分别发酵生产蛋白：将OD 600＝6～8的毕赤酵母种子液接种至含20mg/L血红素的48mL YPD培养基中，接种量2％(2mL/100mL)，在30℃，220rpm下发酵至少60h。应用SDS-PAGE与Bradford蛋白浓度测定试剂盒检测发酵上清中猪肌红蛋白的表达情况。
结果如图2所示，其中，1～9泳道分别代表了P.pastoris X33-SP23 GAP-Mb，X33-HSA GAP-Mb，X33-α-amylase GAP-Mb，X33-nsB GAP-Mb，X33-Inulinase GAP-Mb，X33-Invertase GAP-Mb，X33-Glucoamylase GAP-Mb，X33-Killer protein GAP-Mb，X33-Lysozyme GAP-Mb；右边胶图中的泳道分别为P.pastoris X33-pre-Ost1-Mb，X33-HSA GAP-Mb。
从图2中看出，P.pastoris X33-α GAP-Mb，X33-SP23 GAP-Mb，X33-HSA GAP-Mb，X33-pre-Ost1-Mb可分泌猪肌红蛋白。产量分别为21.90、12.38、8.84、19.01mg/L。
(2)启动子的改造
合成G1启动子序列(核苷酸序列如SEQ ID NO:12所示)，以质粒pGAPZα-A-Mb作为模板，通过酶切酶连的方式，以G1启动子替换GAP启动子，得到重组质粒pG1-Mb。
将重组质粒pG1-Mb，转化至大肠杆菌DH5α中，将转化液涂布于含有Zeocin的LB平板上，在37℃至长出单克隆，将单克隆经菌落PCR和测序验证后，从正确的阳性克隆中提取质粒，将提取的质粒通过电转的方法转化至毕赤酵母X-33菌株，分别构建得到重组菌P.pastoris X33-G1-Mb。
将构建得到的毕赤酵母重组菌株P.pastoris X33-G1-Mb发酵生产蛋白：将OD 600＝6～8的毕赤酵母种子液接种至含有终浓度为20mg/L血红素的48mL YPD培养基中，接种量为2％(2mL/100mL)，在30℃，220rpm下发酵至少60h。应用SDS-PAGE与Bradford蛋白浓度测定试剂盒检测发酵上清中猪肌红蛋白的表达情况。结果如图3所示，利用G1启动子构建得到的重组菌能表达猪肌红蛋白，并且，蛋白产量较含GAP启动子的重组菌的21.90mg/mL提升至46.15mg/L。
实施例3：摇瓶水平发酵培养基及发酵条件优化
1、一级种子液制备：将-80℃保藏的实施例1构建得到的菌种Pichia pastoris X33-α GAP-Mb于平板上划线，挑取单菌落接种于含5mL YPD培养基的50mL无菌摇菌管中，30℃，220rpm，摇床培养16～18h即为一级种子液。
2、二级种子液制备：将一级种子液以1％(1mL/100mL)的接种量接种至含50mL YPD培养基的250mL摇瓶中，30℃，220rpm，摇床培养22h，培养至OD 600＝8～10。
3、发酵条件：
(1)基因工程菌在不同培养基中发酵生产猪肌红蛋白
将二级种子液以2％(2mL/100mL)的接种量接种到含49mL发酵培养基(分别为YPG、BMGY、YPD培养基)的250mL摇瓶中，发酵培养基中含有10g/L的甘油和终浓度为20mg/L的血红素，在30℃、220rpm下发酵至少60h。
结果如图4A所示，在培养基BMGY中培养重组菌株，猪肌红蛋白的产量最高。
(2)基因工程菌在不同碳源下发酵生产猪肌红蛋白
将二级种子液以2％(2mL/100mL)的接种量接种到含49mL发酵培养基(每升培养基中含有20g蛋白胨，10g酵母提取物，4×10 -4g生物素，终浓度为20mg/L的血红素)的250mL摇瓶中，发酵培养基中另外分别添加10g/L的甘油、10g/L葡萄糖、10g/L山梨醇，发酵至少60h。
结果图4B所示，当碳源为甘油时，猪肌红蛋白的产量最高。
(3)基因工程菌在不同氮源下发酵生产猪肌红蛋白
将二级种子液以2％(2mL/100mL)的接种量接种到含49mL发酵培养基(每升培养基中含有10g甘油，10g酵母提取物，4×10 -4g生物素，终浓度为20mg/L的血红素)的250mL摇瓶中，发酵培养基中另外分别添加20g/L的蛋白胨、20g/L玉米浆、20g/L牛肉膏、20g/L磷酸氢二铵、20g/L硫酸铵，发酵至少60h。
结果图4C所示，当氮源为蛋白胨时，猪肌红蛋白的产量最高。
(4)基因工程菌在不同温度下发酵生产猪肌红蛋白
将二级种子液以2％的接种量接种到含49mL发酵培养基(发酵培养基为BMGY)的250mL摇瓶中，发酵培养基中含有10g/L的甘油和终浓度为20mg/L的血红素，发酵至少60h。
结果图5A所示，菌体在30℃的条件下进行发酵时，蛋白表达能力最强。
(5)基因工程菌在不同血红素水平下发酵生产猪肌红蛋白
将二级种子液以2％(2mL/100mL)的接种量接种到含49mL发酵培养基(发酵培养基为BMGY)的250mL摇瓶中，发酵培养基中含有终浓度分别为5，10，20，40mg/L的血红素，发酵至少60h。结果图5B所示，培养基中的血红素终浓度为40mg/L时，猪肌红蛋白产量高于其它条件。
实施例4：在不同溶氧条件下生产猪肌红蛋白(发酵罐水平)
1、一级种子液制备：将-80℃保藏的实施例1构建得到的菌种Pichia pastoris X33-α GAP-Mb于平板上划线，挑取单菌落接种于含5mL YPD培养基的50mL无菌摇菌管中，30℃，220rpm，摇床培养16～18h即为一级种子液。
2、二级种子液制备：将一级种子液以1％(1mL/100mL)的接种量接种至含50mL YPD培养基的250mL摇瓶中，30℃，220rpm，摇床培养22h，培养至OD 600＝8～10。
3、发酵条件：将二级种子液以10％(10mL/100mL)的接种量接种到含1.8L发酵培养基(发酵培养基为BMGY)的5L发酵罐中，发酵培养基中含有10g/L的甘油和终浓度为20mg/L的血红素。pH控制在5.50左右。分别采用DO 20％-Stat、30％-Stat、40％-Stat对猪肌红蛋白的发酵进行控制，在30℃、200-800rpm，1.5VVM的条件下发酵84h。
猪肌红蛋白产量检测结果如图6，DO控制在20％、30％、40％时产量分别为219.13、235.70、103.94mg/L。
实施例5：流加不同浓度血红素生产猪肌红蛋白
(一)Pichia pastoris X33-α GAP-Mb发酵生产猪肌红蛋白
1、一级种子液制备：将-80℃保藏的实施例1构建得到的菌种Pichia pastoris X33-α GAP-Mb、Pichia pastoris X33-α G1-Mb于平板上划线，挑取单菌落接种于含5mL YPD培养基的50mL无菌摇菌管中，30℃，220rpm，摇床培养16～18h即为一级种子液。
2、二级种子液制备：将一级种子液以1％的接种量接种至含50mL YPD培养基的250mL摇瓶中，30℃，220rpm，摇床培养22h，培养至OD 600＝8～10。
3、发酵条件：将种子液以10％的接种量接种到含1.8L发酵培养基的5L发酵罐中，发酵培养基中含有10g/L的甘油和终浓度为20mg/L的血红素。按照30％DO-Stat发酵策略进行发酵，通气量为1.5VVM，搅拌转速为200-800rpm。发酵12h左右，甘油耗尽，此时开始补料。补料的速度主要依靠DO、搅拌进行自动控制，当DO>30％时，开始补加50％(W/V)甘油，甘油流加量刚好满足菌体生长所需，当DO<30％时，搅拌速度提高(初始转速为200rpm，最高转速为800rpm)。流加补料的过程中，加入甘油的同时分别加入了终浓度为50mg/L、100mg/L、150mg/L、200mg/L的血红素，发酵84h。
猪肌红蛋白产量检测结果如图7，血红素终浓度为150mg/L时产量最高，为243.43mg/L，血红素终浓度为50mg/L、100mg/L、200mg/L时，猪肌红蛋白产量分别为218.96、242.92、204.45mg/L此时对应发酵菌株为P.pastoris X33-GAP-Mb。
(二)Pichia pastoris X33-α G1-Mb发酵生产猪肌红蛋白
将Pichia pastoris X33-α G1-Mb按照上述步骤进行种子液的制备、并进行发酵，结果如图8所示。P.pastoris X33-G1-Mb和P.pastoris X33-GAP-Mb两个菌株对应的猪肌红蛋白产量检测结果如图，P.pastoris X33-GAP-Mb两个菌株对应的猪肌红蛋白产量为243.43mg/L，P.pastoris X33-G1-Mb菌株对应猪肌红蛋白的产量为285.42mg/L。
实施例6：盐析-脱盐-阴离子交换层析法分离纯化发酵液中的猪肌红蛋白
发酵液的盐析：用一步法进行沉淀，向发酵上清中缓慢加入硫酸铵粉末并搅拌至硫酸铵浓度达到30％、40％、50％、60％、70％、80％的硫酸铵饱和度，于4℃静置2h；4℃，10000g离心30min收集沉淀，用10mM，pH 9.20的Tris-HCl缓冲液将沉淀复溶，通过SDS-PAGE验证分析。图9为盐析结果，其中，1-6泳道分别对应了30％硫酸铵，40％硫酸铵，50％硫酸铵，60％硫酸铵，70％硫酸铵，80％硫酸铵。从图中可以看出，当硫酸铵浓度为60％时，猪肌红蛋白大量盐析，硫酸铵浓度为80％时，猪肌红蛋白已经不再浓缩。因此，选用了50％，70％两个浓度的硫酸铵进行两阶段盐析。
1、两阶段硫酸铵沉淀(盐析)：向发酵上清中缓慢加入硫酸铵粉末并搅拌至硫酸铵浓度达到50％饱和度，于4℃静置2h；4℃，10000g离心30min收集上清；向上清中加入硫酸铵粉末，至硫酸铵浓度达到70％，于4℃静置过夜；10000g离心30min收集沉淀；用10mM，pH 9.20的Tris-HCl缓冲液将沉淀复溶；
2、脱盐：将步骤1所得的溶液上样到脱盐柱Sephadex G-25中，用10mM，pH 9.20的Tris-HCl缓冲液进行平衡以及洗脱，并通过检测电导率，收集电导率变化前的洗脱峰；
3、阴离子交换层析：将步骤2得到的脱盐样品上样到阴离子交换柱DEAE-Sepharose中，用10mM，pH 9.20的Tris-HCl缓冲液进行平衡，再用1M NaCl缓冲溶液进行梯度洗脱，并通过检测UV 280nm收集 第二个洗脱峰。
将步骤2和步骤3得到的样品分别通过SDS-PAGE验证分析，结果如图2所示，1号泳道为脱盐结果，3号泳道为纯化结果，纯度为81.60％，纯化回收率为48.25％。
实施例7：盐析-脱盐-凝胶过滤层析法分离纯化发酵液中的猪肌红蛋白
1、两阶段硫酸铵沉淀：同实施例3中硫酸铵沉淀步骤；
2、脱盐：将步骤1所得的溶液上样到脱盐柱中，用10mM，pH 9.20的Tris-HCl缓冲液进行平衡以及洗脱，并通过检测电导率，收集电导率变化前的洗脱峰；
3、凝胶过滤层析：将步骤2得到的脱盐样品上样到Superdex凝胶过滤层析柱中，用10mM，pH 9.20的Tris-HCl缓冲液进行平衡，再用10mM，pH 9.20的Tris-HCl缓冲液进行洗脱，并通过检测UV 280nm收集洗脱峰。
将步骤3得到的样品分别通过SDS-PAGE验证分析，结果如图10的2号泳道所示，纯度为100％，纯化回收率为14.84％。
实施例8：浓缩-阴离子交换层析法分离纯化发酵液中的猪肌红蛋白
1、样品制备：用Tris碱调节浓缩后(按照实施例6所述的两阶段硫酸铵沉淀步骤)发酵液的pH至9.20，用0.45μm的滤膜过滤发酵液。之后将发酵液接入到Vivaflow 200膜包回流系统中，截取分子量大于10kDa的肌红蛋白，进行浓缩。膜包回流具体操作步骤如下：将Vivaflow 200膜包按照说明书进行连接，形成一个回流系统。之后在蠕动泵的作用下进行超滤浓缩，得到超滤浓缩液。
2、Q阴离子交换柱填料平衡：用5倍柱体积的buffer A溶液对Q Beads 6FF阴离子填料进行柱平衡。
3、上样：加入1倍柱体积的样品，由于重力作用，样品会自发往下流动。待到液体流尽后，加入buffer A溶液对样品进行平衡。
4、洗脱：用不同浓度的buffer B溶液(分别为10％buffer B、30％-90％buffer B)对样品进行梯度洗脱，收集洗脱液。
将洗脱液通过SDS-PAGE验证分析，结果如图12所示，其中图12A的1泳道对应了纯化结果，纯度为88.04％，最高纯化率可以达到66.05％；图12B对应了梯度洗脱，2泳道对应了20％buffer B洗脱液，1号、3-9号泳道分别对应了10％buffer B(10％盐浓度)、30％-90％buffer B。
实施例9：血红素提取及光学检测
1、酸性丙酮法。将纯化的猪肌红蛋白用酸性丙酮溶液(V 丙酮：V 盐酸＝3：100)萃取40分钟，然后使用1M NaOH将溶液的pH调节至中性。3000g，离心5分钟，得上清液，使用旋转蒸发仪除去丙酮。用1M HCl将所得溶液的pH值调节至5-7，出现血红素沉淀。3000g，离心15分钟，并用蒸馏水洗涤两次。
2、利用光学检测法检测上述方法提取的血红素。全波长扫描(分别加入200μL的纯化液于96孔板中，之后在200～800nm下进行波长扫描)，后确定出特征峰。之后在特征峰下测定相应的响应值，检测结果如图13所示。
结果显示当外源底物血红素的浓度为150mg/L时，肌红蛋白结合上的血红素最多，为0.22mol血红素/mol猪肌红蛋白。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (20)

  1. 一种基因工程菌，其特征在于，以毕赤酵母为宿主，在毕赤酵母中表达猪肌红蛋白，所述猪肌红蛋白的NCBI Reference Sequence为NP_999401.1；编码猪肌红蛋白的基因的核苷酸序列如SEQ ID NO:1所示；所述猪肌红蛋白基因通过含有GAP启动子或G1启动子的表达载体表达；编码所述G1启动子的核苷酸序列如SEQ ID NO:12所示；根据权利要求3所述的基因工程菌，其特征在于，所述表达载体的信号肽为α-factor(核苷酸序列如SEQ ID NO:1所示)、α-amalyse(核苷酸序列如SEQ ID NO:2所示)、Glucoamylase(核苷酸序列如SEQ ID NO:3所示)、Inulinase(核苷酸序列如SEQ ID NO:4所示)、Invertase(核苷酸序列如SEQ ID NO:5所示)、Lysozyme(核苷酸序列如SEQ ID NO:6所示)、Killer protein(核苷酸序列如SEQ ID NO:7所示)、Serum albumin(核苷酸序列如SEQ ID NO:8所示)、sp23(核苷酸序列如SEQ ID NO:9所示)、nsB(核苷酸序列如SEQ ID NO:10所示)或pre-Ost1-alpha factor(核苷酸序列如SEQ ID NO:11所示)。
  2. 根据权利要求1所述的基因工程菌，其特征在于，以毕赤酵母X33或毕赤酵母KM71为宿主。
  3. 一种发酵生产猪肌红蛋白的方法，其特征在于，利用权利要求1～2任一项所述的基因工程菌发酵生产猪肌红蛋白。
  4. 根据权利要求3所述的方法，其特征在于，将所述基因工程菌接种至摇瓶或发酵罐中发酵生产猪肌红蛋白，发酵培养基包括但不限于YPD培养基、YPG培养基或BMGY培养基。
  5. 根据权利要求4所述的方法，其特征在于，在摇瓶发酵条件下，将所述基因工程菌种子液培养至OD 600＝6～10，按反应体系体积的1％～5％添加至发酵体系中，在25～35℃，pH＝4.0～7.0，150～300rpm下发酵至少60h。
  6. 根据权利要求5所述的方法，其特征在于，发酵体系中含有20～40mg/L的血红素。
  7. 根据权利要求5所述的方法，其特征在于，发酵体系中的碳源为10～20g/L的甘油、10～20g/L葡萄糖或10～20g/L山梨醇，氮源为15～25g/L的蛋白胨、15～25g/L玉米浆、15～25g/L牛肉膏、15～25g/L磷酸氢二铵或15～25g/L硫酸铵。
  8. 根据权利要求4所述的方法，其特征在于，在发酵罐发酵条件下，将所述基因工程菌种子液培养至OD 600＝8～10，按反应体系体积的5％～10％的量接种至发酵体系中，在25～35℃，通气量为1.0～2.0VVM，pH＝4.0～7.0，DO控制在20％～30％发酵至少70h。
  9. 根据权利要求8所述的方法，其特征在于，当DO不低于30％，向发酵体系中添加50～150mg/L的血红素。
  10. 根据权利要求8或9所述方法，其特征在于，发酵体系中含有10～30g蛋白胨、5～15 g甘油、5～15g酵母提取物和1×10 -4～5×10 -4g生物素。
  11. 从微生物发酵液中分离纯化猪肌红蛋白的方法，其特征在于，利用如(a)～(c)任一所述方法从发酵液中分离纯化猪肌红蛋白：
    (a)盐析-脱盐-阴离子交换法；
    (b)盐析-脱盐-凝胶过滤层析法；
    (c)浓缩-阴离子交换法；
    所述发酵液为利用权利要求1或2所述基因工程菌发酵生产得到，或是利用权利要求3～10任一所述方法得到的；所述猪肌红蛋白通过添加硫酸铵进行浓缩。
  12. 根据权利要求11所述的方法，其特征在于，向发酵上清中缓慢加入硫酸铵粉末并搅拌至硫酸铵浓度达到50～60％饱和度，于1～4℃静置2h；4℃，5000～10000g离心25～35min收集上清；向上清中加入硫酸铵粉末，至硫酸铵浓度达到60～70％，于1～4℃静置过夜；5000～10000g离心25～35min收集沉淀；用10mM、pH 9.20的Tris-HCl缓冲液将沉淀复溶得到浓缩液。
  13. 根据权利要求12所述的方法，其特征在于，当采用盐析-脱盐-阴离子交换法时，将得到的浓缩液上样到脱盐柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡以及洗脱，检测电导率，收集电导率变化前的洗脱峰，收集得到脱盐样品；将得到的脱盐样品上样到阴离子交换柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡，再用1～2M NaCl缓冲溶液进行梯度洗脱，并通过检测UV 280nm收集第二个洗脱峰，得到纯化后的猪肌红蛋白。
  14. 根据权利要求12所述的方法，其特征在于，
    当采用盐析-脱盐-凝胶过滤层析法时，将得到的浓缩液上样到脱盐柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡以及洗脱，检测电导率，收集电导率变化前的洗脱峰，收集得到脱盐样品；将得到的脱盐样品上样到凝胶过滤层析柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡，再用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行洗脱，并通过检测UV280 nm收集洗脱峰，得到纯化后的猪肌红蛋白。
  15. 根据权利要求12所述的方法，其特征在于，当采用浓缩-阴离子交换法时，利用膜包对发酵液进行浓缩，将得到的浓缩发酵液上样到阴离子交换柱中，用10～15mM，pH 9.0～10.0的Tris-HCl缓冲液进行平衡，再用1～2M NaCl缓冲溶液进行梯度洗脱，收集洗脱浓度为20％NaCl的洗脱液，即为肌红蛋白纯品。
  16. 根据权利要求15所述的方法，其特征在于，所述膜包为：Vivaflow 200切向流过滤膜包。
  17. 根据权利要求15或16所述的方法，其特征在于，需用buffer B溶液进行洗脱，buffer B的氯化钠浓度为10％～90％。
  18. 根据权利要求15或16所述的方法，其特征在于，利用膜包截取分子量大于10kDa的肌红蛋白。
  19. 权利要求1～2任一项所述的基因工程菌在制备含肌红蛋白或肌红蛋白衍生物的产品中的应用。
  20. 权利要求3～18任一项所述方法在生产猪肌红蛋白或其衍生产品方面的应用。
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