CN114107264A - Method for producing aminopeptidase by fermentation - Google Patents
Method for producing aminopeptidase by fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 100
- 230000004151 fermentation Effects 0.000 title claims abstract description 100
- 102000004400 Aminopeptidases Human genes 0.000 title claims abstract description 50
- 108090000915 Aminopeptidases Proteins 0.000 title claims abstract description 50
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 27
- 230000000694 effects Effects 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 17
- 239000000463 material Substances 0.000 claims abstract description 16
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 15
- 239000000600 sorbitol Substances 0.000 claims abstract description 15
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 15
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 15
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 51
- 230000004913 activation Effects 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 21
- 229910052760 oxygen Inorganic materials 0.000 claims description 21
- 239000001301 oxygen Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 101150051118 PTM1 gene Proteins 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 12
- 238000009630 liquid culture Methods 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 238000012262 fermentative production Methods 0.000 claims description 10
- 238000009423 ventilation Methods 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 6
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 6
- 235000011151 potassium sulphates Nutrition 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000006052 feed supplement Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000013589 supplement Substances 0.000 abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 3
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- 238000005457 optimization Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 9
- 239000000203 mixture Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- PKZFFNJDFBBUIS-ZSCHJXSPSA-N (2s)-2-amino-4-methylpentanoic acid;4-nitroaniline Chemical compound CC(C)C[C@H](N)C(O)=O.NC1=CC=C([N+]([O-])=O)C=C1 PKZFFNJDFBBUIS-ZSCHJXSPSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
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Abstract
The invention belongs to the technical field of bioengineering, and particularly provides a method for producing aminopeptidase by fermentation, which takes pichia pastoris as an expression system, optimizes the components of a culture medium of a fermentation tank, adjusts the formula of a supplement, utilizes sorbitol as a carbon source, greatly reduces the production cost, adds zinc sulfate in a bottom material and the supplement, remarkably improves the enzyme activity of aminopeptidase in fermentation liquor, and can reach 2200U/mL after 178h of fermentation and improve the enzyme activity by 37.5 percent compared with that before optimization; the method has low cost and high safety, solves the problem of low yield of aminopeptidase produced by fermentation at present, and realizes high-quality industrial production conversion.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for producing aminopeptidase by fermentation.
Background
Aminopeptidases (APs) are a class of exoproteases that specifically hydrolyze specific amino acid residues from the N-terminus of proteins or polypeptides. Aminopeptidase has important application in improving food flavor, and is often compounded with other proteases to remove bitterness of protein hydrolysate and increase the protein hydrolysis depth; aminopeptidases have also been used to prepare biologically active peptides, to develop functional foods, and the like.
At present, the domestic research on aminopeptidase mainly focuses on the aspects of screening of enzyme-producing strains, characterization of enzymological characteristics, heterologous expression, fermentation optimization and the like. However, the contradiction between the increasing demand for aminopeptidase in the fields of food, medicine and the like and the low yield and high price of aminopeptidase is a problem to be solved. The method can improve the yield of the aminopeptidase and solve the stability problem of each link by an effective method, and is an important subject on the industrial road of the aminopeptidase in China.
Disclosure of Invention
The invention aims to overcome the problems of low aminopeptidase yield and high cost in the prior art.
To this end, the invention provides a process for the fermentative production of an aminopeptidase comprising the steps of:
(1) activating strains: inoculating pichia pastoris into an activation culture medium for activation to obtain a purified strain;
(2) preparing a seed solution: inoculating the activated pichia pastoris strain into a shake flask seed liquid culture medium for shake flask fermentation culture to obtain shake flask seed liquid;
(3) liquid submerged fermentation: inoculating the seed liquid in the shake flask into a fermentation tank for fermentation culture, starting feeding the supplementary material A when the dissolved oxygen does not fall back, and starting feeding the supplementary material B after the supplementary material A is added for 1h until the fermentation is terminated;
wherein, the feed supplement A comprises 20 to 50 percent of sorbitol, 0.5 to 5 per mill of zinc sulfate, 0.2 to 0.8 percent of PTM1 and the balance of water;
the supplementary material B comprises 80-99% of methanol, 0.5-5% of zinc sulfate, 0.2-0.8% of PTM1 and the balance of water;
(4) filtering, concentrating the fermentation liquid, and extracting aminopeptidase.
Specifically, the activation temperature in the step (1) is 25-32 ℃, and the activation time is 60-84 h.
Specifically, in the step (2), the shake flask fermentation culture temperature is 25-32 ℃, the shaking table rotation speed is 150-.
Specifically, the activation medium in the step (1) comprises 1.0-2.5% of glycerol, 0.8-2.0% of peptone, 0.15-2.0% of yeast powder and 0.5-2.0% of agar powder; the seed liquid culture medium in the shake flask in the step (2) comprises 1.0-2.5% of glycerol, 0.8-2.0% of peptone and 0.15-2.0% of yeast powder.
Specifically, in the step (3), the shake flask seed solution is inoculated into a fermentation tank for fermentation culture in an inoculation amount of 5-10%.
Specifically, the culture medium of the fermentation tank in the step (3) comprises 0.51-2.975% of phosphoric acid, 0.8-4.5% of ammonium sulfate, 0.02-0.12% of calcium sulfate, 0.8-2.7% of potassium sulfate, 0.3-2.3% of magnesium sulfate, 0.3-1.2% of potassium hydroxide, 0.5-5% of zinc sulfate, 2-5% of sorbitol and 0.2-0.8% of PTM 1.
Specifically, in the step (3), the fermentation temperature of the fermentation tank is 25-32 ℃, the rotation speed is 200-800rpm/min, the ventilation amount is 0.5-2.5m3/h, and the pH is preferably controlled to be 4.5-5.5 by using ammonia water with the concentration of 20-25% in the whole process.
Specifically, the ratio of the total volume of the fed-batch feed A fed in the step (3) to the total volume of the fermentation tank culture medium is 1: 3.
Specifically, when the supplementing material A flows in the step (3), the supplementing speed is adjusted, and the dissolved oxygen is controlled to be 10-25%; and when the material B is fed in a flowing manner, adjusting the feeding speed and controlling the dissolved oxygen to be 10-30%.
Specifically, the fermentation termination conditions in the step (3) are as follows: and (3) measuring the enzyme activity of the fermentation supernatant every 24 hours after two days of fed-batch feeding B, and stopping fermentation when the increase amplitude between the enzyme activities measured in two consecutive times is less than 200U/ml.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the method for producing the aminopeptidase by fermentation provided by the invention takes pichia pastoris as an expression system, optimizes the culture medium components of a fermentation tank, adjusts the formula of a supplement, utilizes sorbitol as a carbon source, greatly reduces the production cost, and further adds zinc sulfate in a base material and the supplement, so that the enzyme activity of the aminopeptidase in the fermentation liquid is obviously improved, after 178h of fermentation, the enzyme activity of the aminopeptidase can reach 2200U/mL, and is improved by 37.5% compared with that before optimization; the method has low cost and high safety, solves the problem of low yield of aminopeptidase produced by fermentation at present, and realizes high-quality industrial production conversion.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The invention provides a method for producing aminopeptidase by fermentation, which comprises the following steps:
(1) activating strains: inoculating pichia pastoris into an activation culture medium for activation, wherein the activation temperature is 25-32 ℃, and the activation time is 60-84h, so as to obtain a purified strain;
wherein the activation culture medium comprises 1.0-2.5% of glycerol, 0.8-2.0% of peptone, 0.15-2.0% of yeast powder and 0.5-2.0% of agar powder, and is dissolved by adding water, sterilized at 121 ℃ for 30min, cooled and formed into an inclined plane;
(2) preparing a seed solution: inoculating the activated pichia pastoris strain into a shake flask seed liquid culture medium for shake flask fermentation culture at the culture temperature of 25-32 ℃, the rotation speed of a shaking table of 150-;
wherein the shake flask seed liquid culture medium comprises 1.0-2.5% glycerol, 0.8-2.0% peptone and 0.15-2.0% yeast powder, and is sterilized at 121 deg.C for 30min after being dissolved in water;
(3) liquid submerged fermentation: inoculating the seed liquid in the shake flask into a fermentation tank with an inoculation amount of 5-10% for fermentation culture at 25-32 deg.C, rotation speed of 200-800rpm/min, and ventilation amount of 0.5-2.5m3The pH value is controlled between 4.5 and 5.5 by ammonia water with the concentration of 20 to 25 percent in the whole process; feeding the material supplement A when the dissolved oxygen rebounds and does not fall, adjusting the material supplement speed, and controlling the dissolved oxygen to be 10-25%, wherein the ratio of the total volume of the material supplement A to the total volume of the fermentation tank culture medium is 1: 3; 1h after the addition of the supplementary material A is finished, starting to feed the supplementary material B, adjusting the supplementary material speed and controlling the dissolved oxygen to be 10-30%; after feeding the material B for two days, measuring the enzyme activity of the fermentation supernatant every 24 hours, and stopping fermentation when the increase amplitude between the enzyme activities measured for two times is less than 200U/ml;
wherein the culture medium of the fermentation tank comprises 0.51-2.975% of phosphoric acid, 0.8-4.5% of ammonium sulfate, 0.02-0.12% of calcium sulfate, 0.8-2.7% of potassium sulfate, 0.3-2.3% of magnesium sulfate, 0.3-1.2% of potassium hydroxide, 0.5-5% of zinc sulfate, 2-5% of sorbitol and 0.2-0.8% of PTM1, and is sterilized for 30min at 121 ℃ after being dissolved in water;
the supplementary material A comprises 20-50% of sorbitol, 0.5-5% of zinc sulfate, 0.2-0.8% of PTM1 and the balance of water;
the supplementary material B comprises 80-99% of methanol, 0.5-5% of zinc sulfate, 0.2-0.8% of PTM1 and the balance of water;
(4) filtering, concentrating the fermentation liquid, and extracting aminopeptidase.
The effect of the method for producing an aminopeptidase by fermentation of the present invention will be examined below with reference to specific examples.
Example 1:
the method for producing aminopeptidase by fermentation employed in this example comprises the following steps:
(1) activating strains: preparing an activation culture medium which comprises 1.5% of glycerol, 2.0% of peptone, 1.0% of yeast powder and 1.5% of agar powder, adding water to dissolve, sterilizing at 121 ℃ for 30min, and cooling to form a slope;
inoculating pichia pastoris into an activation culture medium for activation under the aseptic condition, wherein the activation temperature is 30 ℃, the activation time is 72 hours, and a clear bacterial colony grows on an inclined plane, so that a purified strain is obtained;
(2) preparing a seed solution: preparing shake flask seed liquid culture medium comprising 1.5% glycerol, 2.0% peptone and 1.0% yeast powder, dissolving in water, and sterilizing at 121 deg.C for 30 min;
under the aseptic condition, selecting an activated pichia pastoris bacterial colony, inoculating the Pichia pastoris bacterial colony into a 500mL triangular flask filled with 100mL shake flask seed liquid culture medium, and carrying out shake flask fermentation culture at the culture temperature of 30 ℃, the rotation speed of a shaking table of 220rpm/min for 24h until the culture liquid in the shake flask is turbid, thereby obtaining shake flask seed liquid;
(3) liquid submerged fermentation: preparing a fermentation tank culture medium which comprises 2.125% of phosphoric acid, 4% of ammonium sulfate, 0.1% of calcium sulfate, 2% of potassium sulfate, 1.8% of magnesium sulfate, 0.8% of potassium hydroxide, 0.5% of zinc sulfate, 4.2% of sorbitol and 0.5% of PTM1, adding water to dissolve the mixture, and sterilizing the mixture for 30min at 121 ℃;
15L of fermentation medium is filled in a 30L fermentation tank, the seed liquid in the shake flask is inoculated into the fermentation tank for fermentation culture by the inoculation amount of 5 percent, the culture temperature is 30 ℃, and the pH value is controlled to be 5.0 by using ammonia water with the concentration of 22 percent in the whole process; when the dissolved oxygen rebounds and does not fall, feeding the material A, adjusting the feeding speed, controlling the dissolved oxygen to be 10-25%, and controlling the total volume of the material A to be 5L; after the addition of the supplementary material A is finished for 1h, entering a methanol induction period, starting to feed the supplementary material B, adjusting the speed of the supplementary material, and controlling the dissolved oxygen to be 10-30%; initial ventilation of fermentation 0.5m3H, stirring speed is 200 rpm/min; when the dissolved oxygen is less than 40%, the air is raised and rotated until the fermentation reaches the maximum ventilation of 2.5m3H, the stirring speed reaches 800 rpm/min; after feeding the material B for two days, measuring the enzyme activity of the fermentation supernatant every 24 hours, and stopping fermentation when the increase amplitude between the enzyme activities measured for two times is less than 200U/ml;
wherein, the feed supplement A comprises 50 percent of sorbitol, 0.5 per mill percent of zinc sulfate, 0.5 percent of PTM1 and the balance of water;
the supplementary material B comprises 99 percent of methanol, 0.5 per mill of zinc sulfate, 0.5 percent of PTM1 and the balance of water;
three batches are fermented by adopting the method, the aminopeptidase enzyme activity in each batch of fermentation process is detected by adopting an aminopeptidase enterprise labeling method of Wuhan Xinhuayang biology GmbH, the aminopeptidase activity unit is defined as 1g of solid enzyme or 1mL of liquid enzyme, 1mmol/L L-leucine p-nitroaniline (Sigma L2158) is hydrolyzed per minute at 50 ℃ and pH7.5 to generate 1 mu mol of p-nitrophenol, namely 1 enzyme activity unit, and the U/g or U/mL is expressed.
The results of enzyme activity measurements are shown in Table 1.
TABLE 1 enzyme Activity of aminopeptidase at different fermentation time points in the batch fermentors
Under the regulation and control of the method provided by the invention, the aminopeptidase is synthesized in a large amount after fermentation for 82 hours, the enzyme production rate is slowed down after 154 hours, the aminopeptidase is fermented to 178 hours and put into a tank, and the activity of the aminopeptidase in the fermentation liquid reaches about 2200U/mL.
Comparative example 1:
the method for producing aminopeptidase by fermentation employed in this example comprises the following steps:
(1) activating strains: preparing an activation culture medium which comprises 1.5% of glycerol, 2.0% of peptone, 1.0% of yeast powder and 1.5% of agar powder, adding water to dissolve, sterilizing at 121 ℃ for 30min, and cooling to form a slope;
inoculating pichia pastoris into an activation culture medium for activation under the aseptic condition, wherein the activation temperature is 30 ℃, the activation time is 72 hours, and a clear bacterial colony grows on an inclined plane, so that a purified strain is obtained;
(2) preparing a seed solution: preparing shake flask seed liquid culture medium comprising 1.5% glycerol, 2.0% peptone and 1.0% yeast powder, dissolving in water, and sterilizing at 121 deg.C for 30 min;
under the aseptic condition, selecting an activated pichia pastoris bacterial colony, inoculating the Pichia pastoris bacterial colony into a 500mL triangular flask filled with 100mL shake flask seed liquid culture medium, and carrying out shake flask fermentation culture at the culture temperature of 30 ℃, the rotation speed of a shaking table of 220rpm/min for 24h until the culture liquid in the shake flask is turbid, thereby obtaining shake flask seed liquid;
(3) liquid submerged fermentation: preparing a culture medium of a fermentation tank, wherein the culture medium comprises 2.125% of phosphoric acid, 4% of ammonium sulfate, 0.1% of calcium sulfate, 2% of potassium sulfate, 1.8% of magnesium sulfate, 0.8% of potassium hydroxide, 4.2% of sorbitol and 0.5% of PTM1, adding water to dissolve the mixture, and sterilizing the mixture for 30min at 121 ℃;
15L of fermentation medium is filled in a 30L fermentation tank, the seed liquid in the shake flask is inoculated into the fermentation tank for fermentation culture by the inoculation amount of 5 percent, the culture temperature is 30 ℃, and the pH value is controlled to be 5.0 by using ammonia water with the concentration of 22 percent in the whole process; after the dissolved oxygen rebounds and does not fall, starting to feed sorbitol, adjusting the feeding speed, controlling the dissolved oxygen to be 10-25%, and controlling the total feeding volume of sorbitol to be 5L; after finishing sorbitol feeding for 1h, entering a methanol induction period, starting to feed methanol, adjusting the feeding speed, and controlling the dissolved oxygen to be 10-30%; initial ventilation of fermentation 0.5m3H, stirring speed is 200 rpm/min; when the dissolved oxygen is less than 40%, the air is raised and rotated until the fermentation reaches the maximum ventilation of 2.5m3H, the stirring speed reaches 800 rpm/min; after fermentation for 178h, the aminopeptidase activity in the fermentation liquor is 1600U/mL by using the method similar to that in example 1, which is far lower than that of the aminopeptidase produced by fermentation in example 1 by using the method provided by the invention.
Comparative example 2:
the method for producing aminopeptidase by fermentation employed in this example comprises the following steps:
(1) activating strains: preparing an activation culture medium which comprises 1.5% of glycerol, 2.0% of peptone, 1.0% of yeast powder and 1.5% of agar powder, adding water to dissolve, sterilizing at 121 ℃ for 30min, and cooling to form a slope;
inoculating pichia pastoris into an activation culture medium for activation under the aseptic condition, wherein the activation temperature is 30 ℃, the activation time is 72 hours, and a clear bacterial colony grows on an inclined plane, so that a purified strain is obtained;
(2) preparing a seed solution: preparing shake flask seed liquid culture medium comprising 1.5% glycerol, 2.0% peptone and 1.0% yeast powder, dissolving in water, and sterilizing at 121 deg.C for 30 min;
under the aseptic condition, selecting an activated pichia pastoris bacterial colony, inoculating the Pichia pastoris bacterial colony into a 500mL triangular flask filled with 100mL shake flask seed liquid culture medium, and carrying out shake flask fermentation culture at the culture temperature of 30 ℃, the rotation speed of a shaking table of 220rpm/min for 24h until the culture liquid in the shake flask is turbid, thereby obtaining shake flask seed liquid;
(3) liquid submerged fermentation: preparing a fermentation tank culture medium which comprises 2.125% of phosphoric acid, 4% of ammonium sulfate, 0.1% of calcium sulfate, 2% of potassium sulfate, 1.8% of magnesium sulfate, 0.8% of potassium hydroxide, 0.5% of zinc sulfate, 4.2% of glycerol and 0.5% of PTM1, adding water to dissolve the mixture, and sterilizing the mixture for 30min at 121 ℃;
15L of fermentation medium is filled in a 30L fermentation tank, the shake flask seed liquid is inoculated into the fermentation tank for fermentation culture by the inoculation amount of 5 percent, the culture temperature is 30 ℃, and the pH value is controlled to be 5.0 in the whole process; after the dissolved oxygen rebounds and does not fall, feeding glycerol, adjusting the feeding speed, controlling the dissolved oxygen to be 10-25%, and controlling the total volume of glycerol fed-batch to be 5L; after 1h of glycerol feeding, entering a methanol induction period, beginning to feed methanol, adjusting the feeding speed, and controlling the dissolved oxygen to be 10-30%; initial ventilation of fermentation 0.5m3H, stirring speed is 200 rpm/min; when the dissolved oxygen is less than 40%, the air is raised and rotated until the fermentation reaches the maximum ventilation of 2.5m3H, the stirring speed reaches 800 rpm/min; after fermentation for 178h, the fermentation liquid is put into a tank, the aminopeptidase activity in the fermentation liquid is detected to be 2186U/mL by adopting the method which is the same as the method in the embodiment 1, the enzyme activity is similar to the enzyme activity of the aminopeptidase which is generated by adopting the method in the embodiment 1, but the sorbitol which is used as a carbon source in the embodiment of the invention is lower than glycerol in price, and the cost can be greatly reduced.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.
Claims (10)
1. A method for producing an aminopeptidase by fermentation, comprising the steps of:
(1) activating strains: inoculating pichia pastoris into an activation culture medium for activation to obtain a purified strain;
(2) preparing a seed solution: inoculating the activated pichia pastoris strain into a shake flask seed liquid culture medium for shake flask fermentation culture to obtain shake flask seed liquid;
(3) liquid submerged fermentation: inoculating the seed liquid in the shake flask into a fermentation tank for fermentation culture, starting feeding material A when the dissolved oxygen does not fall back, and starting feeding material B when the feeding of the material A is finished for 0.5-1h until the fermentation is finished;
wherein, the feed supplement A comprises 20 to 50 percent of sorbitol, 0.5 to 5 per mill of zinc sulfate, 0.2 to 0.8 percent of PTM1 and the balance of water;
the supplementary material B comprises 80-99% of methanol, 0.5-5% of zinc sulfate, 0.2-0.8% of PTM1 and the balance of water;
(4) filtering, concentrating the fermentation liquid, and extracting aminopeptidase.
2. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: the activation temperature in the step (1) is 25-32 ℃, and the activation time is 60-84 h.
3. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: in the step (2), the shake flask fermentation culture temperature is 25-32 ℃, the shaking table rotation speed is 150-300rpm/min, and the culture time is 18-25 h.
4. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: the activation culture medium in the step (1) comprises 1.0-2.5% of glycerol, 0.8-2.0% of peptone, 0.15-2.0% of yeast powder and 0.5-2.0% of agar powder; the seed liquid culture medium in the shake flask in the step (2) comprises 1.0-2.5% of glycerol, 0.8-2.0% of peptone and 0.15-2.0% of yeast powder.
5. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: and (4) inoculating the shake flask seed solution into a fermentation tank for fermentation culture in an inoculation amount of 5-10% in the step (3).
6. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: the culture medium of the fermentation tank in the step (3) comprises 0.51-2.975% of phosphoric acid, 0.8-4.5% of ammonium sulfate, 0.02-0.12% of calcium sulfate, 0.8-2.7% of potassium sulfate, 0.3-2.3% of magnesium sulfate, 0.3-1.2% of potassium hydroxide, 0.5-5% of zinc sulfate, 2-5% of sorbitol and 0.2-0.8% of PTM 1.
7. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: the fermentation culture temperature of the fermentation tank in the step (3) is 25-32 ℃, the rotating speed is 200-800rpm/min, and the ventilation volume is 0.5-2.5m3The pH value is controlled to be 4.5-5.5 in the whole process.
8. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: the ratio of the total volume of the fed-batch A fed-batch in the step (3) to the total volume of the culture medium in the fermentation tank is 1: 3.
9. The process for the fermentative production of an aminopeptidase according to claim 1, wherein: when the feeding material A flows in the step (3), adjusting the feeding speed and controlling the dissolved oxygen to be 10-25%; and when the material B is fed in a flowing manner, adjusting the feeding speed and controlling the dissolved oxygen to be 10-30%.
10. The process for fermentative production of an aminopeptidase according to claim 1, wherein the fermentation termination conditions in the step (3) are: and (3) measuring the enzyme activity of the fermentation supernatant every 24 hours after two days of fed-batch feeding B, and stopping fermentation when the increase amplitude between the enzyme activities measured in two consecutive times is less than 200U/ml.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011086447A2 (en) * | 2010-01-12 | 2011-07-21 | Lupin Limited | Fermentation process for the preparation of recombinant heterologous proteins |
| KR20170078306A (en) * | 2015-12-29 | 2017-07-07 | 샘표 주식회사 | Aspergillus oryzae SMF119 strain having high leucine aminopeptidase activity, derived from Korean traditional fermented dried soybean |
| CN107129978A (en) * | 2017-04-12 | 2017-09-05 | 江南大学 | The fermentation process of heat-resisting lysine aminopeptidase |
| CN109468240A (en) * | 2018-11-15 | 2019-03-15 | 江南大学 | A kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase |
| CN112501041A (en) * | 2020-12-03 | 2021-03-16 | 西安德诺海思医疗科技有限公司 | Pichia pastoris high-density fermentation medium and fermentation method thereof |
| CN112725201A (en) * | 2021-01-19 | 2021-04-30 | 武汉新华扬生物股份有限公司 | Liquid submerged fermentation method of pichia pastoris producing acid protease |
| CN113621550A (en) * | 2021-09-23 | 2021-11-09 | 武汉瀚海新酶生物科技有限公司 | Culture medium for producing alkaline phosphatase by fermentation and preparation method of alkaline phosphatase |
-
2021
- 2021-12-07 CN CN202111482954.7A patent/CN114107264A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011086447A2 (en) * | 2010-01-12 | 2011-07-21 | Lupin Limited | Fermentation process for the preparation of recombinant heterologous proteins |
| KR20170078306A (en) * | 2015-12-29 | 2017-07-07 | 샘표 주식회사 | Aspergillus oryzae SMF119 strain having high leucine aminopeptidase activity, derived from Korean traditional fermented dried soybean |
| CN107129978A (en) * | 2017-04-12 | 2017-09-05 | 江南大学 | The fermentation process of heat-resisting lysine aminopeptidase |
| CN109468240A (en) * | 2018-11-15 | 2019-03-15 | 江南大学 | A kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase |
| CN112501041A (en) * | 2020-12-03 | 2021-03-16 | 西安德诺海思医疗科技有限公司 | Pichia pastoris high-density fermentation medium and fermentation method thereof |
| CN112725201A (en) * | 2021-01-19 | 2021-04-30 | 武汉新华扬生物股份有限公司 | Liquid submerged fermentation method of pichia pastoris producing acid protease |
| CN113621550A (en) * | 2021-09-23 | 2021-11-09 | 武汉瀚海新酶生物科技有限公司 | Culture medium for producing alkaline phosphatase by fermentation and preparation method of alkaline phosphatase |
Non-Patent Citations (8)
| Title |
|---|
| 傅小蒙;孔令聪;裴志花;刘树明;马红霞;: "毕赤酵母表达系统优化策略概述", 中国生物工程杂志, no. 10, 15 October 2015 (2015-10-15), pages 86 - 90 * |
| 刘强: "细菌耐热赖氨酸氨肽酶毕赤酵母表达及发酵优化", 中国优秀硕士论文电子期刊网基础科学辑, no. 3, 15 March 2017 (2017-03-15), pages 13 - 14 * |
| 刘强;田亚平;: "耐热赖氨酸氨肽酶毕赤酵母表达及培养条件优化", 食品与生物技术学报, no. 06, pages 589 - 594 * |
| 宋婷婷 等: "米曲霉AAP新基因的异源表达及其酶学性质研究", 食品与发酵工业, vol. 45, no. 05, 26 December 2018 (2018-12-26), pages 38 - 44 * |
| 宋婷婷: "米曲霉AAP新基因的异源表达及其酶学性质研究", 食品与发酵工业, vol. 45, no. 05, pages 39 * |
| 梁二宾: "氨肽酶产生菌的鉴定、酶的分离纯化及性质研究", 中国优秀硕士论文电子期刊网基础科学辑, no. 5, 15 May 2015 (2015-05-15), pages 42 * |
| 武婕: "毕赤酵母工程菌高密度发酵研究与进展", 中国生物工程杂志, vol. 36, no. 01, pages 108 - 114 * |
| 黄伟谦: "酱油曲霉亮氨酸氨肽酶sLAP1在毕赤酵母中表达及其酶学性质研究", 中国优秀硕士论文电子期刊网基础科学辑, no. 1, 15 January 2015 (2015-01-15), pages 006 - 68 * |
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