CN116463237A - Protein glutaminase production strain and production method and application thereof - Google Patents
Protein glutaminase production strain and production method and application thereof Download PDFInfo
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- 102000009127 Glutaminase Human genes 0.000 title claims abstract description 61
- 108010073324 Glutaminase Proteins 0.000 title claims abstract description 61
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 87
- 230000004151 fermentation Effects 0.000 claims abstract description 87
- 230000000694 effects Effects 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 27
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
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- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 17
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
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- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 14
- 229910052799 carbon Inorganic materials 0.000 description 9
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- 239000000203 mixture Substances 0.000 description 6
- 241000589565 Flavobacterium Species 0.000 description 5
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- 108010030291 alpha-Galactosidase Proteins 0.000 description 2
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
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- 229930006000 Sucrose Natural products 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
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- 125000003368 amide group Chemical group 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
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- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
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- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
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- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
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- 239000002689 soil Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01044—Protein-glutamine glutaminase (3.5.1.44)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a protein glutaminase production strain, a method for preparing protein glutaminase by using the strain and application thereof. The invention discloses a high-yield strain of protein glutaminase, which is named as golden yellow bacillus (Chryseobacterium proteolyticum) 0326-509, and is preserved in China general microbiological culture Collection center (CGMCC) at the 8 th month of 2022, the strain is preserved under the condition of CGMCC No.25600, and the colony of the strain is round, protruding, regular in edge, golden yellow or orange yellow, smooth in surface and sticky. The method for preparing protein glutaminase by fermenting the strain disclosed by the invention can obtain fermentation supernatant protein which has the activity of 7.0U/mL. The strain, the production method and the application of the invention can realize the industrial large-scale production of the protein glutaminase and have wide prospect.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a protein glutaminase production strain, a production method and application thereof.
Background
The Protein Glutaminase (PG) is a novel protein hydrolase, can specifically hydrolyze the amide groups of the glutamine residues of the protein, can effectively improve the solubility, foamability, emulsibility and other various food processing characteristics of the protein, and has wide application prospect in the food industry.
Protein Glutaminase (PG) A protein deaminase-producing strain Chryseobacterium proteolyticum 9670 was originally screened from soil in 2000 by Japanese scholars using Cbz-Gln-Gly as the sole nitrogen source T However, the enzyme activity was very low, and it was only 0.258U/mL (Yamaguchi S, yokoe M.A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp.nov., a newly isolated bacterium from oil. Applied and Environmental Microbiology,2000,66 (8): 3337-3343). The domestic research on protein glutaminase is relatively late, the activity of the strain producing protein glutaminase is very low and far does not meet the industrial production requirement, such as Zhang Yanfang, a plurality of strains producing PG enzyme are reported to be screened, the highest enzyme activity is 0.717U/mL (Zhang Yanfang, gu Caifeng, what can be the same, etc.. The separation, screening and identification of the strain producing protein glutaminase, the food industry science and technology, 2015,36 (1): 170-176.), kang Li, etc. the conditions and carbon source nitrogen sources of the fermentation PG enzyme of the indoxylum gold bacteria are optimized, the highest enzyme activity is 0.594U/mL (Kang Li, niu Yanning, zhang Yanfang, etc.. The fermentation and preliminary separation and application research of protein glutaminase [ J]Journal of Chinese bioengineering, 2014,34 (5): 54-59.), he Can, et al, optimized with orthogonal experimental design for PG enzyme fermentation medium formulation, with a maximum enzyme activity of 0.661U/mL (Brilliant, song)Preferably, huang Di, et al preliminary optimization of protein glutaminase fermentation conditions [ J]Jiangsu agricultural science 2014,42 (03): 218-220.). Huang Jing et al discloses a strain of Bacillus prinus YF810, which comprises adding zeolite with a maximum enzyme activity of 2U/mL (Huang Jing, etc., preparation and application of protein glutaminase-producing strain and fermentation product, grant publication No. CN 107325978B); huang Jing et al disclose a strain of Flavobacterium prion-degrading bacterium A4142 with a maximum enzyme activity of 2.15U/mL (Huang Jing et al, a method for producing protein glutaminase, screening and characteristic analysis, patent application No. 202010927185.6), which is far from the requirement of being applied to industrial production. The industrial production of protein glutaminase, the production cost is the primary factor. The current publications show that the nitrogen source in the seed medium composition is a high cost medium such as polypeptone. As for the seed medium and the fermentation medium, for example, kang Li and He Can, the nitrogen source was polypeptone (Kang Li, niu Yanning, zhang Yanfang, etc.. Fermentation of protein glutaminase, preliminary separation and application research [ J ]]Journal of Chinese bioengineering, 2014,34 (5): 54-59, splendid, song Jia, huang Di, et al preliminary optimization of protein glutaminase fermentation conditions [ J ]]The Jiangsu agricultural science 2014,42 (03): 218-220). Huang Jing and the like, and the nitrogen sources of the fermentation medium formula and the fermentation medium formula are polypeptone (Huang Jing and the like, a protein glutaminase production strain, screening and characteristic analysis method, and patent application No. 202010927185.6). Because polypeptone is expensive, it is only used in laboratory research and cannot be used in industrial production.
Disclosure of Invention
The invention provides a strain of flavobacterium prions with higher activity of protein glutaminase produced. The invention adopts mutation breeding technology to protein glutaminase production strain, and obtains a high-yield strain, namely protein glutaminase production strain (Chryseobacterium proteolyticum) 0326-509) with high enzyme activity, by multiple rounds of NTG mutation and primary re-screening. The strain is gram-negative bacteria, does not produce spores, does not move, and has circular bacterial colony, golden yellow or orange yellow.
The protein glutaminase producing strain is a bacillus thuringiensis (Chryseobacterium proteolyticum) 0326-509 with the following preservation information:
preservation time: 2022 8, 26
Preservation unit name: china general microbiological culture Collection center (China general microbiological culture Collection center)
Preservation number: CGMCC No.25600
Deposit unit address: beijing city, the North Chen Xili No.1, 3 national academy of sciences of China for microbiology
Classification naming: bacillus prion (Chryseobacterium proteolyticum)
The invention relates to a bacillus prion-degrading bacterium 0326-509, which has the following morphological characteristics on an LB plate: the colony is round, protruding, neat in edge, golden yellow or orange, smooth in surface and sticky.
The main physiological and biochemical characteristics of the strain of the flavobacterium prions 0326-509 are shown in table 1:
table 1:0326-509 cell morphology and physiological and biochemical characteristics
The 16s rDNA sequence of the bacillus thuringiensis 0326-509 is shown in the following SEQ ID NO: 1:
TACCTAGGCAGCTCCTGTTACGGTCACCGACTTCAGGTACCCCAGACTTCCATGGCTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGCCATGGCTGATGCGCGATTACTAGCGATTCCAGCTTCATAGAGTCGAGTTGCAGACTCCAATCCGAACTGAGACCAGCTTTCGAGATTAGCATCCAGTCGCCTGGTAGCAGCCCTCTGTACTGGCCATTGTATTACGTGTGTGGCCCAAGGCGTAAGGGCCGTGATGATTTGCTATCCCCACCTTCCTCTCTACTTGCGTAGGCAGTCTCACTAGAGTCCCCAACTGAATGATGGCAACTAGTGACAGGGGTTGCGCTCGTTGCAGGACTTAACCTAACACCTCACGGCACGAGCTGACGACAACCATGCAGCACCTTGAAAAATGTCCGAAGAAAAGCCTATTTCTAAGCCTGTCATTTCCCATTTAAGCCTTGGTAAGGTTAATCGCGTATCATCGAATTAAACCACATAATCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAAACTTGCGTTCGTACTCCCCAGGTGGCTAACTTATCACTTTCGCTTAGTCTCTGAATCCGAAAACCCAAAAACGAGTTAGCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGTCCATCAGCGTCAGTTAAGACATGGTAACCTGCCTTCGCAATTGGTGTTCTAAGTAATATCTATGCATTTCACCGCTACACTACTTATTCCAGCTACCTCTACCTTACTCAAGACCCGCAGTATCAATGGCAGTTTCATAGTTAAGCTATGAGATTTCACCACTGACTTACGAGTCCGCCTACGGACCCTTTAAACCCAATAAATCCGGATAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAACCGGTGCTTATTCGTATAGTACCTTCAGCTACCCTCACGAGGGTAGGTTTATCCCTATACAAAAGAAGTTTACAACCCATAGGGCCGTCATCCTTCACGCGGGATGGCTGGATCAGGCTCTCACCCATTGTCCAATATTCCTCACTGCTGCCTCCCGTAGGAGTCTGGTCCGTGTCTCAGTACCAGTGTGGGGGATCACCCTCTCAGGCCCCCTAAAGATCATTGACTTGGTGAGCCGTTACCTCACCAACTATCTAATCTTGCGCGTGCCCATCTCTATCCACCGGAGTTTTCAATATCTAATGATGCCATCAAATATATTATGGGGTATTAATCTTCCTTTCGAAAGGCTATCCCCCTGATAAAGGTAGGTTGCACACGTGTTCCGCACCCGTGCGCCGCTCTCTAGATCCCGAAAGATCTAT(SEQ ID NO：1)
the invention also provides a method for preparing protein glutaminase by optimizing fermentation by using the strain of golden yellow bacillus (Chryseobacterium proteolyticum) 0326-509.
The preparation method is realized by the following technical scheme:
(1) Strain activation: and (3) streaking a bacterial solution of a protein glutaminase production strain which is preserved at the temperature of minus 80 ℃ namely a strain of golden yellow bacterium (Chryseobacterium proteolyticum) 0326-509 on an LB plate, and inverting the LB plate into a constant temperature incubator for activation.
(2) Seed liquid preparation: scraping activated thalli in an LB plate by an inoculating loop, placing the thalli in a triangular flask filled with sterilized seed culture medium, and placing the triangular flask in a constant temperature shaking table for culture.
(3) Fermentation: the activated seed liquid is inoculated into a triangular flask filled with a sterilized fermentation medium and placed in a constant temperature shaking table for fermentation, and the protein glutaminase is prepared.
In the step (1), the amount of the bacterial liquid is 80-120 mu L, preferably 100 mu L; the culture temperature is 30-40deg.C, preferably 37deg.C; the culture time is 15-18h, and is optimized to 16h.
In the step (2), the diameter of the inoculating loop is 2-4mm, preferably 3mm; the amount of the cells is 1-4 rings, preferably 3 rings; the liquid loading amount of the seed culture medium is 20-30mL, preferably 25mL; the temperature of the shaking table is 28-37 ℃, preferably 30 ℃; the rotation speed of the shaking table is 180-250rpm, preferably 200rpm; the incubation time is 6 to 10 hours, preferably 8 hours.
In the step (2), the seed culture medium comprises Angel yeast peptone FP103 8-12g/L, angel yeast powder FM 802.8-2.2 g/L, magnesium sulfate heptahydrate 2-2.2g/L, and pH6.8-7.2. Preferably, it is: angel yeast peptone FP10310g/L, angel yeast powder FM8022g/L, magnesium sulfate heptahydrate 2.05g/L, pH7.0.
In the step (3), the inoculation amount of the seed liquid is 5-10% (v/v), preferably 7% (v/v); the liquid loading amount of the fermentation medium is 20-30mL, preferably 25mL; the fermentation temperature is 28-37deg.C, preferably 30deg.C; the rotation speed of the shaking table is 180-250rpm, preferably 200rpm; the cultivation time is 14 to 18 hours, preferably 16 hours.
In the step (3), the composition of the fermentation medium is as follows: angel yeast peptone FP103 18-22g/L, potassium dihydrogen phosphate 4.8-5.0g/L, disodium hydrogen phosphate 0.15-0.18g/L, magnesium sulfate heptahydrate 6.7-6.8g/L, ferrous sulfate heptahydrate 0.04-0.06g/L. Preferably, it is: angel yeast peptone FP10320g/L, potassium dihydrogen phosphate 4.89g/L, disodium hydrogen phosphate 0.17g/L, magnesium sulfate heptahydrate 6.75g/L, ferrous sulfate heptahydrate 0.05g/L.
The protein glutaminase prepared by the method is novel protease which has mild effect, high specificity and safety and edibility, and the activity of the protein glutaminase can reach 7.0U/mL.
The invention also provides the protein glutaminase produced and prepared by the method.
The invention also provides a seed culture medium formula and a fermentation culture medium formula which can be used for industrial production of the protein glutaminase.
The composition of the seed culture medium is as follows: 8-12g/L of Angel yeast peptone, 1.8-2.2g/L of Angel yeast powder and 2-2.2g/L of magnesium sulfate heptahydrate; ph=6.8-7.2.
The composition of the fermentation medium is as follows: 18-22g/L of Angel yeast peptone, 4.8-5.0g/L of potassium dihydrogen phosphate, 0.15-0.18g/L of disodium hydrogen phosphate, 6.7-6.8g/L of magnesium sulfate heptahydrate and 0.04-0.06g/L of ferrous sulfate heptahydrate.
Preferably, the high-yield strain, the seed culture medium and the fermentation culture medium are applied in the method, so that the enzyme activity of the fermentation supernatant obtained by production and preparation reaches 7.0U/mL, and the industrial mass production of the protein glutaminase can be realized.
The invention also provides a protein glutaminase production strain, namely golden yellow bacillus (Chryseobacterium proteolyticum) 0326-509, and application of the production preparation method using the strain in preparation of protein glutaminase.
The beneficial effects of the invention include: the protein glutaminase production strain 0326-509 belongs to the genus of the golden yellow bacillus and is obtained by a traditional mutation breeding method, and is a food safety production strain;
the invention also provides a novel seed culture medium formula and a fermentation culture medium formula, wherein the main nitrogen sources of the seed culture medium formula are Angel yeast peptone FP103 and Angel yeast powder FM802, and the main nitrogen source of the fermentation culture medium formula is Angel yeast peptone FP103. Both nitrogen sources are industrial raw materials, are suitable for industrial fermentation, and greatly reduce the industrial cost.
And the fermentation medium is utilized to ferment the strain 0326-509 to prepare the protein glutaminase, the enzyme activity of the fermentation supernatant can reach 7.0U/mL, which is far higher than the enzyme activity of the protein glutaminase produced by the edible strain which is published and reported in China at present, and the industrial production can be realized.
The invention provides an industrial nitrogen source safety yeast peptone and Angel yeast powder. The method is low in price and is very suitable for fermenting and producing protein glutaminase by using the golden yellow bacillus subtilis 0326-509, and the fermentation enzyme activity reaches 7.0U/mL. Can realize industrial production.
The innovation of the invention provides that the enzyme production activity can be effectively improved by greatly improving the content of the magnesium sulfate heptahydrate in the fermentation medium to 6.7-6.8g/L, preferably 6.75 g/L. This is probably due to the fact that magnesium ions are activators of various enzymes, which have an activating effect on the enzymes. The new thought of improving the content of the magnesium sulfate heptahydrate in the fermentation medium can effectively improve the fermentation enzyme activity of the protein glutaminase production strain 0326-509, and provides a substantial advanced technical breakthrough for improving the enzyme production activity. In the prior art and in the published literature, the content of magnesium sulfate heptahydrate in the fermentation medium is low, for example, the content of magnesium sulfate heptahydrate in the fermentation medium for producing xylanase reported by the method of Mo Xiao and the like is 2.2g/L (the fermentation medium for producing xylanase is optimized by using methanol protein (J), light industrial report, 2021,36 (04): 1-8), the content of magnesium sulfate heptahydrate in the fermentation medium for producing naringinase reported by Zhang Taotao and the like is 2.0g/L (Zhang Taotao, zhang, niu Chun and the like), the content of magnesium sulfate heptahydrate in the fermentation medium for producing alpha-galactosidase reported by the method of 2021,49 (07): 170-173+176), liu Dehai and the like is 1.0g/L (Liu Dehai, weight is calm and the like, and the fermentation medium is screened and identified for producing alpha-galactosidase strain and optimized by using the method of producing naringinase (J), and the method of brewing, 2019,38 (12): 80-86).
Drawings
FIG. 1 is a graph showing the addition of MgSO at different concentrations to the fermentation medium 4 ·7H 2 O strain 0326-509.
FIG. 2 shows the fermentation enzyme activity of strain 0326-509 with different nitrogen sources added to the fermentation medium.
FIG. 3 shows the fermentation enzyme activity of the strain 0326-509 with different types of carbon sources added to the fermentation medium.
Detailed Description
The process, conditions, experimental methods, etc. for carrying out the present invention will be described in further detail, comprehensively and accurately with reference to the following specific examples and drawings. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.
The strain according to the present invention and the method for producing protein glutaminase are specifically implemented in the following manner.
Example 1 protein glutaminase was prepared by fermentation using the strain of Flavobacterium prion disinsection 0326-509.
(1) Activation of the strain of Flavobacterium prion 0326-509: the protein glutaminase production strain of the invention, namely the strain of golden yellow bacterium (Chryseobacterium proteolyticum) 0326-509, which is preserved at the temperature of 100 mu L-80 ℃ is streaked on an LB solid culture medium flat plate, and is inversely cultured for 16 hours in a constant temperature incubator at the temperature of 37 ℃. The LB solid medium consists of: 10g/L of tryptone, 8025g/L of Angel yeast powder FM, 10g/L of sodium chloride, 18g/L of agar and pH7.2.
(2) Seed liquid preparation: scraping activated thalli on a three-ring LB plate by an inoculating ring with the diameter of 3mm, inoculating the thalli into a sterilized seed culture medium, bottling 25mL of the seed culture medium into every 250mL triangular flask, sealing by a sealing film, and then placing into a shaking table for culturing for 8 hours at the temperature of 30 ℃ and at the speed of 200 rpm. The seed culture medium consists of: angel yeast peptone FP10310g/L, angel yeast powder FM8022g/L, magnesium sulfate heptahydrate 2.05g/L, pH7.0.
(3) Shaking and fermenting: the seed solution is inoculated into the sterilized fermentation medium according to the inoculum size of 7% (v/v), 25mL of the fermentation medium is bottled in every 250mL triangular flask, and the fermentation medium is put into a shaking table for fermentation at 30 ℃ and 200rpm after sealing by a sealing film for 16h. The fermentation medium consists of: angel peptone FP10320g/L, potassium dihydrogen phosphate 4.89g/L, disodium hydrogen phosphate 0.17g/L, magnesium sulfate heptahydrate 6.75g/L, ferrous sulfate heptahydrate 0.05g/L.
(4) Enzyme activity determination: after the fermentation was completed, the fermentation broth was centrifuged at 10000rpm for 5 minutes at 4℃and the supernatant was diluted 5-fold with PBS buffer having a pH of 6.5, and the diluted supernatant was assayed by a protein glutaminase activity assay method.
The enzyme activity determination method comprises the following steps: mu.L of the supernatant dilution was added to a 96-well plate, and 100. Mu.L of a 6.74g/L Cbz-Gln-Gly solution was added thereto for reaction at 37℃for 30 minutes, followed by addition of 100. Mu.L of a TCA solution to terminate the reaction. The ammonia content of the reacted solution was measured by phenol method, and the absorbance A1 was measured by A630. The control group is to add TCA solution first for reaction for 30min, then add substrate solution, and the rest operations are the same, and the absorbance A2 is measured. One enzyme activity unit (U) is the amount of enzyme required to produce 1. Mu. Mol of ammonia per minute. The calculation formula of the enzyme activity in the fermentation broth is as follows:
enzyme Activity (U/mL) = (A1-A2) ×21×5/17.03/30/K
A1: absorbance values for the experimental group; a2: absorbance values of the control group; k: slope of the ammonia standard curve.
After the strain 0326-509 is fermented for 16 hours, the protein glutaminase activity of the fermentation supernatant can reach 7.0U/mL.
Example 2
The fermentation method and the enzyme activity measurement method were the same as in example 1 of the present invention, except that the content of magnesium sulfate heptahydrate in the fermentation medium formulation was modified to 2.5g/L, 4.5g/L, 6.75g/L and 9.0g/L, respectively. After the strain 0326-509 is fermented for 16 hours, the fermentation liquor OD 600 And protein glutaminase activity of the fermentation supernatant are shown in table 2:
TABLE 2 fermentation broths OD with different contents of magnesium sulfate heptahydrate added to fermentation medium 600 And fermentation enzyme activity
As can be seen from the results in Table 2 of example 2 of the present invention, when the content of magnesium sulfate heptahydrate in the fermentation medium formulation was 6.75g/L, the protein glutaminase activity of the fermentation supernatant was highest after 16 hours of fermentation by strain 0326-509, reaching 7.0U/mL.
Example 3
The fermentation method and the enzyme activity measurement method are the same as in the embodiment 1 of the invention, the adding amount of the nitrogen source in the fermentation medium is kept unchanged, and the nitrogen source in the formula of the fermentation medium is only modified into polypeptone, angel yeast peptone FP103, tryptone and Angel yeast powder FM802. After the strain 0326-509 is fermented for 16 hours, the fermentation liquor OD 600 And protein glutaminase activity of the fermentation supernatant are shown in table 3:
table 3: fermentation broth OD with different nitrogen sources added into fermentation medium 600 And fermentation enzyme activity
As can be seen from the results in Table 3 of example 3 of the present invention, when Angel Yeast peptone FP103 was used as the nitrogen source, the enzyme activity of the protein glutaminase of the fermentation supernatant could reach 7.0U/mL, and the cost could be greatly reduced compared with that of polypeptone.
Example 4
The fermentation method and the enzyme activity measurement method were the same as in example 1 of the present invention, except that 5g/L of different carbon sources including lactose, glucose, maltose and sucrose were added to the fermentation medium, and no carbon source was added to the medium, and after fermentation for 16 hours, the fermentation broth OD 600 And protein glutaminase activity of the fermentation supernatant are shown in table 4:
table 4: adding fermentation liquor OD of different carbon sources into fermentation medium 600 And fermentation enzyme activity
The results of example 4 of the present invention show that the enzyme activity of the protein glutaminase of the fermentation broth supernatant reaches 7.0U/mL when no carbon source is added and lactose is used as the carbon source, so that the cost can be saved while the fermentation broth supernatant has high fermentation enzyme activity when no carbon source is added.
Claims (10)
1. A protein glutaminase production strain is characterized in that the strain is chrysogenum bacteria protein yticum 0326-509 with a preservation number of CGMCC No.25600, preserved in the China general microbiological culture Collection center with a preservation date of 2022, 8 and 26 days, and has a 16S rRNA sequence shown as SEQ ID NO.1, is a gram-negative bacterium, does not produce spores, does not move, and has a circular colony, golden yellow or orange yellow color.
2. A method for the fermentative preparation of a protein glutaminase using a protein glutaminase-producing strain of claim 1, which method comprises the steps of:
(1) Strain activation: streaking the protein glutaminase-producing strain of claim 1 stored at-80 ℃ on an LB plate, inverting the LB plate and activating the LB plate in a constant temperature incubator;
(2) Seed liquid preparation: scraping activated thalli in an LB plate by an inoculating loop, placing the thalli in a triangular flask filled with a sterilized seed culture medium, and placing the triangular flask in a constant temperature shaking table for culture;
(3) Fermentation: the activated seed liquid is inoculated into a triangular flask filled with a sterilized fermentation medium and placed in a constant temperature shaking table for fermentation, and the protein glutaminase is prepared.
3. The method according to claim 2, wherein in the step (1), the amount of the bacterial liquid of the protein glutaminase-producing strain is 80 to 120. Mu.L, the activation temperature is 30 to 40℃and the activation time is 15 to 18 hours.
4. The method according to claim 2, wherein in the step (2), the diameter of the inoculating loop is 2-4mm, the amount of the bacterial cells is 1-4 loops, the temperature of the shaking table is 28-37 ℃, the rotation speed of the shaking table is 180-250rpm, and the culturing time is 6-10 hours.
5. The method of claim 2, wherein in step (2), the seed medium comprises the following components and contents: angel yeast peptone 8-12g/L, angel yeast powder FM802 1.8-2.2g/L, and magnesium sulfate heptahydrate 2-2.2g/L; the pH of the seed medium=6.8-7.2.
6. The method according to claim 2, wherein in the step (3), the seed liquid after activation is inoculated in an amount of 5 to 10% (v/v), the fermentation medium is charged in an amount of 20 to 30mL, the fermentation temperature is 28 to 37 ℃, the shaking table rotation speed is 180 to 250rpm, and the cultivation time is 14 to 18 hours.
7. The method of claim 2, wherein in step (3), the fermentation medium comprises the following components in percentage by weight: 18-22g/L of Angel yeast peptone, 4.8-5.0g/L of potassium dihydrogen phosphate, 0.15-0.18g/L of disodium hydrogen phosphate, 6.7-6.8g/L of magnesium sulfate heptahydrate and 0.04-0.06g/L of ferrous sulfate heptahydrate.
8. The method according to claim 2, wherein the protein glutaminase prepared by the method is a novel deaminase which has mild action, high specificity and safety and edibility, and the activity of the novel deaminase can reach 7.0U/mL.
9. A protein glutaminase produced according to the protocol of any one of claims 2 to 8.
10. Use of a protein glutaminase-producing strain according to claim 1 or of a method according to any one of claims 2 to 8 for the preparation of a protein glutaminase.
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