CN109880810A - The glutamine transaminage mutant that secretion capacity improves - Google Patents
The glutamine transaminage mutant that secretion capacity improves Download PDFInfo
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- CN109880810A CN109880810A CN201910077029.2A CN201910077029A CN109880810A CN 109880810 A CN109880810 A CN 109880810A CN 201910077029 A CN201910077029 A CN 201910077029A CN 109880810 A CN109880810 A CN 109880810A
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- glutamine transaminage
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Abstract
The invention discloses the glutamine transaminage mutant that a kind of secretion capacity improves, and use following culture medium: LB culture medium, YPD culture medium, YNB culture medium, solid medium, fermentation medium.The present invention with streptomyces mobaraensis (Streptomyces mobaraensis) source glutamine transaminage (TGase) and its proenzyme area (pro) based on, by proenzyme area replace with streptomyces hygroscopicus (Streptomyces hygroscopicus) source TGase proenzyme area (pro (S.H)), realize TGase in grade-safe bacterial strain --- Yarrowia lipolytica (Yarrowia lipolyticaPo1h the high efficient expression in) has obtained the superior strain of glutamine transaminage.
Description
Technical field
The present invention relates to a kind of mutant, are mutated more particularly to the glutamine transaminage that a kind of secretion capacity improves
Body.
Background technique
Glutamine transaminage (Transglutaminase, EC 2.3.2.13, TGase), can be catalyzed in peptide chain
γ-carboxamide groups in glutamine residue occurs transacylate with acyl acceptor and reacts, to make to send out between protein or polypeptide
Raw covalent cross-linking.TGase is widely used in food processing field, for example, TGase can make protein and necessary amino acid (such as
Lysine) crosslinking, promote some nutritive values of food.TGase can bond meat mincing blocking, improve the utilization of meat products
Rate improves the elasticity of meat products.In addition, TGase is in medicine, cosmetics, biotechnology research, the neck such as textile industry and leather processing
There is the huge market demand in domain.
Microbe-derived glutamine transaminage is usually secreted in the form of inactive proenzyme (pro-TGase), need through
The excision such as the protease dispase end N- proenzyme area (pro) could convert Viability TGase.Proenzyme area (pro) be located at signal peptide with
Between maturase, folding and secretion to glutamine transaminage have important influence.
Microbe-derived glutamine transaminage production bacterial strain be mainly luxuriant source streptomycete (Streptomyces mobaraensis), but the yield of wild mushroom is generally lower than recombinant bacterium, using technique for gene engineering by glutamine transaminage base
Because being cloned into the hosts such as Escherichia coli, Corynebacterium glutamicum, enable TGase high efficient expression, but most hosts are non-food
The safe bacterial strain of product.In addition, the country is used for the bacterial strain strain Genomic instability of production at present, a large amount of personnel is needed to carry out in production
Rejuvenation work.Therefore, how to stablize in food safety bacterial strain, efficiently express glutamine transaminage, being urgently to solve at present
Certainly the problem of.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of secretion capacity improve glutamine transaminage mutant,
Its with streptomyces mobaraensis (Streptomyces mobaraensis) source glutamine transaminage (TGase) and its proenzyme area
(pro) based on, by proenzyme area replace with streptomyces hygroscopicus (Streptomyces hygroscopicus) source TGase enzyme
Former area (pro (S.H)), realizes TGase in grade-safe bacterial strain --- Yarrowia lipolytica (Yarrowia lipolyticaPo1h the high efficient expression in) has obtained the superior strain of glutamine transaminage.
The present invention is to solve above-mentioned technical problem by following technical proposals: a kind of paddy ammonia that secretion capacity improves
Amide transaminase mutant, which is characterized in that the glutamine transaminage mutant that the secretion capacity improves uses following training
Support base:
LB culture medium: Yeast Extract 5, Tryptone 10, NaCl 10；
YPD culture medium: Yeast Extract 10, Tryptone 20, glucose 20；
YNB culture medium: YNB 6.7, glucose 20；
Solid medium is then the agar for adding 2% in liquid medium；
Fermentation medium: glycerol 15, yeast powder 20, ammonium chloride 2.64, potassium dihydrogen phosphate 0.32, anhydrous magnesium sulfate 0.25,
3.34 × 10-4 of vitamin B1 adjusts pH to 8.0；
The building that proenzyme area replaces efficient expression strain is as follows: the plasmid pET 20b/pro-TGase retained using laboratory is mould
Plate, P3 and P4 are that primer carries out PCR, contain the genetic fragment containing TGase by PCR amplification.
Preferably, the glutamine transaminage mutant that the secretion capacity improves is turned using colorimetric method for determining glutamine
The enzyme activity of adnosine deaminase.
Preferably, the starting strain pro- of YPD inoculation of medium recombinant bacterium pro (S.H) TGase and building early period
TGase。
The positive effect of the present invention is that: the present invention withS. mobaraensisThe glutamine transaminage in source
Based on gene and its proenzyme area (pro), by by proenzyme area replace with streptomyces hygroscopicus (Streptomyces hygroscopicus) source proenzyme area (pro (S.H)), realize glutamine transaminage in grade-safe bacterial strain ---
Yarrowia lipolytica (Yarrowia lipolyticaPo1h the high efficient expression in) has obtained the high yield of glutamine transaminage
Bacterial strain.
Detailed description of the invention
Fig. 1 is the enzyme activity effect diagram of recombinant bacterium of the present invention and starting strain.
Fig. 2 is the effect diagram of secreting, expressing amount of the present invention.
Specific embodiment
Present pre-ferred embodiments are provided with reference to the accompanying drawing, in order to explain the technical scheme of the invention in detail.
The present invention uses following culture medium:
LB culture medium (g/L): Yeast Extract 5, Tryptone 10, NaCl 10.
YPD culture medium (g/L): Yeast Extract 10, Tryptone 20, glucose 20.
YNB culture medium (g/L): YNB 6.7, glucose 20.
Solid medium is then the agar for adding 2% in liquid medium.
Fermentation medium (g/L): glycerol 15, yeast powder 20, ammonium chloride 2.64, potassium dihydrogen phosphate 0.32, anhydrous slufuric acid
Magnesium 0.25,3.34 × 10-4 of vitamin B1 adjust pH to 8.0.
The Activated in Vitro of proTGase: taking 40 μ L fermentation supernatants, and 2 μ L neutral proteinase dispase(0.1 mg/ are added
ML), mixed with vortex shaker, 37 DEG C of 20 min of heat preservation.
The measurement of glutamine transaminage enzyme activity: using the enzyme activity of colorimetric method for determining glutamine transaminage.1 unit
Enzyme activity is defined as: under conditions of 37 DEG C, per minute be catalyzed α-N-CBZ-GLN-GLY synthesize 1 μm of ol Pidolidone-
Enzyme amount (U/mL) used in the mono- light amino acid of γ-.Enzyme activity determination condition: under the conditions of 37 DEG C, 40 μ L fermented supernatant fluids, 100 μ L
30 mM α-N-CBZ-GLN-GLY, 10 min of reaction, and 40 μ L terminators of addition (3M HCl, 12% trichloroacetic acid, 5%
FeCl3) terminate reaction.Light absorption value is measured at 525 nm, and standard curve is drawn by the mono- light amino acid of Pidolidone-γ-, according to
Standard curve calculates enzyme activity.
The building of proenzyme area replacement efficient expression strain: using the plasmid pINA1297/N355Q of laboratory reservation as template,
P1 and P2 is the abbreviation that primer carries out PCR(polymerase chain reaction), contain the 1297 of pro (S.H) proenzyme area by PCR amplification
Expression vector.PCR amplification uses following system: 1 μ L of template, each 25 μ L of 1 μ L, primeSTAR of upstream and downstream primer, distilled water
22 μL.The PCR amplification uses the following conditions: 98 DEG C of 3 min, 98 DEG C of 10 s, 60 DEG C of 5 s, 72 DEG C of 5 min 30
S, 72 DEG C of 20 min, 30 circulations.For the plasmid pET 20b/pro-TGase retained using laboratory as template, P3 and P4 are to draw
Object carries out PCR, contains the genetic fragment containing TGase by PCR amplification.PCR amplification system is same as above, PCR condition are as follows: 98 DEG C
3 min, 98 DEG C of 10 s, 60 DEG C of 5 s, 72 DEG C of 1 min 20 s, 72 DEG C of 10 min, 30 circulations.Two kinds of PCR products
ThroughDpnGlue recycling is carried out after I digestion, recovery product is that 1:2 is mixed with molar ratio, uses One Step Cloning
After Kit is attached, conversionE.coliJM109, bacterium colony PCR screen positive transformant.Choose 2 positive transformants to be inoculated into
In LB liquid medium, 37 DEG C, 12 h are cultivated, the raw work in Shanghai is transferred to be sequenced, sequencing is correct to illustrate recombinant bacterium
PINA1297/pro (S.H) TGase is constructed successfully.By recombinant plasmid pINA1297/pro (S.H) TGase through fast enzyme cuttingNot I
Linearisation converts Yarrowia lipolytica po1h after glue recycling, after auxotroph culture medium YNB screening, obtains recombinant bacterium
po1h/pro(S.H)TGase。
1 primer of table
Glutamine transaminage hypersecretion ability production bacterial strain verifying: by recombinant bacterium pro (S.H) TGase of above-mentioned building with
Early period, the starting strain pro-TGase of building was inoculated in respectively in YPD fluid nutrient medium, and 28 DEG C, 200 rpm cultivate 24 h, secondary
Day transfers in Yarrowia lipolytica fermentation medium by 10% inoculum concentration, and 28 DEG C, 200 rpm shaking flasks (specification: 250 mL) training
Support 120 h.Fermentation liquid is in 4 DEG C, and 4000 rpm are centrifuged 10 min, and supernatant is extracellular crude enzyme liquid, after dispase is activated,
Survey enzyme activity.Detection discovery proenzyme area replaces with pro (S.H), and enzyme activity compared with starting strain improves 106 times afterwards, reaches 11.7 U/mL
(Fig. 1).The result of SDS-PAGE, which further demonstrates proenzyme area and replaces with pro (S.H), can significantly improve glutamine transaminage
Secreting, expressing amount (Fig. 2).
The present invention withS. mobaraensisBased on the aminotransierase gene of glutamine and its proenzyme area (pro) in source,
By by proenzyme area replace with streptomyces hygroscopicus (Streptomyces hygroscopicus) source proenzyme area (pro
(S.H)) glutamine transaminage, is realized in grade-safe bacterial strain --- Yarrowia lipolytica (Yarrowia lipolyticaPo1h the high efficient expression in) has obtained the superior strain of glutamine transaminage.The Yarrowia lipolytica
Using pINA1297 plasmid as carrier, expression contains the aminotransierase gene of glutamine of pro (S.H) proenzyme.
Particular embodiments described above, the technical issues of to solution of the invention, technical scheme and beneficial effects carry out
It is further described, it should be understood that the above is only a specific embodiment of the present invention, is not limited to
The present invention, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be included in this
Within the protection scope of invention.
Claims (3)
1. the glutamine transaminage mutant that a kind of secretion capacity improves, which is characterized in that the paddy that the secretion capacity improves
Glutamine transaminase mutant uses following culture medium:
LB culture medium: Yeast Extract 5, Tryptone 10, NaCl 10；
YPD culture medium: Yeast Extract 10, Tryptone 20, glucose 20；
YNB culture medium: YNB 6.7, glucose 20；
Solid medium is then the agar for adding 2% in liquid medium；
Fermentation medium: glycerol 15, yeast powder 20, ammonium chloride 2.64, potassium dihydrogen phosphate 0.32, anhydrous magnesium sulfate 0.25,
3.34 × 10-4 of vitamin B1 adjusts pH to 8.0；
The building that proenzyme area replaces efficient expression strain is as follows: the plasmid pET 20b/pro-TGase retained using laboratory is mould
Plate, P3 and P4 are that primer carries out PCR, contain the genetic fragment containing TGase by PCR amplification.
2. the glutamine transaminage mutant that secretion capacity as described in claim 1 improves, which is characterized in that the secretion
The glutamine transaminage mutant that ability improves uses the enzyme activity of colorimetric method for determining glutamine transaminage.
3. the glutamine transaminage mutant that secretion capacity as described in claim 1 improves, which is characterized in that the YPD
The starting strain pro-TGase of inoculation of medium recombinant bacterium pro (S.H) TGase and building early period.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961845A (en) * | 2021-03-08 | 2021-06-15 | 上海交通大学 | Method for improving fermentation level of glutamine transaminase by knocking out cslA gene |
| CN114686410A (en) * | 2020-12-25 | 2022-07-01 | 江苏东汇生物科技有限公司 | Glutamine transaminase high-producing strain for enhancing 1-phosphofructokinase gene transcription level and preparation and fermentation methods thereof |
| CN114686389A (en) * | 2020-12-25 | 2022-07-01 | 江苏东汇生物科技有限公司 | Glutamine transaminase high-producing strain for enhancing vgbS gene transcription level and preparation and fermentation methods thereof |
| CN115029290A (en) * | 2022-03-17 | 2022-09-09 | 上海交通大学 | Method for inhibiting expression of non-essential high-abundance protein to improve fermentation level of TG enzyme |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574159A (en) * | 2017-10-26 | 2018-01-12 | 江南大学 | A kind of mutant for the glutamine transaminage expressed in an active |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574159A (en) * | 2017-10-26 | 2018-01-12 | 江南大学 | A kind of mutant for the glutamine transaminage expressed in an active |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686410A (en) * | 2020-12-25 | 2022-07-01 | 江苏东汇生物科技有限公司 | Glutamine transaminase high-producing strain for enhancing 1-phosphofructokinase gene transcription level and preparation and fermentation methods thereof |
| CN114686389A (en) * | 2020-12-25 | 2022-07-01 | 江苏东汇生物科技有限公司 | Glutamine transaminase high-producing strain for enhancing vgbS gene transcription level and preparation and fermentation methods thereof |
| CN114686410B (en) * | 2020-12-25 | 2023-11-21 | 泰兴市东圣生物科技有限公司 | Glutamine transaminase high-yield strain for enhancing transcription level of 1-phosphofructokinase gene and preparation and fermentation methods thereof |
| CN114686389B (en) * | 2020-12-25 | 2024-05-07 | 泰兴市东圣生物科技有限公司 | Glutamine transaminase high-yield strain for enhancing transcription level of vgbS gene and preparation and fermentation methods thereof |
| CN112961845A (en) * | 2021-03-08 | 2021-06-15 | 上海交通大学 | Method for improving fermentation level of glutamine transaminase by knocking out cslA gene |
| CN112961845B (en) * | 2021-03-08 | 2022-06-28 | 上海交通大学 | Method for improving fermentation level of glutamine transaminase by knocking out cslA gene |
| CN115029290A (en) * | 2022-03-17 | 2022-09-09 | 上海交通大学 | Method for inhibiting expression of non-essential high-abundance protein to improve fermentation level of TG enzyme |
| CN115029290B (en) * | 2022-03-17 | 2024-05-28 | 上海交通大学 | Method for inhibiting expression of nonessential high-abundance proteins to increase fermentation level of TG enzyme |
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