CN109811001A - The mutant of high efficient expression glutamine transaminage - Google Patents
The mutant of high efficient expression glutamine transaminage Download PDFInfo
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- CN109811001A CN109811001A CN201910077027.3A CN201910077027A CN109811001A CN 109811001 A CN109811001 A CN 109811001A CN 201910077027 A CN201910077027 A CN 201910077027A CN 109811001 A CN109811001 A CN 109811001A
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- glutamine transaminage
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- efficient expression
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Abstract
The invention discloses a kind of mutant of high efficient expression glutamine transaminage (TGase), building process include: using laboratory save plasmid pINA1297/N355Q as template, using P1 and P2 be primer progress PCR obtain containing streptomyces hygroscopicus (Streptomyces hygroscopicus) source TGase proenzyme area (pro (S.H)) gene；WithS. mobaraensisGenome is template, and amplification obtainsS. mobaraensis TGase gene；Pro (S.H) is connect with TGase gene by fusion DNA vaccine, is expressed in grade-safe bacterial strain --- Yarrowia lipolytica (Yarrowia lipolyticaPo1h), the superior strain of TGase has been obtained.
Description
Technical field
The present invention relates to a kind of mutant, more particularly to a kind of mutant of high efficient expression glutamine transaminage.
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-MTG), need to be through egg
The excision such as the white enzyme dispase end N- proenzyme area (pro) could convert Viability MTG.Proenzyme area (pro) is located at signal peptide and maturation
Between enzyme, 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 MTG high efficient expression, but most hosts are non-food stuff
Safe bacterial strain.In addition, the country is used for the bacterial strain strain Genomic instability of production at present, a large amount of personnel are needed to carry out in production multiple
Unskilled labourer is made.Therefore, how to stablize in food safety bacterial strain, efficiently express glutamine transaminage, being urgently to be resolved at present
The problem of.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of mutant of high efficient expression glutamine transaminage, withS. mobaraensisBased on the aminotransierase gene of glutamine and its proenzyme area (pro) in source, by the way that proenzyme area is replaced
For streptomyces hygroscopicus (Streptomyces hygroscopicus) source proenzyme area (pro (S.H)), realize glutamine
Transaminase is in grade-safe bacterial strain --- Yarrowia lipolytica (Yarrowia lipolyticaPo1h the high efficient expression in),
The superior strain of glutamine transaminage is obtained.
The present invention is to solve above-mentioned technical problem by following technical proposals: a kind of high efficient expression glutamine turns ammonia
The mutant of enzyme, which is characterized in that the plasmid that the mutant of the high efficient expression glutamine transaminage is retained with laboratory
PINA1297/N355Q is template, and P1 and P2 are that primer carries out PCR, contains the 1297 of pro (S.H) proenzyme area by PCR amplification
Expression vector.
Preferably, the PCR amplification uses following system: 1 μ L of template, each 1 μ L, primeSTAR 25 of upstream and downstream primer
μ L, 22 μ L of distilled water.
Preferably, the PCR amplification is using the following conditions: 98 DEG C of 3 min, 98 DEG C of 10 s, 60 DEG C of 5 s, and 72 DEG C
5 min 30 s, 72 DEG C of 20 min, 30 circulations.
Preferably, the mutant of the high efficient expression glutamine transaminage uses colorimetric method for determining glutamine transaminage
Enzyme activity.
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 proMTG: 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-mTG retained using laboratory as template, P3 and P4 are primer
PCR is carried out, the genetic fragment containing MTG is contained 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 product warpsDpnGlue recycling is carried out after I digestion, recovery product is that 1:2 is mixed with molar ratio, uses One Step Cloning Kit
After being attached, conversionE.coliJM109, bacterium colony PCR screen positive transformant.Choose 2 positive transformants and is inoculated into LB liquid
In body culture 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) MTG is constructed successfully.By recombinant plasmid pINA1297/pro (S.H) MTG through fast enzyme cuttingNotI linearisation, glue recycling
Yarrowia lipolytica po1h is converted afterwards, after auxotroph culture medium YNB screening, is obtained recombinant bacterium po1h/pro (S.H)
MTG。
1 primer of table
The verifying of glutamine transaminage hypersecretion ability production bacterial strain: by recombinant bacterium pro (S.H) MTG of above-mentioned building with before
Phase, the starting strain pro-MTG of building was inoculated in respectively in YPD fluid nutrient medium, and 28 DEG C, 200 rpm cultivate 24 h, and next day presses
10% inoculum concentration is transferred in Yarrowia lipolytica fermentation medium, and 28 DEG C, 200 rpm shaking flasks (specification: 250 mL) culture
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, surveys
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(figure
1).The result of SDS-PAGE further demonstrate proenzyme area replace with pro (S.H) can significantly improve glutamine transaminage point
Secrete expression quantity (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 (4)
1. a kind of mutant of high efficient expression glutamine transaminage, which is characterized in that the high efficient expression glutamine turns ammonia
For the plasmid pINA1297/N355Q that the mutant of enzyme is retained using laboratory as template, P1 and P2 are that primer carries out PCR, pass through PCR
Amplification contains 1297 expression vectors in pro (S.H) proenzyme area.
2. the mutant of high efficient expression glutamine transaminage as described in claim 1, which is characterized in that the PCR amplification
Using following system: 1 μ L of template, upstream and downstream primer each 25 μ L of 1 μ L, primeSTAR, 22 μ L of distilled water.
3. the mutant of high efficient expression glutamine transaminage as described in claim 1, which is characterized in that the PCR amplification
Using 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.
4. the mutant of high efficient expression glutamine transaminage as described in claim 1, which is characterized in that the high efficient expression
The mutant of glutamine transaminage uses the enzyme activity of colorimetric method for determining glutamine transaminage.
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