CN114686409A - Method for enhancing expression of superoxide dismutase gene and increasing glutamine transaminase yield - Google Patents
Method for enhancing expression of superoxide dismutase gene and increasing glutamine transaminase yield Download PDFInfo
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- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
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- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
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Abstract
The invention discloses a glutamine transaminase high-yield strain TGS101 for enhancing the transcription level of a Superoxide dismutase (Superoxide dismutase. Ni) gene SMDS _2075 and a preparation and fermentation method thereof, wherein based on genomics, an endogenous Superoxide dismutase. Ni gene SMDS _2075 is overexpressed by utilizing an artificial strong promoter kasOp in Streptomyces mobaraensis C2 to obtain a mutant strain TGS101 of the high-yield glutamine transaminase, and the yield of the glutamine transaminase can be obviously improved. The final yield of the glutamine transaminase fermentation of the engineering strain TGS101 obtained by the invention is increased by 31.30% compared with a control strain in a laboratory shake flask level, and the fermentation cost is greatly reduced.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a method for improving the expression of superoxide dismutase (Superoxidadedimutase. Ni) gene SMDS _2075 and increasing the yield of glutamine transaminase.
Background
Transglutaminase (TGase) is produced by Streptomyces mobaraensis (Streptomyces mobaraensis), an enzyme that catalyzes acyl transfer reactions, which catalyze intramolecular or intermolecular crosslinking of proteins, linkage between proteins and amino acids, and hydrolysis of glutamine groups within protein molecules, thereby improving the structural and functional properties of proteins. The TGase derived from microorganisms belongs to extracellular enzyme, can be directly secreted into a culture medium by fermentation microorganisms, has the molecular weight of 37.9kD, consists of 331 amino acids, exists in the form of pre-pro-TGase in cells, and is transported to the outside of the cells in the form of precursor protein of the pro-TGase by transmembrane transport and recognition of signal peptide. Pro-TGase has no catalytic activity, and is cleaved by metalloprotease TAMEP to form FRAP-TGase, and then cleaved by serine protease SM-TAP to form the final mature TGase.
TGase is widely applied as a protein cross-linking agent due to the advantages of good stability, safe use and the like, and can be used for meat processing in the field of food to obviously improve the texture of low-meat products, for example, the covalent cross-linking generated by the acyl transfer reaction catalyzed by TGase can enhance the texture, gel strength, elasticity and water-retaining property of meat products without adverse effect; casein, lactoglobulin and the like contained in the dairy product are good substrates of TGase, because the TGase is easy to combine with the casein due to the low-degree tertiary structure, random spiral arrangement and disulfide bonds of the casein, and the TGase can improve the rheological property of the casein under the condition of not changing the functional characteristics of the casein and catalyze the casein to form a micelle structure; the black pepper essential oil capsule is prepared by a complex coacervation method by adopting TGase as a cross-linking agent, and the cross-linking agent has good nuclear protection effect on the black pepper essential oil capsule, and can be used for transporting active ingredient terpene or for systemic delivery of other bioactive ingredients and the like. Due to the reaction characteristic of TGase, the TGase has great application prospect in the textile industry, the biotechnology field and the medicine field.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a glutamine transaminase high-producing strain TGS101 for enhancing the transcription level of a Superoxide dismutase (Superoxide dismutase. Ni) gene SMDS _2075 and a preparation and fermentation method thereof, wherein the yield of the glutamine transaminase can be improved by over-expressing the endogenous Superoxide dismutase. Ni gene SMDS _2075 by using an artificial strong promoter kasOp in Streptomyces mobaraensis C2 based on genomics.
The superoxydismutase. Ni gene SMDS _2075 overexpression mutant strain and the transglutaminase high-producing strain referred to in the present invention are both TGS 101.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a Streptomyces mobaraensis C2 which is obtained by strain mutagenesis of Taixing east sage biotechnology limited, the classification name of the strain is Streptomyces mobaraensis C2, the strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2020194, the preservation date is 2020, 6 and 10.
The invention also provides a strain TGS101 with high TGase yield, which is obtained by enhancing the transcription level of a Superoxide dismutase.
Furthermore, the strain is obtained by over-expressing a Superoxide diversity. Ni gene SMDS _2075 derived from Streptomyces mobaraensis C2 by Streptomyces mobaraensis C2.
Further, the strain is obtained by over-expressing an endogenous Superoxide diversity. Ni gene SMDS _2075 in Streptomyces mobaraensis C2 by using an artificial strong promoter kasOp.
Superoxide mutase. Ni gene SMDS _2075 of the strain is over-expressed.
"overexpression" refers to inserting a copy of Superoxide diversity. Ni gene SMDS _2075 from Streptomyces mobaraensis C2 on chromosome of recipient bacterium Streptomyces mobaraensis C2, and enhancing the transcription level of Superoxide diversity. Ni gene SMDS _ 2075.
The strain contains an artificial strong promoter kasOp for over-expressing an expression cassette of a Superoxide diversity. Ni gene SMDS _2075 derived from Streptomyces mobaraensis C2.
The expression cassette contains artificial strong promoter kasOp, Superoxide mutase. Ni gene SMDS _2075 and transcription termination sequence.
The sequence of the Superoxide diversity. Ni gene SMDS _2075 is shown in SEQ ID No. 1.
The present invention also provides an expression cassette comprising: artificial strong promoter kasOp, Superoxide mutase. Ni gene SMDS _2075, transcription termination sequence.
The invention also provides application of the expression cassette in efficient screening of the strain TGS 101.
The invention also provides a method for enhancing the transcription level of Superoxide mutase Ni gene SMDS _2075 to improve the fermentation level of glutamine transaminase, which comprises the following steps:
the method comprises the following steps: designing and constructing an integrated plasmid vector pTDS101 for over-expressing Superoxide mutase. Ni gene SMDS _ 2075; the sequence of the Superoxide diversity. Ni gene SMDS _2075 is shown in SEQ ID No. 1;
step two: inserting a copy of Superoxide diversity. Ni gene SMDS _2075 derived from Streptomyces mobaraensis C2 on a receptor Streptomyces mobaraensis C2 chromosome by using an integrative plasmid vector pTDS101 (phi C31 integration site, pSET152 derived and provided with a kasOp promoter), and screening a recombinant mutant strain TGS101 with gene overexpression through resistance and PCR verification;
step three: inoculating spores of a transglutaminase high-producing strain TGS101 obtained by overexpression of activated Superoxide diversity. Ni gene SMDS _2075 into a seed culture medium, culturing for 20-24h (preferably 24h) under the conditions of 25-35 ℃, 180-220rpm (preferably 30 ℃, 200rpm), transferring the inoculated amount of 8-15% (preferably 10%) into a fermentation culture medium, fermenting for 28-32h (preferably 28h) under the conditions of 25-35 ℃, 180-220rpm (preferably 30 ℃, 200rpm), collecting the fermentation broth, and performing enzyme activity determination.
In the first step, the construction method of the integrative plasmid vector pTDS101 is as follows: obtaining a 396bp PCR fragment of a Superoxide dismutase. Ni gene SMDS _2075 sequence through PCR amplification, and connecting the PCR fragment into an NdeI/EcoRI site of the integrative plasmid pDR3-K by an enzyme digestion connection method to obtain the integrative plasmid vector pTDS 101.
In the third step, the seed culture medium comprises 1-3 w/v% of glycerol, 0.5-1 w/v% of yeast extract, 2-3 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.5w/v％，K2HPO4·3H2O0.1-0.5 w/v%, pH 7.4; preferably, the concentration is 2 w/v% of glycerol, 0.6 w/v% of yeast extract, 2.5 w/v% of fish meal peptone and MgSO4·7H2O 0.2w/v％，K2HPO4·3H2O 0.2w/v％， pH 7.4。
In the third step, the fermentation medium comprises 1-3 w/v% of glycerol, 0.5-1 w/v% of yeast extract, 2-3 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.5w/v％，K2HPO4·3H20.1-0.5 w/v% of O, 0.1-0.3 w/v% of fermentation accelerator and pH 7.4; preferably, 2 w/v% glycerol, 0.6 w/v% yeast extract, 2.5 w/v% fish meal peptone, MgSO4·7H2O 0.2w/v％，K2HPO4·3H2O0.2 w/v%, fermentation accelerator 0.1 w/v%, pH 7.4.
The invention also provides a preparation method of the glutamine transaminase high-producing strain TGS101, which comprises the following steps:
the method comprises the following steps: designing and constructing an integrated plasmid vector pTDS101 for over-expressing an endogenous Superoxide dismutase. Ni gene SMDS _ 2075; the sequence of the Superoxide diversity. Ni gene SMDS _2075 is shown in SEQ ID No. 1;
step two: a copy of Superoxide mutase. Ni gene SMDS _2075 (from Streptomyces mobaraensis C2) was inserted into the recipient Streptomyces mobaraensis C2 chromosome using an integrative plasmid vector pTDS101(Φ C31 integration site, pSET152 derived with kasOp promoter), and a recombinant mutant strain TGS101 with gene overexpression was selected by resistance and PCR verification.
In the first step, the construction method of the integrative plasmid vector pTDS101 is as follows: obtaining a 396bp PCR fragment of the Superoxide mutase Ni gene SMDS _2075 sequence through PCR amplification, and connecting the fragment into NdeI/EcoRI sites of an integrative plasmid pDR3-K by an enzyme digestion connection method to obtain an integrative plasmid vector pTDS 101.
The invention also provides a Superoxide diversity. Ni gene SMDS _2075, wherein the sequence of the Superoxide diversity. Ni gene SMDS _2075 is shown as SEQ ID No. 1.
The invention also provides a gene sequence for expressing the Superoxide diversity Ni gene SMDS _2075, wherein the gene sequence is a nucleotide sequence of an artificial strong promoter kasOp or a nucleotide sequence with more than 90% of homology with the promoter kasOp, and the nucleotide sequence of the promoter kasOp is shown as SEQ ID NO. 2.
The invention also provides a construction method of the plasmid vector pTDS101, which comprises the steps of obtaining a 396bp PCR fragment of the Superoxide diversity. Ni gene SMDS _2075 sequence through PCR amplification, and connecting the PCR fragment to NdeI/EcoRI sites of the integrative plasmid pDR3-K by an enzyme digestion connection method to obtain the integrative plasmid vector pTDS 101.
The invention also provides application of the Streptomyces mobaraensis C2 in screening of a high-producing strain TGS101 of transglutaminase.
The invention also provides application of the high-yield glutamine transaminase bacterial strain TGS101 in improving the fermentation yield of glutamine transaminase.
The plasmid pDR3-K according to the invention is described in SCI database literature "Xinjuan Ning, Xinran Wang, stabilizing Wu, Qianjin Kang and Linquan Bai:identificationand Engineering of Post-PKS Modification Bottlenecks for analysis of P-3Titer Improvement in Actinosynnema prediction subsp.m. prediction mAC 31280, Biotechnology journal 2017,12, 1700484.
The strain Streptomyces mobaraensis C2 is obtained by strain mutagenesis of Taixing east Sheng Biotechnology Limited company, is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2020194, accession number 2020.6.10.
The beneficial effects of the invention include: by respectively inserting a copy of a Superoxide mutase. Ni gene SMDS _2075 derived from Streptomyces mobaraensis C2 into a chromosome of Streptomyces mobaraensis C2 by using an integrative vector pDR3-K in Streptomyces mobaraensis C2, the enzyme activity is improved by 31.30 percent compared with that of a control strain at the laboratory shake flask level. The invention can obviously improve the fermentation yield of the TGase and greatly reduce the fermentation cost.
Drawings
FIG. 1 is a schematic diagram of construction of an SMDS _2075 gene overexpression plasmid;
FIG. 2 is a schematic diagram showing the fermentation yields of a Superoxide diversity. Ni gene SMDS _2075 enhanced expression mutant strain and a control strain TGase;
FIG. 3 is a SDS-PAGE result of the Superoxide mutase Ni gene SMDS _2075 enhanced expression mutant strain and a control strain. Wherein, M represents a protein Marker; lane 1 represents the SDS-PAGE result of Streptomyces mobaraensis C2; lane 2 represents the results of SDS-PAGE of mutant strains with enhanced expression of Superoxide diversity ni gene SMDS _ 2075.
Detailed Description
The invention is further described in detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1
This example is a specific process for preparing Superoxide diversity. ni gene SMDS _2075 overexpressed mutant strain TGS101, specifically including the following steps:
the first step is as follows: construction of plasmid pTDS 101: a gene fragment of SMDS _2075(396bp) was obtained by PCR amplification using Streptomyces mobaraensis C2 genomic DNA as a template and primers SMDS _2075-F/R with NdeI/EcoRI cleavage sites introduced at both ends. The amplified digested fragment was inserted into NdeI/EcoRI site of plasmid pDR3-K to obtain plasmid pTDS 101.
The endonuclease recognition sites (restriction sites) involved in the first step are as follows:
NdeI recognition site: an EcoRI recognition site:
5'...CA^TATG...3' 5'...G^AATTC...3'
3'...GTAT^AC...5' 3'...CTTAA^G...5'
the primer sequences used in the first step were:
PCR system and conditions used for gene fragment preparation in the first step:
and (3) PCR reaction system: 30ng of DNA template, 20pmol of primer F/R, 5 mu L of 50% DMSO, 10nmol of dNTP, 25 mu L of buffer solution and 1 unit of Taq DNA polymerase, and adding pure water to make up to 30 mu L;
PCR conditions were as follows: 5min at 95 ℃; circulating for 30 times at 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 30 s; 10min at 72 ℃.
The second step is that: a mutant strain with over-expressed SMDS _2075 gene is obtained by inserting a copy of Superoxide dismutase. Ni gene SMDS _2075 (derived from Streptomyces mobaraensis C2) on the chromosome of a recipient strain Streptomyces mobaraensis C2 by using an integrative plasmid vector pTDS101 (phi C31 integration site, pSET152 derived, with a kasOp promoter), and screening a correct zygote through resistance and PCR verification, wherein the mutant strain comprises the following steps:
1) transforming the gene-overexpressed plasmid pTDS101 into a host ET12567(pUZ8002), inoculating the corresponding ET12567(pUZ8002) into LB containing three antibiotics Apr, Kan and Chl, culturing at 37 ℃ for 20h, and then rinsing the thalli with fresh LB solution to remove the antibiotics in the culture; simultaneously preparing a C2 spore pre-germination solution, collecting C2 spores growing for 7 days, thermally shocking at 50 ℃ for 10min, adding a 2 XYT culture medium, pre-germinating at 37 ℃ for 2h, and rinsing with a fresh LB culture medium for 2 times;
the LB culture medium comprises the following components: tryptone 1 w/v%, yeast extract 0.5 w/v%, NaCl 1 w/v%, pH 7.0; the 2 XYT culture medium comprises the following components: tryptone 1.6 w/v%, yeast extract 1 w/v%, NaCl 0.5 w/v%, pH 7.0.
2) The C2 spore pre-germination fluid was mixed with the previously prepared host bacterium ET12567(pUZ8002) (the ratio of recipient bacterium cells C2 and donor bacterium ET12567(pUZ8002) was about 1: 10) after being homogenized, the mixture is spread on an ISP4 solid culture medium containing 10mM magnesium ions and is inversely cultured for 16h at the temperature of 30 ℃;
the above-mentionedThe components of ISP4 solid medium are: soluble starch 1 w/v%, MgSO4·7H2O 0.1w/v％、(NH4)2SO4 0.2w/v％、FeSO4·7H2O 0.0001w/v％、K2HPO40.1 w/v％、NaCl 0.1w/v％、CaCO30.2 w/v％、MnCl2·4H2O 0.0001w/v％、ZnSO4·7H2O0.0001 w/v%, agar 2 w/v%, pH 7.0-7.4.
3) After 16h, the plate is taken out, two antibiotics of apramycin (with the final concentration of 50 mu g/mL) and nalidixic acid (with the final concentration of 25 mu g/mL) are respectively added into 1mL of sterile water, are uniformly mixed, are covered on a joint transfer plate, are dried by air and are transferred to a 30 ℃ incubator for inverted culture. Generally, after 3-5 days, a zygote grows out from the flat plate, the zygote is transferred to a Gaoshi I solid culture medium containing two antibiotics, namely apramycin and nalidixic acid for amplification culture, and a mutant strain with a Superoxide dismutase Ni gene SMDS _2075 doubled is obtained through mycelium PCR verification and screening;
the solid culture medium of Gao's I number comprises the following components: soluble starch 2 w/v%, MgSO4·7H2O 0.05w/v％、KNO3 0.1w/v％、FeSO4·7H2O 0.001w/v％、K2HPO40.05 w/v%, NaCl 0.05 w/v%, agar 2 w/v%, pH 7.2-7.4.
In the second step, the zygospore mycelium was used as a DNA template, and the PCR system and conditions used for screening mutants were verified by PCR using primers smds _2075-F/R with NdeI/EcoRI cleavage sites introduced at both ends:
and (3) PCR system: 10-100 ng of DNA template, 10pmol of primer F/R, 2 mu L of 50% DMSO and 10 mu L of 2 xMix buffer solution, and adding pure water to make up to 20 mu L;
the 2 xmix buffer comprises the following components: taq DNA Polymerase (recombiant) 0.05 units/. mu. L, MgCl24 mM、dNTPs(dATP、dCTP、dGTP、dTTP)0.4mM；
PCR conditions were as follows: 10min at 95 ℃; circulating for 30 times at 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 30 s; 10min at 72 ℃.
Example 2
This example is a process of producing TGase by fermentation of mutant TGS101 with overexpression of Superoxide diversity. ni gene SMDS _2075, and the specific steps are as follows: respectively coating the strain TGS101 with overexpression of Superoxide mutase. Ni on a Gauss I culture medium for activation, culturing for 7d at 30 ℃, scraping a flat spore, inoculating the flat spore into a seed culture medium, culturing for 24h at 30 ℃ and 200rpm, transferring 10 percent of inoculum size into a fermentation culture medium, fermenting for 28h at 30 ℃ and 200rpm, and collecting fermentation liquor for enzyme activity determination and SDS-PAGE detection.
Example 3
The embodiment is a method for detecting the enzyme activity of TGase by using a colorimetric method, which specifically comprises the following steps:
1) 200 mu L of the supernatant of the fermentation broth diluted 20 times was placed in two test tubes, one of which was added 200. mu.L of water as a control, and the other was added 2mL of solution A preheated at 37 ℃ for reaction at 37 ℃ for 10min, and then 2mLB solution was added to terminate the reaction.
2) The absorbance of the fermentation broth was measured at 525nm of a spectrophotometer using a 1cm quartz cuvette. Finally will OD525And substituting the formula obtained by conversion of the standard curve to calculate the enzyme activity of the TGase.
The solution preparation method comprises the following steps:
solution A: 9.688g of tris (hydroxymethyl) aminomethane, 2.780g of hydroxylamine hydrochloride, 1.229g of reduced glutathione and 4.048 g of a substrate Na-CBZ-GLN-GLY were weighed into a beaker, 350mL of water was added, the pH was adjusted to 6.0, and the volume was adjusted to 400mL by adding water.
And B, liquid B: 3mol/L hydrochloric acid, 12% trichloroacetic acid and 5% FeCl3Dissolved in 0.1mol/LHCl and the three solutions are mixed homogeneously in equal amounts.
FIG. 2 is a schematic diagram of relative fermentation yields of Superoxide mutase Ni gene SMDS _2075 expression-enhanced mutant strain and a control strain TGase. The results show that the yield of the mutant strain is improved by 31.30 percent compared with the wild strain under the laboratory shake flask level.
FIG. 3 is a schematic diagram of SDS-PAGE detection results of a Superoxide mutant gene SMDS _2075 expression-enhanced mutant strain and a control strain C2. Wherein, M represents a protein Marker; lane 1 represents the SDS-PAGE result of Streptomyces mobaraensis C2; lane 2 represents the results of SDS-PAGE of the mutant with enhanced expression of Superoxide mutase. Ni gene SMDS _ 2075. The results show that the content of mature TGase of the mutant strain is increased compared with that of the control strain.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
SEQUENCE LISTING
<110> Jiangsu Donghui Biotech Co., Ltd
<120> method for increasing glutamine transaminase yield by enhancing expression of superoxide dismutase gene
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 396
<212> DNA
<213> Artificial sequence
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atgctttccc gcctgttcgc ccccaaggtg aaggtcagcg cccactgcga cctgccctgc 60
ggcgtgtacg acccggccca ggcccgcatc gaggcggaat cggtcaaggc cgtccaggag 120
aagtaccagg ccaacgagga cccccacttc cgtgcccgcg ccacgatcat caaggagcag 180
cgcgcggagc tcgccaagca ccacgtctcg gtgctgtgga gcgactactt caaggcgccg 240
cacttcgaga agtaccccca gttgcaccag ctggtcaatg acaccctgaa ggcgctgagc 300
gccgccaagg cgtcgaccga cccgaagacg ggcgagaagg cgctggagct catcgccgag 360
atcgaccgta tcttctggga gaccaagaag gcgtaa 396
<210> 2
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tgttcacatt cgaacggtct ctgctttgac aacatgctgt gcggtgttgt aaagtcgtgg 60
ccaggagaat acgacagcgt gcaggactgg gggagtt 97
<210> 3
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<212> DNA
<213> Artificial sequence
<400> 3
atatcatatg atgctttccc gcctgttcgc 30
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence
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atatgaattc ttacgccttc ttggtctccc 30
Claims (14)
1. A glutamine transaminase highly producing strain TGS101 obtained by enhancing the transcription level of the Superoxide dismutase ni gene SMDS _ 2075.
2. The transglutaminase producing strain TGS101 as claimed in claim 1, wherein said strain is obtained after overexpression of Superoxide mutase ni gene SMDS _2075 derived from streptomyces mobaraensis C2 using artificial strong promoter kasOp in streptomyces mobaraensis C2;
the Streptomyces mobaraensis C2 is classified as Streptomyces mobaraensis C2 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2020194, the preservation date is 2020, 6 and 10.
3. The transglutaminase producing strain TGS101 of claim 1, wherein said strain is overexpressing the Superoxide dismutase.ni gene SMDS _ 2075; the sequence of the Superoxide diversity. Ni gene SMDS _2075 is shown in SEQ ID NO. 1.
4. The transglutaminase producing strain TGS101 as claimed in claim 1, wherein said strain comprises an expression cassette for overexpression of a Superoxide mutase Ni gene SMDS _2075 derived from Streptomyces mobaraensis C2 from an artificial strong promoter kasOp.
5. The transglutaminase producing strain TGS101 as claimed in claim 4, wherein said expression cassette comprises an artificial strong promoter kasOp, a Superoxide mutase. Ni gene SMDS _2075, and a transcription termination sequence.
6. A method for enhancing the transcription level of Superoxide diversity. Ni gene SMDS _2075 to improve the fermentation level of glutamine transaminase, which is characterized by comprising the following steps:
the method comprises the following steps: designing and constructing an integrated plasmid vector pTDS101 for over-expressing Superoxide mutase. Ni gene SMDS _ 2075; the sequence of the Superoxide mutase.Ni gene SMDS _2075 is shown as SEQ ID No. 1;
step two: inserting a copy of a Superoxide diversity. Ni gene SMDS _2075 derived from Streptomyces mobaraensis C2 as claimed in claim 2 into a recipient strain Streptomyces mobaraensis C2 chromosome by using an integrative plasmid vector pTDS101, and screening to obtain a recombinant mutant strain TGS101 with gene overexpression through resistance and PCR verification;
step three: inoculating spores of a glutamine transaminase high-producing strain TGS101 with over-expressed cultured Superoxide dismutase Ni gene SMDS _2075 into a seed culture medium, culturing for 20-24h under the conditions of 25-35 ℃ and 180-220rpm, transferring 8-15% of inoculum size into a fermentation culture medium, fermenting for 28-32h under the conditions of 25-35 ℃ and 180-220rpm, collecting fermentation liquor and carrying out enzyme activity detection.
7. The method of claim 6, wherein in step one, the integrative plasmid vector pTDS101 is constructed by: obtaining a 396bp PCR fragment of a Superoxide dismutase. Ni gene SMDS _2075 sequence through PCR amplification, and connecting the PCR fragment into an NdeI/EcoRI site of the integrative plasmid pDR3-K by an enzyme digestion connection method to obtain the integrative plasmid vector pTDS 101.
8. The method of claim 6, wherein the seed medium comprises glycerol 1-3 w/v%, yeast extract 0.5-1 w/v%, fish meal peptone 2-3 w/v%, MgSO4·7H2O 0.1-0.5w/v％，K2HPO4·3H2O 0.1-0.5w/v％，pH 7.4；
The fermentation culture medium comprises 1-3 w/v% of glycerol, 0.5-1 w/v% of yeast extract, 2-3 w/v% of fish meal peptone and MgSO 24·7H2O 0.1-0.5w/v％，K2HPO4·3H20.1-0.5 w/v% of O, 0.1-0.3 w/v% of fermentation accelerator and pH 7.4.
9. A preparation method of a glutamine transaminase high-producing strain TGS101 is characterized by comprising the following steps:
the method comprises the following steps: designing and constructing an integrated plasmid vector pTDS101 for over-expressing Superoxide mutase. Ni gene SMDS _ 2075; the sequence of the Superoxide diversity. Ni gene SMDS _2075 is shown in SEQ ID No. 1;
step two: a copy of the Superoxide mutant. Ni gene SMDS _2075 from Streptomyces mobaraensis C2 as claimed in claim 2 was inserted into the recipient strain Streptomyces mobaraensis C2 chromosome using the integrative plasmid vector pTDS101 and screened for the recombinant mutant strain TGS101 with gene overexpression by resistance and PCR validation.
10. The method for preparing transglutaminase producing strain TGS101 of claim 9, wherein in step one, the integrative plasmid vector pTDS101 is constructed by: obtaining a 396bp PCR fragment of a Superoxide dismutase. Ni gene SMDS _2075 sequence through PCR amplification, and connecting the PCR fragment into an NdeI/EcoRI site of the integrative plasmid pDR3-K by an enzyme digestion connection method to obtain the integrative plasmid vector pTDS 101.
11. A gene sequence for expressing Superoxide diversity ni gene SMDS 2075, wherein the gene sequence is a nucleotide sequence of an artificial strong promoter kasOp or a nucleotide sequence having more than 90% homology with the promoter kasOp, and the nucleotide sequence of the promoter kasOp is represented by SEQ ID No. 2.
12. Use of the high-producing strain of transglutaminase TGS101 of claim 1 for increasing the fermentation yield of transglutaminase.
13. An expression cassette comprising: artificial strong promoter kasOp, Superoxide mutase. Ni gene SMDS _2075, transcription termination sequence.
14. Use of the expression cassette of claim 13 for efficient screening of strain TGS 101.
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