CN105087510A - Fermentation preparation method of recombinant Cu/Zn-SOD - Google Patents

Fermentation preparation method of recombinant Cu/Zn-SOD Download PDF

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CN105087510A
CN105087510A CN201510585981.5A CN201510585981A CN105087510A CN 105087510 A CN105087510 A CN 105087510A CN 201510585981 A CN201510585981 A CN 201510585981A CN 105087510 A CN105087510 A CN 105087510A
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sod
fermentation
recombinant
carry out
purification
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林娟
吕橄
李仁宽
叶秀云
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Fuzhou University
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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a fermentation preparation method of recombinant Cu/Zn-SOD. According to the method, pichia pastoris engineering bacteria GS115-pPIC9K capable of effectively expressing spotted deer cornu cervi pantotrichum source Cu/Zn-SOD genes are used as production strains for seed culture under the seed culture conditions that the culture medium is a YPD culture medium; the pH is 6.0; the temperature is 30 DEG C; the rotating speed is 200r/min; and the culture time is 15h; then, fermentation is performed; the fermentation culture medium is a BMMY culture medium, and the pH is 6.0; the inducted enzyme production conditions include the inoculation amount being 5 percent, the initial methanol adding amount being 1 percent, the methanol supplementing amount being 1 percent every 24h, the rotating speed being 200r/min, the culture time being 96h at 30 DEG C and the recombinant SOD activity being 1876U/mL; and fermentation liquid is subjected to separation and purification to obtain electrophoresis pure Cu/Zn-SOD. Through the fermentation production of engineering microbes, a great amount of Cu/Zn-SOD can be prepared. the method has the advantages that the relaying on natural resources can be reduced; the adverse influence due to animal diseases can be eliminated; and the application safety of superoxide dismutase is improved.

Description

The fermentation preparation of a kind of recombinant C u/Zn-SOD
Technical field
The invention belongs to bioengineering field, be specifically related to the fermentation preparation of a kind of recombinant C u/Zn-SOD.
Technical background
Superoxide-dismutase (SOD, EC1.15.1.1) be first antioxidase played a role in body active oxygen radical cleaning reaction process, SOD can remove human free radical, delaying senility, and there is good prevention and therapy effect the aspect such as tumour, cardiovascular and cerebrovascular diseases, eye disease, acquired immune deficiency syndrome (AIDS), diabetes, inflammation of other diseases caused by free radical as serious harm HUMAN HEALTH.Simultaneously because SOD has good senile-resistant efficacy, also can be applicable in the industries such as makeup, functional foodstuff, dental products, daily chemical products.
Different according to the kind of bind metal ion, superoxide-dismutase can be divided into Four types: copper-zinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), iron superoxide dismutase (Fe-SOD) and nickel superoxide dismutase (Ni-SOD).Wherein Cu/Zn-SOD is extensively present in eukaryotic cells matter, chloroplast(id) and the peroxisomes such as most Mammals and fungi, be occurring in nature distribution the most a kind of superoxide-dismutase, be also the superoxide-dismutase be most widely used at present simultaneously.
To be that China's one-level emphasis is rare watch for animals spotted deer, and pilose antler is that a kind of length has fine hair and rare medicinal herbs containing spotted deer blood, has high pharmaceutical use.Cui Kai etc. (2012) purifying from antler growth period deer blood obtains the deer blood SOD that Rate activity is 4122.74U/mg, and deer blood SOD contains 2 subunits; Zhang Lanjie etc. (2005) are separated and obtain Rate activity from erythrocytes of sika deer blood is 13707.27U/mg copper-zinc superoxide dismutase, and relative molecular mass is about 32kDa, and relative subunit molecules quality is 17kDa.Superoxide-dismutase in the antioxygenation of pilose antler and deer blood has certain relation.
Most of SOD product is mainly derived from animal blood, internal organ etc. in the market, due to the reason such as limited starting material, purification difficult, causes that the purity of SOD is low, output is few; Particularly along with mad cow disease, bird flu, foot and mouth disease and the report frequently by pernicious transmissible diseases such as synzoic SARS all over the world, the increased risk of production animal source blood products; In addition, production cost is too increased to increasing of product purity requirement.The SOD of natural microbial and plant origin also because its kind is few, expression amount is low, enzyme molecular weight is large, low with the homology of SOD in animal and human body, thus make it be difficult to be widely used.Therefore, finding the SOD foreign gene of high-quality animal-origin, build efficient expression system, is the important channel realizing SOD industrialization production.
Summary of the invention
The object of the invention is to, for the purity of SOD is low in existing SOD product, output is few, provide the fermentation preparation of a kind of high purity recombinant C u/Zn-SOD.With the Pichia yeast engineering GS115-pPIC9K of high expression spotted deer antler sources Cu/Zn-SOD gene for producing bacterial strain, utilize microbial project method fermentative production restructuring SOD.By building efficient expression system, SOD industrialization can be realized and produce, the dependence to natural resources can be reduced, eliminating the negative impact that Animal diseases are brought, improving the application security of superoxide-dismutase.
For achieving the above object, the present invention adopts following technical scheme:
The fermentation preparation of spotted deer antler source recombinant C u/Zn-SOD is: with the Pichia yeast engineering GS115-pPIC9K of energy high expression spotted deer antler sources Cu/Zn-SOD gene for producing bacterial strain, seed culture adopts YPD substratum (pH6.0), 30 DEG C, 200r/min cultivates 15h; Fermention medium is BMMY(pH6.0); Induction condition of enzyme production is: inoculum size 5%, and initial methyl alcohol addition 1% supplements 1% methyl alcohol every 24h, and 200r/min, 30 DEG C of cultivation 96h, restructuring SOD vigor are 1876U/mL; Fermentation liquor aperture is that the microfiltration of ceramic membrane of 100nm is degerming, is the hollow fiber column ultrafilter of 10kDa with molecular weight cut-off, to remove the materials such as small molecular protein, metal ion and pigment in fermented liquid, and by its concentrated 3-5 doubly; After ultrafiltration, sample adopts HiTrapDEAEFF Weak anion-exchange chromatography post to carry out separation and purification, and adsorption-buffering liquid is pH8.0,50mmol/LTris-HCl, 0-1.0mol/LNaCl gradient elution, and flow velocity is 1mL/min; Collecting elution peak component utilizes TSK-G3000SW to carry out gel-filtration, adopts the phosphate buffered saline buffer of pH7.0,0.1mol/L (containing 0.1mol/LNa 2sO 4) carry out wash-out, flow velocity 0.5mL/min, obtain the pure Cu/Zn-SOD component of electrophoresis.
SOD Rate activity after purifying is 17647.1U/mg, and the rate of recovery is 36.84%, and purification is 10.54 times.
beneficial effect is:
The present invention adopts engineered method to build the Pichia yeast engineering of energy high expression spotted deer antler sources Cu/Zn-SOD gene, by the fermentative production of engineered microbes, prepares Cu/Zn-SOD in a large number; The dependence to natural resources can not only be reduced, eliminate the negative impact that Animal diseases are brought, improve the application security of superoxide-dismutase; And the enforcement of superoxide-dismutase industrialization will reduce production cost and the market value of this enzyme greatly, make its range of application more wide, more be conducive to serving fitness-for-all, also will bring good economic and social benefit simultaneously.
Accompanying drawing explanation
The growing state of Fig. 1 Pichia yeast engineering in different seed culture medium; Ordinate zou is OD value after fermented liquid dilutes 50 times;
The impact that Fig. 2 different culture media initial pH value grows Pichia yeast engineering; Ordinate zou is OD value after fermented liquid dilutes 50 times;
Fig. 3 different fermentations substratum is on the impact of restructuring SOD expression amount;
The initial pH of Fig. 4 different B MMY substratum is on the impact of restructuring SOD expression amount;
The different Cu of Fig. 5 2+concentration is on the impact of restructuring SOD expression amount;
The different Zn of Fig. 6 2+concentration is on the impact of restructuring SOD expression amount;
The different methyl alcohol addition of Fig. 7 is on the impact of restructuring SOD expression amount;
Fig. 8 different vaccination amount is on the impact of restructuring SOD expression amount;
Fig. 9 leavening temperature is on the impact of restructuring SOD expression amount;
Figure 10 HiTrapDEAEFF Weak anion-exchange chromatography elution curve;
Figure 11 DEAEFF weak anionic exchanges each chromatographic peak electrophorogram; Note: 1.ProteinMarker; 2. sample; 3.D 1; 4.D 2; 5.D 3; 6.D 4;
Figure 12 TSKgelG3000SW high productivity computing elution curve;
The each chromatographic peak electrophorogram of Figure 13 TSKgelG3000SW efficient gel; Note: 1.ProteinMarker; 2.DEAE-D 2; 3.T 1; 4.T 2; 5.T 3; 6.T 4.
Embodiment
The present invention's the following example further illustrates, but protection scope of the present invention is not limited to the following example.
experiment material and reagent
1, bacterial strain: the Pichia yeast engineering GS115-pPIC9K expressing spotted deer antler sources Cu/Zn-SOD gene is built voluntarily by enzyme engineering institute of University of Fuzhou.Engineering bacteria construction process is shown in national inventing patent " a kind of superoxide-dismutase and preparation method thereof " (ZL200910112388.3).
The structure of Pichia yeast engineering GS115-pPIC9K: mRNA reverse transcription is cDNA by separation and Extraction total serum IgE from spotted deer antler, gets PCR primer and carries out electrophoresis detection after pcr amplification, reclaim goal gene; Preparation double-strand cDNA carries out subclone, gained goal gene is carried out determined dna sequence, and compares in NCBI receipt storehouse, obtain Cu/Zn-SOD gene; Cu/Zn-SOD encoding sequence warp xhoi He notafter I double digestion with xhoi He notthe pPIC9K plasmid of I double digestion connects, and obtains yeast recombinant expression plasmid pPIC9K-SOD; By the recombinant plasmid pPIC9K-SOD warp prepared saci enzyme is cut, and obtains linearization plasmid pPIC9K-SOD, transforms Pichia pastoris GS115, selects positive colony and carry out abduction delivering.
2, main agents:
Yeast extract and Tryptones are purchased from OXOID company; Vitamin H, without amino yeast nitrogen (YNB), Tutofusin tris (Tris) purchased from Sangon Biotech (Shanghai) Co., Ltd.; Methyl alcohol, glucose and other conventional medication are domestic analytical reagent.
3, substratum:
YP substratum: yeast extract 1%, Tryptones 2%, 121 DEG C of sterilizing 20min;
YPD substratum (pH6.0): add the 10*D of 10% in YP substratum after sterilization;
10*D:20% glucose, 115 DEG C of sterilizing 30min;
YPM substratum: add 1% pure methyl alcohol in YP substratum after sterilization;
YPDM substratum: add 1% glucose on the basis of YPM substratum;
BMGY substratum: 0.5g yeast extract, 1g Tryptones, is dissolved in 35mL water, 121 DEG C of sterilizing 20min; 10*YNB5mL, 1mol/L phosphate buffered saline buffer 5mL, 10* glycerine 5mL, 500* vitamin H 0.1mL is added under gnotobasis;
BMMY substratum (pH6.0): 0.5g yeast extract, 1g Tryptones, is dissolved in 40mL water, 121 DEG C of sterilizing 20min; 10*YNB5mL, 1mol/L phosphate buffered saline buffer 5mL, 500* vitamin H 0.1mL, pure methyl alcohol 0.5mL is added under gnotobasis;
10*YNB: dissolve 13.4gYNB in 100mL water, filtration sterilization, deposits for 4 DEG C;
500* vitamin H: filtration sterilization after 0.02% vitamin H dissolves at 50 DEG C, deposits for 4 DEG C;
10* glycerine: 10% glycerine filtration sterilization, deposits for 4 DEG C.
4, chromatographic column:
HiTrapDEAEFF weak anionic chromatographic column, purchased from American GE company;
TSK-gelG3000SW high productivity computing post, purchased from eastern ソ mono-Co., Ltd. of Japan (TOSOH).
5, SOD vigour-testing method: build up scientific & technical corporation's superoxide-dismutase testing cassete specification sheets (article No.: A001-1 hydroxylamine assay) operation according to Nanjing.
Enzyme activity unit is defined as: SOD amount corresponding when SOD inhibiting rate reaches 50% in every milliliter of reaction solution is a SOD unit of activity (U).
Enzyme activity unit calculates:
In formula:
SOD---SOD unit of activity (U/mL);
OD contrasts---light absorption value under control tube 550mn;
OD sample---light absorption value under sample hose 550mn;
V is total---reaction solution cumulative volume (mL);
V sample---sample liquid cumulative volume (mL).
condition optimizing is tested:
1) optimization of Pichia yeast engineering GS115-pPIC9K seed culture medium
Pichia yeast engineering is inoculated in respectively in YPD and BMGY substratum, in 30 DEG C, cultivate 48h under 200r/min condition, every 2h sampling and measuring OD600, compare the growing state of Pichia yeast engineering in YPD and BMGY; Result (Fig. 1) shows, and no matter be YPD or BMGY substratum, Pichia yeast engineering all enters logarithmic phase at 11h, and growth tendency is consistent; But because YPD medium component is relatively simple, preparation is convenient, therefore selects the seed culture medium that YPD substratum is cultivated as Pichia yeast engineering in subsequent experimental.The seed culture time chooses 15h, and now seed is in logarithmic phase mid-term.
Regulate the initial pH of YPD substratum to be respectively 4,5,6,7,8, in 30 DEG C, cultivate under 200r/min condition, sample every 2h and measure OD600, result is as shown in Figure 2.When the initial pH of seed culture medium is 6,7, pichia spp growth better, cultivates 15h, and the OD600 value of seed liquor dilutes 50 times for 0.293().
2) the optimization of Pichia yeast engineering GS115-pPIC9K fermention medium
On enzymatic productivity, the SOD vigor that cultivation 96h, BMMY substratum records is 483.9U/mL, far above YPM substratum (126.0U/mL) and YPDM substratum (109.2U/mL); But in thalli growth amount, be then YPDM>YPM>BMMY, when not biomass growth is the highest, enzyme activity also the highest (Fig. 3).Therefore, adopt BMMY substratum as the enzymatic production substratum of Pichia yeast engineering.
When the initial pH of BMMY substratum is 6, the vigor of restructuring SOD is the highest, and the initial pH therefore regulating BMMY substratum is 6.0(Fig. 4).
Under the prerequisite of not adding YNB, in BMMY substratum, add CuSO 4solution, makes Cu 2+final concentration is respectively 0,5,500,2000,10000 μm of ol/L, not add Cu 2+measured by blank group fermentation 96h, SOD vigor is 100%, fermentation 120h.Cu 2+there is promoter action, Cu to the expression of restructuring SOD 2+final concentration is that the experimental group fermentation 96h of 500 μm of ol/L records SOD vigor for not add Cu 2+3.34 times (Fig. 5) of blank group.
Equally, under the prerequisite of not adding YNB, in BMMY substratum, add ZnSO 4solution, makes Zn 2+final concentration is respectively 100,200,500,1000,2000 μm of ol/L, not add Zn 2+measured by blank group fermentation 96h, SOD vigor is 100%, fermentation 96h.Work as Zn 2+when concentration is 1000 μm of ol/L, restructuring SOD vigor is blank group 1.81 times (Fig. 6).
Pichia spp is methanotrophic yeast, and methyl alcohol can be utilized as its sole carbon source, under the prerequisite that other culture condition are constant, is optimized methyl alcohol addition in substratum (0.25%, 0.5%, 1%, 2%, 3%); And add identical quantity of methyl alcohol every 24h, sampling and measuring SOD vigor after fermentation 96h, is defined as 100% with the highest group of enzyme activity, calculates enzyme activity.Fig. 7 shows, and when methyl alcohol addition is 1%, pichia spp SOD expression amount is the highest.
3) the induction optimization culture conditions of Pichia yeast engineering GS115-pPIC9K
inoculum size is on the impact of restructuring SOD expression amount
Under the prerequisite that other culture condition are constant, SOD expression amount of recombinating when inoculum size is 5% reaches maximum, therefore determines that optimum inoculation amount is 5%(Fig. 8);
temperature is on the impact of restructuring SOD expression amount
Restructuring SOD expression amount reaches the highest 30 DEG C time, and thalline weight in wet base reaches maximum at 32 DEG C; When temperature is higher than 35 DEG C, SOD expression amount and Fungal biodiversity decline all rapidly.Determine that leavening temperature is 30 DEG C (Fig. 9).
4) the separation and purification of recombinant C u/Zn-SOD
Fermentation liquor aperture is that the microfiltration of ceramic membrane of 100nm is degerming, is the hollow fiber column ultrafilter of 10kDa with molecular weight cut-off, to remove the materials such as small molecular protein, metal ion and pigment in fermented liquid, and by its concentrated 3-5 doubly.
After ultrafiltration, sample adopts HiTrapDEAEFF Weak anion-exchange chromatography post to carry out separation and purification.Adopt pH8.0,50mmol/LTris-HCl as adsorption-buffering liquid, elution process adopts 0-1.0mol/LNaCl gradient elution, and flow velocity is 1mL/min, and often 2mL collected by pipe.Purity is identified to the measured in solution SOD vigor in collection tube and SDS-PAGE.
The relative molecular weight subunit of restructuring SOD is 17kDa, Figure 11 display, and ultrafiltration and concentration liquid mainly exists an obvious object band (greatly near 17kDa) and four assorted bands (size is respectively: 22kDa, 34kDa, 70kDa, 200kDa); Penetrate peak D 1electrophoretic band is had in 17kDa position, may for there is no the target protein adsorbed; D 2peak is that 17kDa place has obvious band at molecular weight, and illustration purpose albumen can by wash-out in 0.3mol/LNaCl concentration; And D 3only there is a more shallow band at peak at 34kDa place, D 4there is not target protein band in peak.
Collect the D that SOD vigor is higher 2chromatographic peak component utilizes TSK-G3000SW gel chromatographic columns to carry out separation and purification further.With the phosphate buffered saline buffer of pH7.0,0.1mol/L (containing 0.1mol/LNa 2sO 4) carry out wash-out, flow velocity 0.5mL/min, wash-out result as shown in figure 12, collects each elution peak T 1, T 2, T 3, T 4, measure its SOD vigor and carry out SDS-PAGE and identify purity (Figure 13).
Figure 13 shows, T 1, T 2and T 4substantially there is not target stripe in peak, and the test of SOD vigor also shows without SOD vigor; Target protein mainly concentrates on T 3peak (swimming lane 5), and T 3peak molecules of interest amount is that 17kDa and theoretical value match, and shows that restructuring SOD has reached electrophoresis pure.
Filter (TSKgelG3000SW) 4 purification procedures through micro-filtration, ultrafiltration, Weak anion-exchange chromatography (DEAE), efficient gel, obtain electrophoretically pure restructuring SOD; SOD Rate activity after purifying is 17647.1U/mg, and the rate of recovery is 36.84%, and purification is 10.54 times.
Embodiment 1
With can the Pichia yeast engineering GS115-pPIC9K of high expression spotted deer antler sources Cu/Zn-SOD gene for producing bacterial strain, seed culture adopts YPD substratum (pH6.0), 30 DEG C, 200r/min cultivates 15h; Fermention medium is BMMY(pH6.0); Induction condition of enzyme production is: inoculum size 5%, and initial methyl alcohol addition 1% supplements 1% methyl alcohol every 24h, and 200r/min, 30 DEG C of cultivation 96h, restructuring SOD vigor are 1876U/mL; Fermentation liquor aperture is that the microfiltration of ceramic membrane of 100nm is degerming, is the hollow fiber column ultrafilter of 10kDa with molecular weight cut-off, to remove the materials such as small molecular protein, metal ion and pigment in fermented liquid, and by its concentrated 3-5 doubly; After ultrafiltration, sample adopts HiTrapDEAEFF Weak anion-exchange chromatography post to carry out separation and purification, and adsorption-buffering liquid is pH8.0,50mmol/LTris-HCl, 0-1.0mol/LNaCl gradient elution, and flow velocity is 1mL/min; Collecting elution peak component utilizes TSK-G3000SW to carry out gel-filtration, adopts the phosphate buffered saline buffer of pH7.0,0.1mol/L (containing 0.1mol/LNa 2sO 4) carry out wash-out, flow velocity 0.5mL/min, obtain the pure Cu/Zn-SOD component of electrophoresis.
SOD Rate activity after purifying is 17647.1U/mg, and the rate of recovery is 36.84%, and purification is 10.54 times.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

Claims (4)

1. a fermentation preparation of recombinant C u/Zn-SOD, is characterized in that: comprise the following steps:
(1) produce bacterial strain with the Pichia yeast engineering GS115-pPIC9K of energy high expression spotted deer antler sources Cu/Zn-SOD gene, carry out seed culture; The condition of seed culture is: YPD substratum, pH6.0,30 DEG C, and 200r/min cultivates 15h;
(2) then ferment; Fermention medium is BMMY substratum, pH6.0, and induction condition of enzyme production is: inoculum size 5%, and initial methyl alcohol addition 1% supplements 1% methyl alcohol every 24h, and 200r/min, 30 DEG C of cultivation 96h, restructuring SOD vigor are 1876U/mL;
(3) fermentation liquor microfiltration of ceramic membrane, hollow fiber column ultrafilter, HiTrapDEAEFF Weak anion-exchange chromatography post carry out separation and purification;
(4) collect elution peak component, utilize TSK-G3000SW to carry out gel-filtration, obtain the pure Cu/Zn-SOD of electrophoresis.
2. the fermentation preparation of recombinant C u/Zn-SOD according to claim 1, it is characterized in that: described step (3) is specially: the microfiltration of ceramic membrane by fermentation liquor aperture being 100nm is degerming, be the hollow fiber column ultrafilter of 10kDa with molecular weight cut-off, and by its concentrated 3-5 doubly; After ultrafiltration, sample adopts HiTrapDEAEFF Weak anion-exchange chromatography post to carry out separation and purification, and adsorption-buffering liquid is pH8.0,50mmol/LTris-HCl, 0-1.0mol/LNaCl gradient elution, and flow velocity is 1mL/min.
3. the fermentation preparation of recombinant C u/Zn-SOD according to claim 1, is characterized in that: in step (4), the condition of gel-filtration is: adopt pH7.0,0.1mol/L, containing 0.1mol/LNa 2sO 4phosphate buffered saline buffer carry out wash-out, flow velocity 0.5mL/min, obtain the pure Cu/Zn-SOD component of electrophoresis.
4. the fermentation preparation of recombinant C u/Zn-SOD according to claim 1, is characterized in that: the pure Cu/Zn-SOD Rate activity of the electrophoresis that step (4) obtains is 17647.1U/mg, and the rate of recovery is 36.84%, and purification is 10.54 times.
CN201510585981.5A 2015-09-15 2015-09-15 Fermentation preparation method of recombinant Cu/Zn-SOD Pending CN105087510A (en)

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Cited By (2)

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CN105861537A (en) * 2016-05-20 2016-08-17 沈阳医学院 Study of anthropogenic SOD expression by recombinant pichia pastoris
CN106967733A (en) * 2017-04-06 2017-07-21 福建省水产研究所 Haliotis discus hannai Ino superoxide dismutase and its preparation method and application

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105861537A (en) * 2016-05-20 2016-08-17 沈阳医学院 Study of anthropogenic SOD expression by recombinant pichia pastoris
CN106967733A (en) * 2017-04-06 2017-07-21 福建省水产研究所 Haliotis discus hannai Ino superoxide dismutase and its preparation method and application

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Application publication date: 20151125