CN102533838A - Bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof - Google Patents
Bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of food industry biology and relates to a bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof. The vector utilizes a bacillus subtilis vector pHB201 as a framework, the pHB201 enzyme cutting site is modified first through an enzyme cutting method to obtain a pHB-hc vector, composition type strong promoter P43, composition type strong promoter PamyE, molecular chaperone PrsA of an Sec path and neutral protease signal peptide SnprB are obtained by cloning in bacillus subtilis 168 genome through a polymerase chain reaction (PCR), and a bacillus subtilis secretion type expression vector pHBSR is obtained in construction. Lipoxygenase gene obtained from anabaenagenome DNA in amplification is inserted into autonomously constructed expression vector pHBSR and then electrically converted into a bacillus subtilis host WB800 to achieve secreting expression of the recombination lipoxygenase, and the largest enzyme activity in fermentation liquid can reach 76U/ml.
Description
Technical field
The invention belongs to the foodstuffs industry biological technical field, relate to a kind of Bacillus subtilus expression vector and application thereof of efficient secretory expression reorganization lipoxygenase.
Background technology
Potassium bromate is as the quality improver that bakes industrial bread cake mix, and especially American-European abroad successful Application has the history of decades.As a kind of oxygenant at a slow speed, improve structure of dough and rheological, strengthen biceps and elasticity, make baked goods obtain satisfied result.China also earlier with potassium bromate as flour quality improver, list GB2760 in and use hygienic standard, 0.03g/kg limits the quantity of.But must not have residual after regulation bakes.Think in early days that potassium bromate can divide and take off after baking, but the eighties Japan and Britain, and through the discovery that studies for a long period of time, potassium bromate has residue after baking, animal is had carcinogenic toxicity.Afterwards, the FAO/WHO combination food additive Committee of Experts (JECFA) cancelled the ADI value that potassium bromate uses in flour in 1994, and the European Community also in foodstuff additive and numbering list thereof, has cancelled potassium bromate.The decision of China Ministry of Health was from July 1st, 2005, and the cancellation potassium bromate uses in wheat-flour as the flour treatment agent.
At present, the most frequently used bleaching of flour agent both at home and abroad is a Lucidol.Its bleaching mechanism is Lucidol hydrolysis under the effect of air and enzyme, emits the oxygen of status nascendi, thereby the unsaturated fat-soluble pigment in the oxidation flour makes it lose color.Researcher discovers that long-term excessive use Lucidol is deleterious to human body.The phenylformic acid that produces after the Lucidol hydrolysis will decompose in liver behind the entering human body.The long-term edible burden of liver that increases the weight of, pathological change can appear in liver, kidney when serious, and growth and life-span all will be affected; Residual undecomposed Lucidol in the flour can produce benzene radical, and then can form benzene, phenol, biphenyl in wheaten food heating making processes, these products are all toxic, and health is had bad influence; Free-radical oxidn can quicken human senility, causes atherosclerosis, even brings out multiple disease.In the national standard of China in the regulation flour the maximum usage quantity of Lucidol be 0.06g/kg.
Therefore adopt novel enzyme preparation and other safety, natural composition, developing with the zymin is that the new and effective flour quality improver that can substitute potassium bromate, Lucidol of main body becomes one of emphasis direction that food-processing studies.
(Lipoxygenase EC1.13.11.12), is called for short lipoxygenase to lipoxygenase; Belong to oxydo-reductase; That but catalysis contains is suitable-and suitable-1, the unsaturated fatty acids (like linolic acid, linolenic acid etc.) of 4-pentadiene structure and ester form hydroperoxide [1-2], and the labile hydrogen superoxide of generation can form disulfide linkage by the oxidation gluten protein; Make the protein molecule polymerization, thereby reach the effect that strengthens gluten.Lipoxygenase can also destroy the double bond structure of carrotenoid through coupled reaction, makes bleaching of flour.Therefore, lipoxygenase has strong muscle of flour and the effect that brightens concurrently, can reduce or the use of instead of chemical gluten-strengthening agent potassium bromate and chemical bleaching agent Lucidol, reduces its harm to HUMAN HEALTH.But since at present lipoxygenase mainly from animal vegetable tissue separation and Extraction obtain, yield poorly, purity is the major cause of its large-scale application of restriction inadequately.The separation and Extraction of lipoxygenase, structural analysis and zymologic property research are the mains direction of studying of present Chinese scholars.For High-efficient Production that how to realize lipoxygenase and industrial applications, present domestic correlative study is very few.Food belongs to large Industrial products with enzyme, and separation and Extraction means are cheaply generally adopted in downstream, can not guarantee to remove fully such as intestinal bacteria thermal source material; If adopt complicated purification process, then cost just becomes the restrictive factor of application.Subtilis is a gram positive bacterium, is that Traditional industrial is produced bacterium, and the secretory protein ability is strong, and cell wall structure simply and not contains intracellular toxin, and U.S. FDA is classified it as GRAS (Generally Recognized As Safe) level other mikrobe.The Bacillus subtilus expression system has been successfully used to the expression of multiple protein, and major part is the enzyme with industrial application value, especially proteolytic enzyme and glycase.Though the research Bacillus subtilus about exogenous protein expression also lags far behind intestinal bacteria,, in the research of food genetically engineered, have more the incomparable meliority of intestinal bacteria then obviously being superior to intestinal bacteria aspect expression and the secretion activity albumen.Be applied to the engineered research of food about Bacillus subtilus in recent years, more and more come into one's own.Along with molecular biology and engineered develop rapidly, have reason to believe that the Bacillus subtilus expression system will be more perfect, become one of best host of protokaryon protein expression.
Summary of the invention
The objective of the invention is above-mentioned deficiency, a kind of Bacillus subtilus expression vector of efficient secretory expression reorganization lipoxygenase is provided to prior art.
Another object of the present invention provides the application of the Bacillus subtilus expression vector of this efficient secretory expression reorganization lipoxygenase.
The object of the invention can be realized through following technical scheme:
The construction process of a kind of Bacillus subtilus secretion expression carrier pHBSR comprises following steps successively:
I. carrier construction pHB-hc: synthetic a pair of primer P1:SEQ ID NO.2/P2:SEQ ID NO.3, annealing makes it to match and forms double-strandedly, and two ends form the sticky end of ClaI, XhoI; After the ClaI/HindIII enzyme is cut the big fragment of the 5.4kb that obtains is connected, obtain carrier pHB-hc with annealing double-stranded product and pHB201 are first;
Ii carrier construction pHP43: with subtilis 168 genomes is template; P43-F:SEQ ID NO.4/P43-R:SEQ IDNO.5 is a primer; Pcr amplification obtains the P43 promotor; The P43 promotor is inserted behind the XhoI/ClaI double digestion in the carrier pHB-hc of same double digestion, obtains carrier pHP43;
Iii. carrier construction pHP43R: with Bacillus subtilus 168 genomes is template, and P3:SEQ ID NO.6/P4:SEQ ID NO.7 is primer, and pcr amplification obtains Bacillus subtilus glycase E promoter gene PamyE; With Bacillus subtilus 168 genomes is template, and P5:SEQ ID NO.8/P6:SEQ ID NO.9 is a primer, and pcr amplification obtains Bacillus subtilus Chaperones Molecular Sec Secretory Pathway Chaperones Molecular PrsA; With P3 is upstream primer, is downstream primer with P6, is template with PCR reaction product PamyE and PrsA gene fragment mixture, the expression cassette PamyE-PrsA that the PrsA that utilizes the method amplification of PCR to obtain promotor PamyE mediation expresses; Spend Starch phosphorylase after carrier pHP43 cuts with the ClaI enzyme and handle, be connected acquisition carrier pHP43R through the T4 ligase enzyme with the PamyE-PrsA fragment of cutting through the ClaI enzyme;
Iv. make up secretion expression carrier pHBSR: with SnprB-F:SEQ ID NO.10/SnprB-R:SEQ ID NO.11 is primer; With Bacillus subtilus 168 genomic dnas is template pcr amplification SnprB signal peptide; Obtain the SnprB signal peptide gene through the reaction of EcoRI/BamHI double digestion, be connected acquisition secretion expression carrier pHBSR through the T4DNA enzyme with the pHP43R carrier that same double digestion is handled.
Bacillus subtilus secretion expression carrier pHBSR according to above-mentioned method structure.
The composing type recombinant expression vector pHBSR-ana-LOX of a kind of secreting, expressing reorganization lipoxygenase is to be the XhoI restriction enzyme site gained that the fat oxygenase gene ana-LOX of SEQID NO.1 inserts described secretion expression carrier pHBSR with sequence.
The construction process of described composing type recombinant expression vector pHBSR-ana-LOX comprises following steps:
I. be template with the Anabaena PCC 7120 genomic dna, clone's Anabaena PCC 7120 fat oxygenase gene ana-LOX, its sequence is SEQ ID NO.1, connects the cloning vector into pMD19-T, the correct back of order-checking called after pMD19-ana-LOX;
II. make up Bacillus subtilus secretion expression carrier pHBSR according to the method described above;
III. plasmid pMD19-ana-LOX and expression vector pHBSR are carried out the XhoI enzyme respectively and cut processing, the ana-LOX gene fragment of the 1368bp that recovery is obtained is connected with the linear big fragment of pHBSR and obtains composing type recombinant expression vector pHBSR-ana-LOX.
The application of described composing type recombinant expression vector pHBSR-ana-LOX in producing the reorganization lipoxygenase.
A kind of method of utilizing subtilis to produce the reorganization lipoxygenase is that pHBSR-ana-LOX composing type recombinant expression vector electricity is transformed the anabena lipoxygenase recombination bacillus subtilis genetic engineering bacterium that Bacillus subtilus host WB800 obtains containing Anabaena PCC 7120 fat oxygenase gene ana-LOX; This genetic engineering bacterium is inoculated in the LB substratum that contains paraxin/Oxacyclotetradecane,erythromycin deriv, cultivates centrifugal collection supernatant under 37 ℃, 180rpm condition.
Beneficial effect:
The present invention has made up a kind of novel Bacillus subtilus secretion expression carrier pHBSR; Utilize this carrier; Functional LOX gene in the anabena genome of clone's acquisition is inserted this carrier; Realized high reactivity secreting, expressing,, realized that LOX substitutes chemicaladditives harmful in the flour course of processing and becomes to take a firm foundation for realizing a large amount of, the high vigor reorganization of the outer production of food grade fermenting bacillus subtilis born of the same parents LOX in Bacillus subtilus.
Description of drawings
The acquisition of Fig. 1 carrier pHB-hc
The acquisition of Fig. 2 carrier pHP43
The acquisition of Fig. 3 carrier pHP43R
The acquisition of Fig. 4 carrier pHBSR
Fig. 5 P1, the P2 double-stranded product synoptic diagram of annealing
Fig. 6 anabena lipoxygenase expression vector establishment process of recombinating
Embodiment
Embodiment 1 anabena (Anabaena sp.PCC 7120) fat oxygenase gene clone
Centrifugal collection anabena (Anabaena sp.PCC 7120) thalline is given birth to the genomic dna that worker's genome DNA extracting reagent kit extracts anabena with Shanghai.Genome sequence according to NCBI announces is designed two primer: ana-LOX-F (SEQ IDNO.12) and ana-LOX-R (SEQ ID NO.13), is template with the genomic dna of anabena, utilizes pcr amplification to obtain the ana-LOX gene, and reaction system is following:
The PCR program is: 94 ℃ of 2min; 30 * (94 ℃ of 45s; 58 ℃ of 50s; 72 ℃ of 4min); 72 ℃ of 10min.
After the 1368bp gene fragment rubber tapping recovery with the pcr amplification acquisition, connect into the pMD19-T cloning vector, called after pMD 19-ana-LOX after order-checking is correct preserves subsequent use under-20 ℃ of conditions.
Analyze sequencing result with computer software DNAMAN, anabena fat oxygenase gene (ana-LOX), sequence is SEQID NO.1, the protein of forming by 455 amino acid of encoding, its aminoacid sequence is SEQ ID NO.14.
The structure of embodiment 2 secretion expression carrier pHBSR
2.1 the acquisition of carrier pHB-hc (Fig. 1)
In order to make up the Bacillus subtilus expression vector that can secrete the foreign protein product, choosing intestinal bacteria/Bacillus subtilus shuttle vectors pHB201 is skeleton, and this carrier is a stable cloning vector, and its skeleton derives from Bacillus subtilus carrier pTA1060.MCS is arranged in fusion gene cat86::lacZ α, and because it is not suitable for expressing, can (Fig. 1) be excised in its promotor P59, cat86::lacZ α zone.
Design a pair of primer P1 (SEQ ID NO.2), P2 (SEQ ID NO.3), the formation that makes it to match is double-stranded, and two ends form the sticky end of ClaI, XhoI.Reaction system is following:
In 200 μ L PCR thin-walled tubes, add:
Behind the mixing, place 94 ℃ of sex change of water-bath 2 minutes, naturally cooling makes its annealing, forms the protruding terminus of HindIII, ClaI, like Fig. 5.
PHB201 is successively cut off with ClaI, HindIII, reclaim the big fragment of 5.4kb.
16 ℃ of connections are spent the night, all transformed into escherichia coli DH5 α competent cell.It is dull and stereotyped to coat the LB that contains paraxin, 37 ℃ of incubated overnight.Picking list bacterium colony is to liquid LB substratum, and shaking culture is extracted plasmid, restriction enzyme digestion and electrophoresis, sequence verification.With this plasmid called after pHB-hc.
2.2 the acquisition of carrier pHP43 (Fig. 2)
Design P43-F (SEQ ID NO.4); P43-R (SEQ ID NO.5) introduces a plurality of restriction enzyme sites for primer and at downstream primer; Constituting MCS (MSC), is that the template pcr amplification obtains the P43 promotor with subtilis 168 genomes, and reaction system is following:
The PCR program is: 94 ℃ of 2min; 30 * (94 ℃ of 45s; 58 ℃ of 50s; 72 ℃ of 4min); 72 ℃ of 10min.
After the PCR rubber tapping recovery with the amplification acquisition, connect into the pMD19-T cloning vector, called after pMD19-T-P43 after order-checking is correct.XhoI/ClaI double digestion pMD19-T-P43 obtains P43 promoter gene fragment; Spend the night 16 ℃ of connections with the T4 ligase enzyme with the identical double digestion pHB-hc carrier of process; Transformed into escherichia coli DH5 α competent cell is coated the flat board that contains paraxin, 37 ℃ of overnight cultures.Choose single bacterium colony shaking culture, extract plasmid in a small amount, restriction enzyme digestion and electrophoresis is identified, called after pHP43 preserves subsequent use under-20 ℃ of conditions.
2.3 the acquisition of carrier pHP43R (Fig. 3)
Design primer P3 (SEQ ID NO.6), P4 (SEQ ID NO.7) are template with Bacillus subtilus 168 genomes, utilize the method amplification of PCR to obtain Bacillus subtilus glycase E promoter gene PamyE, and the PCR condition is identical among reaction system and condition and the embodiment 2.2.
Design primer P5 (SEQ ID NO.8), P6 (SEQ ID NO.9); With Bacillus subtilus 168 genomes is template; Utilize the method amplification of PCR to obtain Bacillus subtilus Chaperones Molecular Sec Secretory Pathway Chaperones Molecular PrsA, the PCR condition is identical among reaction system and condition and the embodiment 2.2.
With P3 (SEQ ID NO.6) is upstream primer; With P6 (SEQ ID NO.9) is downstream primer; With PCR reaction product PamyE and PrsA gene fragment mixture is template; The PCR condition is identical among the expression cassette PamyE-PrsA that the PrsA that utilizes the method amplification of PCR to obtain promotor PamyE mediation expresses, reaction system and condition and embodiment 2.2.
After the PCR rubber tapping recovery with the amplification acquisition, connect into the pMD19-T cloning vector, called after pMD19-T-PamyE-PrsA after order-checking is correct preserves subsequent use under-20 ℃ of conditions.
PMD19-T-PamyE-PrsA cuts with the ClaI enzyme, and rubber tapping obtains the PamyE-PrsA gene fragment respectively after reclaiming.Spend Starch phosphorylase after the pHP43 carrier is cut with the ClaI enzyme and handle, be connected acquisition pHP43R carrier through the T4 ligase enzyme, under-20 ℃ of conditions, preserve subsequent use with the PamyE-PrsA fragment.
2.4 the acquisition of carrier pHBSR (Fig. 4)
With SnprB-F (SEQ ID NO.10)/SnprB-R (SEQ ID NO.11) is primer, is template pcr amplification SnprB signal peptide with Bacillus subtilus 168 genomic dnas, and the PCR condition is identical among reaction system and condition and the embodiment 2.2.The PCR product connects into the pMD19-T carrier, and the correct back of order-checking called after pMD19-T-SnprB preserves subsequent use under-20 ℃ of conditions.PMD19-T-SnprB obtains the SnprB signal peptide gene through the reaction of EcoRI/BamHI double digestion; Be connected acquisition secretion expression carrier pHBSR with the pHP43R carrier that same double digestion is handled through T4 DNA enzyme; And, under-20 ℃ of conditions, preserve subsequent use through the restriction enzyme digestion and electrophoresis checking.
Plasmid pMD19-ana-LOX and expression vector pHBSR are carried out the XhoI enzyme respectively cut processing.Give birth to worker PCR product purification test kit gene fragment (ana-LOX) and the linearizing pHBSR plasmid of purifying 1368bp respectively with Shanghai.Connect transformed into escherichia coli DH5 α behind two kinds of gene fragment purifying with the T4 ligase enzyme.From transforming on the flat board the several bacterium colonies of picking at random, insert the LB liquid nutrient medium, shaking culture is extracted plasmid in a small amount, electrophoresis, the plasmid that lags behind with electrophoresis is that template is carried out the PCR checking, confirms that delivering to Shanghai after the successful connection gives birth to worker's order-checking.
Embodiment 4 reorganization pHPSB-ana-LOX expression vectors transform Bacillus subtilus host WB800 and secreting, expressing lipoxygenase.
PHBSR-ana-LOX composing type recombinant expression vector electricity is transformed into the WB800 competent cell, coats the two anti-dull and stereotyped enterprising row filter of paraxin (10 μ g/mL)/Oxacyclotetradecane,erythromycin deriv (10 μ g/mL).With the single bacterium colony that grows on two anti-flat boards, connect bacterium in the LB of paraxin/Oxacyclotetradecane,erythromycin deriv substratum, cultivate under 37 ℃, 180rpm condition.Centrifugal collection supernatant is to measure the LOX enzyme end to live with the linolic acid.The secreting, expressing amount of pHBSR-ana-LOX recombinant bacterial strain is the highest, and enzyme work reaches 76U/mL.
Claims (6)
1. the construction process of a Bacillus subtilus secretion expression carrier pHBSR is characterized in that comprising successively following steps:
I. carrier construction pHB-hc: synthetic a pair of primer P1:SEQ ID NO.2/P2:SEQ ID NO.3, annealing makes it pairing and forms double-strandedly, and two ends form the sticky end of ClaI, XhoI; The double-stranded product of annealing is cut the big fragment of the 5.4kb that obtains with pHB201 through the ClaI/HindIII enzyme be connected, obtain carrier pHB-hc;
Ii carrier construction pHP43: with subtilis 168 genomes is template; P43-F:SEQ ID NO.4/P43-R:SEQ IDNO.5 is a primer; Pcr amplification obtains the P43 promotor; The P43 promotor is inserted behind the XhoI/ClaI double digestion in the carrier pHB-hc of same double digestion, obtains carrier pHP43;
Iii. carrier construction pHP43R: with Bacillus subtilus 168 genomes is template, and P3:SEQ ID NO.6/P4:SEQ ID NO.7 is primer, and pcr amplification obtains Bacillus subtilus glycase E promoter gene PamyE; With Bacillus subtilus 168 genomes is template, and P5:SEQ ID NO.8/P6:SEQ ID NO.9 is a primer, and pcr amplification obtains Bacillus subtilus Chaperones Molecular Sec Secretory Pathway Chaperones Molecular PrsA; With P3 is upstream primer, is downstream primer with P6, is template with PCR reaction product PamyE and PrsA gene fragment mixture, the expression cassette PamyE-PrsA that the PrsA that utilizes the method amplification of PCR to obtain promotor PamyE mediation expresses; Spend Starch phosphorylase after carrier pHP43 cuts with the ClaI enzyme and handle, be connected acquisition carrier pHP43R through the T4 ligase enzyme with the PamyE-PrsA fragment of cutting through the ClaI enzyme;
Iv. make up secretion expression carrier pHBSR: with SnprB-F:SEQ ID NO.10/SnprB-R:SEQ ID NO.11 is primer; With Bacillus subtilus 168 genomic dnas is template pcr amplification SnprB signal peptide; Obtain the SnprB signal peptide gene through the reaction of EcoRI/BamHI double digestion, be connected acquisition secretion expression carrier pHBSR through T4 DNA enzyme with the pHP43R carrier that same double digestion is handled.
2. the Bacillus subtilus secretion expression carrier pHBSR that makes up according to the described method of claim 1.
3. the composing type recombinant expression vector pHBSR-ana-LOX of a secreting, expressing reorganization lipoxygenase is characterized in that being is the XhoI restriction enzyme site gained of the described secretion expression carrier pHBSR of fat oxygenase gene ana-LOX insertion claim 2 of SEQ ID NO.1 with sequence.
4. the construction process of the described composing type recombinant expression vector of claim 3 pHBSR-ana-LOX is characterized in that comprising following steps:
I. be template with the Anabaena PCC 7120 genomic dna, clone's Anabaena PCC 7120 fat oxygenase gene ana-LOX, its sequence is SEQ ID NO.1, connects the cloning vector into pMD19-T, the correct back of order-checking called after pMD19-ana-LOX;
II. make up Bacillus subtilus secretion expression carrier pHBSR according to the described method of claim 1;
III. plasmid pMD19-ana-LOX and Bacillus subtilus secretion expression carrier pHBSR are carried out the XhoI enzyme respectively and cut processing, the ana-LOX gene fragment of the 1368bp that recovery is obtained is connected with the linear big fragment of pHBSR and obtains composing type recombinant expression vector pHBSR-ana-LOX.
5. the application of the described composing type recombinant expression vector of claim 3 pHBSR-ana-LOX in producing the reorganization lipoxygenase.
6. a method of utilizing subtilis to produce the reorganization lipoxygenase is characterized in that it being that pHBSR-ana-LOX composing type recombinant expression vector electricity is transformed the anabena lipoxygenase recombination bacillus subtilis genetic engineering bacterium that Bacillus subtilus host WB800 obtains containing Anabaena PCC 7120 fat oxygenase gene ana-LOX; This genetic engineering bacterium is inoculated in the LB substratum that contains paraxin/Oxacyclotetradecane,erythromycin deriv, cultivates centrifugal collection supernatant under 37 ℃, 180rpm condition.
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| CN104591407A (en) * | 2015-01-19 | 2015-05-06 | 南京农业大学 | Method for degrading triphenylmethane dye by utilizing recombinational lipoxygenase |
| CN104830887A (en) * | 2015-05-13 | 2015-08-12 | 武汉华美生物工程有限公司 | Secreted expression vector of acetylcholin esterase and building method and application of secreted expression vector |
| WO2015176641A1 (en) * | 2014-05-21 | 2015-11-26 | 中国科学院上海生命科学研究院 | Method for promoting soluble expression of recombinant extremely thermostable α-amylase |
| CN105695472A (en) * | 2016-04-28 | 2016-06-22 | 中国科学院上海高等研究院 | Bacillus subtilis fermentation production promoter and application method thereof |
| CN106834311A (en) * | 2017-01-24 | 2017-06-13 | 南京农业大学 | A kind of acid lipoxygenase and preparation method and application |
| CN107881181A (en) * | 2017-12-14 | 2018-04-06 | 河北省微生物研究所 | Neutral protease gene, albumen, bacillus subtilis and the preparation and application built using molecular chaperones DnaK |
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| CN110564662A (en) * | 2019-09-30 | 2019-12-13 | 南京农业大学 | Construction method of integrated bacillus subtilis for efficiently expressing acetaldehyde dehydrogenase |
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| CN104591407A (en) * | 2015-01-19 | 2015-05-06 | 南京农业大学 | Method for degrading triphenylmethane dye by utilizing recombinational lipoxygenase |
| CN104830887B (en) * | 2015-05-13 | 2018-07-03 | 武汉华美生物工程有限公司 | The secreted expression carrier and its construction method of a kind of acetylcholinesterase and application |
| CN104830887A (en) * | 2015-05-13 | 2015-08-12 | 武汉华美生物工程有限公司 | Secreted expression vector of acetylcholin esterase and building method and application of secreted expression vector |
| CN105695472A (en) * | 2016-04-28 | 2016-06-22 | 中国科学院上海高等研究院 | Bacillus subtilis fermentation production promoter and application method thereof |
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| CN106834311B (en) * | 2017-01-24 | 2020-11-06 | 南京农业大学 | Acidic lipoxygenase, and preparation method and application thereof |
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| CN107881181A (en) * | 2017-12-14 | 2018-04-06 | 河北省微生物研究所 | Neutral protease gene, albumen, bacillus subtilis and the preparation and application built using molecular chaperones DnaK |
| CN108570459A (en) * | 2018-04-10 | 2018-09-25 | 南京农业大学 | A kind of method of high-efficiency fermenting production recombinant bacteria laccase |
| CN108841772A (en) * | 2018-07-16 | 2018-11-20 | 江南大学 | A kind of bacillus subtilis engineering bacteria of high efficient expression alpha-amylase |
| CN108841772B (en) * | 2018-07-16 | 2021-03-30 | 江南大学 | Bacillus subtilis engineering bacterium for efficiently expressing alpha-amylase |
| CN113366113A (en) * | 2019-01-30 | 2021-09-07 | 诺维信公司 | Co-expression of homologous folding enzymes |
| CN110564662A (en) * | 2019-09-30 | 2019-12-13 | 南京农业大学 | Construction method of integrated bacillus subtilis for efficiently expressing acetaldehyde dehydrogenase |
| CN110564662B (en) * | 2019-09-30 | 2022-03-25 | 南京农业大学 | Construction method of integrated bacillus subtilis for efficiently expressing acetaldehyde dehydrogenase |
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