CN104140984A - Method for producing L(+)-tartaric acid by improving cell permeability - Google Patents
Method for producing L(+)-tartaric acid by improving cell permeability Download PDFInfo
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- CN104140984A CN104140984A CN201410379113.7A CN201410379113A CN104140984A CN 104140984 A CN104140984 A CN 104140984A CN 201410379113 A CN201410379113 A CN 201410379113A CN 104140984 A CN104140984 A CN 104140984A
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Abstract
The invention discloses a method for producing L(+)-tartaric acid by improving cell permeability. The method comprises the following steps of (1) culturing reconstitution cells expressing cis-epoxysuccinate hydrolase in a fermentation mode; (2) permeabilizing the reconstitution cells by taking triton-series surface active agents as permeabilization agents; (3) producing the L(+)-tartaric acid by means of the reconstitution cells subjected to the permeable processing. The cell permeability is improved by performing surface active agent processing on the reconstitution cells, and conversion time is shortened by over twelve times. By means of further immobilization processing, use stability of the cis-epoxysuccinate hydrolase is improved, the utilization rate of the cells is increased, and more than ten bathes of immobilized cells can be stably utilized in a cyclic mode.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of production L (+)-tartaric method.
Background technology
L (+)-tartrate is extensively present in occurring in nature, particularly grape and tamarind, is called as " Threaric acid ", is important foodstuff additive, medical resolving agent, printing and dyeing reserving agent, photographic developer, metal ion screener etc.Microbe transformation method is the main method that current L (+)-tartrate is produced, and, with the microorganism containing cis-Epoxysuccinic acid hydratase, conversion of substrate cis-form epoxy succinic acid is L (+)-tartrate.
Prior art has built and has expressed the reconstitution cell of cis-Epoxysuccinic acid hydratase, but directly with reconstitution cell, producing L (+)-tartrate mainly exists following 2 problems:
(1) though the high efficient expression of the cis-Epoxysuccinic acid hydratase of reconstitution cell, because cis-Epoxysuccinic acid hydratase belongs to intracellular enzyme, the chance contacting with substrate is limited by the permeability of cytolemma, causes its catalytic performance low.
(2) reconstitution cell can only be used once, and utilization ratio is low, and cost is high, pollutes large.
Tensio-active agent, refers to and has fixing hydrophilic and oleophilic group, on the surface of solution, can align, and the material that can make surface tension significantly decline.It is a large class organic compound, and their character has characteristic, and application very flexibly, extensively, has very large practical value and theory significance.
Fixation of microbial cell is the new technology that 20th century zymetology sixties field emerges, utilize the means of chemistry or physics free cell to be positioned to the area of space limiting, be beneficial to intracellular a kind of enzyme or plurality of enzymes and carry out biocatalysis, and keep cytoactive, repeatedly used a kind of technology.Compare with traditional free cell, cell density that immobilized cell has unit volume is high, the easy advantage such as recovery of thalline.At present both at home and abroad also not about first using tensio-active agent to carry out permeabilized processing to reconstitution cell, and then utilize the reconstitution cell after processing to carry out the report that L (+)-tartrate is produced, therefore utilize tensio-active agent to improve cell permeability, in conjunction with immobilization technology, improve cell rate of utilization, significant to L (+)-tartaric production.
Summary of the invention
The object of this invention is to provide a kind of new cell permeability that improves and produce L (+)-tartaric method, to overcome all or part of defect of prior art.
For achieving the above object, the technical solution used in the present invention is: the present invention produces by improving cell permeability the reconstitution cell that L (+)-tartaric method comprises the steps: (1) fermentation culture expression cis-Epoxysuccinic acid hydratase; (2) use Triton series of surfactants, as penetrating dose, described reconstitution cell is carried out to permeabilized processing; (3) utilize the reconstitution cell of permeabilized processing to produce L (+)-tartrate.
Further, the present invention, before the reconstitution cell that utilizes permeabilized processing is produced L (+)-tartrate, first uses kappa-carrageenan immobilization by the reconstitution cell of this permeabilized processing.
Further, the present invention uses the immobilized method of kappa-carrageenan as follows the reconstitution cell of permeabilized processing:
The reconstitution cell suspension of permeabilized processing is added in kappa-carrageenan solution and mixed, after cooled and solidified, with Klorvess Liquid, soak and fix, wherein, the final concentration of kappa-carrageenan is 20-60g/l, and the final concentration of the reconstitution cell of permeabilized processing is 10-50g/l.
Further, the final concentration of kappa-carrageenan of the present invention is 30-50g/l, and the final concentration of the reconstitution cell of permeabilized processing is 20-30g/l.
Further, the present invention is in described step (2), the method of described reconstitution cell being carried out to permeabilized processing is: the reconstitution cell that step (1) is cultivated suspends with the physiological saline that contains Triton series of surfactants, and the activity of described tensio-active agent is 0.01-50 g/L; Then standing or low rate mixing suspension 10 min-24 h.
Further, the activity of tensio-active agent of the present invention is 0.1-10 g/L, standing or low rate mixing suspension 30 min-5 h.
Further, tensio-active agent of the present invention is nonionogenic tenside.
Further, nonionogenic tenside of the present invention is following any one: sorbitan fatty acid ester series of surfactants (sapn series, trade(brand)name Span), polysorbate series of surfactants (tween series, trade(brand)name Tween), polyoxyethylene glycol is to iso-octyl phenyl ether series of surfactants (Triton series, trade(brand)name Triton), alkylphenol polyoxyethylene series of surfactants (trade(brand)name OP), ethoxylated dodecyl alcohol series of surfactants (trade(brand)name AEO).
Further, nonionogenic tenside of the present invention is preferably polyoxyethylene glycol to iso-octyl phenyl ether series of surfactants.
Further, nonionogenic tenside of the present invention is triton x-100.
With respect to prior art, the obtained beneficial effect of the present invention is:
1. the cis-Epoxysuccinic acid hydratase reconstitution cell that utilization of the present invention is processed through tensio-active agent is produced L (+)-tartrate, compare the highest the shortening more than 12 times of transformation time of cis-Epoxysuccinic acid hydratase reconstitution cell with the reconstitution cell of processing without tensio-active agent.
2. the present invention can further utilize immobilization technology to improve stability and the utilization ratio of this reconstitution cell, and the substrate conversion efficiency after immobilization reconstitution cell is used 10 batches also has 82%, and the substrate conversion efficiency of reconstitution cell after using 3 batches is 0%.Visible, when utilizing reconstitution cell to prepare tartrate, use the conversion batch conversion batch far above use on-fixed reconstitution cell of immobilization reconstitution cell.
3. the present invention has filled up both at home and abroad about utilizing tensio-active agent and immobilization reconstitution cell to produce L (+)-tartaric technological gap.
the preservation information of biological material specimens
The biological material specimens of preservation: rhodococcus USA-AN012 BK-98(
rhodococcussp.USA-AN012 BK-98);
Depositary institution: (be called for short: CGMCC) at China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica (postcode: 100101);
Preservation date: on July 13rd, 2006;
Preservation registration number: CGMCC No. 1756
Note: this rhodococcus USA-AN012 BK-98 (
rhodococcussp. USA-AN012 BK-98) in the patent No. Chinese patent specification sheets that is ZL200710002155.9 of Granted publication on the 27th January in 2010, record.
Embodiment
With specific embodiment, further illustrate technical scheme of the present invention below.
Cis-form epoxy succinic acid salt involved in the present invention is the salt that cis-form epoxy succinic acid and various positively charged ion form, and positively charged ion includes but are not limited to ammonium ion, potassium ion, sodium ion, magnesium ion and calcium ion etc.
In following examples of the present invention, relevant detection method is as follows:
(1) detection method of reconstitution cell transformation time: 0.1 g is through 4 oC high speed centrifugation (5000 * g, 10 min) reconstitution cell of results is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.When cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, required timing definition is the restructuring transformation time (unit represents with h).
(2) detection method of immobilization reconstitution cell transformation time: get immobilization reconstitution cell (the 3*3*3 mm being prepared from through the reconstitution cell of 4 oC high speed centrifugations (5000 * g, 10 min) results by 0.1 g
3gel square), by 50 ml concentration, be after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.When cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, required timing definition is the transformation time (unit represents with h) of immobilization reconstitution cell.
(3) detection method of tartrate content: get the ammonium meta-vanadate of 2.5 ml 10 g/L in the volumetric flask of 25 ml, the tartrate standard solution, the 1 ml 1 mol/L sulphuric acid soln that add successively certain volume, jolting colour developing, and be settled to scale with distilled water, measure nitrite ion at the absorbance at 480 nm places, with tartrate add-on, the absorbance at nitrite ion 480 nm places is made to linear regression graph, obtain typical curve.According to above-mentioned steps, with the sample solution of certain volume, substitute the tartrate standard solution of certain volume, derived sample nitrite ion, at the absorbance at 480 nm places, according to aforesaid standards curve, can calculate the content of sample solution unresolvable tartaric acid.
(4) immobilization reconstitution cell physical strength measuring method: immobilization reconstitution cell gel is cut into 10*10*10 mm
3cubes, be placed on balance, with the counterweight of suitable weight to its front gradually evenly pressurization until flatten when no longer rebounding or breaking, the average mechanical intensity of the numerical representation method immobilization reconstitution cell when breaking on balance, the g/cm of unit
2represent.
(5) measuring method of enantiomeric excess value: 0.1 g reconstitution cell or the immobilization reconstitution cell being prepared from by 0.1 g reconstitution cell, after suspending with 50 ml 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0), oscillatory reaction under 30 oC.With distilled water, by after 400 times of reaction solution dilutions, detect, and calculate L (+)-tartaric enantiomeric excess value by high pressure liquid chromatography (Agilent 1200), its concrete testing conditions is: chiral column Astec CLC (150 * 4.6mm); Sample size 20 μ l; 30 ℃ of column temperatures; Moving phase 3mM CuSO
4(pH 3.2); Flow velocity 1ml/min; Detect wavelength 254nm.
embodiment 1:
By rhodococcus USA-AN012 BK-98 be seeded to seed culture medium (1% glucose, 0.5% peptone, 0.5% extractum carnis, 1% sodium-chlor, pH7.5) in, 30 ℃ of shaking culture 24 h.With EZ-10 pillar genome DNA extraction test kit (being purchased from Bio Basic Inc. company), according to product description, extract the genomic dna of rhodococcus USA-AN012 BK-98.Design two primers (being primer 1 and primer 2), the nucleotide sequence of primer 1 is as shown in SEQ ID NO:1, and the nucleotide sequence of primer 2 is as shown in SEQ ID NO:2.The genomic dna of the rhodococcus USA-AN012 BK-98 that extracts of take is template, utilizes primer 1 and primer 2, and pcr amplification obtains the gene of rhodococcus USA-AN012 BK-98 cis-Epoxysuccinic acid hydratase.Its PCR reaction system is: distilled water 40 μ l, 10 * PCR buffer, 5 μ l, 10 mmol/L dNTPs 1 μ l, the 10 mmol/L primers 1 of 1 μ l, the 10 mmol/L primer 2s of 1 μ l, rhodococcus USA-AN012 BK-98 genomic dna 1 μ l, Taq enzyme 1 μ l, the reaction system of totally 50 μ l.Its PCR program is: after 94 ℃ of preheating 5 min, and 94 ℃ of 50 s, 55 ℃ of 30 s, 72 ℃ of 1 min, 30 circulations, last 72 ℃ are extended 10 min.This PCR fragment and pTrc99A carrier (being purchased from Amersham Biosciences company) are used respectively to restriction enzyme
ncoi and
bamhI cuts 10 h in 37 ℃ of enzymes.Run agarose gel electrophoresis, by Wizard PCR fragment, reclaim test kit (being purchased from Promega company) and reclaim after two object fragments of purifying, use T
4dNA ligase connects 4 h in 16 ℃, to connect product conversion escherichia coli jm109 competent cell and (be purchased from Beijing Ding Guo biotechnology limited liability company, its genotype is recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, Δ (lac-proAB)/F ' [traD36, proAB+, lacIq, lacZ Δ M15]), coat containing (1% peptone on the LB agar plate of 50 μ g/ml penbritins, 0.5% yeast extract, 1% sodium-chlor, 1.5% agar powder, pH7.0), by the template in its PCR reaction system of bacterium colony PCR(, be 1 μ l bacterium liquid, all the other PCR reaction systems and program are same as described above) screening positive clone, obtain the genetic engineering bacterium of rhodococcus USA-AN012 BK-98 cis-Epoxysuccinic acid hydratase.
The genetic engineering bacterium of rhodococcus USA-AN012 BK-98 cis-Epoxysuccinic acid hydratase obtained above is seeded to containing (1% peptone in the LB substratum of 50 μ g/ml penbritins, 0.5% yeast extract, 1% sodium-chlor, pH7.0), as cell concentration (OD
600) while reaching 0.4-0.6, adding final concentration is the isopropylthio-β-D-galactoside of 0.2mM, 37 ℃ of shaking culture 12h.4 ℃, the centrifugal 10min of 5000 * g collects thalline, obtains cis-Epoxysuccinic acid hydratase reconstitution cell.
By the above-mentioned reconstitution cell of 0.1 g, by 50 ml concentration, be after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 22.4 h.
embodiment 2:
By the method for embodiment 1, prepare cis-Epoxysuccinic acid hydratase reconstitution cell.Get this reconstitution cell of 1g, after suspending with the physiological saline that 10 ml are 5 g/L tensio-active agent TritonX-100 containing activity, standing or low rate mixing suspension 30 min, the reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.1 h.
embodiment 3:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 11.5 h.
embodiment 4:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 5.3 h.
embodiment 5:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.5 h.
embodiment 6:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.3 h.
embodiment 7:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.2 h.
embodiment 8:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein surfactant concentration is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 3 h.
embodiment 9:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-100 Processing Example 1, wherein the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 6.5 h.
embodiment 10:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 18.9 h.
embodiment 11:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 7.3 h.
embodiment 12:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 4.6 h.
embodiment 13:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 3.2 h.
embodiment 14:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 3.1 h.
embodiment 15:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 3.2 h.
embodiment 16:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 4.6 h.
embodiment 17:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent TritonX-45 Processing Example 1, the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 13.1 h.
embodiment 18:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 21.9 h.
embodiment 19:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 17.6 h.
embodiment 20:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.5 h.
embodiment 21:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 11.6 h.
embodiment 22:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 11 h.
embodiment 23:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 8.6 h.
embodiment 24:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 9.2 h.
embodiment 25:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Span80 Processing Example 1, the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 16.9 h.
embodiment 26:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 20.6 h.
embodiment 27:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 19.6 h.
embodiment 28:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 17.7 h.
embodiment 29:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 16.7 h.
embodiment 30:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required change time of reconstitution cell is 16.6 h.
embodiment 31:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.6 h.
embodiment 32:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 16.6 h.
embodiment 33:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween20 Processing Example 1, the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 22.6 h.
embodiment 34:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 20.5 h.
embodiment 35:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 17.9 h.
embodiment 36:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 16.2 h.
embodiment 37:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 15.5 h.
embodiment 38:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 15.3 h.
embodiment 39:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.1 h.
embodiment 40:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 15.5 h.
embodiment 41:
Press the method for embodiment 2, the reconstitution cell obtaining by tensio-active agent Tween80 Processing Example 1, the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 23.7 h.
embodiment 42:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 20.7 h.
embodiment 43:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 19.9 h.
embodiment 44:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 15.5 h.
embodiment 45:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.5 h.
embodiment 46:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.2 h.
embodiment 47:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 15 h.
embodiment 48:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 16 h.
embodiment 49:
Press the method for embodiment 2, the reconstitution cell obtaining by Surfactant OP-10 Processing Example 1, the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 21.5 h.
embodiment 50:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 0.01 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 21.9 h.
embodiment 51:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 0.05 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.4 h.
embodiment 52:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 0.1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 12.3 h.
embodiment 53:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 0.5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 10 h.
embodiment 54:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 1 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 7.3 h.
embodiment 55:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 5 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 6.7 h.
embodiment 56:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 10 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 10.4 h.
embodiment 57:
Press the method for embodiment 2, the reconstitution cell obtaining by surfactant A EO-9 Processing Example 1, the activity of tensio-active agent is 50 g/L, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 14.6 h.
embodiment 58:
Press the method for embodiment 2, the reconstitution cell obtaining by same tensio-active agent TritonX-100 Processing Example 1, wherein standing the or low rate mixing suspension time is 10 min, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.9 h.
embodiment 59:
Press the method for embodiment 2, the reconstitution cell obtaining by same tensio-active agent Processing Example 1, wherein standing the or low rate mixing suspension time is 1 h, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 1.8 h.
embodiment 60:
Press the method for embodiment 2, the reconstitution cell obtaining by same tensio-active agent Processing Example 1, wherein standing the or low rate mixing suspension time is 5 h, all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.1 h.
embodiment 61:
Press the method for embodiment 2, with same tensio-active agent, process reconstitution cell, wherein standing the or low rate mixing suspension time is 10 h, and all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.1 h.
embodiment 62:
Press the method for embodiment 2, with same tensio-active agent, process reconstitution cell, wherein standing the or low rate mixing suspension time is 15 h, and all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.3 h.
embodiment 63:
Press the method for embodiment 2, with same tensio-active agent, process reconstitution cell, wherein standing the or low rate mixing suspension time is 20 h, and all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.4 h.
embodiment 64:
Press the method for embodiment 2, with same tensio-active agent, process reconstitution cell, wherein standing the or low rate mixing suspension time is 24 h, and all the other conditions are constant.The reconstitution cell of getting after the above-mentioned tensio-active agent of 0.1 g is processed is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of reconstitution cell is 2.4 h.
embodiment 65:
The reconstitution cell that 1 g embodiment 1 is obtained, with after 10 ml physiological saline, standing or low rate mixing suspension 1 h, then join in 30 ml 54 g/L kappa-carrageenan solution, under the condition of 42 oC, mix, cooling under the condition of 4 oC, solidify rear use 0.3 mol/L Klorvess Liquid and under the condition of 4 oC, fix 10 h.Get partial gel, gel is cut into 3*3*3 mm
3fritter, successively respectively with the washing of distilled water and physiological saline, being fixed reconstitution cell.In the immobilization reconstitution cell of above-mentioned preparation, the final concentration of kappa-carrageenan is 40 g/L, and the final concentration of reconstitution cell is 25 g/L.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 21.1 h, and the physical strength of immobilization reconstitution cell is 1561 g/cm
2.
embodiment 66:
The reconstitution cell that 1 g embodiment 1 is obtained, after suspending with the physiological saline that 10 ml are 5 g/L TritonX-100 containing activity, standing or low rate mixing suspension 1 h, then join in 30 ml 54 g/L kappa-carrageenan solution, under the condition of 42 oC, mix, cooling under the condition of 4 oC, solidify rear use 0.3 mol/L Klorvess Liquid and under the condition of 4 oC, fix 10 h.Get partial gel, gel is cut into 3*3*3 mm
3fritter, successively respectively with the washing of distilled water and physiological saline, being fixed reconstitution cell.In the immobilization reconstitution cell of above-mentioned preparation, the final concentration of kappa-carrageenan is 40 g/L, and the final concentration of reconstitution cell is 25 g/L.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell in the present embodiment is 1.7 h, compared with the transformation time of the control group of not processing with tensio-active agent in embodiment 65, shorten 12 times, the physical strength of immobilization reconstitution cell is 1564 g/cm
2.
embodiment 67:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein the add-on of reconstitution cell is 0.4 g, and the final concentration of reconstitution cell is 10 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.5 h, and the physical strength of immobilization reconstitution cell is 1703 g/cm
2.
embodiment 68:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein reconstitution cell add-on is 0.6 g, and the final concentration of reconstitution cell is 15 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in this enforcement, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.2 h, and the physical strength of immobilization reconstitution cell is 1668 g/cm
2.
embodiment 69:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein reconstitution cell add-on is 0.8 g, and the final concentration of reconstitution cell is 20 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in this enforcement, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 1.8 h, and the physical strength of immobilization reconstitution cell is 1609 g/cm
2.
embodiment 70:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein reconstitution cell add-on is 1.2 g, and the final concentration of reconstitution cell is 30 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 1.8 h, and the physical strength of immobilization reconstitution cell is 1518 g/cm
2.
embodiment 71:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein reconstitution cell add-on is 1.6 g, and the final concentration of reconstitution cell is 40 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.1 h, and the physical strength of immobilization reconstitution cell is 1485 g/cm
2.
embodiment 72:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein reconstitution cell add-on is 2 g, and the final concentration of reconstitution cell is 50 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.4 h, and the physical strength of immobilization reconstitution cell is 1427 g/cm
2.
embodiment 73:
By the method for embodiment 66, prepare immobilization reconstitution cell, the concentration of the 30 ml kappa-carrageenan solution that wherein add is 26.7 g/L, and the final concentration of kappa-carrageenan is 20 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.5 h, and the physical strength of immobilization reconstitution cell is 1048 g/cm
2.
embodiment 74:
By the method for embodiment 66, prepare immobilization reconstitution cell, the concentration of the 30 ml kappa-carrageenan solution that wherein add is 40 g/L, and the final concentration of kappa-carrageenan is 30 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.2 h, and the physical strength of immobilization reconstitution cell is 1369 g/cm
2.
embodiment 75:
By the method for embodiment 66, prepare immobilization reconstitution cell, the concentration of the 30 ml kappa-carrageenan solution that wherein add is 66.7 g/L, and the final concentration of kappa-carrageenan is 50 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.3 h, and the physical strength of immobilization reconstitution cell is 1789 g/cm
2.
embodiment 76:
By the method for embodiment 66, prepare immobilization reconstitution cell, the concentration of the 30 ml kappa-carrageenan solution that wherein add is 80 g/L, and the final concentration of kappa-carrageenan is 60 g/L, and all the other conditions are constant.Getting above-mentioned immobilization reconstitution cell (wherein, containing the reconstitution cell 0.1g before immobilization), is after 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) suspends by 50 ml concentration, oscillatory reaction under 30 oC, the content of monitoring reaction solution unresolvable tartaric acid.Result shows, in the present embodiment, when cis-form epoxy succinic acid sodium generates tartaric transformation efficiency and reaches 90%, the required transformation time of immobilization reconstitution cell is 2.5 h, and the physical strength of immobilization reconstitution cell is 2037 g/cm
2.
embodiment 77:
With the reconstitution cell that 0.1 g embodiment 1 obtains, in 50 ml concentration, be in 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0), under 30 oC constant temperatures, low rate mixing transforms 22.4 h, and it is 99.9% that transformation efficiency reaches 90%, L (+)-tartrate enantiomeric excess value.4 oC high speed centrifugation (5000 * g, 10 min) reclaim reconstitution cell, it is dropped in 50 ml 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) again, oscillatory reaction under 30 oC, reconstitution cell transforms after 22.4 h, transformation efficiency is that 25%, L (+)-tartrate enantiomeric excess value is 99.7%.Continue to repeat above step (filtered and recycled reconstitution cell transforms 22.4 h under above-mentioned condition), at reconstitution cell, reused after 3 times, transformation efficiency is 0% substantially.
embodiment 78:
Get the reconstitution cell that 0.1 g embodiment 1 obtains, after physiological saline suspension with 1 ml containing 5 g/L TritonX-100, standing or low rate mixing suspension 1 h, adding 50 ml concentration is in 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0), under 30 oC constant temperatures, low rate mixing transforms 1.8 h, and it is 99.9% that transformation efficiency reaches 90%, L (+)-tartrate enantiomeric excess value.4 oC high speed centrifugation (5000 * g, 10 min) reclaim cell, it is dropped in 50 ml 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) again, oscillatory reaction under 30 oC, reconstitution cell transforms after 1.8 h, transformation efficiency is that 24%, L (+)-tartrate enantiomeric excess value is 99.7%.Continue to repeat above step (filtered and recycled cell transforms 1.8 h under above-mentioned condition), the reconstitution cell after tensio-active agent is processed has been reused after 3 times, and transformation efficiency is 0% substantially.Above result shows, the transformation time of the reconstitution cell after processing with tensio-active agent TritonX-100 in the present embodiment shortens more than 12 times compared with untreated control group in embodiment 77, but the reconstitution cell of no matter whether processing with tensio-active agent, all cannot circulate and repeatedly use.
embodiment 79:
By the method for embodiment 66, prepare immobilization reconstitution cell, wherein, before immobilization, the consumption of reconstitution cell is 0.1 g, immobilization reconstitution cell is joined to pH is 8.0, concentration is in the 50 ml cis-form epoxy succinic acid sodium of 1 mol/L, under 30 oC constant temperatures, low rate mixing transforms 1.7 h, and it is 99.9% that transformation efficiency reaches 90%, L (+)-tartrate enantiomeric excess value.Filtered and recycled immobilization reconstitution cell, it is dropped in 50 ml 1 mol/L cis-form epoxy succinic acid sodium (pH 8.0) again, oscillatory reaction under 30 oC, reconstitution cell transforms after 1.7 h, transformation efficiency is that 90.5%, L (+)-tartrate enantiomeric excess value is 99.8%.Continue to repeat, after above step (filtered and recycled immobilization reconstitution cell transforms 1.7 h under above-mentioned condition) 8 times, at immobilization reconstitution cell, to have reused after 10 times, transformation efficiency is that 82%, L (+)-tartrate enantiomeric excess value is 99.6%.In the present embodiment, the substrate conversion efficiency after immobilization reconstitution cell is used 10 batches also has 82%; And in embodiment 77 and 78, the substrate conversion efficiency after reconstitution cell is used 3 batches is 0%.Visible, when utilizing reconstitution cell to prepare tartrate, use the conversion batch conversion batch far above use on-fixed reconstitution cell of immobilization reconstitution cell.
The cis-Epoxysuccinic acid hydratase genetic engineering bacterium that above embodiment is used, wherein, pTrc99A is expression vector,
e. colijM109 is host cell.The present invention also can adopt other expression vector, for example pET15b, pET22b, pET28a, pET39b, pP
rOeX HTb or pGEX-4T-2 etc.; Host cell can also be used
e. colidH5 α,
e. colibL21 (DE3),
e. colitG1 or
e. colitop10 etc.These cis-Epoxysuccinic acid hydratase genetic engineering bacteriums that build are carried out to above-mentioned tensio-active agent and immobilization processing, do not compare with the reconstitution cell of immobilization processing with making above-mentioned tensio-active agent, its transformation time and batch conversion results and above embodiment are basically identical.
<110> Hangzhou Bao Jing Biological Co., Ltd.
Mono-kind of <120> produces L(+ by improving cell permeability)-tartaric method
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 44
<212> DNA
<213> artificial sequence
<400> 1
ccatgggcca tcaccatcac catcacatgc aactgaacaa tgcg 44
<210> 2
<211> 26
<212> DNA
<213> artificial sequence
<400> 2
ggatcctcaa tcgataccgg cagttc 26
Claims (9)
1. by improving cell permeability, produce L (+)-tartaric method, it is characterized in that, comprise the steps: the reconstitution cell of (1) fermentation culture expression cis-Epoxysuccinic acid hydratase; (2) use Triton series of surfactants, as penetrating dose, described reconstitution cell is carried out to permeabilized processing; (3) utilize the reconstitution cell of permeabilized processing to produce L (+)-tartrate.
2. according to claim 1 by improving cell permeability production L (+)-tartaric method, it is characterized in that: before the reconstitution cell that utilizes permeabilized processing is produced L (+)-tartrate, first the reconstitution cell of this permeabilized processing is used to kappa-carrageenan immobilization.
3. according to claim 2ly by improving cell permeability, produce L (+)-tartaric method, it is characterized in that, use the immobilized method of kappa-carrageenan as follows the reconstitution cell of permeabilized processing:
The reconstitution cell suspension of permeabilized processing is added in kappa-carrageenan solution and mixed, after cooled and solidified, with Klorvess Liquid, soak and fix, wherein, the final concentration of kappa-carrageenan is 20-60g/l, and the final concentration of the reconstitution cell of permeabilized processing is 10-50g/l.
4. according to claim 3 by improving cell permeability production L (+)-tartaric method, it is characterized in that: the final concentration of described kappa-carrageenan is 30-50g/l, and the final concentration of the reconstitution cell of permeabilized processing is 20-30g/l.
5. according to claim 1 and 2 by improving cell permeability production L (+)-tartaric method, it is characterized in that, in described step (2), the method of described reconstitution cell being carried out to permeabilized processing is: the reconstitution cell that step (1) is cultivated suspends with the physiological saline that contains Triton series of surfactants, and the activity of described tensio-active agent is 0.01-50 g/L; Then standing or low rate mixing suspension 10 min-24 h.
6. according to claim 5 by improving cell permeability production L (+)-tartaric method, it is characterized in that: the activity of described tensio-active agent is 0.1-10 g/L standing or low rate mixing suspension 30 min-5 h.
7. according to claim 1 and 2 by improving cell permeability production L (+)-tartaric method, it is characterized in that: described tensio-active agent is nonionogenic tenside.
8. according to claim 7 by improving cell permeability production L (+)-tartaric method, it is characterized in that, described nonionogenic tenside is that sorbitan fatty acid ester series of surfactants, polysorbate series of surfactants, polyoxyethylene glycol are to iso-octyl phenyl ether series of surfactants, alkylphenol polyoxyethylene series of surfactants or ethoxylated dodecyl alcohol series of surfactants.
9. according to claim 8 by improving cell permeability production L (+)-tartaric method, it is characterized in that: described nonionogenic tenside is triton x-100.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109956501A (en) * | 2019-03-25 | 2019-07-02 | 安徽海蓝生物科技有限公司 | It is a kind of production L- (+)-tartaric acid during sodium tungstate recovery method |
| CN110079561A (en) * | 2019-03-25 | 2019-08-02 | 安徽海蓝生物科技有限公司 | A kind of coupled method prepares L (+)-tartaric acid technique |
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