CN103882000A - Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof - Google Patents
Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof Download PDFInfo
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Abstract
The invention discloses a cis-epoxysuccinate hydrolase immobilization method. According to the immobilization method, the immobilization of cis-epoxysuccinate hydrolase to an affinity chromatography medium is realized through enabling a histidine tag and cis-epoxysuccinate hydrolase to be subjected to fusion expression and using interaction between histidine imidazolyl and metal ion Ni<2+> in the affinity chromatography medium. According to the immobilization method, the activity and yield of an enzyme are high, the obtained immobilized enzyme is good in stability and high in catalytic efficiency, the step of enzyme separation and purification is omitted, and the operation is simple.
Description
Technical field
The present invention relates to bioengineering field, relate in particular to a kind of process for fixation of cis-Epoxysuccinic acid hydratase, its immobilized enzyme, and method based on this immobilized enzyme production chirality tartrate or its salt.
Background technology
Along with chiral drug continuous application clinically, the disassemble technique of chiral drug is also gradually improved.Chirality tartrate, as a kind of important chiral resolving agent, has good market outlook and using value.At present, in the production process of antitubercular agent Tibutol, L-(+)-tartrate has been obtained successful Application as unique, irreplaceable chiral resolving agent.Meanwhile, some the tartaric acid derivatives class medicines take chirality tartrate as precursor have also entered clinical, as metoprolol tartrate tablets.In addition, chirality tartrate is also important foodstuff additive and industrial chemicals, is widely used in the fields such as food, printing and dyeing, plating, leather.
The tartaric production method of chirality mainly contains synthetic four kinds of extraction method, chemical resolution method, saccharic fermentation method and enzyme process.Wherein, enzyme process is synthetic take petrochemicals toxilic acid as initial feed, make cis-form epoxy succinic acid salt by hydrogen peroxide epoxidation, then under the effect of L-(+)-type or D-(-)-type cis-Epoxysuccinic acid hydratase (ESH), hydrolysis generates L-(+)-type or D-(-)-type tartrate.Since the inferior reported first of Japanese scholars assistant rattan English in 1974 can adopt enzyme process to prepare L-(+)-tartrate, due to advantages such as its transformation efficiency are high, specificity is strong, security is good, be applied to L-(+)-tartaric industrial production.
The ESH having reported at present produces bacterial classification to be had: false pseudomonas bacillus (Pseudomonas sp.), root nodule bacterium (Rhizobium validum), Alcaligenes (Alcaligenes sp.), coryneform bacteria (Corynebacterium sp.), Nocardia bacteria (Nocardia tartaricans), rhodococcus (Rhodococcus rhodochrous), achromobacter (Achromobaeter sp.), wherein coryneform bacteria and promise cassette bacterium are L-(+)-tartaric industrial production bacterial classifications.During due to free cell catalysis, the apparent activity of ESH is very low, has caused the catalytic efficiency of enzyme very low.Mao Qingjing has studied the impact that N.tartaricans permeability of cell membrane is lived on the apparent enzyme of ESH, result shows, N.tartaricans cell is after ultrasonic disruption, ESH specific activity intact cell has improved more than 40 times, but after cytoclasis, endogenous proteinase can affect stability (East China University of Science's journal of ESH, 2003,29 (3): 264-267).The people such as Rosenberg have studied the impact that tensio-active agent is lived on the apparent enzyme of ESH, find that N.tartaricans cell is after 0.02% (w/v) Sodium desoxycholate is processed, ESH apparent activity can improve 20 times (Biotechnol.Lett, 1999,21:491-495).
In order further to improve catalytic efficiency and the utilization ratio of enzyme, immobilized cell technique is introduced to L-(+)-tartaric production process.Sun Zhihao etc. adopt gelatin embedding N.tartaricans first, obtain the immobilized cell that ESH vigor is higher (biotechnology journal, 1995,11 (4): 372-376).Zhang Jianguos etc. are take carrageenin as carrier immobilized Corynebacterium sp.JZ-1 strain cell, after activation treatment, the total yield of ESH enzyme activity is more than 100%, and thermostability, pH stability are all improved, in 1500L enzyme post, move continuously 90d, ESH enzyme (the biotechnology journal that substantially remains unchanged alive, 2000,16 (2): 188-192).Vikartovsk á etc. are by the N.tartaricans ATCC31191 cell calcium pectinate Immobilized in Microcapsule containing ESH, after immobilization, the apparent enzyme activity of ESH is 75.8U/mg stem cell (Artif.Cells Blood Substit.Immobil.Biotechnol, 2004,32 (1): 77-89).Afterwards,
deng employing sodiun alginate-sulfate cellulose-polyethylene (SA-CS/PMCG) Immobilized in Microcapsule N.tartaricans, research is found, this method can further improve the apparent activity (91.5U/mg stem cell) of N.tartaricans ESH, shorten the bio-transformation time, improve L-(+)-tartaric production efficiency (Enzyme Microb.Technol, 2005,36:118-126).
Compared with free cell, immobilized cell has effectively overcome the defects such as enzyme utilization ratio is low, catalytic efficiency is low, but still cannot thoroughly remove the diffusion barrier effect of cytolemma.The immobilized enzyme reactor that development in recent years is got up not only can solve the diffusion limitation effect of cytolemma, can also significantly improve stability and the catalytic efficiency of enzyme.Cui's balls etc., by ESH gene and carbohydrate binding modules CBM30 amalgamation and expression, are then immobilized in ESH in cellulose media, have improved the stability (CN102703419A) of ESH.But due to sequence and the textural difference of CBM module self, caused its difference that affects on different proteins (or enzyme) molecule, therefore suitability is poor.
Summary of the invention
For the defect of above-mentioned prior art, the object of the invention is to propose that a kind of enzymic activity yield is high, the process for fixation of the cis-Epoxysuccinic acid hydratase of good stability, its immobilized enzyme and the method based on this immobilized enzyme production chirality tartrate or its salt.
In order to achieve the above object, the present invention is by the following technical solutions:
First aspect, the invention provides a kind of process for fixation of cis-Epoxysuccinic acid hydratase, and the method comprises the steps:
(1) N-terminal of cis-Epoxysuccinic acid hydratase gene or C-terminal are connected with histidine-tagged, build the fusion expression vector of the two;
(2), by step (1) gained fusion expression vector Transformation Engineering bacterium, obtain recombinant bacterial strain;
(3) step (2) gained recombinant bacterial strain is cultivated and abduction delivering, obtained recombinant expressed engineering bacteria;
(4) collect recombinant expressed engineering bacteria, and carry out bacterial cell disruption, solid-liquid separation, collects supernatant liquor;
(5) step (4) gained supernatant liquor is through Ni
2+chelating chromatography media carries out chelatropic reaction, obtains immobilized cis Epoxysuccinic acid lytic enzyme.
In above-mentioned process for fixation, as preferably, in step (1), described histidine-tagged by 4～8, be preferably 6 Histidines and form;
In specific embodiments, described histidine-taggedly formed by 4,5,6,7 or 8 Histidines.
Preferably, the pBV220 carrier that the carrier is carrier of described fusion expression vector is temperature-induced type.
As preferably, the described engineering bacteria of step (2) is intestinal bacteria E.coli.
As preferably, in step (3), described cultivation for by recombinant bacterial strain at 30～37 ℃, preferably at 35～37 ℃, to be more preferably cultured to OD600 at 37 ℃ be 0.6～1.0, be preferably 0.7～0.9, more preferably 0.8;
In specific embodiments, described cultivation is that recombinant bacterial strain is cultivated at 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃ or 37 ℃, to OD600 be 0.6,0.7,0.8,0.9 or 1.0.
Preferably, described abduction delivering is: culture temperature is risen to 40～42 ℃, preferably 42 ℃, and inducing culture 4～6h, is preferably 5h.
In specific embodiments, described abduction delivering is: culture temperature is risen to 40 ℃, 40.5 ℃, 41 ℃, 41.5 ℃ or 42 ℃, inducing culture 4h, 5h or 6h.
As preferably, in step (4), described bacterial cell disruption adopts any one in high pressure cell fragmentation, ultrasonic disruption or N,O-Diacetylmuramidase fragmentation, preferably adopts ultrasonic disruption;
Preferably, before described bacterial cell disruption, wash described thalline with phosphate buffered saline buffer; Further preferably the pH of described phosphate buffered saline buffer is 7.2～7.6, is preferably 7.4, and concentration is 18～22mM, preferred 20mM;
Preferably, described solid-liquid separation is undertaken by centrifugal; Further preferably, described centrifugal condition is: 2～8 ℃, preferably at 4 ℃, and with 8,000～12,000g, preferably 10, the centrifugal 15～30min of rotating speed of 000g, preferably 20min.
As preferably, in step (5), described Ni
2+the solid phase carrier of chelating chromatography media is any one in agarose, dextran or Mierocrystalline cellulose, preferably agarose microbeads solid phase carrier;
Preferably, at described supernatant liquor process Ni
2+before chelating chromatography media, described Ni
2+chelating chromatography media first carries out balance with the phosphate buffered saline buffer containing 0.08～0.12M, excellent 0.1M NaCl; Further preferably, the pH of described phosphate buffered saline buffer is 7.2～7.6, is preferably 7.4, and concentration is 18～22mM, preferred 20mM;
In specific embodiments, the content of NaCl in phosphate buffered saline buffer is 0.08M, 0.09M, 0.1M, 0.11M or 0.12M; The pH of described phosphate buffered saline buffer is 7.2,7.3,7.4,7.5 or 7.6, and concentration is 18mM, 19mM, 20mM, 21mM or 22mM.
Preferably, at described supernatant liquor process Ni
2+after chelating chromatography media, wash with phosphate buffered saline buffer; Further preferably, the pH of described phosphate buffered saline buffer is 7.2～7.6, is preferably 7.4, and concentration is 18～22mM, preferred 20mM.
In specific embodiments, the pH of described phosphate buffered saline buffer is 7.2,7.3,7.4,7.5 or 7.6, and its concentration is 18mM, 19mM, 20mM, 21mM or 22mM.
Described supernatant liquor is through Ni
2+when chelating chromatography media, imidazolyl and the metal ion Ni of the Histidine in amalgamation and expression albumen in supernatant liquor (cis-Epoxysuccinic acid hydratase and Histidine)
2+interact, thus by containing the amalgamation and expression proteopexy of cis-Epoxysuccinic acid hydratase on affinity chromatography medium.
Above-mentioned process for fixation both can be used for the immobilization of L-type cis-Epoxysuccinic acid hydratase, can be used for again the immobilization of D-type cis-Epoxysuccinic acid hydratase.
Second aspect, the invention provides a kind of immobilized cis Epoxysuccinic acid lytic enzyme, is made by preparation method described in first aspect present invention.
The third aspect, the invention provides a kind of method of producing chirality tartrate or its salt, and the method is: cis-form epoxy succinic acid or its salt under the effect of immobilized cis Epoxysuccinic acid lytic enzyme described in second aspect, enzyme digestion reaction occur and obtains;
Preferably, the temperature of described enzyme digestion reaction is 30～40 ℃, is preferably 32～38 ℃, more preferably 37 ℃;
Preferably, the pH value of described enzyme digestion reaction is 7～9, is preferably 8;
Preferably, described chirality tartrate is chirality sodium tartrate, chirality soluble tartrate, chirality Seignette salt or chirality tartarus;
Preferably, described chirality tartrate or its salt are L-type or D type.
In preferred embodiments, the method for described production chirality tartrate or its salt is: by described immobilized cis Epoxysuccinic acid lytic enzyme filling chromatography column; Then aqueous solution loading to the described chromatography column that is 7～9 by pH, be preferably 8 cis-form epoxy succinic acid or its salt carries out enzyme digestion reaction, between the reaction period, the temperature of chromatography column is controlled to 30～40 ℃, preferably 32～38 ℃, more preferably 37 ℃; Finally collect reaction solution and get final product.
In specific embodiment, adopt peristaltic pump or AKTA to carry out loading, in loading process, adjust loading speed and carry out to react fully; Preferably, by loading rate-controlling at 0.5～1BV/min, 1BV/min more preferably.In the time that substrate transfer rate is 1BV/min, the transformation efficiency of enzyme digestion reaction substrate has reached more than 99.5%.
In specific embodiment, recirculated water bath control for the temperature of described chromatography column.
In above-mentioned preferred embodiment, further preferably, the concentration of the aqueous solution of described cis-form epoxy succinic acid or its salt is 0.8～1.6M, be preferably 1.0～1.4M, 1.2M more preferably.
In the method for above-mentioned production chirality tartrate or its salt, in the time that described immobilized cis Epoxysuccinic acid lytic enzyme is L-type, the tartrate of producing or its salt are L-(+)-tartrate or its salt; In the time that described immobilized cis Epoxysuccinic acid lytic enzyme is D type, the tartrate of producing or its salt are D-(-)-tartrate or its salt.
The present invention, by carrying out amalgamation and expression by histidine-tagged with cis-Epoxysuccinic acid hydratase, utilizes the metal ion Ni in Histidine imidazolyl and affinity chromatography medium
2+between interaction, realized the immobilization of cis-Epoxysuccinic acid hydratase on affinity chromatography medium; The enzymic activity yield of this process for fixation is high, gained immobilized enzyme good stability, catalytic efficiency high (substrate conversion efficiency reaches more than 99.5%), and omitted the purification procedures of enzyme, simple to operate.
Accompanying drawing explanation
Fig. 1 is the thermostability comparison of immobilization ESH of the present invention and free ESH; Wherein, "●" represents immobilization ESH of the present invention, and "○" represents free ESH.
Fig. 2 is the pH stability comparison of immobilization ESH of the present invention and free ESH; Wherein, "●" represents immobilization ESH of the present invention, and "○" represents free ESH.
Fig. 3 is the stability in storage comparison of immobilization ESH of the present invention and free ESH; Wherein, "●" represents immobilization ESH of the present invention, and "○" represents free ESH.
Fig. 4 uses immobilization ESH of the present invention to prepare the schematic diagram of chirality sodium tartrate.
Embodiment
Further illustrate technical scheme of the present invention below in conjunction with specific embodiment.
The affine immobilization of embodiment 1 L-type of the present invention cis-Epoxysuccinic acid hydratase
Adopt following steps:
(1) introduce the histidine-tagged sequence of 6 Histidines at the C end of coding L-type cis-Epoxysuccinic acid hydratase gene, take the pBV220 carrier of temperature-induced type as carrier is carrier, vector construction hexose transport protein;
(2) single bacterium colony of picking recombinant plasmid transformed on flat board, access 5mL contains the LB substratum of 100 μ L/mL penbritins, in 37 ℃, 200rpm incubated overnight;
(3) getting above-mentioned bacterium liquid transfers into the fresh LB substratum (4% inoculum size) containing 100 μ L/mL penbritins, in 37 ℃, 200rpm continues to cultivate, in the time that OD600 reaches 0.8 ± 0.1, temperature is risen to 42 ℃, continue after inducing culture 5h, at 4 ℃, centrifugal collection thalline under 8000 × g condition;
(4) the 20mM phosphate buffered saline buffer that is 7.4 with pH washing thalline, removes residual nutrient media components.The resuspended thalline of 20mM phosphate buffered saline buffer that is 7.4 with pH, is made into 20% bacteria suspension, and with the broken somatic cells of ultrasonic disruption instrument, after fragmentation, at 4 ℃, centrifugal 20min under 10000 × g condition, collects supernatant liquor, obtains crude enzyme liquid.
(5) suitable crude enzyme liquid dilution being made to its protein concn is 10mg/mL.To fill in the agarose metal-chelating medium of chromatography column, good by 20mM phosphate buffered saline buffer (pH=7.4) balance containing 0.1M NaCl.By crude enzyme liquid loading to this chromatography column, until medium absorption is saturated.Then the further drip washing of 20mM phosphate buffered saline buffer that is 7.4 with pH, removes non-specific adsorption albumen, being fixed cis-Epoxysuccinic acid hydratase.
The stability study of embodiment 2 immobilized cis Epoxysuccinic acid of the present invention lytic enzyme
Take the prepared immobilization ESH of embodiment 1 as stability study object, carry out respectively as follows the research of thermostability, pH stability and stability in storage.
(1) thermostability: immobilization ESH is placed in to (45～60 ℃) under different temperature condition, measures its residual ESH enzyme activity every 5min, until its vigor is down to half, measure its transformation period, simultaneously with dissociating ESH in contrast.
Result shows, the thermostability of immobilization ESH has obtained improving significantly, has strengthened the anti-adversity ability of ESH to reaction system microenvironment.As shown in Figure 1, the vigor transformation period of immobilization ESH under 45 ℃, 50 ℃, 55 ℃ and 60 ℃ of conditions respectively free ESH improved 2.86,3.75,5.40 and 7.00 times.
Cui's ball etc. has been prepared a kind of cellulose fixed ESH by amalgamation and expression CBM30 carbohydrate binding modules, and study its stability under 30～40 ℃ of conditions, result shows, the transformation period of cellulose fixed ESH under 40 ℃ of conditions is 81.53min, and under equal conditions, the transformation period of free ESH is only 26.55min(CN102703419A).Agarose immobilization ESH prepared by the present invention is at 40 ℃ and have a satisfactory stability below, and its transformation period is greater than 4h, and in the time that temperature reaches 45 ℃, more than its transformation period still can reach 50min.
(2) pH stability: immobilization ESH is placed under different pH conditions (pH3.0～10.0), measures its residual ESH enzyme activity every 5min, until its vigor is down to half, measure its transformation period, simultaneously with dissociating ESH in contrast.
As shown in Figure 2, immobilization ESH is under pH10.0,9.5,6.0 and 5.5 conditions for result, and the free ESH of stability has improved respectively 3.62,3.24,3.75 and 4.80 times.
(3) stability in storage: immobilization ESH is placed under pH7.5 and room temperature (25 ℃) condition, measure at regular intervals its residual ESH enzyme activity, until its vigor is down to half, analyze the Natural Attenuation cycle that its enzyme is lived, simultaneously with dissociating ESH in contrast.
As shown in Figure 3, the stability in storage of immobilization ESH has obtained good improvement to result, and free ESH has improved 11.6 times, and the transformation period of enzyme activity has reached more than 50 days.
Embodiment 3 uses immobilized enzyme production chirality sodium tartrate of the present invention
With AKTA be 8.0 by pH, the substrate cis-form epoxy succinic acid sodium solution loading that concentration is 1.2M is to immobilized enzyme chromatography post, control temperature of reaction is 37 ℃ simultaneously, makes bottoms stream through immobilized enzyme reaction column, its Production Flow Chart schematic diagram is as shown in Figure 4.
In the time that substrate transfer rate is 1BV/min, the transformation efficiency of substrate cis-form epoxy succinic acid sodium has reached more than 99.5%, and the disclosed cellulose fixed ESH of patent documentation CN102703419A transforms while preparing chirality tartrate, and its substrate conversion efficiency is only 75%.
Applicant's statement, the present invention illustrates process for fixation, its immobilized enzyme and the method for the production of chirality tartrate or its salt thereof of cis-Epoxysuccinic acid hydratase of the present invention by above-described embodiment, but the present invention is not limited to above-described embodiment.Person of ordinary skill in the field should understand, any improvement in the present invention, within all dropping on protection scope of the present invention and open scope.
Claims (10)
1. a process for fixation for cis-Epoxysuccinic acid hydratase, is characterized in that, comprises the steps:
(1) N-terminal of cis-Epoxysuccinic acid hydratase gene or C-terminal are connected with histidine-tagged, build the fusion expression vector of the two;
(2), by step (1) gained fusion expression vector Transformation Engineering bacterium, obtain recombinant bacterial strain;
(3) step (2) gained recombinant bacterial strain is cultivated and abduction delivering, obtained recombinant expressed engineering bacteria;
(4) collect recombinant expressed engineering bacteria, and carry out bacterial cell disruption, solid-liquid separation, collects supernatant liquor;
(5) step (4) gained supernatant liquor is through Ni
2+chelating chromatography media carries out chelatropic reaction, obtains immobilized cis Epoxysuccinic acid lytic enzyme.
2. process for fixation according to claim 1, is characterized in that, in step (1), described histidine-tagged by 4～8, be preferably 6 Histidines and form;
Preferably, the pBV220 carrier that the carrier is carrier of described fusion expression vector is temperature-induced type.
3. process for fixation according to claim 1 and 2, is characterized in that, the described engineering bacteria of step (2) is intestinal bacteria E.coli.
4. according to the process for fixation described in claim 1-3 any one, it is characterized in that, in step (3), described cultivation for by recombinant bacterial strain at 30～37 ℃, preferably at 35～37 ℃, to be more preferably cultured to OD600 at 37 ℃ be 0.6～1.0, be preferably 0.7～0.9, more preferably 0.8;
Preferably, described abduction delivering is: culture temperature is risen to 40～42 ℃, preferably 42 ℃, and inducing culture 4～6h, is preferably 5h.
5. according to the process for fixation described in claim 1-4 any one, it is characterized in that, in step (4), described bacterial cell disruption adopts any one in high pressure cell fragmentation, ultrasonic disruption or N,O-Diacetylmuramidase fragmentation, preferably adopts ultrasonic disruption;
Preferably, before described bacterial cell disruption, wash described thalline with phosphate buffered saline buffer; Further preferably, the pH of described phosphate buffered saline buffer is 7.2～7.6, is preferably 7.4, and concentration is 18～22mM, preferred 20mM;
Preferably, described solid-liquid separation is undertaken by centrifugal; Further preferably, described centrifugal condition is: 2～8 ℃, preferably at 4 ℃, and with 8,000～12,000g, preferably 10, the centrifugal 15～30min of rotating speed of 000g, preferably 20min.
6. according to the process for fixation described in claim 1-5 any one, it is characterized in that, in step (5), described Ni
2+the solid phase carrier of chelating chromatography media is any one in agarose, dextran or Mierocrystalline cellulose, preferably agarose microbeads solid phase carrier;
Preferably, at described supernatant liquor process Ni
2+before chelating chromatography media, described Ni
2+chelating chromatography media first carries out balance with the phosphate buffered saline buffer containing 0.08～0.12M, excellent 0.1M NaCl; Further preferably, the pH of described phosphate buffered saline buffer is 7.2～7.6, is preferably 7.4, and concentration is 18～22mM, preferred 20mM;
Preferably, at described supernatant liquor process Ni
2+after chelating chromatography media, wash with phosphate buffered saline buffer; Further preferably, the pH of described phosphate buffered saline buffer is 7.2～7.6, is preferably 7.4, and concentration is 18～22mM, preferred 20mM.
7. an immobilized cis Epoxysuccinic acid lytic enzyme, is characterized in that, is made by preparation method described in claim 1～6 any one.
8. a method of producing chirality tartrate or its salt, is characterized in that, cis-form epoxy succinic acid or its salt under the effect of immobilized cis Epoxysuccinic acid lytic enzyme described in claim 7, enzyme digestion reaction occur and obtains;
Preferably, the temperature of described enzyme digestion reaction is 30～40 ℃, is preferably 32～38 ℃, more preferably 37 ℃;
Preferably, the pH value of described enzyme digestion reaction is 7～9, is preferably 8;
Preferably, described chirality tartrate is chirality sodium tartrate, chirality soluble tartrate, chirality Seignette salt or chirality tartarus;
Preferably, described chirality tartrate or its salt are L-type or D type.
9. method as claimed in claim 8, is characterized in that, by described immobilized cis Epoxysuccinic acid lytic enzyme filling chromatography column; Then aqueous solution loading to the described chromatography column that is 7～9 by pH, be preferably 8 cis-form epoxy succinic acid or its salt carries out enzyme digestion reaction, between the reaction period, the temperature of chromatography column is controlled to 30～40 ℃, preferably 32～38 ℃, more preferably 37 ℃; Finally collect reaction solution and get final product.
10. method as claimed in claim 9, is characterized in that, the concentration of the aqueous solution of described cis-form epoxy succinic acid or its salt is 0.8～1.6M, be preferably 1.0～1.4M, 1.2M more preferably.
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