CN102604922B - Novel maleate cis-trans isomerase and application thereof - Google Patents
Novel maleate cis-trans isomerase and application thereof Download PDFInfo
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- CN102604922B CN102604922B CN2011100262975A CN201110026297A CN102604922B CN 102604922 B CN102604922 B CN 102604922B CN 2011100262975 A CN2011100262975 A CN 2011100262975A CN 201110026297 A CN201110026297 A CN 201110026297A CN 102604922 B CN102604922 B CN 102604922B
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Abstract
The invention discloses the discovery and application of a novel maleate cis-trans isomerase. The full-length gene sequence (SEQ No.1) of a protein probably having the activity of the maleate cis-trans isomerase is found in a Rhodococcus sp. RHA1 whole genome, a gene 5' is subjected to deletion transformation after it is found that the corresponding protein does not have the activity of the maleate cis-trans isomerase, the deletion length is 126-234bp under the premise that the reading frame is not changed, and a group of gene sequences which are collectively called SEQ No. 22 of an enzyme protein molecule having the activity of the maleate cis-trans isomerase is obtained. Recombinant plasmids with the gene sequences of SEQ No. 22 are constructed, and the recombinant plasmids are introduced into the colon bacillus to obtain a genetically-engineered strain. The genetically-engineered strain is used for fermenting and expressing a soluble protein with the activity of the maleate cis-trans isomerase, and the whole bacterial cell sap or immobilized bacterial cell sap or broken bacterial cell sap can used for transforming maleic acid into fumaric acid under a mild condition. The engineering bacteria of the isomerase can be used for producing the fumaric acid on a large scale.
Description
Technical field
The present invention relates to bioengineering field, the expression that relates to a kind of maleic acid isomerase reaches the application that is converted into anti-top enedioic acid at maleic acid.
Background technology
Maleic acid is toxilic acid, can generate MALEIC ANHYDRIDE by benzene oxicracking under catalyzer and high temperature and certain pressure usually, is obtained by hydrolysis by the latter again, also has by the butane oxidation dehydrogenation to obtain.
Be used for preparing production fumaric acid (FUMARIC ACID TECH GRADE) exactly one of industrial toxilic acid important purposes, fumaric acid is as an important industrial chemicals, can be used for producing numerous Chemicals, as: resin, tackiness agent, softening agent, medicine and fine chemicals intermediate, foodstuff additive, etc.Industrial have several different methods to produce fumaric acid.Its main source is in the presence of catalyzer benzene (or butylene) oxidation to be generated maleic acid (or MALEIC ANHYDRIDE), gets through isomerization again.Benzene (or butylene of 80%) and excess air are carried out oxidizing reaction generate MALEIC ANHYDRIDE in fluidized-bed or fixed-bed reactor, the acid solution that is recycled absorbs into maleic acid; Through decolorization filtering, maleic acid carries out isomerization under the thiourea catalyst effect again, and reactant washs after filtration, and being drying to obtain FUMARIC ACID TECH GRADE is fumaric acid.Isomerization catalyst also adopts the hydrochloric acid of ammonium persulphate-brometo de amonio mixture or metal-salt, amine salt, mercaptan and 10-20%.Changed in the chemical process of fumaroyl (fumaric acid) by maleic acid (toxilic acid), need to adopt high temperature, strong acid and metal catalyst, the a large amount of acid waste waters that produce in the production process, bring problems for the later stage sewage disposal, the reaction of high temperature is by the mass consumption that causes the energy, do not meet the industrial development strategy of the energy-saving and emission-reduction that country advocates.Adopt biocatalysis technology, can realize that maleic acid changes anti-top enedioic acid at normal temperatures and pressures, key wherein is exactly to adopt the maleic acid isomerase.
Carbohydrate such as sucrose, glucose, maltose also can make FUMARIC ACID TECH GRADE through the fermentation of black root bacterium.With the method for carbohydrate fermentation, the 1t product need consume grain 8t, and is very uneconomical economically, domestic research replaces the grain fermentation with whiteruss, is carbon source with the more liquid wax of C16-C18 content, ferments through 80-88h, liquid wax transformation efficiency is about 50%, and extraction yield is more than 50%.Also having with the furfural is raw material, gets through the sodium chlorate oxidation.
Since the seventies in last century, people have just found that multiple microorganism has the maleic acid isomerase activity, and it is studied, but owing to the isomerase activity of finding hangs down reasons such as reaching poor stability, never have industrial applications.Develop rapidly along with the gene order-checking technology, a large amount of microbial genome sequences are checked order and are announced, we adopt gene to dig the ore deposit technology, [Michael P.McLeod from the full genome of having announced of Rhodococcus sp.RHA1, Rene ' L. Warren, William W.L.Hsiao, etal, The complete genome of Rhodococcus sp.RHA1 provides insights into a catabolic powerhouse PNAS, 2006,103 (42 :) 15582-87], found the protein gene sequence that the maleic acid isomerase activity may be arranged of this article report, and at expression in escherichia coli, the complete sequence albumen of expression, exist with the inclusion body form, do not have isomerase activity.Be template with this gene, after transforming by genetically engineered and protein engineering, we have obtained to have the zymoprotein molecule of isomerase activity.
The method of present disclosed maleic acid isomerase preparation mainly is screen to obtain from microorganism, as Pseudomonas, Alcaligenes, and Serratia, Proteus, Arthrobacter etc., domestic do not have a pertinent literature report; External main literature is:
1、Yasuo?Kato，Jinsaku?Yamagishi，AND?Yasuhisa?Asano?Maleate?cis-trans?Isomerase?from?Arthrobacter?sp.TPU?5446?Journal?of?Fermentation?and?Bioencineering?1995，80(6)：610-612.
2、Toshiaki?Nakajima-Kambe，Takehiro?Nozue，Masaharu?Mukouyama，Tadaatsu?Nakahara?Bioconversion?of?maleic?acid?to?fumaric?acid?by?Pseudomonas?alcaligenes?strain?XD-1?Journal?of?Fermentation?and?Bioencineering?1997，84(2)：165-168.
3、Kazuhisa?Hatakeyama，Makoto?Goto，Yasukazu?Uchida，Miki?Kobayashi，Masato?Terasawa?and?Hideaki?Yukawa?Molecular?analysis?of?maleate?cis-trans?isomerase?from?thermophilic?bacteria?Biosci.Biotechnol.Biochem.2000，64(3)：569-576
4、Sosaku?Ichikawa，Tomoko?Iino，Seigo?Sato，Tadaatsu?Nakahara，Sukekuni?Mukataka?Improvement?of?production?rate?and?yield?of?fumaric?acid?from?maleic?acid?by?heat?treatment?of?Pseudomonas?alcaligenes?strain?XD-1?Biochemical?Engineering?Journal?2003，13：7-13
Summary of the invention:
The purpose of this invention is to provide class maleic acid isomerase and an application thereof, comprise specifically that the acquisition of maleic acid isomerase gene and engineering bacteria make up and tautomerize to application method in the FUMARIC ACID TECH GRADE at maleic acid.
1, the present invention prepares the method for maleic acid isomerase, may further comprise the steps:
1.1 by gene engineering method, obtain comprising the gene order that has as SEQ NO.2;
1.2 SEQ NO.2 gene is inserted into pET32 (a), makes up the bacillus coli gene engineering;
1.3 the intestinal bacteria with step 1.2 makes up carry out fermentation culture, abduction delivering with the suitable culture base, the big portion of target protein is present in the cell with soluble activity form;
2, maleic acid isomerization of the present invention forms the FUMARIC ACID TECH GRADE method, may further comprise the steps:
2.1 the cultivation thalline with step 1.3 obtains joins in maleic acid ammonium (sodium) solution of suitable concentration, control temperature of reaction and pH carry out isomerization reaction, are reflected at 24～48 hours and finish.
2.2 isomerization reaction endpoint determination simple and easy method is: get 0.5mL step 2.1 conversion fluid, add hcl acidifying and white precipitate occurs, and with 100 microlitre ethyl acetate extractings, the point silica gel thin-layer plate, and exhibition layer (developing agent methyl alcohol: ethylene dichloride=1: 4v/v), obviously observe maleic acid point under the ultraviolet lamp and disappear to produce tangible FUMARIC ACID TECH GRADE spot, illustrate that conversion reaction finishes.
3, further, the method for preparing the maleic acid isomerase comprises:
3.1 obtain gene order as SEQ NO.2 with PCR method;
3.2 make up the recombinant plasmid of the gene order that contains SEQ NO.2, and recombinant plasmid imported intestinal bacteria, obtain fermented bacterium;
3.3 3.2 bacterial classifications that make up gained are fermented, obtain thalline.Cryopreservation, standby.
3.4 more specifically, in 3.1 steps, be template with maleic acid isomerase gene SEQ NO.1, under the prerequisite that does not change reading frame, 5 ' disappearance 126-234bp, according to the synthetic corresponding pcr amplification primer of disappearance needs, 5 ' end and 3 ' end have suitable enzyme cutting clone site, meet the structure of expression vector.
3.5 more specifically, the gene order of SEQ NO.2 after NdeI and BamHI enzyme are cut, is inserted the NdeI/BamHI site construction recombination plasmid of pET (32a), and recombinant plasmid is imported the host bacterium, obtain fermented bacterium; Host cell is e. coli bl21, Rosetta or Origami, and preferred bacterial strain is BL21.
3.6 fermentation process is more specifically: fermented bacterium is inoculated in the substratum of 50ml LB (100ug/ml Amp, 34ug/mlChloroamphenicol) and activates.Be inoculated in 5000ml LB (two anti-concentration the are the same) substratum 37 ℃, 200rpm cultivation in 1: 100 ratio; Be cultured to A
6001.0 about add 0.5mM IPTG inductor (can be the mixture of lactose and IPTG), 25 ℃, 200rpm inducing culture 8～12h.Centrifugal collection bacterium under 5～15 ℃, 6000rpm condition, 4 ℃ of preservations are standby.
4, further, maleic acid isomerization formation FUMARIC ACID TECH GRADE method comprises:
The wet thallus of 3.6 gained is joined in maleic acid ammonium (sodium) solution, and control pH carries out isomerization reaction, is reflected at 24～48 hours and finishes.
Thalline can be spared the matter fragmentation, also cell adds entirely, suitable additional proportion is that the thalline of 1L fermented liquid gained joins in suitable maleic acid ammonium (sodium) solution of 0.5～1.5L, and more excellent ratio is 1: 1, and maleic acid ammonium (sodium) concentration that transforms solution is 10%; The scope of control pH is 5.2～7.5, and more excellent scope is 6.5 ± 0.2; Temperature is 25-40 ℃, and optimum temps is at 30 ± 2 ℃; Conversion reaction was finished at 24～48 hours, generally finished at 36 hours.The isomerization reaction end point determination is carried out according to 2.2.The FUMARIC ACID TECH GRADE ammonium (sodium) that isomerization produces obtains FUMARIC ACID TECH GRADE (fumaric acid) after acidifying.
Description of drawings
Accompanying drawing 1 makes up synoptic diagram for reorganization isomerase plasmid pET32 (a)/isomerase
Accompanying drawing 2 is the agarose electrophoresis figure in the isomerase engineering bacteria building process
Accompanying drawing 3 is converted into the thin-layer chromatography surveillance map of FUMARIC ACID TECH GRADE ammonium (sodium) reaction process under the isomerase effect for maleic acid ammonium (sodium)
Below in conjunction with embodiment, the present invention will be described to the detailed description of preferred embodiments of the present invention.But this explanation should not be understood as limitation of the present invention, and those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from technological thought of the present invention, all belongs to the defined scope of claim of the present invention.
1, the structure of engineering bacteria and isomerase protein expression (gene orders of Δ 144 SEQ NO.1)
2, maleic acid ammonium (sodium) isomerization FUMARIC ACID TECH GRADE ammonium (sodium)
Embodiment
The structure of embodiment one engineering bacteria and isomerase protein expression (gene orders of Δ 144 SEQ NO.1)
Be template with SEQ NO.1 sequence DNA, make pcr amplification with primer shown in the table 1, upstream primer comprises the NdeI site, downstream primer comprises the BamHI site), obtain the gene as SEQ NO.2 (Δ 144SEQ NO.1), the gene that obtains is carried out sequencing analysis after, be cloned on pET32 (a) carrier, the insertion site is NdeI/BamHI, makes up plasmid p ET32 (a)/isomerase, and flow process sees that accompanying drawing 1, agarose electrophoresis the results are shown in accompanying drawing 2.
Table 1, the used PCR primer of SEQ.1 genetic modification
The recombinant vectors for preparing is imported e. coli bl21, Rosetta or Origami with ordinary method be present in endobacillary genetic engineering bacterium to make up the reorganization isomerase with soluble form, filtering out and set up successful genetic engineering bacterium, wherein is that the albumen soluble-expression of reorganization Zoopagales of host bacterium is better relatively with the e. coli bl21.Be not less than 25% engineering bacteria with the target protein expression amount, as production labor journey bacterium bacterial classification, and preserve with glycerol stock or iced milk dry strain form.
Fermented bacterium is inoculated in the substratum of 50ml LB (100ug/ml Amp, 34ug/ml Chloroamphenicol) and activates.Be inoculated in 5000ml LB (two anti-concentration the are the same) substratum 37 ℃, 200rpm cultivation in 1: 100 ratio; Be cultured to A
6001.0 about add 0.5mM IPTG inductor (can be the mixture of lactose and IPTG), 30 ℃, 200rpm inducing culture 8～12h.4 ℃ of fermented liquids preserve or under 5～15 ℃, 6000rpm condition centrifugal collection bacterium, 4 ℃ of preservations are standby.
Embodiment two maleic acid ammonium (sodium) isomerization FUMARIC ACID TECH GRADE ammoniums (sodium)
1, the preparation of maleic acid ammonium (sodium) solution
Take by weighing the 148g MALEIC ANHYDRIDE, add 750mL dd H
2O adds ammoniacal liquor 30ml and adds the 60gNaOH solid again, and adjusting pH is 6.5 ± 0.2, uses dd H again
2O is settled to 1500mL, gets final product.
2, isomerization transformation experiment
Get fermented liquid 2L, centrifugal gained thalline adds in above-mentioned maleic acid ammonium (sodium) solution that configures, stirring at room, and control pH value maintains 6.5 ± 0.2, and reaction 34h finishes.Carry out TLC according to 2.2 methods and detect, and carry out HPLC and analyze, the result does not all have the toxilic acid residue, has realized that maleic acid ammonium (sodium) transforms to FUMARIC ACID TECH GRADE ammonium (sodium).FUMARIC ACID TECH GRADE ammonium (sodium) separates out through acidifying that to obtain FUMARIC ACID TECH GRADE be fumaric acid.
The thin-layer chromatography monitoring is as accompanying drawing 3 in the reaction process.
React HPLC after finishing in 4 hours and 34 hours (analytical column: Eclipse XDB-C18 column (4.6 * 150mm). moving phase: HOAc (pH=3) and methanol (v/v, 70/30) flow velocity 0.4ml/min, detect wavelength: 220nm) analytical results is seen accompanying drawing 4 and accompanying drawing 5 respectively.
Claims (6)
1. new maleic acid isomerase gene, it is:
DNA with sequence shown in the SEQ NO.1 is template, the Δ 144SEQ NO.1 gene that above downstream primer obtains as pcr amplification, wherein upstream primer is: 5 '-GGAATTCCATATGAAGGGCCACAACATGG-3 ', downstream primer is: 5 '-CGGGATCCTACTGAACAGTGGTGGC-3 '.
2. method for preparing the maleic acid isomerase is characterized in that:
(1) obtains the described maleic acid isomerase gene of claim 1 with PCR method;
(2) make up the recombinant plasmid that contains the said gene sequence, and recombinant plasmid is imported intestinal bacteria, obtain the genetically engineered fermented bacterium;
(3) bacterial classification with step (2) structure gained ferments, and obtains thalline, and cryopreservation is standby,
Concrete fermentation process is: fermented bacterium is inoculated in the 50ml LB substratum that contains 100ug/mlAmp and 34ug/mlChloroamphenicol activates, be inoculated in the same 5000ml LB substratum of two anti-concentration 37 ℃, 200rpm cultivation in 1: 100 ratio; Be cultured to A
6001.0 about add 0.5mM IPTG inductor, 25 ℃, 200rpm inducing culture 8～12h, centrifugal collection bacterium under 5～15 ℃, 6000rpm condition, 4 ℃ of preservations are standby.
3. the method for preparing the maleic acid isomerase according to claim 2, described step (2) is specially: the gene order that step (1) is obtained is after NdeI and BamHI enzyme are cut, insert the NdeI/BamHI site construction recombination plasmid of pET32a, and recombinant plasmid imported the host bacterium, obtain fermented bacterium; Described host bacterium is e. coli bl21, Rosetta or Origami.
4. the application of the maleic acid isomerase of the described method preparation of claim 2 is characterized in that the maleic acid isomery is turned to FUMARIC ACID TECH GRADE, comprising:
The thalline that claim 2 step (3) is obtained joins in maleic acid ammonium or the maleic acid sodium solution, and control pH carries out isomerization reaction, is reflected at 24～48 hours and finishes.
5. application according to claim 4, it is characterized in that: the broken or full cell of the even matter of thalline adds, additional proportion is that the thalline of 1L fermented liquid gained joins in suitable the maleic acid ammonium or maleic acid sodium solution of 0.5～1.5L, and maleic acid ammonium or maleic acid na concn are 10%; The scope of control pH is 5.2～7.5, and temperature is 25-40 ℃, and isomerization reaction was finished at 24～48 hours, and FUMARIC ACID TECH GRADE ammonium or FUMARIC ACID TECH GRADE sodium that isomerization produces obtain FUMARIC ACID TECH GRADE after acidifying.
6. application according to claim 5, described pH scope are 6.5 ± 0.2, and temperature is at 30 ± 2 ℃, and the isomerization reaction time is 36 hours.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1235641A (en) * | 1996-11-01 | 1999-11-17 | 索罗蒂亚公司 | Improved preparation of L-aspartic acid |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1235641A (en) * | 1996-11-01 | 1999-11-17 | 索罗蒂亚公司 | Improved preparation of L-aspartic acid |
Non-Patent Citations (8)
| Title |
|---|
| Improvement of production rate and yield of fumaric acid from maleic acid by heat treatment of Pseudomonas alcaligenes strain XD-1;Sosaku Ichikawa等;《Biochemical Engineering Journal》;20031231;第13卷;7-13 * |
| Kazuhisa Hatakeyama等.Molecular Analysis of Maleate cis-trans Isomerase from thermophilic bacteria.《Biosci.Biotechnol.Biochem.》.2000,第64卷(第3期),569-576. |
| Michael P. McLeod等.The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.《PNAS》.2006,第103卷(第42期),15582–15587. |
| Molecular Analysis of Maleate cis-trans Isomerase from thermophilic bacteria;Kazuhisa Hatakeyama等;《Biosci.Biotechnol.Biochem.》;20001231;第64卷(第3期);569-576 * |
| Sosaku Ichikawa等.Improvement of production rate and yield of fumaric acid from maleic acid by heat treatment of Pseudomonas alcaligenes strain XD-1.《Biochemical Engineering Journal》.2003,第13卷7-13. |
| The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse;Michael P. McLeod等;《PNAS》;20061017;第103卷(第42期);15582–15587 * |
| 刘祥涛等.顺丁烯二酸异构酶工程菌发酵条件优化.《应用与环境生物学报》.2011,第17卷(第4期),558-562. |
| 顺丁烯二酸异构酶工程菌发酵条件优化;刘祥涛等;《应用与环境生物学报》;20110825;第17卷(第4期);558-562 * |
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