CN103602708B - A kind of using microbe cell catalysis legal system is for the method for hydroxy aldehyde - Google Patents
A kind of using microbe cell catalysis legal system is for the method for hydroxy aldehyde Download PDFInfo
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Abstract
The present invention relates to a kind of using microbe cell catalysis legal system for the method for hydroxy aldehyde, comprising: S1, build the Gluconobacter oxvdans engineering bacteria that film binding acetaldehyde desaturase knocks out; S2, prepares the resting cell of described Gluconobacter oxvdans engineering bacteria; S3, the described resting cell that the substrate binary primary alconol being dissolved in organic solvent is suspended in the aqueous solution is oxidized to hydroxy aldehyde.According to method of the present invention, resting cell can be utilized to be oxidized binary primary alconol compounds as catalyst selectivity, thus to prepare multiple hydroxy aldehyde.Compared with preparing hydroxy aldehyde with chemical process, few according to the side reaction of method of the present invention, there is reaction conditions gentleness, environmentally friendly, easy and simple to handle, be easy to the advantages such as amplification.The biphase catalytic system of the aqueous solution-organic solvent used in method of the present invention, can remove the suppression of product hydroxy aldehyde to cytoactive, and product hydroxy aldehyde is mainly distributed in organic phase, is easy to product separation.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to the method for a kind of using microbe cell catalysis legal system for hydroxy aldehyde.
Background technology
Aldehyde is the important compound of a class, can be used as natural flavor additives useful is applied in foodstuffs industry, can be used as essence and flavoring agent for cosmetic industry, the intermediate that also can be used as organic synthesis is applied in the compound probability of medicine, agricultural chemicals, spices, dyestuff, sensitive materials, liquid crystal material etc.Some have the hydroxy aldehyde compound of the double properties of alcohols and aldehydes, and the application in spices, food, cosmetic industry and organic synthesis field is particularly extensive, such as salicylic aldehyde.Salicylic aldehyde, also known as salicylaldhyde, can be used as the raw material manufacturing the flavouring agent such as tonka bean camphor, vanillin food grade,1000.000000ine mesh and tetrahydro-isoquinolin, UV light absorber, sterilant, disinfectant and brightening agent.In recent years, spices and medicine industry increasing to the demand without chloro-salicylic aldehyde, the attention rate without the research and development of chloro-salicylic aldehyde's new synthetic process promotes day by day.
At present, the method both at home and abroad mainly through being oxidized primary alconol realizes the production of aldehyde, wherein again based on chemical catalysis.Hydroxy aldehyde is by the selectivity of binary primary alconol, incomplete oxidation and obtaining.Because aldehyde is easier than alcohol oxidized, in order to the aldehyde controlling to synthesize is not further oxided, chemical catalysis condition controls harsh, and process is loaded down with trivial details.In addition, chemical catalysis is difficult to the regioselective conversion realizing polyhydroxy-alcohol.Biocatalysis has the features such as efficiency is high, specificity is strong, selectivity is high, reaction conditions is gentle, environmental friendliness.Especially when aldehyde uses as foods and cosmetics, the aldehyde that biological process is produced can be seen as crude substance, is worth to have significantly to promote.Up to now, the kind of the aldehyde of the biological process synthesis reported is very limited, the mainly aldehyde of some simple functions group, acetaldehyde is synthesized as utilized motion pseudomonas, utilize Saccharomyces to be oxidized short chain fatty alcohol and generate corresponding alkanoic, utilize alcoholdehydrogenase, coenzyme and peroxidase to form compound system synthesis long-chain aldehyde etc.At present also there is no bibliographical information to cross to have the biological process synthetic method of the hydroxy aldehyde of alcohols and aldehydes dual-use function group.
Summary of the invention
The present invention aims to provide the method for a kind of using microbe cell catalysis legal system for hydroxy aldehyde, thus optionally polyvalent alcohol is oxidized to hydroxy aldehyde.
Using microbe cell catalysis legal system of the present invention, for the method for hydroxy aldehyde, comprising: S1, builds the Gluconobacter oxvdans engineering bacteria that film binding acetaldehyde desaturase knocks out; S2, prepares the resting cell of described Gluconobacter oxvdans engineering bacteria; S3, the described resting cell that the substrate binary primary alconol being dissolved in organic solvent is suspended in the aqueous solution is oxidized to hydroxy aldehyde.
Preferably, make film binding acetaldehyde dehydrogenase gene reticent by the method for homologous recombination and gene disruption, thus obtain the described Gluconobacter oxvdans engineering bacteria that oxidation of aldehydes approach is blocked.
Preferably, the preparation of described Gluconobacter oxvdans engineering bacteria comprises the following steps: S11, with the genomic dna carrying the original bacteria of acetaldehyde dehydrogenase gene for template carries out the interior segments that pcr amplification obtains acetaldehyde dehydrogenase gene, described original bacteria is Gluconobacter oxvdans DSM2003; S12, carries out endonuclease reaction respectively by the interior segments of described acetaldehyde dehydrogenase gene and plasmid vector, the product of endonuclease reaction is carried out connection and obtains ligation reaction; S13, by described ligation reaction Transformed E .coliJM109 competent cell; S14, being cloned into the suitable restriction enzyme site XhoI of the inside of the acetaldehyde dehydrogenase gene on carrier selection, being connected with the PCR primer of the Kam gene cut through XhoI enzyme, being converted into competence E.coliJM109, obtaining recombinant plasmid by plate screening; S15, engage being used for three parents containing the E.coliJM109 knocking out mutational vector as donor bacterium, recipient bacterium is Gluconobacter oxvdans DSM2003, acetaldehyde-dehydrogenase enzyme fragment containing Kam gene is incorporated on the karyomit(e) of Gluconobacter oxvdans DSM2003, screens three close zygotes by Cefoxitin, Km; S16, the genome extracting Gluconobacter oxvdans DSM2003 and film binding acetaldehyde desaturase deletion mycopremna is respectively used as pcr template, obtains the Gluconobacter oxvdans engineering bacteria that described film binding acetaldehyde desaturase knocks out.
Preferably, described plasmid vector is suicide plasmid pSUP series.The plasmid vector selected is suicide plasmid, shortly can not exist after entering Gluconobacter oxvdans DSM2003, only have the acetaldehyde-dehydrogenase enzyme fragment containing Kam gene to be incorporated into Gluconobacter oxvdans DSM2003 on karyomit(e) just tool Km resistance, therefore can screen three close zygotes by Cefoxitin, Km.
Preferably, the preparation of described resting cell comprises the following steps: S21, and described in fermentation culture, Gluconobacter oxvdans engineering bacteria obtains cell fermentation liquid; S22, by described cell fermentation liquid harvested by centrifugation bacterial sediment, obtains described resting cell with the phosphate buffered saline buffer described bacterial sediment that suspends.
Preferably, described organic solvent is selected from: hexanaphthene, normal hexane, normal heptane, octane-iso, n-dodecane, oleic acid.
Preferably, the described aqueous solution is the phosphate buffered saline buffer of 100mmol/L.
Preferably, the volume ratio of described organic solvent and the described aqueous solution is 1:1.
Preferably, described binary primary alconol is aromatic dicarboxylic primary alconol.
Preferably, described binary primary alconol is selected from: saligenol, p-Hydroxybenzylalcohol, a salicylic alcohol, p-hydroxyphenylethanol, benzoglycols.
Preferably, the concentration being dissolved in the described binary primary alconol of described organic solvent is 10-30g/L.
Preferably, the temperature of described oxidizing reaction is 20 ~ 40 DEG C.
Preferably, the temperature of described oxidizing reaction is 28 ~ 30 DEG C.
Preferably, under oscillating condition or under aeration-agitation condition, described oxidizing reaction is carried out.
According to method of the present invention, resting cell can be utilized to be oxidized binary primary alconol compounds as catalyst selectivity, thus to prepare multiple hydroxy aldehyde.Compared with preparing hydroxy aldehyde with chemical process, few according to the side reaction of method of the present invention, there is reaction conditions gentleness, environmentally friendly, easy and simple to handle, be easy to the advantages such as amplification.The biphase catalytic system of the aqueous solution-organic solvent used in method of the present invention, can remove the suppression of product hydroxy aldehyde to cytoactive, and product hydroxy aldehyde is mainly distributed in organic phase, is easy to product separation.
Accompanying drawing explanation
Fig. 1 is the HPLC detected result of conversion fluid.Wherein, be substrate saligenol at 5.078min place.
Fig. 2 is the GC detected result of conversion fluid.Wherein, being octane-iso at 2.4min place, is interior mark phenylethyl alcohol at 3.2min place, is product salicylic aldehyde at 3.0min place.
Fig. 3 is the process schematic of Gluconobacter oxvdans engineering bacteria catalysis saligenol accumulation salicylic aldehyde.
Catalytic result when Fig. 4 is the saligenol adding different concns.
Embodiment
Below in conjunction with accompanying drawing, provide preferred embodiment of the present invention, and be described in detail.
In the present invention, resting cell refers to will be collected after be cultured to certain hour on growth medium, by appropriate means and carries out the microorganism cells that washs with damping fluid, is the microorganism cells removed nutritive ingredient, stop growth.
In following embodiment of the present invention, the binary primary alconol of use comprises the aromatic dicarboxylic primary alconols such as saligenol, p-Hydroxybenzylalcohol, a salicylic alcohol, p-hydroxyphenylethanol, benzoglycols, styryl carbinol.
In following embodiment of the present invention, the testing conditions that liquid chromatography (HPLC) detects the transformation efficiency of saligenol is as follows: chromatographic column is the SB-AQ of Agilent, flow velocity 0.8ml/min, column temperature: 30 DEG C, moving phase is methyl alcohol: water=50:50(v/v), determined wavelength 215nm.
The testing conditions that gas-chromatography (GC) detects the yield of salicylic aldehyde is as follows: chromatographic column is the DB-1 capillary chromatographic column of Agilent, injector temperature is 250 DEG C, detector temperature is 250 DEG C, flow velocity is 1.0ml/min, splitting ratio 50:1, heating schedule is 100 DEG C and keeps 1min, is then warming up to 180 DEG C with the speed of 8 DEG C/min, keeps 1min.
embodiment 1the structure of the Gluconobacter oxvdans engineering bacteria that the structure film binding acetaldehyde desaturase of the Gluconobacter oxvdans engineering bacteria that film binding acetaldehyde desaturase knocks out knocks out can reference (BiotechnologyandBioprocessEngineering, 2012,17(6): 1156-1164.), concrete grammar is as follows:
(1) according to Gluconobacter oxvdans 621H(G.oxydans621H) film binding acetaldehyde desaturase (GOX0587) gene order design primer aldhA-F(SEQIDNo.1): 5'-TACTGCAGTCTGCCATGCCGCCGGAAGC-3'; : 5'-AACTGCAGTGGGCTTTTCGGTGTTCTGGATGA-3'(JaRa is biological) and add PstI restriction enzyme site, the interior segments of amplification target gene 2000bp aldhA-R(SEQIDNo.2).
(2) after target gene interior segments and suicide vector pSUP202 being cut with PstI enzyme respectively, connect with T4 ligase enzyme, be converted into competence E.coliJM109, PstI restriction enzyme site is just in time on the Amp site of carrier, when exogenous sequences is after this site insertion vector, can obtain by the flat board containing Amp and the plate screening containing Cm the E.coliJM109 containing recombinant plasmid.
(3) being cloned into the suitable restriction enzyme site XhoI of the inside of the target gene on carrier selection, being connected with the PCR primer of cutting Kam gene through XhoI enzyme, being converted into competence E.coliJM109, by Kam resistant panel screening recombinant plasmid.The Kam gene primer of amplified band XhoI restriction enzyme site is Kam-XhoIF(SEQIDNo.3): 5'-AATCTCGAGGCGAGGTATGTAGGCGGTGC-3'; Kam-XhoIR(SEQIDNo.4):
5'-CGCCTCGAGTTAGAAAAACTCATCGAGCA-3'。Primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
(4) engage being used for three parents containing the JM109 knocking out mutational vector as donor bacterium, recipient bacterium is G.oxydansDSM2003(Germany Culture Collection).Knock out mutational vector and can enter G.oxydansDSM2003 under the help helping bacterium, by the homologous fragment thereon with target gene, therefore can with the DNA fragmentation generation double exchange of this gene on G.oxydansDSM2003 karyomit(e), be incorporated into containing the target gene fragment of Kam gene on the karyomit(e) of G.oxydansDSM2003.Plasmid pSUP202 is suicide plasmid, shortly can not exist after entering G.oxydansDSM2003, only have the target gene fragment containing Kam gene to be incorporated into G.oxydansDSM2003 on karyomit(e) just tool Km resistance, therefore can screen three close zygotes by Cefoxitin, Km.
(5) genome of G.oxydansDSM2003 and film binding acetaldehyde desaturase deletion mycopremna is extracted respectively, as pcr template.Article one, the complementary on PCR primer and Km resistant gene carrier: Kam-XhoI-R(SEQIDNo.5):
5'-CGCCTCGAGTTAGAAAAACTCATCGAGCA-3' or Kam-XhoI-F(SEQIDNo.6): 5'-AATCTCGAGGCGAGGTATGTAGCGGTGC-3', another primer and target aldhA upstream region of gene (or downstream) complementary: ALDH-F(SEQIDNo.7): 5'-GCGAATTCATTCATCGGGAGACATACTTGC-3' or ALDH-R(SEQIDNo.8): 5'-AATCTAGAGCTTTCGGTCCAGCACAGG-3'.Primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.When Km gene be inserted into target gene inner time, the PCR primer of length-specific just there will be, and PCR primer is connected to T-carrier and checks order, thus determines whether be inserted with foreign vector in the goal gene of mutants which had.The Gluconobacter oxvdans engineering bacteria correctly knocking out film binding acetaldehyde desaturase is labeled as G.oxydansaldhA::Km.
embodiment 2the preparation of the resting cell of the genetic engineering bacterium of Gluconobacter oxvdans
Culture medium prescription (g/L): sorbyl alcohol 80.0g, yeast powder extract 20.0g, potassium primary phosphate 1.0g five water magnesium sulfate 0.5g, is dissolved in 1000mL distilled water.And it is for subsequent use after 15 minutes in 121 DEG C of sterilizings.
From sorbyl alcohol solid plate, single colony inoculation of picking G.oxydansaldhA::Km is in the culture medium A test tube of the gentamicin sulphate containing 25 μ g/mL, at 30 DEG C, the shaking table of 250rpm is cultivated about 20h as seed liquor, then transfer in the 500ml triangular flask containing the same substratum of 100ml in the ratio of 10%, under 250rpm and 30 DEG C condition, shaking culture 24h.
By fermented liquid in the centrifugal 10min of 8000rpm, collect bacterial sediment, and with brine thalline twice, centrifugally remove supernatant, be suspended in the phosphoric acid buffer (pH6.0) of 0.1M by the thalline of wet thallus 20g/L, obtain the resting cell for catalyzed reaction.
embodiment 3the conversion of saligenol and the detection of product
In 50ml triangular flask, add the octane-iso of 5ml final concentration 100mmol/l saligenol and 5ml, then add the resting cell 20g/l obtained in embodiment 2, in 30 DEG C, cultivate in the constant-temperature table of 200rpm, termination reaction after reaction 8h.By reaction solution in the centrifugal 10min of 12000rpm, and with the membrane filtration of 0.22 μm, collect filtrate, use liquid chromatography (HPLC) and gas-chromatography (GC) to detect the transformation efficiency of saligenol and the yield of salicylic aldehyde respectively.According to the detected result of Fig. 1 and Fig. 2, after 8h, the productive rate of salicylic aldehyde can reach 86.2%, and no coupling product produces (Fig. 3).
embodiment 4saligenol concentration is on the impact of conversion reaction
In 50ml triangular flask, add phosphoric acid buffer (0.1M, pH6.0) and 5ml octane-iso that 5ml contains 20g/l cell, add the saligenol substrate of a certain amount of (10-30g/l), in 150rpm, at 30 DEG C, react 8h.When concentration of substrate is less than or equal to 10g/l, saligenol is almost converted into salicylic aldehyde completely, and productive rate is greater than 95%.If increase concentration of substrate further, substrate produces cytoactive and suppresses, and causes the initial velocity reacted to start the (see figure 4) that declines.The preferred concentration of saligenol is 10g/l.
embodiment 5the selection of organic solvent in saligenol conversion reaction
Phosphoric acid buffer (0.1M, pH6.0) and 5ml organic solvent (hexanaphthene, normal hexane that 5ml contains 20g/l cell is added in 50ml triangular flask, normal heptane, octane-iso, n-dodecane oleic acid), add the saligenol substrate of final concentration 10g/l, in 150rpm, at 28 DEG C, react 8h.As shown in Table 1, the reaction of Gluconobacter oxydans engineering bacteria resting cell catalysis saligenol, can carry out adding in the two-phase system that different organic solvents formed.Wherein, preferred octane-iso, in this system, output is the highest.
Table 1
embodiment 6the conversion of other aromatic dicarboxylic primary alconol
In 50ml triangular flask, add phosphoric acid buffer (0.1M, pH6.0) and 5ml octane-iso that 5ml contains 20g/l cell, add the different aromatic dicarboxylic primary alconol of 5g/l respectively, in 150rpm, at 30 DEG C, react 8h.With the catalytic efficiency of saligenol (diathesin) for reference (being set to 100%), measure the relative conversion results of other substrate.As shown in table 2, the Gluconobacter oxydans engineering bacteria resting cell provided in invention the aromatic dicarboxylic primary alconol of other structure such as catalysis p-Hydroxybenzylalcohol, a salicylic alcohol, p-hydroxyphenylethanol (tyrosol), styryl carbinol, benzoglycols can generate corresponding hydroxy aldehyde.
Table 2
In sum, use method provided by the invention, by cultivating microorganism separately, the microorganism cells that a large amount of activity is high can be obtained, prepare the catalyzed reaction of resting cell for dibasic alcohol, be beneficial to regulation and control cell concn first, to improve the efficiency of reaction, second catalyzed reaction liquid composition is simple, and impurity is few, is beneficial to product separation purifying.The invention provides the method building the Gluconobacter oxvdans that film binding acetaldehyde desaturase knocks out simultaneously, not only block the peroxidation of product hydroxy aldehyde, inhibit the generation of by product, improve reaction efficiency, also for the structure of Gluconobacter oxvdans gene knockout engineering bacteria provides reference.Catalystic converter system have employed water-organic solvent two-phase, can remove the suppression of product hydroxy aldehyde to cytoactive, and product hydroxy aldehyde is mainly distributed in organic phase, is easy to be separated, and finally realizes the High-efficient Production of hydroxy aldehyde.
Above-described, be only preferred embodiment of the present invention, and be not used to limit scope of the present invention, the above embodiment of the present invention can also make a variety of changes.Namely every claims according to the present patent application and description are done simple, equivalence change and modify, and all fall into the claims of patent of the present invention.The not detailed description of the present invention be routine techniques content.
Claims (6)
1. using microbe cell catalysis legal system is for a method for hydroxy aldehyde, it is characterized in that, the method comprises: S1, builds the Gluconobacter oxvdans engineering bacteria that film binding acetaldehyde desaturase knocks out; S2, prepares the resting cell of described Gluconobacter oxvdans engineering bacteria; S3, the described resting cell that the substrate binary primary alconol being dissolved in organic solvent is suspended in the aqueous solution is oxidized to hydroxy aldehyde; Described organic solvent is selected from: hexanaphthene, oleic acid; Described binary primary alconol is selected from: saligenol, a salicylic alcohol; The concentration being dissolved in the described binary primary alconol of described organic solvent is 10-30g/L.
2. method according to claim 1, is characterized in that, makes film binding acetaldehyde dehydrogenase gene reticent by the method for homologous recombination and gene disruption, thus obtains the described Gluconobacter oxvdans engineering bacteria that oxidation of aldehydes approach is blocked.
3. method according to claim 2, it is characterized in that, the preparation of described Gluconobacter oxvdans engineering bacteria comprises the following steps: S11, with the genomic dna carrying the original bacteria of acetaldehyde dehydrogenase gene for template carries out the interior segments that pcr amplification obtains acetaldehyde dehydrogenase gene, described original bacteria is Gluconobacter oxvdans DSM2003; S12, carries out endonuclease reaction respectively by the interior segments of described acetaldehyde dehydrogenase gene and plasmid vector, the product of endonuclease reaction is carried out connection and obtains ligation reaction; S13, by described ligation reaction Transformed E .coliJM109 competent cell; S14, being cloned into the suitable restriction enzyme site XhoI of the inside of the acetaldehyde dehydrogenase gene on carrier selection, being connected with the PCR primer of the Kam gene cut through XhoI enzyme, being converted into competence E.coliJM109, obtaining recombinant plasmid by plate screening; S15, engage being used for three parents containing the E.coliJM109 knocking out mutational vector as donor bacterium, recipient bacterium is Gluconobacter oxvdans DSM2003, acetaldehyde-dehydrogenase enzyme fragment containing Kam gene is incorporated on the karyomit(e) of Gluconobacter oxvdans DSM2003, screens three close zygotes by Cefoxitin, Km; S16, the genome extracting Gluconobacter oxvdans DSM2003 and film binding acetaldehyde desaturase deletion mycopremna is respectively used as pcr template, obtains the Gluconobacter oxvdans engineering bacteria that described film binding acetaldehyde desaturase knocks out.
4. method according to claim 3, is characterized in that, described plasmid vector is suicide plasmid pSUP series.
5. method according to claim 1, is characterized in that, the preparation of described resting cell comprises the following steps: S21, and described in fermentation culture, Gluconobacter oxvdans engineering bacteria obtains cell fermentation liquid; S22, by described cell fermentation liquid harvested by centrifugation bacterial sediment, obtains described resting cell with the phosphate buffered saline buffer described bacterial sediment that suspends.
6. method according to claim 1, is characterized in that, the volume ratio of described organic solvent and the described aqueous solution is 1:1.
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| Characterization of Enzymes in the Oxidation of 1,2-Propanediol to D-(2)-Lactic Acid by Gluconobacter oxydans DSM 2003;Liujing Wei等;《Mol Biotechnol》;20101231;第46卷(第1期);第28页右栏第3-4段，第29页左栏第1段 * |
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