CN113583927A - Recombinant bacillus subtilis, transaminase prepared by same and application of transaminase - Google Patents
Recombinant bacillus subtilis, transaminase prepared by same and application of transaminase Download PDFInfo
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- CN113583927A CN113583927A CN202110772022.XA CN202110772022A CN113583927A CN 113583927 A CN113583927 A CN 113583927A CN 202110772022 A CN202110772022 A CN 202110772022A CN 113583927 A CN113583927 A CN 113583927A
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C12Y206/01—Transaminases (2.6.1)
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Abstract
The invention relates to the technical field of drug synthesis and enzyme engineering, in particular to recombinant bacillus subtilis, transaminase prepared by the same and application thereof, wherein the recombinant bacillus subtilis is prepared by mixing the following components in percentage by weight: the biological enzyme gene shown in 1 is connected to BamHI and ZraI sites of pHT43 plasmid, and then is transformed into Bacillus subtilis A CC6633 for expression, thus obtaining the recombinant Bacillus subtilis. The recombinant bacillus subtilis is adoptedThe transaminase prepared by the bacillus subtilis is used for synthesizing sitagliptin, a compound II is used as a starting raw material, and is converted into the sitagliptin by the transaminase in the presence of buffer solution, cosolvent, coenzyme PLP and amino donor, the reaction route is as follows,
Description
Technical Field
The invention relates to the technical field of drug synthesis and enzyme engineering, in particular to recombinant bacillus subtilis and prepared transaminase.
Background
Sitagliptin (Sitagliptin) has the chemical name of (3R) -3-amino- [3- (trifluoromethyl) -5,6,7, 8-tetrahydro-1, 2, 4-triazolo [4,3-a ] pyrazin-7-yl ] -4- (2,4, 5-trifluorophenyl) butan-1-one (structural formula is shown below).
Sitagliptin phosphate (sitagliptin phosphate) is a novel anti-type II diabetes drug, is taken orally, one tablet for each time, is prepared by 100mg once, is successfully developed and developed by Moshadong in 2006-10 months, is approved by the US FDA to be on the market, and is the first dipeptidyl peptidase IV (DPP-4) inhibitor drug for treating type II diabetes in history.
Both chemical preparation methods and biological preparation methods of sitagliptin are reported, wherein ATA177 transaminase is most widely used for preparing sitagliptin, and optimization of ATA117 transaminase is researched by various enterprises and colleges at home and abroad. For example, in CN103608355A, the daemonorops company uses resin-immobilized ATA117 to reuse the transaminase ATA117 for many times, thereby reducing the production cost. In CN109777813A, Zhejiang industrial university co-immobilizes transaminase-PLP to prepare sitagliptin, and the method has the advantages of good stability, long service life of immobilized enzyme, good tolerance of organic solvent and repeated utilization.
At present, reports about transaminase mainly in the aspects of improvement of enzyme activity, enzyme immobilization and the like have few reports on the research on the degradation of high-COD wastewater caused by enzyme catalysis.
In addition, the enzyme catalysis of sitagliptin is the last step of reaction, and the enzyme catalyst prepared by escherichia coli has bacterial endotoxin residue, so a new high-efficiency expression vector and green pollution-free expression cells are needed for preparing transaminase.
Disclosure of Invention
The invention aims to provide a recombinant bacillus subtilis, a prepared transaminase and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of recombinant bacillus subtilis comprises the following steps: the peptide as shown in SEQ ID NO: the biological enzyme gene shown in 1 is connected to BamHI and ZraI sites of pHT43 plasmid, and then is transformed into Bacillus subtilis A CC6633 for expression, thus obtaining the recombinant Bacillus subtilis.
The recombinant bacillus subtilis obtained by the preparation method is adopted.
The transaminase prepared by the recombinant bacillus subtilis has a nucleotide sequence shown in SEQ ID NO. 1.
The preparation method of the transaminase comprises the following steps: the inventionThe recombinant bacillus subtilis is inoculated into an LB liquid culture medium, the inoculation amount is 1-5% of the volume of the culture medium, and the recombinant bacillus subtilis is used as a seed solution after overnight culture in a shaking table with the speed of 220rpm at 37 ℃; inoculating fresh seed liquid into TB culture medium, fermenting and culturing at 37 deg.C, and culturing in 220rpm shaking table to OD600Adding IPTG (isopropyl-beta-D-thiogalactoside) with the concentration of 0.5mM into the culture medium at 0.6-0.8, and inducing for 22h at 18 ℃; after the induction of the fermentation liquor is finished, centrifuging for 5min at 4 ℃ and 12000rpm, and respectively collecting thalli and supernatant; adding saturated ammonium sulfate into the supernatant for precipitation, centrifuging at 4 ℃ and 12000rpm for 5min, collecting the precipitate, and freeze-drying to obtain the crude transaminase.
The application of the transaminase in the synthesis of sitagliptin provided by the invention comprises the following specific synthesis methods:
compound II is used as a starting material, and is converted into compound I, namely sitagliptin, by transaminase catalysis in the presence of buffer solution, cosolvent, coenzyme PLP (pyridoxal phosphate) and amino donor, wherein the reaction route is as follows,
the cosolvent is selected from any one of methanol, ethanol, butyl acetate and DMSO, and is preferably DMSO;
the buffer solution is preferably 1M isopropyl amine hydrochloride buffer solution with the pH value of 8.0-8.5; the amino donor is isopropylamine in isopropylamine hydrochloride buffer.
The volume ratio of the cosolvent to the buffer solution is preferably 1: 100;
the mass ratio of the biological enzyme (containing buffer solution) to the coenzyme PLP is preferably 10: 1-10.
When the compound II is subjected to a biological enzyme catalytic reaction, the reaction temperature is preferably 20-40 ℃.
The application of the recombinant bacillus subtilis in treating the enzyme-catalyzed wastewater generated in the synthesis of sitagliptin comprises the following steps:
(1) the recombinant bacillus subtilis collects thalli after enzyme is secreted;
(2) recovering an organic cosolvent from enzyme catalysis wastewater during sitagliptin synthesis through reduced pressure distillation, and then adding the thallus collected in the step (1), wherein the mass ratio of the thallus to the wastewater is 1:10 to 100 parts; the reaction temperature is 30-40 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a recombinant bacillus subtilis and transaminase prepared by the same, and sitagliptin is catalytically synthesized by the transaminase. The transaminase is expressed as exocrine expression, cell disruption is not needed, and the bacillus subtilis does not generate bacterial endotoxin, is safe and has no pollution.
Moreover, the strain producing the enzyme does not need to be wasted after secreting the enzyme, can be further used for biodegrading the biological wastewater after the enzyme catalysis, and can be recycled, so that the production cost is greatly reduced.
Drawings
FIG. 1 is a schematic diagram of vector construction in example 1 of the present invention;
FIG. 2 shows the results of liquid phase detection in example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of recombinant Bacillus subtilis
The preparation method of the engineering bacteria of the recombinant bacillus subtilis comprises the following steps:
the peptide as shown in SEQ ID NO: 1, designing an upstream primer F, R and a downstream primer F, R (shown as SEQ ID NO: 2-3) for PCR amplification, wherein the PCR conditions are as follows: 3min at 98 ℃, 30s at 95 ℃, 90s at 55 ℃, 90s at 72 ℃ and 35 cycles; PCR amplification System: 1.5. mu.L of template, 1.5. mu.L of each of the upstream and downstream primers, 20.5. mu.L of sterilized double distilled water, 25. mu.L of PrimerSTAR Mix; and after the amplification, performing gel running to confirm that NO errors exist, constructing a recombinant vector pHT43 containing a target sequence (shown as SEQ ID NO: 1) by using BamHI and ZraI double enzyme digestion vectors, and transferring the recombinant vector pHT43 into an expression cell Bacillus subtilis ATCC6633(ATCC strain bank) after gel running confirmation to obtain an expression strain, namely recombinant Bacillus subtilis.
EXAMPLE 2 preparation of transaminase
The expression strain prepared in example 1 was inoculated into LB liquid medium in an amount of 1-5% by volume of the medium, and cultured overnight in a shaker at 37 ℃ and 220rpm to obtain a seed solution. Inoculating fresh seed liquid into TB culture medium, fermenting and culturing at 37 deg.C, and culturing in 220rpm shaking table to OD600IPTG was added to the medium at 0.6-0.8 mM, and induction was carried out at 18 ℃ for 22 hours at an IPTG concentration of 0.5 mM. After the induction of the fermentation liquor is finished, the fermentation liquor is centrifuged for 5min at 4 ℃ and 12000rpm, and thalli and supernatant are respectively collected. Adding saturated ammonium sulfate into the supernatant for precipitation, centrifuging at 4 ℃ and 12000rpm for 5min, collecting the precipitate, and freeze-drying to obtain the crude transaminase.
Example 3 preparation of sitagliptin
A250 mL Erlenmeyer flask was charged with 95mL of buffer (1M isopropylamine hydrochloride, pH8.0), 1g of crude enzyme powder prepared in example 2, and 5mL of DMSO containing 50mM of Compound II and 5mL of glycerol were added during the reaction, and finally pyridoxal phosphate was added to a final concentration of 1mM, isopropylamine in isopropylamine hydrochloride was used as an amino donor, and after 16 hours of reaction at 30 ℃, the reaction was terminated, followed by extraction with ethyl acetate, and the yield of Compound I was 92% as determined by liquid phase assay (see FIG. 2), and the purity was 95.32% after further purification.
Example 4 biodegradation of reaction waste Water
The enzyme-catalyzed wastewater of example 3 was distilled under reduced pressure to remove the organic phase, and the cells collected in example 2 were added to dilute the wastewater (wastewater ddH) per 50mL2Diluting O to COD10000mg/L), adding 1g of wet thallus, carrying out biodegradation reaction at 35 ℃ and 180rpm, controlling the pH value to be 6.0-8.0, reacting for 12h, stopping reaction, and centrifugally collecting thallusThe COD value detected by the detection is about 938mg/mL, and the COD reduction rate is more than 90 percent.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Jiangsu alpha pharmaceutical Co., Ltd
<120> recombinant bacillus subtilis, transaminase prepared by using same and application of transaminase
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1011
<212> DNA
<213> Bio enzyme Gene Sequence (Artificial Sequence)
<400> 1
atgcaccatc accatgccac aatggataaa gtgtttgcag gctatgcaga acgtcaggca 60
gttctggaag caagtaaaaa tccgttagcc aaaggtgttg cctggattca gggcgaactg 120
gttccgttac atgaagcacg cattccgtta ctggatcagg gctttatgca tagcgatctg 180
acctatgata ccccgagcgt ttgggatggt cgcttttttc gtctggaaga tcatctgaat 240
cgcttagaag cctcttgtaa aaaaatgcgc ttaagaatgc cgctgccgcg cgaagaagtt 300
attaaaacct tagttgatat ggttgccaaa tcaggtattc gtgatgcaat ggtggaactg 360
attgtgaccc gcggcctgac cggcgttcgt ggcgccaaac cggaagaact gctgaataat 420
aatctgtata tgtttattca gccgtatgtt tgggttatgg acccggatgt tcagtatacc 480
ggtggtcgtg ccattgtggc acgtaccgtg cgtcgtgtgc cgccgggtag tattgatccg 540
accattaaaa atctacagtg gggtgattta gtgcgcggcc tgtttgaagc caatgatcgc 600
ggcgcaacct atccgtttct gaccgatggc gatgccaatt taaccgaagg ctcaggcttt 660
aatgttgtgc tgattaaaga tggcgtgctg tataccccgg atcgcggtgt gttacagggc 720
attacccgca aatcagttat tgatgcagca cgctcttgtg gctatgaaat tcgcgttgaa 780
catgttccga ttgaagcaac ctatcaggca gatgaaattt taatgtgtac caccgccggc 840
ggtattatgc cgattaccac cttagatgat aaaccggtga aagatggtaa agtgggtccg 900
attaccaaag caatttggga tcgctattgg gccatgcatt gggaagatga atttagcttt 960
aaaattaatt atctggaaga aaagaagaaa aagaaaaaga agaaaaagtg a 1011
<210> 2
<211> 28
<212> DNA
<213> primer F (Artificial sequence)
<400> 2
cgcggatcca tgcaccatca ccatgcca 28
<210> 3
<211> 34
<212> DNA
<213> primer R (Artificial sequence)
<400> 3
gacgtcgaag aaaaagaaaa agaagaaaaa gtga 34
Claims (9)
1. A preparation method of recombinant bacillus subtilis is characterized by comprising the following steps: the peptide as shown in SEQ ID NO: the biological enzyme gene shown in 1 is connected to BamHI and ZraI sites of pHT43 plasmid, and then is transformed into Bacillus subtilis A CC6633 for expression, thus obtaining the recombinant Bacillus subtilis.
2. A recombinant Bacillus subtilis obtained by the production method according to claim 1.
3. A transaminase enzyme produced using the recombinant bacillus subtilis of claim 2, which is characterized by: the transaminase nucleotide sequence is shown in SEQ ID NO. 1.
4. The process for the preparation of the transaminase of claim 3, characterized in that: inoculating the recombinant bacillus subtilis of claim 2 into an LB liquid culture medium, wherein the inoculation amount is 1-5% of the volume of the culture medium, and the recombinant bacillus subtilis is used as a seed solution after being cultured in a shaker at 220rpm under the condition of 37 ℃; inoculating fresh seed liquid into TB culture medium, fermenting and culturing at 37 deg.C, and culturing in 220rpm shaking table to OD600Adding IPTG (isopropyl-beta-D-thiogalactoside) with the concentration of 0.5mM into the culture medium at 0.6-0.8, and inducing for 22h at 18 ℃; after the induction of the fermentation liquor is finished, centrifuging for 5min at 4 ℃ and 12000rpm, and respectively collecting thalli and supernatant; adding saturated ammonium sulfate into the supernatant for precipitation, centrifuging at 4 ℃ and 12000rpm for 5min, collecting the precipitate, and freeze-drying to obtain the crude transaminase.
5. The use of the transaminase enzyme of claim 3 for the synthesis of sitagliptin, which is characterized in that: compound II as initial material is converted into compound I, sitagliptin, with transaminase catalysis in the presence of buffering liquid, cosolvent, coenzyme PLP and amino donor,
6. use of a transaminase according to claim 5 in the synthesis of sitagliptin, characterized in that: the cosolvent is selected from any one of methanol, ethanol, butyl acetate and DMSO; the buffer solution is 1M isopropyl amine hydrochloride buffer solution with the pH value of 8.0-8.5; the amino donor is isopropylamine in isopropylamine hydrochloride buffer.
7. Use of a transaminase according to claim 5 in the synthesis of sitagliptin, characterized in that: the volume ratio of the cosolvent to the buffer solution is 1: 100.
8. Use of a transaminase according to claim 5 in the synthesis of sitagliptin, characterized in that: during the catalytic reaction, the reaction temperature is 20-40 ℃.
9. The use of recombinant bacillus subtilis in treating enzyme-catalyzed wastewater generated in the synthesis of sitagliptin according to claim 2, which comprises the following steps:
(1) the recombinant bacillus subtilis collects thalli after enzyme is secreted;
(2) recovering an organic cosolvent from enzyme catalysis wastewater during sitagliptin synthesis through reduced pressure distillation, and then adding the thallus collected in the step (1), wherein the mass ratio of the thallus to the wastewater is 1:10 to 100 parts; the reaction temperature is 30-40 ℃.
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| CN202110772022.XA CN113583927A (en) | 2021-07-08 | 2021-07-08 | Recombinant bacillus subtilis, transaminase prepared by same and application of transaminase |
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| CN202110772022.XA CN113583927A (en) | 2021-07-08 | 2021-07-08 | Recombinant bacillus subtilis, transaminase prepared by same and application of transaminase |
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