CN110776572B - 7 beta-HSDH enzyme mutant and preparation method thereof - Google Patents
7 beta-HSDH enzyme mutant and preparation method thereof Download PDFInfo
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- CN110776572B CN110776572B CN201911114498.3A CN201911114498A CN110776572B CN 110776572 B CN110776572 B CN 110776572B CN 201911114498 A CN201911114498 A CN 201911114498A CN 110776572 B CN110776572 B CN 110776572B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01201—7-Beta-hydroxysteroid dehydrogenase (NADP+) (1.1.1.201)
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- C07K2319/00—Fusion polypeptide
Abstract
The invention relates to a fusion protein of 7 beta-HSDH enzyme and DPS, which mutates glycine at 159 th site of the 7 beta-HSDH enzyme into histidine and lysine at 165 th site into histidine, wherein the amino acid SEQUENCE of the 7 beta-HSDH enzyme is shown as SEQUENCE NO.1, so as to obtain a 7 beta-HSDH enzyme mutant with the amino acid SEQUENCE of SEQUENCE NO.2, and the 7 beta-HSDH enzyme mutant and DPS protein from Thermoanaerobacter pseudethanolicus are subjected to fusion expression to obtain the fusion protein of the 7 beta-HSDH enzyme and the DPS. Compared with wild 7 beta-HSDH enzyme, the thermal stability of the 7 beta-HSDH enzyme and the DPS fusion protein obtained by the invention is obviously improved, and the enzyme activity is improved by 70%.
Description
Technical Field
The invention relates to the technical field of fusion proteins, in particular to a fusion protein of 7 beta-HSDH enzyme and DPS.
Background
Tauroursodeoxycholic acid (TUDCA Ursodeoxycholic acid) is a main pharmacological active ingredient in bear gall, and is mainly obtained by means of artificial extraction of bear gall at present. Tauroursodeoxycholic acid is mainly used for treating diseases related to bile acid metabolism disorder clinically, but because of the scarcity of bear gall resources and the particularity of sources, the yield of tauroursodeoxycholic acid (TUDCA) is far from meeting the clinical medication requirements. TUDCA can be extracted from bear gall, and its preparation method includes chemical synthesis method and biotransformation method. The chemical synthesis method for preparing tauroursodeoxycholic acid (TUDCA) has complex process and low yield, and the activity and the thermal stability of 7 beta-HSDH enzyme in the biotransformation method need to be further improved so as to meet the requirement of industrial production.
Disclosure of Invention
The invention analyzes and compares the thermal stability and the enzymatic activity of 4 types of 7 beta-HSDH enzymes from Ruminococcus torques, Ruminococcus gnavus, Collinlla aerofaciens and Clostridium sardiniense, carries out point mutation design on the 7 beta-HSDH enzyme from the Ruminococcus gnavus, carries out fusion expression on the 7 beta-HSDH enzyme mutant and DPS protein, improves the activity and the thermal stability of the 7 beta-HSDH enzyme and is convenient for subsequent industrial catalytic application.
A7 beta-HSDH enzyme mutant, wherein the amino acid SEQUENCE of the 7 beta-HSDH enzyme mutant is shown as SEQUENCE ID NO. 2.
The preparation method of the 7 beta-HSDH enzyme mutant comprises the following steps: the 159 th glycine of the 7 beta-HSDH enzyme with the amino acid SEQUENCE shown as SEQUENCE NO.1 is mutated into histidine, and the 165 th lysine is mutated into histidine, so as to obtain the 7 beta-HSDH enzyme mutant with the amino acid SEQUENCE shown as SEQUENCE NO. 2.
The 7 beta-HSDH enzyme is derived from the genus of Ruminococcus gnavus.
And carrying out fusion expression on the 7 beta-HSDH enzyme mutant and DPS protein from Thermoanaerobacter pseudoethanolica species to obtain the fusion protein of the 7 beta-HSDH enzyme and the DPS.
The preparation method of the fusion protein of the 7 beta-HSDH enzyme and the DPS comprises the steps of constructing the 7 beta-HSDH enzyme mutant and the DPS protein on an escherichia coli pGEX6P-1 vector by a PCR or gene synthesis method, transforming an escherichia coli BL21(DE3) competent cell by the obtained recombinant vector, and carrying out induced expression on the recombinant protein to obtain the fusion protein of the 7 beta-HSDH enzyme and the DPS.
Compared with wild 7 beta-HSDH enzyme, the thermal stability and the enzyme activity of the fusion protein of the 7 beta-HSDH and the DPS obtained by the invention are obviously higher than the activity, and the enzyme activity is improved by 70 percent.
Description of the drawings:
FIG. 1 is a plasmid map of pGEX 6P-1-N-GST-3C-7. beta. -HSDH mutant.
FIG. 2 is a plasmid map of pGEX 6P-1-N-GST-3C-DPS-linker-7. beta. -HSDH.
FIG. 3 shows the result of SDS-PAGE analysis of 7 β -HSDH enzyme (Ruminococcus torque); lane 1: 2ug of protein of interest, lane 2: 10ug of the protein of interest.
FIG. 4 shows the results of SDS-PAGE analysis of 7. beta. -HSDH (Ruminococcus gnavus); lane 1: 2ug of protein of interest, lane 2: 10ug of the protein of interest.
FIG. 5 is the result of SDS-PAGE electrophoretic analysis of 7 β -HSDH enzyme (Collinsella aerofaciens); lane 1: 2ug of protein of interest, lane 2: 10ug of the protein of interest.
FIG. 6 shows the results of SDS-PAGE analysis of 7. beta. -HSDH enzyme (Clostridium sardiniense); lane 1: 2ug of protein of interest, lane 2: 10ug of the protein of interest.
FIG. 7 shows the SDS-PAGE analysis of the fusion protein of 7. beta. -HSDH enzyme and DPS; lane 1: 2ug of protein of interest, lane 2: 10ug of the protein of interest.
FIG. 8 shows the results of thermal stability analysis of 7. beta. -HSDH enzyme and the fusion protein of 7. beta. -HSDH enzyme and DPS in 4 species. The 7 beta-HSDH enzyme derived from the Ruminococcus torques is the curve No.1 in the figure, the 7 beta-HSDH enzyme derived from the Ruminococcus gnavus 7 beta-HSDH enzyme is the curve No.2 in the figure, the 7 beta-HSDH enzyme derived from the Collinsella aerofacies is the curve No. 3 in the figure, the 7 beta-HSDH enzyme derived from the Clostridium sardiniense is the curve No. 4 in the figure, and the fusion protein of the 7 beta-HSDH enzyme and the DPS is the curve No. 5 in the figure.
FIG. 9 shows the results of the assays of the activities of 7. beta. -HSDH enzyme and the fusion protease of 7. beta. -HSDH enzyme with DPS in 4 species.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, all technical means described herein are known to those skilled in the art. Furthermore, the embodiments are to be regarded as illustrative rather than limiting the scope of the invention. The spirit and scope of the present invention are limited only by the appended claims.
Materials (I) and (II)
1.1 expression vector: (1) pGEX6P-1-N-GST-3C-7 beta-HSDH, the plasmid map of which is shown in 1;
(2) pGEX6P-1-N-GST-3C-DPS-linker-7 beta-HSDH was constructed by the company itself, and its plasmid map is shown in FIG. 2.
1.2 competent cells: a TransT1 competent cell, BL21 competent cell.
1.3 culture medium and reagents: LB medium, IPTG inducer, L-glutamhiene (reduced), SYPRO Orange dye, NADP, TUDCA, plasmid miniprep kit (TIANGEN), general agarose gel recovery kit (TIANGEN)
1.4 Experimental facility SPH-211C temperature control shaking table, AKTA-FPLC rapid protein purification liquid phase, Agilent LC-MS mass spectrum analyzer, Biometra TProfessional PCR instrument, LRH-70F biochemical incubator, MD SpectraMax Plus384 continuous wavelength microplate reader, ABI7500.
Second, method
2.17 beta-HSDH enzyme Point mutation design
The whole gene sequence DNA of 4 wild type 7 beta-hydroxysteroid dehydrogenases (7 beta-HSDH) from the species Ruminococcus torques, Ruminococcus gnavus, Collinsella aerofaciens and Clostridium sardiniense is obtained by PCR or gene synthesis respectively.
The 7 beta-HSDH amino acid SEQUENCE derived from the genus of Ruminococcus gnavus is shown as SEQUENCE NO. 1. The 7 beta-HSDH whole gene sequence from the genus Ruminococcus gnavus is subjected to point mutation design, glycine at the 159 th position is mutated into histidine, and lysine at the 165 th position is mutated into histidine. Through a site-directed mutagenesis method, a 7 beta-HSDH enzyme (G159H, K165H) mutant SEQUENCE is obtained, and is shown as SEQUENCE NO. 2.
2.27 beta-HSDH expression vector construction
GST is used as an affinity tag, and a 7 beta-HSDH enzyme (G159H, K165H) mutation sequence derived from the genus of the genus Caminococcus gnavus and a DPS protein gene derived from the genus of the genus Thermoanaerobacter pseudo-ethanolica are introduced into an Escherichia coli pGEX6P-1 vector by utilizing a PCR and seamless cloning method to construct a recombinant plasmid: pGEX6P-1-N-GST-3C-7 beta-HSDH and pGEX6P-1-N-GST-3C-DPS-linker-7 beta-HSDH.
Meanwhile, the whole gene sequence DNA of 4 wild type 7 beta-hydroxysteroid dehydrogenases (7 beta-HSDH) from the species Ruminococcus torques, Ruminococcus gnavus, Collinlla aerofaciens and Clostridium sardiniense is introduced into an Escherichia coli pGEX6P-1 vector to construct a recombinant plasmid: pGEX6P-1-N-GST-3C-7 beta-HSDH
2.37 beta-HSDH protein expression
Coli BL21(DE3) was prepared and the recombinant plasmid: pGEX6P-1-N-GST-3C-7 beta-HSDH and pGEX6P-1-N-GST-3C-DPS-linker-7 beta-HSDH were transformed into E.coli BL21(DE3) competent cells, and the target protein was obtained from 2L of liquid medium at a culture temperature of 15 ℃ and a final IPTG concentration of 0.5mM for 16 hours.
2.47 beta-HSDH enzyme and DPS fusion protein purification and SDS-PAGE electrophoretic analysis
4 kinds of 7 beta-HSDH enzyme, 7 beta-HSDH enzyme mutant and DPS fusion protein are purified by using an affinity chromatography method, and the expression and purification effects of the 4 kinds of 7 beta-HSDH enzyme, 7 beta-HSDH enzyme mutant and DPS fusion protein are analyzed by using SDS-PAGE electrophoresis. As shown in fig. 3, 4, 5, 6 and 7.
2.57 beta-HSDH enzyme and DPS fusion protein thermal stability analysis
And (3) carrying out thermal stability analysis on the 7 beta-HSDH enzyme, the 7 beta-HSDH enzyme and the DPS fusion protein by using SYPRO Orange dye, and adding a protein purification buffer solution, a target protein and the SYPRO Orange dye into a 96-well microplate. The program is set as follows: 95 ℃ for 2min, 25 ℃ for 2min, the temperature rise rate was set at 1 ℃ per minute. As shown in fig. 8.
2.67 Activity analysis of beta-HSDH enzyme and DPS fusion protease
The enzyme activity of the obtained 7. beta. -HSDH enzyme, 7. beta. -HSDH enzyme and DPS fusion protein was analyzed, and a buffer, a target protein, NADP and TUDCA were added to a 96-well reaction plate to measure the change in absorbance at 340 nM.
Third, result analysis
3.1 analysis of protein expression level
According to experimental determination, under the same expression condition, the expression quantities of the four 7 beta-HSDH enzymes, the 7 beta-HSDH enzyme mutant and the DPS fusion protein are different, the expression quantity of the 7 beta-HSDH enzyme and the DPS fusion protein is the highest, and the expression quantity of the 7 beta-HSDH enzyme from the genus of Ruminococcus gnavus is the second expression quantity, which is shown in Table 1.
3.2 analysis of thermal stability results
According to experimental determination, the Tm average value of the 7 beta-HSDH enzyme and the DPS fusion protein is 64.43 ℃, the Tm average value of the 7 beta-HSDH enzyme derived from Ruminococcus torques is 55.01, the Tm average value of the 7 beta-HSDH enzyme derived from Ruminococcus gnavus is 55.85, the Tm average value of the 7 beta-HSDH enzyme derived from Collinsella aerofaciens is 54.58, the Tm average value of the 7 beta-HSDH enzyme derived from Clostridium sardiniense is 54.07, and the thermal stability of the 7 beta-HSDH enzyme and the DPS fusion protein is improved by 20 percent compared with that of the 7 beta-HSDH enzyme of 4 species, as shown in Table 1.
3.3 analysis of enzyme Activity results
According to experimental determination, the activity of 7 beta-HSDH enzyme derived from the genus Ruminococcus torques is 41.1U/mg, the activity of 7 beta-HSDH enzyme derived from the genus Ruminococcus gnavus is 68.8U/mg, the activity of 7 beta-HSDH enzyme derived from the genus Collinsella aerofaciens is 30.5U/mg, the activity of 7 beta-HSDH enzyme derived from the genus Clostridium sardiniense is 34.1U/mg, the activity of 7 beta-HSDH enzyme and DPS fusion protease is 120.8U/mg, the activity of 7 beta-HSDH enzyme and DPS fusion protease is obviously higher than that of 7 beta-HSDH enzyme derived from the 4 species, and the enzyme activity is improved by 70%. See table 1 and fig. 9.
TABLE 1 comparison of expression level, thermostability, and enzyme Activity of each protein
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the present invention pertains, the architecture form can be flexible and varied without departing from the concept of the present invention, and a series of products can be derived. But rather a number of simple derivations or substitutions are made which are to be considered as falling within the scope of the invention as defined by the appended claims.
Sequence listing
<120> fusion protein of 7 beta-HSDH enzyme and DPS
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 263
<212> PRT
<213> Ruminococcus gnavus
<400> 1
Met Thr Leu Arg Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Arg Leu Ala Lys Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Lys Glu Leu
35 40 45
Gly Glu Glu Leu Lys Asn Thr Tyr Glu Ile Asp Tyr Lys Val Val Lys
50 55 60
Ala Asp Phe Ser Leu Pro Asp Ala Thr Asp Lys Ile Phe Ala Ala Thr
65 70 75 80
Glu Asn Leu Asp Met Gly Phe Met Ala Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Met Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Thr Glu Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Thr Ala Gln Thr Pro Glu Glu Val Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ser Gly Glu Arg Asn Lys Ala Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Asp Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Gln Glu
260
<210> 2
<211> 263
<212> PRT
<213> Ruminococcus gnavus
<400> 2
Met Thr Leu Arg Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Arg Leu Ala Lys Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Lys Glu Leu
35 40 45
Gly Glu Glu Leu Lys Asn Thr Tyr Glu Ile Asp Tyr Lys Val Val Lys
50 55 60
Ala Asp Phe Ser Leu Pro Asp Ala Thr Asp Lys Ile Phe Ala Ala Thr
65 70 75 80
Glu Asn Leu Asp Met Gly Phe Met Ala Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Met Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala His Lys
145 150 155 160
Ala Phe Ile Leu His Met Thr Glu Ala Val Ala Cys Glu Thr Glu Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Thr Ala Gln Thr Pro Glu Glu Val Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ser Gly Glu Arg Asn Lys Ala Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Asp Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Gln Glu
260
Claims (3)
1. A7 beta-HSDH enzyme mutant is characterized in that the amino acid SEQUENCE of the 7 beta-HSDH enzyme mutant is shown as SEQUENCE ID NO. 2.
2. A method for preparing a mutant of 7 β -HSDH enzyme according to claim 1, wherein glycine 159 and lysine 165 of 7 β -HSDH enzyme having the amino acid SEQUENCE shown in SEQUENCE No.1 are mutated to histidine, respectively, to obtain a mutant of 7 β -HSDH enzyme having the amino acid SEQUENCE shown in SEQUENCE ID No. 2.
3. The method for preparing a mutant 7 β -HSDH enzyme according to claim 2, wherein the 7 β -HSDH enzyme is derived from the genus runinococcus gnavus.
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| CN112029740A (en) * | 2020-09-15 | 2020-12-04 | 江西邦泰绿色生物合成生态产业园发展有限公司 | 7 beta hydroxysteroid dehydrogenase mutant and application thereof |
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