CN106701708B - 7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1 - Google Patents
7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1 Download PDFInfo
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- CN106701708B CN106701708B CN201710008586.XA CN201710008586A CN106701708B CN 106701708 B CN106701708 B CN 106701708B CN 201710008586 A CN201710008586 A CN 201710008586A CN 106701708 B CN106701708 B CN 106701708B
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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)
- C12Y101/01159—7-Alpha-hydroxysteroid dehydrogenase (1.1.1.159)
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Abstract
The present invention relates to hydroxysteroid dehydrogenases, and in particular to a kind of 7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1.The nucleotide sequence of the gene is as shown in SEQ ID NO.2; encode a kind of 7alpha-Hydroxysteroid dehydrogenase; its amino acid sequence is as shown in SEQ ID NO.1; chenodeoxycholic acid (CDCA) can be catalyzed, Taurochenodeoxycholic Acid (TCDCA) generates 7- Ketolithocholsaeure (7K-LCA), ox sulphur 7- Ketolithocholsaeure (T7K-LCA); catalytic activity to CDCA or TCDCA is more than 2 times of existing 7 α-HSDH of Clostridium sardiniense, has very big industrial application value.
Description
Technical field
The present invention relates to hydroxysteroid dehydrogenases, and in particular to a kind of 7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1.
Background technique
The asymmetric reduction of carbonyl is always one of the hot spot for chemically reacting research.Although current chemical method has been achieved with
Certain achievement, but chemical method often there is catalyst type and Limited Number, stereoselectivity be high, auxiliary reagent
It is expensive and the disadvantages of be not easily recycled.And enzymatic reaction not only has high efficiency, chemo-selective, regioselectivity also and has height
Stereoselectivity.The enzymatic that hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) mediates is anti-
Should have relatively stringent stereoselectivity and " no " stringent substrate specificity.For example, early in the early 1980s
Scientist just has begun 7 α-, 7 β-HSDH the joint epimerism conversion chenodeoxycholic acid for attempting to generate using microorganism
(Chenodeoxycholic acid, CDCA) synthesizes ursodesoxycholic acid (Ursodesoxycholic acid, UDCA).And dissociate
Enzyme can be with catalyzed combination state bile acid --- Taurochenodeoxycholic Acid (Taurochenodeoxycholic acid, TCDCA)
It is converted into Tauro ursodesoxy cholic acid (Tauroursodeoxycholic acid, TUDCA).
The substrate of HSDH is not solely restricted to steroid compound, and document report HSDH can also be catalyzed alkyl and replace monocycle
The carbonyl asymmetric reduction of the substances such as ketone, two cyclic ketones classes.Outstanding catalysis quality possessed by HSDH determines that it turns in biology
Change field has larger application potential.In recent years, scientific research personnel has gradually recognized 7 α-, 7 β-HSDH in field of bioconversion institute
The huge applications potentiality having.Currently, the 7 α-HSDH that the function of registering in GenBank has been acknowledged share 5, they distinguish
From Bacteroides fragilis, Clostridium scindens, Clostridium sordellii,
Clostridium absonum and Escherichia coli；From Clostridium absonum and Collinsella
7 β-HSDH the genes of aerofaciens have also successfully been cloned.
Can the activity and thermal stability of enzyme be to determine put into industrial important parameter, and existing hydroxy steroid is de-
The activity and stability of hydrogen enzyme can't meet industrial demand simultaneously, and therefore, it is necessary to further find and develop newly
Hydroxysteroid dehydrogenase suitable for industrial mass production.
Summary of the invention
The present invention provides a kind of 7alpha-Hydroxysteroid dehydrogenase gene, the 7alpha-Hydroxysteroid dehydrogenase of gene coding
Catalytic activity to CDCA or TCDCA is more than 2 times of existing 7 α-HSDH of Clostridium sardiniense, has very big industrial application value.
The claimed technical solution of the present invention is as follows:
A kind of 7alpha-Hydroxysteroid dehydrogenase, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1.
Encode the gene of the 7alpha-Hydroxysteroid dehydrogenase.
The nucleotide sequence of the gene is as shown in SEQ ID NO.2.
A kind of expression cassette, which is characterized in that include any gene.
A kind of carrier, which is characterized in that include any gene or the expression cassette.
A kind of recombinant cell, which is characterized in that include any gene or the expression cassette or the carrier.
The method for preparing the 7alpha-Hydroxysteroid dehydrogenase, which is characterized in that in the inducible protein expression that can succeed
Under the conditions of cultivate the recombinant cell and isolated 7alpha-Hydroxysteroid dehydrogenase.
A kind of catalyst, which is characterized in that its effective component includes the 7alpha-Hydroxysteroid dehydrogenase.
The catalyst further includes that can be improved enzymatic efficiency when using simultaneously with the 7alpha-Hydroxysteroid dehydrogenase
Or increase other reagents of enzyme stability.
A method of realizing the carbonyl asymmetric reduction of chemical substance, which is characterized in that solid using the 7 Alpha-hydroxy class
Alcohol dehydrogenase or the catalyst carry out catalysis under the conditions of 15-37 DEG C, pH 6.0-11.0 with reaction substrate and react.
The present invention protects from Sichuan black bear and is incubated for the excrement of a healthy black bear in base using macro genomic sequencing technique
Just a kind of new 7alpha-Hydroxysteroid dehydrogenase gene is isolated in sample, is named as 7 α-HSDHY1-a-1, nucleotides sequence
Column are as shown in SEQID NO.2.The gene encodes a kind of new 7alpha-Hydroxysteroid dehydrogenase, amino acid sequence such as SEQ ID
Shown in NO.1, can be catalyzed chenodeoxycholic acid (CDCA), Taurochenodeoxycholic Acid (TCDCA) 7 hydroxyls epimerism, make
It generates ursodesoxycholic acid (UDCA), the intermediate 7- Ketolithocholsaeure (7K-LCA) of Tauro ursodesoxy cholic acid (TUDCA), ox sulphur 7-
Ketolithocholsaeure (T7K-LCA).
7 α-HSDH Y1-a-1 the genes that the present invention separates, 7 α-HSDHY1-a-1 zymoprotein of expression product, with Sardinia
7alpha-Hydroxysteroid dehydrogenase in clostridium Clostridium absonum is compared, and is had preferably than activity.With CDCA
Or TCDCA is substrate, the activity of 7 α-HSDHY1-a-1 zymoproteins of the invention is Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase
More than 2 times, have very big industrial application value.
Carrier provided by the invention, can be cloning vector, comprising needed for 7 α-HSDHY1-a-1 genes and plasmid replication
Other elements；It is also possible to expression vector, comprising 7 α-HSDHY1-a-1 genes and other members of albumen successful expression can be made
Part.
Recombinant cell provided by the invention can be the recombinant cell comprising cloning vector, such as E.coli DH5 α；?
It can be the cell comprising expression vector, cultivate cell under suitable condition, for example, suitable IPTG is added, 16 DEG C of inductions 7
The expression of α-HSDHY1-a-1 zymoprotein.
Catalyst provided by the invention, effective component include 7 α-HSDHY1-a-1 zymoproteins of the invention.The catalysis
Agent can also use simultaneously with other suitable catalyst, thus improve enzymatic efficiency or in same reaction system successively
Carry out two kinds of catalysis reactions.
7 α-HSDHY1-a-1 zymoproteins of the invention can be catalyzed under 15-37 DEG C, the reaction condition of pH 6.0-11.0
TCDCAC7Alpha-hydroxy carbonyl asymmetric reduction reaction.
Detailed description of the invention
The sequence alignment result of 7 α-HSDH genes and 7 α-HSDH gene of Clostridium sardiniense that Fig. 1 present invention separates；
Wherein, 1 be existing 7 α-HSDH gene of Clostridium sardiniense nucleotide sequence, 2 be 7 α-that separate of the present invention
The nucleotide sequence of HSDH gene.
The agarose gel electrophoresis figure for the 7 α-HSDH genes that Fig. 2 present invention separates.
The SDS-PAGE electrophoresis of Fig. 37 α-HSDHY1-a-1 zymoproteins of the invention.
The SDS-PAGE electrophoresis of 7 α-HSDH zymoprotein of Fig. 4 Clostridium sardiniense.
Specific embodiment
The present invention is further described combined with specific embodiments below, it is to be understood that, following embodiments are only as explanation
And explanation, it does not limit the scope of the invention in any way.
Biomaterial:
E.coli DH5 α, E.coli BL21 cell is the preservation of this laboratory, commercially available.
Experiment reagent:
Faeces DNA genome extraction kit is bought from Qiagen company, Germany, article No.: 51604；
Ago-Gel QIAquick Gel Extraction Kit is bought from Omega, article No.: D2500-01；
Mono- step directed cloning kit of PCR is bought from left bank albumen Science and Technology Ltd., article No.: NR001；
Vector pGEX -6p-1, is purchased from Shanghai Sangon Biotech Company；
Plasmid extraction kit OMEGA Plasmid Mini Kit I is bought from OMEGA company, article No.: D6943；
Lysis buffer (lysis buffer) is prepared and is obtained, and the PBS of 10mM pH7.3 contains PMSF 0.1mM, bright peptide
Plain Leupeptin 0.5mg/mL；
Glutathione Sepharose 4B is bought from GE Healthcare, article No.: 10223836；
PreScission Protease is bought from GenScript company, article No.: Z02799-100；
BCA kit is bought from Beyotime company, article No.: P0006；
TCDCA is bought from lark prestige scientific & technical corporation, article No.: 330776.
DNA extracts used black bear excrement, this laboratory freezes.
Not specified biological chemical reagent in following embodiment is this field conventional reagent, can be according to this field
Conventional method prepare and or it is commercially available, specification be the pure grade in laboratory.
The separation of 1. 7 α-HSDH gene of embodiment
1. the discovery of gene
Using the spoon of sterilization treatment, the excrement sample of Sichuan black bear protection and a healthy black bear for being incubated for base is taken
Product, and laboratory is transported in dry ice preservation back.This black bear excrement is extracted using Qiagen faeces DNA genome extraction kit
Total DNA transfers to Shanghai Major Biological Medical Technology Co., Ltd. to carry out macro gene order-checking.With existing 7 α of Clostridium sardiniense-
Hydroxysteroid dehydrogenase (7 α-HSDH) coding gene sequence (Genebank No.AET80685) and macro gene order-checking number
According to comparing, it was found that a kind of 7 new α-HSDH genes are named as 7 α-HSDHY1-a-1, nucleotide sequence such as SEQ ID
Shown in NO.2.Sequence alignment result shows this 7 new α-HSDH genes and existing 7 α-HSDH gene sequence of Clostridium sardiniense
The consistency of column is 59.18% (Fig. 1).
2. the separation of gene
(1) design of primers: the 7 new α-HSDHY1-a-1 gene order design primers that sequencing is obtained, obtained primer
Nucleotide sequence is as follows:
Y1-a-1-f:ATGAAAATTTTAAATAACAAAATAGCTTTAGT
Y1-a-1-r:TTAATCTTGCTCAACAACTTTACGT
(2) PCR amplification: using the total DNA of black bear excrement as template, the primer obtained using step (1), according to following PCR
System and program are expanded.
PCR system:
2 μ L of 5xFastPfu buffer ... ... ... .. ...
2.5mM dNTPs…………….….……2μL
Y1-a-1-f(5μM)……………...…..…2μL
Y1-a-1-r(5μM)………….............…2μL
Template DNA ... ... ... ... ... 20ng
FastPfu polymerase ... ... ... ... ... .1 μ L
Mend ddH2O is extremely ... ... ... ... .. ... ..20 μ L
PCR program:
A.94 DEG C initial denaturation 3min, 5 circulations；
B.94 DEG C denaturation 30sec, 52 DEG C of annealing 30sec, 72 DEG C of extension 1min, 27 recycle；
C.72 DEG C extension 10min.
(3) agarose gel electrophoresis: using 1% agarose gel electrophoresis detect pcr amplification product, as a result as shown in Fig. 2,
There is the band that size is about 800bp.
(4) gel extraction: cutting purpose band in the UV lamp, using Omega Ago-Gel QIAquick Gel Extraction Kit, according to
Operating procedure in kit specification recycles target gene fragment.
The expression of 2.7 α-HSDHY1-a-1 gene of embodiment
1. vector construction:
It is used in the mono- step directed cloning kit of PCR of left bank albumen Science and Technology Ltd. purchase, is illustrated according to kit
The sequence of 7 α-HSDHY1-a-1 genes is connected on vector pGEX -6p-1 by the operating procedure in book.
2. Transformed E .coli DH5 α competent cell
1) competent cell E.coli DH5 α placement is melted on ice.
2) the resulting linked system of step 1 is added in the E.coli DH5 α competence melted, on ice 30min.
3) 42 DEG C of heat treatment 90s.
4) 2min is stood on ice.
5) 600 μ L of LB culture medium is added, 37 DEG C of shaking table temperature, shaking table shakes fast 150rpm, time 45min.
6) 200 μ L bacterium solutions are drawn, ampicillin (Amp is coated on+) on resistance LB plating medium.
7) it is incubated overnight for 37 DEG C.
3. positive clone identification:
(1) white single colonie is selected, LB/Amp is inoculated in+Fluid nutrient medium in, 200rpm, 37 DEG C cultivate 8 hours,
8000rpm is centrifuged 5min and obtains thallus.
(2) use OMEGA Plasmid Mini Kit I plasmid extraction kit, to specifications in operating procedure
Extract plasmid.
(3) cloning primer Y1-a-1-f and Y1-a-1-r are used, according to the PCR system and program progress PCR in embodiment 1
Verifying, determines that genetic fragment is successively inserted into pGEX-6p-1 carrier.
(4) it will identify that correct recombinant plasmid send Shanghai Sangon Biotech Company to be sequenced, sequencing result compared into correctly recombination matter
Grain pGEX-6p-1/7 α-HSDHY1-a-1 is used as expression vector.
4. the expression of 7 α-HSDHY1-a-1 genes
(1) plasmid Transformed E .coli BL21 cell
E.coliBL21 competent cell is taken out in a.-80 DEG C to place on ice.
B. 2 μ L expression vector pGEX-6p-1/7 α-HSDHY1-a-1 are added, place 30min on ice.
C.42 DEG C heat treatment 90s.
D. 2min is placed on ice.
E. it recovers, 600 μ L LB culture mediums, in 37 DEG C, 150rpm shaking table culture 45min is added.
F. 200 μ L bacterium solutions are drawn, Amp is coated on+On LB plating medium.
G.37 DEG C overnight incubation.
(2) protein expression and purifying
A. strain is inoculated in sterile LB liquid medium, ampicillin final concentration of 50 μ g/mL, 37 DEG C,
180rpm shaking table culture.
B. when bacterium solution OD600 ≈ 0.8, it is added the isopropylthiogalactoside (IPTG) of final concentration of 0.2mM, 16 DEG C
Overnight induction (12h).8000rpm, 5min collect thallus.
C. add the ratio of 30mL Lysis buffer that thallus, carrying out ultrasonic bacteria breaking to clarification is resuspended in 1L cultivating system.
12000rpm is centrifuged 20min.Take supernatant.
D. by supernatant in conjunction with Glutathione Sepharose 4B, the ratio that filler uses is that every liter of cultivating system makes
With 5mL filler, 4 DEG C combine 2h.Gently vertical reverse suspension.
E. after combining, 500rpm, 5min precipitation filling.Filler rinses 3-5 column volume, removal with 4 DEG C of pre-cooling PBS
Foreign protein.
F. PreScission Protease enzyme cutting buffering liquid is added, PreScission Protease enzyme is added.
G.4 DEG C digestion is stayed overnight.After digestion, supernatant is released from chromatographic column.
H. gained sample is subjected to SDS-PAGE, identifies its molecular size range and purity, uses BCA
The concentration of kit measurement purifying protein.
As a result as shown in figure 3, the 7 α-HSDHY1-a-1 zymoproteins obtained are in single band, purity is very high.With identical side
Method carries out protein expression to 7 α-HSDH of Clostridium sardiniense, as a result as shown in figure 4, also obtaining the Clostridium sardiniense 7 of single band
α-HSDH。
The Function Identification of 3. 7 α-HSDHY1-a-1 gene of embodiment
Enzyme activity determination:
At room temperature, 158uL 50mM Tris-HCl (PH=8.0) solution, 20uL 50mM are added into cuvette
NADP+Solution, then the 7 α-HSDHY1-a-1 zymoprotein solution that 2 step 4 of 2uL embodiment obtains are added thereto, it is eventually adding
The DMSO solution of the CDCA or TCDCA of 20uL 50mM, sufficiently piping and druming mix.It is immediately placed in spectrophotometer and returns to zero after mixing.
Start timing, reads the changing value of light absorption at a 340nm per minute.
At the same time, according to the enzyme activity of above-mentioned identical method measurement 7 α-HSDH of Clostridium sardiniense.
The result shows that the product of 7 isolated α-HSDHY1-a-1 genes of the invention is 7 alpha-hydroxysteroid dehydrogenations
Enzyme, can be catalyzed chenodeoxycholic acid (CDCA), Taurochenodeoxycholic Acid (TCDCA) 7 hydroxyls epimerism, make its generation
Ursodesoxycholic acid (UDCA), the intermediate 7- Ketolithocholsaeure (7K-LCA) of Tauro ursodesoxy cholic acid (TUDCA), ox sulphur 7- ketone stone gallbladder
Sour (T7K-LCA).The enzyme is as shown in table 1 to the Rate activity of CDCA, TCDCA.
1. 7 α-HSDHY1-a-1 of the present invention of table are compared with the Rate activity of 7 α-HSDH of Clostridium sardiniense
Rate activity (U/mg) definition: under the above-described reaction conditions, enzyme activity unit number contained by every milligram of albumen.
1 data of table are shown, compared with 7 α-HSDH of Clostridium sardiniense, 7 α-HSDHY1-a-1 of the invention have higher enzyme
Living, the Rate activity to CDCA or TCDCA is more than 2 times of 7 α-HSDH of Clostridium sardiniense.
SEQUENCE LISTING
<110>University Of Chongqing
<120>new 7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1
<130> P1632010-CQD-CQ-TXH
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 268
<212> PRT
<213> Artificial Sequence
<220>
<223>amino acid sequence of 7alpha-Hydroxysteroid dehydrogenase Y1-a-1 of the present invention
<400> 1
Met Lys Ile Leu Asn Asn Lys Ile Ala Leu Val Thr Ser Ala Thr Arg
1 5 10 15
Gly Ile Gly Leu Ala Thr Ala Ile Lys Leu Ala Glu Asn Gly Ala Thr
20 25 30
Val Tyr Met Gly Val Arg Arg Leu Glu Ala Thr Gln Glu Ile Cys Asp
35 40 45
Arg Tyr Ala Lys Glu Gly Leu Val Met Lys Thr Val Phe Phe Asp Ala
50 55 60
Tyr Lys Val Asp Ser Tyr Lys Glu Met Ile Asp Thr Ile Ile Glu Lys
65 70 75 80
Glu Gly Lys Ile Asp Ile Leu Val Asn Asn Phe Gly Thr Gly Arg Pro
85 90 95
Glu Thr Asp Ser Asp Leu Val Asn Gly Ser Glu Glu Ser Phe Phe Glu
100 105 110
Leu Phe Glu Arg Asn Val Gly Ser Val Tyr Arg Ile Ser Lys Leu Val
115 120 125
Val Pro His Met Ile Glu Asn Gly Gly Gly Ser Ile Val Asn Ile Ser
130 135 140
Ser Ile Gly Gly Ser Val Pro Asp Ile Ser Arg Ile Gly Tyr Gly Val
145 150 155 160
Ser Lys Ser Gly Val Asn Asn Ile Thr Gln Gln Ile Ala Met Gln Tyr
165 170 175
Ala Lys Asn Lys Val Arg Cys Asn Ala Val Leu Pro Gly Met Ile Ala
180 185 190
Thr Asp Ala Val Ala Ser Asn Met Pro Lys Glu Phe Gln Arg Ser Phe
195 200 205
Leu Ser His Val Pro Leu Asn Arg Met Gly Asn Pro Glu Asp Ile Ala
210 215 220
Asn Ala Val Leu Phe Phe Ala Ser Glu Asn Ser Ser Tyr Ile Thr Gly
225 230 235 240
Ser Ile Leu Glu Val Ser Gly Gly Tyr His Leu Gly Thr Pro Gln Tyr
245 250 255
Ala Asp Phe Val Gly Arg Lys Val Val Glu Gln Asp
260 265
<210> 2
<211> 807
<212> DNA
<213> Artificial Sequence
<220>
<223>nucleotide sequence of 7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1 of the present invention
<400> 2
atgaaaattt taaataacaa aatagcttta gtaacttcag caactagagg aattggttta 60
gcaactgcta taaaattagc tgaaaatgga gctacagtat atatgggtgt tagaaggtta 120
gaagctactc aagaaatatg tgataggtat gcaaaagaag gtttagttat gaaaacagtc 180
ttctttgatg cttataaagt agatagctat aaagaaatga ttgatacaat cattgaaaaa 240
gaaggtaaaa ttgatatatt agttaataac tttggaacag gaagaccaga aacagattca 300
gacttagtaa atggaagtga agaatcattc tttgaattat tcgaacgtaa tgtgggaagt 360
gtttatagaa tctccaaatt agttgttcct catatgatag aaaatggtgg aggaagtata 420
gttaatatat cttcaatcgg aggtagtgtt ccagatatat ctagaatagg atacggcgtt 480
tcaaagtccg gagttaacaa tataactcaa caaatagcta tgcaatatgc aaagaacaaa 540
gtaagatgta atgcagtatt gccaggaatg attgctactg atgctgttgc atcaaacatg 600
cctaaagaat tccaaagatc ttttttatct catgttccat taaatagaat gggtaatcct 660
gaagatatag caaatgcagt tttattcttc gctagcgaaa attcatctta tataactggt 720
tccatattag aagtttctgg tggatatcat ctcggaactc ctcaatatgc cgattttgta 780
ggacgtaaag ttgttgagca agattaa 807
<210> 3
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223>forward primer of 7 α-HSDH gene Y1-a-1 of the PCR amplification present invention
<400> 3
atgaaaattt taaataacaa aatagcttta gt 32
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>reverse primer of 7 α-HSDH gene Y1-a-1 of the PCR amplification present invention
<400> 4
ttaatcttgc tcaacaactt tacgt 25
Claims (10)
1. a kind of 7alpha-Hydroxysteroid dehydrogenase, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1.
2. encoding the gene of 7alpha-Hydroxysteroid dehydrogenase described in claim 1.
3. gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.2.
4. a kind of expression cassette, which is characterized in that include gene described in claim 2 or 3.
5. a kind of carrier, which is characterized in that include gene described in claim 2 or 3 or expression cassette as claimed in claim 4.
6. a kind of recombinant cell, which is characterized in that include gene described in claim 2 or 3 or expression as claimed in claim 4
Carrier described in box or claim 5.
7. the method for preparing 7alpha-Hydroxysteroid dehydrogenase described in claim 1, which is characterized in that can successfully induce
Recombinant cell as claimed in claim 6 and isolated 7alpha-Hydroxysteroid dehydrogenase are cultivated under conditions of protein expression.
8. a kind of catalyst, which is characterized in that its effective component includes 7alpha-Hydroxysteroid dehydrogenase described in claim 1.
9. catalyst according to claim 8, which is characterized in that further include solid with 7 Alpha-hydroxy class described in claim 1
Alcohol dehydrogenase can be improved enzymatic efficiency or increase other reagents of enzyme stability when using simultaneously.
10. a kind of method for the carbonyl asymmetric reduction for realizing chemical substance, which is characterized in that use described in claim 17
Catalyst described in alpha-hydroxysteroid dehydrogenase or claim 8 or 9 and reaction substrate are in 15-37 DEG C, pH 6.0-11.0 item
Catalysis reaction is carried out under part.
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| CN107841489B (en) * | 2017-11-14 | 2020-02-18 | 重庆大学 | Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M |
| CN115386557A (en) * | 2022-10-14 | 2022-11-25 | 安徽大学 | Preparation method of 7 alpha-hydroxysteroid dehydrogenase and catalytic conversion application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103502442A (en) * | 2010-12-16 | 2014-01-08 | 细胞制药有限公司 | Novel 7 Beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid |
| CN104382941A (en) * | 2014-10-28 | 2015-03-04 | 上海凯宝药业股份有限公司 | Artificial bear gall powder and preparation method thereof |
| CN106282138A (en) * | 2016-09-22 | 2017-01-04 | 重庆大学 | Clostridium sardiniense 7 α hydroxysteroid dehydrogenase mutant T145S |
-
2017
- 2017-01-05 CN CN201710008586.XA patent/CN106701708B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103502442A (en) * | 2010-12-16 | 2014-01-08 | 细胞制药有限公司 | Novel 7 Beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid |
| CN105441399A (en) * | 2010-12-16 | 2016-03-30 | 细胞制药有限公司 | Novel 7 Beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid |
| CN104382941A (en) * | 2014-10-28 | 2015-03-04 | 上海凯宝药业股份有限公司 | Artificial bear gall powder and preparation method thereof |
| CN106282138A (en) * | 2016-09-22 | 2017-01-04 | 重庆大学 | Clostridium sardiniense 7 α hydroxysteroid dehydrogenase mutant T145S |
Non-Patent Citations (4)
| Title |
|---|
| 7α-羟基类固醇脱氢酶晶体结构中辅酶NADP(H)的识别位点分析;谭君等;《中国化学会第30届学术年会摘要集-第二十五分会：化学信息学与化学计量学》;20160731;第1页 |
| Comparative analysis of the gut microbiota of black bears in China using high-throughput sequencing;Can Song et al.;《Mol Genet Genomics》;20161227;第1-8页 |
| Discovery of tauroursodeoxycholic acid biotransformation enzymes from the gut microbiome of black bears using metagenomics;Can Song et al.;《Scientific Reports》;20170424;第7卷;第1-8页 |
| 基于宏基因组序列的黑熊肠道微生物组的应用基础研究;宋璨;《中国博士学位论文全文数据库 医药卫生科技辑》;20180615;E057-2 |
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