CN107980064A - A kind of preparation method of urso and its alternation enzyme processed 2 - Google Patents
A kind of preparation method of urso and its alternation enzyme processed 2 Download PDFInfo
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- CN107980064A CN107980064A CN201780001726.9A CN201780001726A CN107980064A CN 107980064 A CN107980064 A CN 107980064A CN 201780001726 A CN201780001726 A CN 201780001726A CN 107980064 A CN107980064 A CN 107980064A
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- 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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Abstract
The present invention relates to a kind of 7 β steroid dehydrogenases of method and its preparation that urso is prepared using biological enzyme technology.This method is using 3 α hydroxyls, 7 oxo, 5 β cholanic acids as substrate, under the conditions of existing for NADP, glucose, glucose dehydrogenase and buffer solution, urso is prepared with 77 oxo of β steroid dehydrogenases 3 α hydroxyls of enzymatic, 5 β cholanic acids, wherein 7 β steroid dehydrogenases derive from enterococcusEnterococcus silesiacus.This method is easy to operate, reaction condition it is gentle it is easily-controllable, the reaction time is short, to the high conversion rate of substrate up to more than 99.6%, the content of the product obtained is more than 98.5%.
Description
Technical field
It is more particularly to a kind of to be prepared using biological enzyme technology the present invention relates to molecular biology and biological technical field
The method and its preparation 7 β-steroid dehydrogenase of urso.
Background technology
Urso is the principle active component of rare Chinese medicine bear gall, have increase bile acid secretion and make bile into
Change, the cholesterol be conducive in gall stone is divided gradually to dissolve, reduce bile cholesterol and cholesterol ester and other effects, it is main to use
In treatment cholelith disease.It is well known that bear gall is a kind of very rare resource, reason is that the classical pathway that it is obtained is main
It is to rely on the method that artificial breeding bear living takes courage.At present, this cycle is long, yield is low and the gradual quilt of inhuman classical pathway
Artificial synthesis is substituted.
Originally, the artificial synthesis of urso is mostly chemical method, and is widely used in industrial production.But
Chemical method there are operating condition is harsh, selectivity is low, pollution environment, using a large amount of organic solvents, there are organic solvent residual,
The shortcomings of poisonous and harmful.In order to solve shortcomings existing for chemical method, people look for another way, and find more preferable production ways.
Chinese invention patent application CN105368828A discloses a kind of using-7-5 β of oxo of whole-cell catalytic 3 Alpha-hydroxy of preparation-cholane
The method of sour urso, but such a method need to carry out cell fermentation culture, there are the reaction time is long, cumbersome, production
The shortcomings of thing is complicated.
The content of the invention
It is an object of the invention to provide a kind of new preparation method of urso, to solve in above-mentioned background technology
The organic solvent residual present in existing preparation method mentioned, condition are harsh, the reaction time is long, cumbersome, pollution environment
The shortcomings of, invention also provides the biology enzyme that the new preparation process is applicable in.
To achieve the above object, inventor gropes by long-term substantial amounts of experiment, it is attempted in the failure for undergoing up to a hundred times
Afterwards, the biology enzyme that urso is prepared suitable for extracellular biological catalysis is filtered out finally, and is carried out in this sequence basis
Optimization, obtains activity and improves and remove the mutant enzyme that substrate suppresses, and a kind of new urso is prepared so as to develop
Method, it is characterised in that：Using-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid as substrate, in NADP, glucose, glucose dehydrogenase
And under the conditions of buffer solution is existing, prepares bear with 7 β-- 7-5 β of oxo of 3 Alpha-hydroxy of steroid dehydrogenase enzymatic-cholanic acid and go
Oxycholic acid, 7 β-steroid dehydrogenase derive from enterococcus Enterococcus silesiacus, the glucose dehydrogenation
Enzyme source is in bacillus megaterium Bacillus megaterium, its gene order such as SEQ ID NO：Shown in 3, entirely urging
Change in reaction system, the concentration of the substrate is 50~100mg/mL, and the concentration of the NADP is 0.01~0.25mg/mL, institute
The concentration for stating glucose is 30~50mg/mL.
The specific existence form of two kinds of enzymes used in the above method includes liquid enzyme liquid, solid-state and various immobilizations
Enzyme, can be not purified thick enzyme form or the form through partial purification or Economical Purification.
Preferably, control the catalytic process temperature be 25~35 DEG C, pH value be 7.5~8.5 under conditions of carry out.
Preferably, the buffer solution is 50~100mM kaliumphosphate buffers.
Preferably, above-mentioned preparation method further includes following purification step：Treat the catalytic process after reaction, adjust pH
It is worth for 10.5~12.5, removes insoluble matter, then adjust pH value as 1.0~2.0,50-60 DEG C of water-bath, stirs 20~30min, treat cold
But afterwards filtering, be dried in vacuo after washing three times finished product up to urso.
It is highly preferred that above-mentioned preparation method further includes following purification step：By the finished product 10- of the urso of acquisition
0.5-1h is stirred at reflux under 50-60 DEG C of water bath condition of 20 times of absolute ethyl alcohols, heat filtering, takes filtrate to carry out vacuum-concentrcted to 1/
4-1/5 volumes, add 5-10 times of pure water stirring 1h and are crystallized, filter, filter cake is dried in vacuo, up to urso
Highly finished product.
Preferably, 7 β used in above-mentioned preparation method-steroid dehydrogenase is the protein of following (a) or (b)：
(a) its amino acid sequence such as SEQ ID NO：Protein shown in 2,
(b) (a) limit amino acid sequence in by substitution, lack or add one or several amino acid and
Have in the presence of NADP by substrate of-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid than amino acid sequence such as SEQ ID NO：Shown in 2
The protein as derived from (a) of 7 high β of parent-steroid dehydrogenase enzymatic activity.
It is highly preferred that 7 β-steroid dehydrogenase and such as SEQ ID NO：Amino acid sequence shown in 2 compare selected from
There is at least one mutation at least one following sites：66th, the 67th, the 90th, the 91st, the 150th, the 153rd
Position and the 200th.
It is highly preferred that 7 β-steroid dehydrogenase has at least one following mutation：D66N、L67K、Y90W、V91A、
A150I, I153K and D200N.
Present invention also offers one kind 7 β-steroid dehydrogenase, it is characterised in that：7 β-steroid dehydrogenase enzyme source
In enterococcus Enterococcus silesiacus, ursodeoxycholic is prepared for being catalyzed-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid
Acid, 7 β-steroid dehydrogenase are the protein of following (a) or (b)：
(a) its amino acid sequence such as SEQ ID NO：Protein shown in 2,
(b) (a) limit amino acid sequence in by substitution, lack or add one or several amino acid and
Have in the presence of NADP by substrate of-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid than amino acid sequence such as SEQ ID NO：Shown in 2
The protein as derived from (a) of 7 high β of parent-steroid dehydrogenase enzymatic activity.
Preferably, 7 β-steroid dehydrogenase and such as SEQ ID NO：Amino acid sequence shown in 2 is compared selected from extremely
There is at least one mutation at few following sites：66th, the 67th, the 90th, the 91st, the 150th, the 153rd
And the 200th.
Preferably, 7 β-steroid dehydrogenase has at least one following mutation：D66N、L67K、Y90W、V91A、
A150I, I153K and D200N.
Beneficial effect：
1st, compared with the preparation method of existing urso, method provided by the invention has easy to operate, reaction
Mild condition is easily-controllable, the reaction time is short, without using organic solvent, it is nontoxic and pollution-free and of low cost the advantages of, be proven,
The reaction duration of method provided by the invention only need 8~15 it is small when, its high conversion rate to substrate is obtained up to more than 99.6%
Product content more than 98.5%.
2nd, the present invention has filtered out 7 β-steroid dehydrogenase base that urso is prepared suitable for extracellular biological catalysis
Cause, and optimized in this sequence basis, obtain activity and improve and remove the mutant enzyme that substrate suppresses, these mutant
Enzyme shows high selectivity so that this method will not form accessory substance, the high catalytic activity of these mutant enzymes and high specificity
So that the cost of ursodeoxycholic acid-enzyme hydrolysis method large-scale production is lower, there is higher industrial application value.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, and following embodiments are the solutions to the present invention
Release, the person that is not specified actual conditions the invention is not limited in following embodiments, in embodiment, routinely condition or manufacturer suggest
Condition carry out.
The specific implementation process of the preparation method of urso provided by the invention is as follows：
- 7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid is suspended in 50~100mM kaliumphosphate buffers (pH8.0), with 10M's
NaOH adjusts pH to 8.0, add final concentration of 30~50mg/mL glucose and with the NaOH of 10M adjusting pH to 7.8~
8.0,7 β-steroid dehydrogenase and glucose dehydrogenase are added, is eventually adding the NADP of final concentration of 0.01~0.25mg/mL,
Final substrate concentrations are 50~100mg/mL, react and are carried out in 25~35 DEG C of temperature, 200~400rpm and pH7.5~8.5, reaction
Time is 8~15h.Extract reaction solution at regular intervals with 50~100 times of phase dilution of flowing, sample introduction progress liquid phase after micro porous filtration
Analysis.Liquid phase detection is analytical column using 5 μm of 250 × 4.6mm of NX-C18 110A of Phenomenx Gemini, and mobile phase is
Acetonitrile:Buffer solution (take sodium dihydrogen phosphate 0.78g, dissolve in 1L water, be 3 with phosphoric acid tune pH value, you can):Methanol=30:37:
40, with spare after 0.45um membrane filtrations.Column temperature is 40 DEG C, Composition distribution (RID), flow velocity 0.8mL/min.Wait to be catalyzed
After reaction, the pH value that the NaOH adjustment reaction solutions of 5~10M are added in the case of quick stirring is 10.5~12.5 to journey,
Filtrate is filtered to take, filtrate added drop-wise hydrochloric acid to pH value is 1.0~2.0, and 50-60 DEG C of water-bath, stirs 20~30min, after cooling mistake
Filter, be dried in vacuo after washing three times finished product up to urso.Again by finished product with 10-20 times of 50-60 DEG C of absolute ethyl alcohol
0.5-1h is stirred at reflux under water bath condition, heat filtering, takes filtrate to carry out vacuum-concentrcted to 1/4-1/5 volumes, add 5-
10 times of pure water stirring 1h are crystallized, and are filtered, filter cake are dried in vacuum overnight, up to the highly finished product of urso.
The specific existence form of two kinds of enzymes used in the above method includes liquid enzyme, solid-state enzyme and various immobilizations
Enzyme, can be not purified thick enzyme form or the form through partial purification or Economical Purification.
Embodiment 1
The preparation of coexpression recombinant plasmid pET22b-BHSDH5-GDH containing parental gene
Will derive from enterococcus (Enterococcus silesiacus) 7 β-steroid dehydrogenase genes BHSDH5 and
Glucose dehydrogenase gene GDH from bacillus megaterium (Bacillus megaterium) is utilized respectively primer pair 5'
CGCCATATGATGTATACAGATTTAAAAGA3' and 5'CCGGAATTCTTAGCCTCTTCCCGTTTGGA3' and primer pair
5'CCGGAATTCAAGGAGATATACATATGATGTATACAGATTTAAAAGA3' and 5'
CCGCTCGAGTTAGCCTCTTCCCGTTTGGA3' after PCR amplification acquisition PCR product by digestion by handling, at the same time
Nde I and the EcoR I site and EcoR I sites and Xho I sites of expression vector pET22b (+) are inserted into, obtains common table
Up to recombinant plasmid pET22b-BHSDH5-GDH.Through DNA sequencing, the nucleosides of 7 β of the parent-steroid dehydrogenase being cloned is determined
Acid sequence such as SEQ ID NO：Shown in 1, its amino acid sequence such as SEQ ID NO：Shown in 2；Determine the parent's grape being cloned
The nucleotide sequence of glucocorticoid dehydrogenase such as SEQ ID NO：Shown in 3, its amino acid sequence such as SEQ ID NO：Shown in 4.
Embodiment 2
The preparation of coexpression recombinant plasmid containing 7 β-steroid dehydrogenase enzyme mutant
Rite-directed mutagenesis is carried out to 7 β-steroid dehydrogenase parent by inverse PCR technique, it is anti-by designing in mutated site
To primer, purpose fragment is expanded using upstream and downstream mutant primer, and corresponding mutation is introduced on primer, with recombinant plasmid
PET22b-BHSDH5-GDH carries out inverse PCR as template, and PCR product is transformed into large intestine after the processing of Dpn I enzymic digestions template
Bacillus Rosetta (de3), picking colony send sequencing after the screening of Amp.Mutational site and design of primers are as shown in table 1.
PCR system is：TaKaRa EX Taq HS 0.25ul；10×Ex Taq Buffer 5ul；Template plasmid 1ul；
dNTP(2.5mM each)4ul；Sense primer 1ul；Anti-sense primer 1ul；Sterile water up to 50ul.
PCR programs are：98 DEG C of 2min first；Then 98 DEG C of 10s, 50-65 DEG C of 30s, 72 DEG C of 7min, 30 circulations；Finally
72℃10min。
Table 1
Embodiment 3
The preparation of enzyme liquid
Parent prepared by embodiment 1 and embodiment 2 and mutant coexpression recombinant plasmid are transferred to Escherichia coli respectively
Rosetta (de3), then the recombination bacillus coli of acquisition is seeded in the LB culture mediums (Amp containing 100 μ g/mL) of small size,
After 30~37 DEG C are incubated overnight, it is transferred to 1~5% inoculum concentration in the LB culture mediums of certain volume (containing 100 μ g/mL's
Amp), continue to cultivate OD at 30~37 DEG C600Reach the isopropyl-β-D- sulphur of 0.6~1.0 final concentration of 0.1mM~1mM of addition
For galactoside (IPTG), thalline is collected by centrifugation after 20~37 DEG C of 8~20h of induced expression.Fermentation thalli is suspended in certain body
In the kaliumphosphate buffer (pH8.0) of 50 long-pending~100mM and ultrasonic wave breaks born of the same parents, centrifuges up to glucose dehydrogenase and 7 is contained
β-steroid dehydrogenase parent or the crude enzyme liquid with 7 β-steroid dehydrogenase enzyme mutant, available for enzyme activity measure and
It is prepared by the living things catalysis of urso.
Embodiment 4
The measure of enzyme activity
The enzyme activity determination method of 7 β-steroid dehydrogenase：Using-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid as substrate, at one
- 7-5 β of oxo of 150mM3 Alpha-hydroxies-cholanic acid of 10uL, the dilution enzyme liquid of 100uL, NADP+ ends are added in the reaction system of 3mL
Concentration is 0.2mM, reacts certain time at pH8.0 and 25 DEG C, and light absorption value increase is measured at 340nm.
The enzyme activity determination method of glucose dehydrogenase：Using glucose as substrate, added in the reaction system of a 3mL
The 50mM glucose of 100uL, the dilution enzyme liquid of 100uL, the final concentration of 0.2mM of NADPH, in pH8.0 and 25 DEG C of timing of reaction one
Between, light absorption value reduction is measured at 340nm.
The measurement result of enzyme activity is as shown in table 2, and wherein GDH is glucose dehydrogenase, and 7 β-HSDH take off for 7 β-steroids
Hydrogen enzyme.
Table 2
Embodiment 5
The preparation of urso
With reference to the specific implementation process of the preparation method of foregoing urso, the crude enzyme liquid prepared using embodiment 3, enzyme
The input amount of liquid accounts for the stereometer of whole reaction system ,-7-5 β of oxo of 3 Alpha-hydroxy of control substrate-cholanic acid with the weight of enzyme liquid
Final concentration of 100mg/mL, remaining each design parameter is as shown in table 3.Measured after 8~15h of reaction, substrate conversion efficiency exists
More than 99.6%, for finished product content more than 98.5%, yield is 86~92%.
Table 3
Embodiment 6
The preparation of urso
Total system 1L ,-7-5 β of oxo of the 3 Alpha-hydroxy-cholanic acid for taking 50g contents to be 99%, is suspended in the potassium phosphate of 100mM
Buffer solution (pH8.0), with adding the glucose of final concentration 45g/L after the adjusting pH to 8.0 of 10M NaOH, and sequentially adds
0.1g7 β-steroid dehydrogenase freeze-dried powder (D200N mutant enzymes) and 0.07g glucose dehydrogenase freeze-dried powders, are eventually adding end
Concentration is the NADP, Final substrate concentrations 50g/L of 0.15g/L.Reaction 12h is carried out in 25 DEG C, 250rpm and pH8.0 or so, is turned
Rate is up to 99.8%.After reaction, it is 12.5 to add 10M NaOH to pH in the case of quick stirring, filters to take filtrate,
Filtrate added drop-wise hydrochloric acid to pH value is 1.0, and 55 DEG C of water-bath, stirs 30min, and filtering after cooling, be dried in vacuo after washing three times
Obtain the finished product 55.8g of urso.The finished product of obtained urso 800ml absolute ethyl alcohols are dissolved again, 60 DEG C
1h, heat filtering are stirred under water bath condition, and filter cake is washed with a small amount of ethanol, takes filtrate to carry out being concentrated under reduced pressure into 200ml, adds
2L pure water stirs 1h, filtering, and filter cake is washed with water three times, and gained filter cake is by obtaining the refined of urso after vacuum drying
Product 50.6g.
Claims (10)
- A kind of 1. preparation method of urso, it is characterised in that：Using-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid as substrate, Under the conditions of NADP, glucose, glucose dehydrogenase and buffer solution are existing, with 7 β -3 α of steroid dehydrogenase enzymatic-hydroxyl - 5 β of the oxo of base-7-cholanic acid prepares urso, and 7 β-steroid dehydrogenase derives from enterococcus Enterococcus Silesiacus, the glucose dehydrogenase derive from bacillus megaterium Bacillus megaterium, its gene order is such as SEQ ID NO：Shown in 3, in whole catalystic converter system, the concentration of the substrate is 50~100mg/mL, the NADP's Concentration is 0.01~0.25mg/mL, and the concentration of the glucose is 30~50mg/mL.
- 2. the preparation method of urso according to claim 1, it is characterised in that：The catalytic process is controlled in temperature Spend for 25~35 DEG C, pH value carries out under conditions of being 7.5~8.5.
- 3. the preparation method of urso according to claim 1, it is characterised in that：The buffer solution for 50~ 100mM kaliumphosphate buffers.
- 4. the preparation method of urso according to claim 1, it is characterised in that the preparation method further include as Lower purification step：Treat the catalytic process after reaction, it is 10.5~12.5 to adjust pH value, removes insoluble matter, then adjust pH Be worth for 1.0~2.0,50-60 DEG C of water-bath, stir 20~30min, filtering after cooling, be dried in vacuo after washing three times to obtain the final product The finished product of urso.
- 5. the preparation method of urso according to any one of claims 1 to 4, it is characterised in that 7 β-class is solid Alcohol dehydrogenase is the protein of following (a) or (b)：(a) its amino acid sequence such as SEQ ID NO：Protein shown in 2,(b) pass through substitution in the amino acid sequence that (a) is limited, lack or add one or several amino acid and deposited in NADP Have under by substrate of-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid than amino acid sequence such as SEQ ID NO：Parent shown in 2 is high 7 β-steroid dehydrogenase enzymatic activity the protein as derived from (a).
- 6. the preparation method of urso according to claim 5, it is characterised in that 7 β-steroid dehydrogenase with Such as SEQ ID NO：Amino acid sequence shown in 2 is compared has at least one mutation at least one following sites：The 66, the 67th, the 90th, the 91st, the 150th, the 153rd and the 200th.
- 7. the preparation method of urso according to claim 6, it is characterised in that 7 β-steroid dehydrogenase tool There are at least one following mutation：D66N, L67K, Y90W, V91A, A150I, I153K and D200N.
- 8. one kind 7 β-steroid dehydrogenase, it is characterised in that：7 β-steroid dehydrogenase derives from enterococcus Enterococcus silesiacus, urso is prepared for being catalyzed-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid, and 7 β- Steroid dehydrogenase is the protein of following (a) or (b)：(a) its amino acid sequence such as SEQ ID NO：Protein shown in 2,(b) pass through substitution in the amino acid sequence that (a) is limited, lack or add one or several amino acid and deposited in NADP Have under by substrate of-7-5 β of oxo of 3 Alpha-hydroxy-cholanic acid than amino acid sequence such as SEQ ID NO：Parent shown in 2 is high 7 β-steroid dehydrogenase enzymatic activity the protein as derived from (a).
- 9. 7 β according to claim 8-steroid dehydrogenase, it is characterised in that 7 β-steroid dehydrogenase and such as SEQ ID NO：Amino acid sequence shown in 2 is compared has at least one mutation at least one following sites：66th, 67, the 90th, the 91st, the 150th, the 153rd and the 200th.
- 10. 7 β according to claim 9-steroid dehydrogenase, it is characterised in that 7 β-steroid dehydrogenase has extremely Few following mutation：D66N, L67K, Y90W, V91A, A150I, I153K and D200N.
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| PCT/CN2017/070605 WO2018126469A1 (en) | 2017-01-09 | 2017-01-09 | Method for preparing ursodeoxycholic acid and enzyme 2 for use in preparation thereof |
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| CN108546691A (en) * | 2018-05-09 | 2018-09-18 | 华东理工大学 | 7 beta-hydroxy sterol dehydrogenase mutants and its application in preparing ursodesoxycholic acid |
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| CN115521964B (en) * | 2022-09-19 | 2024-04-12 | 湖北共同生物科技有限公司 | Preparation method of steroid hormone drug intermediate |
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