CN107267479B - A kind of acyltransferase and its mutant catalyzing and synthesizing Simvastatin - Google Patents
A kind of acyltransferase and its mutant catalyzing and synthesizing Simvastatin Download PDFInfo
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- CN107267479B CN107267479B CN201710554484.8A CN201710554484A CN107267479B CN 107267479 B CN107267479 B CN 107267479B CN 201710554484 A CN201710554484 A CN 201710554484A CN 107267479 B CN107267479 B CN 107267479B
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Abstract
The present invention provides a kind of acyltransferase and preparation method thereof for catalyzing and synthesizing Simvastatin.The acyltransferase is amino acid sequence shown in SEQID NO:2 at 161,163,180, the mutant of the mutation of 235 progress；The features such as novel acyltransferase enzyme activity is 12 times of wild-type enzyme, and the acyltransferase that the present invention constructs has enzyme at low cost, and transformation time is short, technological operation is simple, the extensive prospect with large-scale industrial application.
Description
Technical field
The invention belongs to genetic engineerings and enzyme engineering field, are related to a kind of acyl group transfer for catalyzing and synthesizing Simvastatin
Enzyme and its mutant.
Background technique
Simvastatin, trade name simvastatin (Zocor), the entitled Simvastatin of English, Simvastatin is Statins
(statin) blood lipid-lowering medicine is HMG CoA (HMG-COA) reductase inhibitor, inhibits endogenous gallbladder solid
The synthesis of alcohol is lipid regulating agent.Documents and materials, which show to have, reduces that Hyperlipidemia Rabbits serum, liver, total gallbladder is solid in aorta
Alcohol (content of TC) reduces C-VLDL (VLDL-C), low density lipoprotein cholesterol (LDL-C) level
Effect.It is clinically used for treatment hypercholesterolemia, coronary heart disease, controls the content of albumen alcohol and prevention vascular diseases in blood.
Simvastatin is developed by United States Merck company, which obtains U.S. FDA batch in December, 1991 in Initial Public Offering in 1988
It is quasi-.The simvastatin (Zocor) of Merck in 2000 comes second place on global marketing list, and sales volume is 52.8 hundred million dollars,
Reach within 2001 66.7 hundred million dollars of summit, 2002 are 55.8 hundred million dollars, have the huge market demand.
The method of the Simvastatin of industry production at present is mainly chemical synthesis, fermentation method and production conversion method, mainly
Intermediate can obtain Monascin J(by fermentation and be also known as Monascin, Monacolin J, and No. CAS: 132748-10-8).
Chemical synthesis is also known as Monascin, Monacolin J by intermediate Monascin J(), pass through chemical catalysis
The series reactions such as protection, deprotection, hydrolysis, esterification obtain Simvastatin, it is obvious that such method is limited to step
Various, reagent type consumes greatly more, and product separates the defects of complicated, and the best approach of nonproductive Simvastatin.
Fermentation method mainly using the Aspergillus terreus bacterial strain of the genetic engineering transformation of building, produces Simvastatin using fermentation method,
But the yield of Simvastatin does not show apparent effect compared with traditional fermentation process.
Biotransformation method, it is selectively lower currently with the esterase catalyzed conversion zone of existing commercialization, still it can make
The problems such as complicated at the purification step of the product of reaction.
Summary of the invention
In order to solve problem above, the first purpose of this invention is to provide a kind of acyl group for catalyzing and synthesizing Simvastatin
Transferase.
Second purpose of the invention is to provide the core that the acyl group transfer undergraduate course of Simvastatin is catalyzed and synthesized described in a kind of coding
Nucleotide sequence.
Third purpose of the present invention is to provide a kind of mutant of acyltransferase for catalyzing and synthesizing Simvastatin.
4th purpose of the invention is to provide the mutation that the acyltransferase of Simvastatin is catalyzed and synthesized described in a kind of coding
The nucleotide sequence of body.
5th purpose of the invention is to provide a kind of side for preparing and catalyzing and synthesizing the acylase mutant of Simvastatin
Method.
6th purpose of the invention is to provide the method using acylase catalyzed synthesis Simvastatin.
Technical solution of the invention is as follows:
A kind of acyltransferase catalyzing and synthesizing Simvastatin, the acyltransferase are ammonia shown in SEQ ID NO:2
Base acid sequence.
A kind of such as above-mentioned acyltransferase for catalyzing and synthesizing Simvastatin, nucleotide sequence is as shown in SEQ NO:1.
A kind of mutant of such as above-mentioned acyltransferase for catalyzing and synthesizing Simvastatin, the amino acid sequence of the mutant
As shown in SEQ ID NO.4；Compared with amino acid sequence SEQ ID NO.2, taken in the amino acid sequence SEQ ID NO:4
Band tetra- mutant of D161N, Y163F, G180S, A235S；Wherein, D161N represents the 164th amino acid residue from asparagus fern ammonia
Acid becomes asparagine, and Y163F represents the 163rd amino acids residue and becomes phenylalanine from tyrosine, and G180S represents the 180th
Amino acids residue becomes serine from glycine, and A235S represents the 235th amino acids residue and becomes serine from alanine.
According to the mutant of the above-mentioned acyltransferase for catalyzing and synthesizing Simvastatin, nucleotide sequence such as SEQ ID
Shown in NO.3.
A method of the above-mentioned acylase mutant for catalyzing and synthesizing Simvastatin is prepared, is included the following steps:
(1) engineering strain of acyltransferase, the SEQID NO:1 nucleotides sequence that full genome is synthesized are constructed first
The biological enzyme genes segment of column is cloned into pET29a(+ in efficient expression vector), the engineering strain of acyltransferase is constructed,
The acyltransferase is recombinated through digestion to expression vector pET29a(+), host cell BL21(DE3 is transformed into) in；
(2) positive colony is chosen and is transferred in LB liquid medium after being activated in LB liquid medium, cultivated, most afterwards through sending out
Somatic cells are collected in ferment culture；
(3) somatic cells are crushed, the acyltransferase is obtained.
Preferably, the digestion is reassembled as with primers F 1 and R1 being respectively forward and reverse primer, fallibility PCR, structure are carried out
Mutant library is built, the primer nucleotide sequences are as follows: F1:5'-GGAATTCCATATGCAGGATATCGAAC-3'；R1:5'-
CCCAAGCTTTCAGTCGTTGCGCAGT-3', the primer both ends are respectively provided with NdeI and HindIII restriction enzyme site.
A method of preparing Simvastatin using above-mentioned acyltransferase: step includes:
(1) it synthesizes Simvastatin side chain: 3- mercapto-propionate, ethyl acetate, triethylamine, above-mentioned being added into reaction kettle
The acyltransferase of preparation；Stirring is opened, is cooled to 0~5 DEG C, 2,2- dimethyl-butyrylchlorine is added dropwise, synthesizes Simvastatin side chain；
(2) not that crin acid, pungent described in above-mentioned steps acylase catalyzed synthetic reaction: is added into reaction kettle
Statin side chain, acid solution are cut down, controls pH value of reaction system 9.0~10.0；Above-mentioned acyltransferase, temperature control is added
At 25~45 DEG C, it is stirred to react 48~60h；Reaction solution is filtered；Filtering finishes, and temperature controls the decompression at 30~50 DEG C, vacuum
Degree≤- 0.09MPa, it is dry, obtain intermediate；
(3) prepared by Simvastatin crude product: intermediate made from above-mentioned steps being added in methylene chloride and is dissolved；Temperature control
At 0~25 DEG C, methylene chloride and Loprazolam mixed solution are added dropwise into filtrate；It is added dropwise, temperature is controlled 0~25
DEG C stirring 1~4h；Reacting liquid temperature is adjusted to 25~45 DEG C, is concentrated under reduced pressure into solid, it is thick to obtain Simvastatin by end of reaction
Product.
Preferably, the acid solution is one of hydrochloric acid, sulfuric acid, acetic acid.
Beneficial effect: the present invention uses the biological enzyme activity of novel acyltransferase for 12 times of wild-type enzyme, and synthesis turns
Rate reaches 75%.The acyltransferase thermal stability and storage stability are more preferable, and activity is high, which is applied to produce pungent cut down
Statin, and reaction environment is friendly, and production cost is low, and transformation time is short, technological operation is simple, and reaction system impurity content is low,
Yield is improved, is easily post-processed, the extensive prospect with large-scale industrial application.
Specific implementation method
The screening of 1 acyltransferase of embodiment
A. terreus is derived from reported acyltransferase LovD() it is template, BLAST ratio is carried out on NCBI
It is right, select sequence of the similitude between 40-70% therefrom to carry out gene chemical synthesis and inducing expression, and for Simvastatin
Enzyme process screening.Screening system is as follows: reaction system (concentration of substrate 1g/L)
The enzyme that can be used for Simvastatin enzymatic clarification is successfully screened, which is from fungal sp.
The a certain agnoprotein of NO.14919, coding nucleotide sequence is as shown in SEQ NO:2, the amino acid sequence of coding such as SEQ
Shown in NO:1.
However the reactivity of the enzyme is lower, needs to be oriented evolution to meet industrial applications demand.
The foundation of embodiment 2, mutant library
With the expression plasmid of full genome synthesis, (by Changzhou, Ji Yu Bioisystech Co., Ltd is synthesized, and clone has volume on plasmid
Amino acid sequence shown in code SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2) it is template, with primers F 1 and R1
Respectively forward and reverse primer carries out fallibility PCR, constructs mutant library.
Primer sequence is as follows: F1:5'-GGAATTCCATATGCAGGATATCGAAC-3'；R1:5'-
CCCAAGCTTTCAGTCGTTGCGCAGT-3', both ends are respectively provided withNdeI andHindIII restriction enzyme site.
Fallibility PCR reaction system is as follows: 2.5 μ L, 10mM dGTP of 10* PCR buffer, 0.5 μ L, 10mM dTTP
0.5 μ L, 10mM dCTP, 2.5 μ L, 10mM dATP, 2.5 μ L, 1mM MnCl22.5 μ L, 55mM MgCl2 2.5 μ L, 10 μM of F1
1 μ L, 10 μM of 1 μ L, Template(10ng/ μ L of R1) 1 μ L, Taq DNA polymerase(5U/ μ L, takara) 0.2 μ L,
ddH2O is mended to 25 μ L.
Fallibility PCR is carried out on Bio-Rad T100 thermal cycler, and PCR program is as follows: 94 DEG C of 5min；94℃
30s, 58 DEG C of 30s, 72 DEG C of 15 min, 30 circulations；72℃ 10min；15℃ forever.
Above-mentioned PCR reaction solution is subjected to 1% agarose gel electrophoresis, the segment (concrete operations of gel extraction 1200bp size
See TIANGEN Biotech's Ago-Gel QIAquick Gel Extraction Kit operating instruction), and through restriction enzymeNdeI andHindAfter III digestion, (Novagen) carrier is attached, and is converted with the pET29a(+ by same double digestion)
BL21(DE3 it) (is purchased from TIANGEN Biotech (Beijing) Co., Ltd.), is shifted after being inverted and be incubated overnight in 37 DEG C to get to acyl group
Enzyme mutant library, the monoclonal grown are used for screening active ingredients.
The activation and inducing expression of 3 mutant library of embodiment
From picking monoclonal on the plate of above-mentioned overnight incubation to equipped with 1mL, containing final concentration of 50 μ g/mL sulfuric acid card, that is mould
In 96 deep-well plates of the LB liquid medium of element, stayed overnight in 37 DEG C, 220rpm shaken cultivation.Next day is incubated overnight from above-mentioned
100 μ L culture solutions are drawn in 96 hollow plates, are added to LB liquid of the fresh 1mL containing final concentration of 50 μ g/mL kanamycin sulfate
In 96 deep-well plates of body culture medium, 37 DEG C, after 220rpm shaken cultivation 4h, the IPTG that final concentration of 1mM is added is induced, with
Continue to cultivate 20h at 30 DEG C afterwards.400 monoclonals of picking altogether are used for screening active ingredients.
Bacterium solution after above-mentioned induction is centrifuged 15min in 4 DEG C, 4000rpm, abandons supernatant.100 μ L 50mM phosphoric acid of thallus
Sodium buffer (pH7.0) suspends, and the lysozyme of final concentration of 0.5g/l is added, in 37 DEG C of processing 1h, 4 DEG C, 4000rpm centrifugation
30min takes 50 μ L supernatants to be transferred to 96 new orifice plates, the template as screening active ingredients.
4 HPLC method of embodiment measures Simvastatin content
Chromatographic column ZORBAX SB-C18(4.6mm × 250mm, 5 μm)；Mobile phase is methanol/water (gradient: 75% methanol liter
To 95% methanol, 15min), flow velocity 1.0ml/min；Detection wavelength 238nm；30 DEG C of column temperature.With Monconlin J, DMB-S-
MMP and Simvastatin standard items are control.
The screening active ingredients of 5 mutant of embodiment
In above-mentioned 96 orifice plates for containing 50 μ L mutant enzyme supernatants, 10%DMSO, 5mmol Monconlin J are sequentially added
With 10mmol DMB-S-MMP, in 30 DEG C of reaction 30min, then in 100 DEG C of processing 10min inactivators, 4000rpm is centrifuged
10min carries out HPLC detection after taking supernatant to dilute 10 times, screens mutant with the production quantity of Simvastatin, wild-type enzyme is raw
At Simvastatin as control.
Find there is the production quantity of the Simvastatin of 1 mutant to be apparently higher than wild type control through HPLC analysis, this is prominent
Variant number is clone No. 280.Then this clone is expanded culture, to verify whether its activity significantly improves.
The expansion culture and activity verifying of 6 enzyme mutant of embodiment
LB Liquid Culture of the 5mL containing final concentration of 50 μ g/mL kanamycin sulfate is inoculated by mutant clone 280
In the test tube of base, 37 DEG C, 220rpm shaken cultivation stay overnight.Next day is transferred to 100mL containing final concentration of 50 μ g/mL in 1% ratio
In the LB liquid medium of kanamycin sulfate, the IPTG of final concentration of 1mM is added in 37 DEG C, 220rpm 3~4h of shaken cultivation,
Then in 30 DEG C, 200rpm overnight induction.
Bacterium solution after induction is centrifuged 10min, thallus 20mL pH7.0 50mM sodium phosphate buffer in 4 DEG C, 8000rpm
After washing twice, 10mL pH7.0 50mM sodium phosphate buffer is added and suspends.Ultrasound is then carried out under condition of ice bath
Broken (ultrasonic power 200W, ultrasonic 3S/ interval 5S, ultrasonic 10min).Sample after ultrasound is centrifuged in 4 DEG C, 12000rpm
20min, supernatant are freeze-dried, and obtain freeze-dried powder as Activity determination.
Reaction condition is with example 5, and HPLC testing conditions are with embodiment 4, and freeze-dried powder buffer solution is at 1mg/mL concentration, instead
Answering system is 100mL.The results show that the amount of the Simvastatin of No. 280 generations of clone will be significantly higher than the production of wild-type enzyme generation
The amount of object, activity are about 12 times of wild type.
It then gives these three clones to sequencing, and is compared with wild type DPE amino acid sequence, discovery clone 280 takes
D161N, Y163F, G180S, A235S tetra- mutation of band (as shown in SEQ ID NO.3, gene order such as SEQ ID NO.4 institute
Show).Wherein, the amino acid residue that D161N represents the 164th becomes asparagine from aspartic acid, and Y163F represents the 163rd
Amino acid residue becomes phenylalanine from tyrosine, and G180S represents the 180th amino acids residue and becomes serine from glycine,
A235S represents the 235th amino acids residue and becomes serine from alanine.
Embodiment 7: the fermentation preparation of biological enzyme
The present invention is applied
A) shake flask fermentation
Shake-flask seed culture medium ingredient: yeast extract: 5g/L；Peptone: 10g/L, NaCl:10g/L；Kanamycins
(referred to as blocking that) 50ug/ml.
Shake-flask seed culture medium preparation: taking 5g yeast powder extracts, 10g peptone, and 10gNaCl is dissolved in 800ml distillation
In water, pH to 7 is adjusted, is settled to 1000ml, 121 DEG C of holding 15min with distilled water, in 121 DEG C of holding 15min, solution is cooling
It is added after to 60 DEG C or less and blocks that final concentration 50ug/ml.
Fermentation step:
It takes original seed strain to cross on LB plate, 37 DEG C, is inverted overnight incubation.On plate picking monoclonal be inoculated in
In 3ml seed culture medium (10ml test tube), OD600 grows to 0.5-1 after 37 DEG C, 200rpm shaken cultivation 16-20 hours.It is connect with 1%
Kind amount is seeded in 300ml seed culture medium (1L triangular flask), and OD600 grows to 1- after 37 DEG C, 200rpm shaken cultivation 4-8 hours
2。
B) fermented and cultured
Culture medium is divided into fermentation medium and fed-batch medium
Fermentation medium is that M9 medium component is as follows: Na2HPO46 g/L, KH2PO43g/L, MgSO4▪7H2O
0.246g/L, (NH4)2SO4 2.24 g/L, NaCl 0.5g/L, glucose 20g/L.
Fermentation medium configuration: Na2HPO4、KH2PO4、MgSO4▪7H2O、(NH4)2SO4, NaCl, glucose stirring and dissolving,
30min is kept at 121 DEG C, stand-by after cooling, kanamycins uses non-velum filteration degerming.Then by sterile kanamycins
It is added in fermentation medium.
Fed-batch cultivation based component is as follows: glucose 600g/L
Fed-batch medium configuration: glucose is dissolved in water, 115 DEG C of holding 30min, stand-by after cooling.
Ferment tank control
Whole process controls 20% or more DO, ventilating ratio 1:1-4(VVM), 37 DEG C of fermented and cultured temperature control, pH7.0, culture
Dissolved oxygen mutation in 8 hours starts feed supplement, OD600=30 or so induction, the final concentration of 1mM of IPTG, 30 DEG C of inducing temperature control, culture
Put tank within 21 hours.
C) fermentation liquor treatment and enzyme preparation
After fermentation, fermentation liquid is sent into centrifuge and carries out bacterium solution separation, concentration thallus uses homogeneous crusher machine, bacterium solution
In 50~200g/L, cracking pressure control obtains supernatant, supernatant warp after 30~60Mpa, clasmatosis liquid centrifugation for concentration control
After crossing ultrafiltration concentration, enzyme solution is obtained, after being lyophilized, obtains acyltransferase dry powder.
Embodiment 8: Enzyme catalyzed synthesis Simvastatin method includes the following steps:
(1) it synthesizes Simvastatin side chain: 3- mercapto-propionate, ethyl acetate, triethylamine, above-mentioned being added into reaction kettle
The acyltransferase of preparation；Stirring is opened, is cooled to 0~5 DEG C, 2,2- dimethyl-butyrylchlorine is added dropwise, synthesizes Simvastatin side chain；
(2) not that crin acid, pungent described in above-mentioned steps acylase catalyzed synthetic reaction: is added into reaction kettle
Statin side chain, acid solution are cut down, controls pH value of reaction system 9.0~10.0；Above-mentioned acyltransferase, temperature control is added
At 25~45 DEG C, it is stirred to react 48~60h；Reaction solution is filtered；Filtering finishes, and temperature controls the decompression at 30~50 DEG C, vacuum
Degree≤- 0.09MPa, it is dry, obtain intermediate；
(3) prepared by Simvastatin crude product: intermediate made from above-mentioned steps being added in methylene chloride and is dissolved；Temperature control
At 0~25 DEG C, methylene chloride and Loprazolam mixed solution are added dropwise into filtrate；It is added dropwise, temperature is controlled 0~25
DEG C stirring 1~4h；Reacting liquid temperature is adjusted to 25~45 DEG C, is concentrated under reduced pressure into solid, it is thick to obtain Simvastatin by end of reaction
Product.
In the present embodiment, the acid solution is one of hydrochloric acid, sulfuric acid, acetic acid.
The present invention uses the biological enzyme activity of novel acyltransferase for 12 times of wild-type enzyme, and Synthesis conversion reaches 75%.
The acyltransferase thermal stability and storage stability are more preferable, and activity is high, which are applied to production Simvastatin, and react
Environmental-friendly, production cost is low, and transformation time is short, technological operation is simple, and reaction system impurity content is low, improves yield, Yi Hou
Processing, the extensive prospect with large-scale industrial application.
SEQ ID NO:1
MQDIERAFEQAVESGQIPGVVLMAKDRSGAKINYTRCYGSRTARLDNAPAETTIMEVDSPMRLASACKI
ITTVMAMQCVERKLLRLDEDVSGILPEVGNMMVLEGFDSDSRPRMRKPEAVVTLRSLLTHTSGISYIVEHPDLMRYR
DLGHIARPDAGKVVDRYNYPLVSDPGRCWSYGPGLEWAGKLVERVTGLSLEEYLQQNICAPLGVADMTFKLQQRPDM
RARRADMSRRDAEGVPRNEDASYYRADPEDCFGGMGIFASPRAFMAVLHSLLAKDGKLLAAGTIETMFQPQLDAVCE
QSLNDEIDARQQTNHGGLLPRTGIRRSHGLGGLMILENCDGMDWRRQGSMSWGGFPNLYWCIDPEAGICILIAFQLI
PWADPQCVELGCAFERAIYQQLRND
SEQ ID NO:2
ATGCAGGATATCGAACGCGCGTTTGAACAGGCAGTTGAATCTGGTCAGATTCCGGGCGTTGTTCTGATG
GCAAAAGATCGTAGCGGCGCAAAAATCAACTACACCCGCTGCTACGGTAGCCGTACCGCACGTCTGGATAACGCACC
GGCTGAAACCACCATTATGGAGGTCGATTCTCCGATGCGTCTGGCAAGCGCCTGCAAAATTATCACCACCGTTATGG
CCATGCAGTGCGTTGAACGTAAACTGCTGCGTCTGGACGAAGACGTTTCAGGTATTCTGCCGGAAGTTGGCAACATG
ATGGTCCTGGAAGGCTTTGATAGCGATAGCCGTCCGCGTATGCGTAAACCGGAAGCAGTTGTTACCCTGCGTAGTCT
GCTGACCCATACCAGCGGTATTAGCTACATCGTCGAACATCCGGATCTGATGCGTTATCGCGATCTGGGTCATATTG
CACGTCCGGACGCAGGTAAAGTTGTTGATCGCTACAACTATCCGCTGGTAAGCGATCCGGGTCGTTGTTGGAGTTAC
GGTCCGGGTCTGGAATGGGCAGGTAAACTGGTTGAACGCGTTACCGGTCTGAGTCTGGAAGAATACCTGCAGCAAAA
TATTTGCGCACCGCTGGGCGTTGCGGATATGACCTTCAAACTGCAGCAACGTCCGGATATGCGCGCTCGTCGCGCAG
ATATGAGTCGTCGCGACGCAGAAGGCGTTCCGCGTAACGAAGACGCAAGCTATTATCGCGCCGATCCGGAAGATTGC
TTTGGCGGTATGGGTATCTTTGCAAGTCCGCGCGCATTTATGGCAGTTCTGCATAGCCTGCTGGCAAAAGACGGTAA
ACTGCTGGCAGCAGGTACCATTGAAACCATGTTCCAGCCGCAACTGGACGCAGTTTGCGAACAAAGCCTGAACGACG
AAATTGACGCGCGTCAACAAACCAATCACGGCGGTCTGCTGCCGCGTACCGGTATTCGTCGTAGTCACGGTCTGGGC
GGTCTGATGATTCTGGAAAACTGCGACGGTATGGATTGGCGTCGTCAAGGTAGTATGAGTTGGGGCGGTTTTCCGAA
CCTGTATTGGTGCATTGATCCGGAAGCGGGTATTTGCATCCTGATCGCGTTTCAACTGATTCCGTGGGCAGATCCGC
AGTGCGTTGAACTGGGTTGCGCATTTGAACGCGCCATTTATCAGCAACTGCGCAACGACTGA
SEQ ID NO:3
MQDIERAFEQAVESGQIPGVVLMAKDRSGAKINYTRCYGSRTARLDNAPAETTIMEVDSPMRLASACKI
ITTVMAMQCVERKLLRLDEDVSGILPEVGNMMVLEGFDSDSRPRMRKPEAVVTLRSLLTHTSGISYIVEHPDLMRYR
DLGHIARPDAGKVVNRFNYPLVSDPGRCWSYGPSLEWAGKLVERVTGLSLEEYLQQNICAPLGVADMTFKLQQRPDM
RARRADMSRRDSEGVPRNEDASYYRADPEDCFGGMGIFASPRAFMAVLHSLLAKDGKLLAAGTIETMFQPQLDAVCE
QSLNDEIDARQQTNHGGLLPRTGIRRSHGLGGLMILENCDGMDWRRQGSMSWGGFPNLYWCIDPEAGICILIAFQLI
PWADPQCVELGCAFERAIYQQLRND
SEQ ID NO:4
ATGCAGGATATCGAACGCGCGTTTGAACAGGCAGTTGAATCTGGTCAGATTCCGGGCGTTGTTCTGATG
GCAAAAGATCGTAGCGGCGCAAAAATCAACTACACCCGCTGCTACGGTAGCCGTACCGCACGTCTGGATAACGCACC
GGCTGAAACCACCATTATGGAGGTCGATTCTCCGATGCGTCTGGCAAGCGCCTGCAAAATTATCACCACCGTTATGG
CCATGCAGTGCGTTGAACGTAAACTGCTGCGTCTGGACGAAGACGTTTCAGGTATTCTGCCGGAAGTTGGCAACATG
ATGGTCCTGGAAGGCTTTGATAGCGATAGCCGTCCGCGTATGCGTAAACCGGAAGCAGTTGTTACCCTGCGTAGTCT
GCTGACCCATACCAGCGGTATTAGCTACATCGTCGAACATCCGGATCTGATGCGTTATCGCGATCTGGGTCATATTG
CACGTCCGGACGCAGGTAAAGTTGTTAATCGCTTCAACTATCCGCTGGTAAGCGATCCGGGTCGTTGTTGGAGTTAC
GGTCCGAGTCTGGAATGGGCAGGTAAACTGGTTGAACGCGTTACCGGTCTGAGTCTGGAAGAATACCTGCAGCAAAA
TATTTGCGCACCGCTGGGCGTTGCGGATATGACCTTCAAACTGCAGCAACGTCCGGATATGCGCGCTCGTCGCGCAG
ATATGAGTCGTCGCGACTCGGAAGGCGTTCCGCGTAACGAAGACGCAAGCTATTATCGCGCCGATCCGGAAGATTGC
TTTGGCGGTATGGGTATCTTTGCAAGTCCGCGCGCATTTATGGCAGTTCTGCATAGCCTGCTGGCAAAAGACGGTAA
ACTGCTGGCAGCAGGTACCATTGAAACCATGTTCCAGCCGCAACTGGACGCAGTTTGCGAACAAAGCCTGAACGACG
AAATTGACGCGCGTCAACAAACCAATCACGGCGGTCTGCTGCCGCGTACCGGTATTCGTCGTAGTCACGGTCTGGGC
GGTCTGATGATTCTGGAAAACTGCGACGGTATGGATTGGCGTCGTCAAGGTAGTATGAGTTGGGGCGGTTTTCCGAA
CCTGTATTGGTGCATTGATCCGGAAGCGGGTATTTGCATCCTGATCGCGTTTCAACTGATTCCGTGGGCAGATCCGC
AGTGCGTTGAACTGGGTTGCGCATTTGAACGCGCCATTTATCAGCAACTGCGCAACGACTGA
Claims (3)
1. a kind of mutant for catalyzing and synthesizing Simvastatin acyltransferase, it is characterised in that: the nucleotide sequence of the mutant
As shown in SEQ ID NO.4；Compared with nucleotide sequence SEQ ID NO.2, taken in the nucleotide sequence SEQ ID NO:4
Band tetra- mutant of D161N, Y163F, G180S, A235S；Wherein, D161N represents the 161st amino acid residue from asparagus fern ammonia
Acid becomes asparagine, and Y163F represents the 163rd amino acids residue and becomes phenylalanine from tyrosine, and G180S represents the 180th
Amino acids residue becomes serine from glycine, and A235S represents the 235th amino acids residue and becomes serine from alanine；
This catalyzes and synthesizes the mutant of the acyltransferase of Simvastatin, and amino acid sequence is as shown in SEQ ID NO.3.
2. a kind of method that the mutant using Simvastatin acyltransferase described in claim 1 catalyzes and synthesizes Simvastatin,
Step includes: synthesis Simvastatin side chain: 3- mercapto-propionate, ethyl acetate, triethylamine, above-mentioned system being added into reaction kettle
Standby acyltransferase；Stirring is opened, is cooled to 0~5 DEG C, 2,2- dimethyl-butyrylchlorine is added dropwise, synthesizes Simvastatin side chain；
(2) not that crin acid, Simvastatin side described in above-mentioned steps acylase catalyzed synthetic reaction: are added into reaction kettle
Chain, acid solution control pH value of reaction system 9.0~10.0；The mutation of acyltransferase described in claim 1 is added
Body, temperature are controlled at 25~45 DEG C, are stirred to react 48~60h；Reaction solution is filtered；Filtering finishes, and temperature is controlled 30~50
DEG C, decompression, vacuum degree≤- 0.09MPa is dry, obtains intermediate；(3) prepared by Simvastatin crude product: in made from above-mentioned steps
Mesosome, which is added in methylene chloride, to be dissolved；Temperature controls at 0~25 DEG C, and methylene chloride is added dropwise into filtrate and Loprazolam is mixed
Close solution；It is added dropwise, 0~25 DEG C of 1~4h of stirring；Reacting liquid temperature is adjusted to 25~45 DEG C, is concentrated under reduced pressure by end of reaction
Solid obtains Simvastatin crude product.
3. the method that the mutant of Simvastatin acyltransferase according to claim 2 catalyzes and synthesizes Simvastatin,
Be characterized in that: the acid solution is one of hydrochloric acid, sulfuric acid, acetic acid.
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Denomination of invention: Acyltransferase for catalytic synthesis of simvastatin and its mutant Effective date of registration: 20220318 Granted publication date: 20190712 Pledgee: Hangzhou United Rural Commercial Bank Co.,Ltd. Xiasha sub branch Pledgor: Hangzhou Hongyou Pharmaceutical Technology Co.,Ltd. Registration number: Y2022980002812 |