CN105586325A - Lovastatin acyltransferase mutant - Google Patents
Lovastatin acyltransferase mutant Download PDFInfo
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- CN105586325A CN105586325A CN201610062451.7A CN201610062451A CN105586325A CN 105586325 A CN105586325 A CN 105586325A CN 201610062451 A CN201610062451 A CN 201610062451A CN 105586325 A CN105586325 A CN 105586325A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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Abstract
The invention discloses a lovastatin acyltransferase mutant, a gene sequence for coding the mutant, and a method for preparing simvastatin from the mutant. Compared with the wild type acyltransferase derived from Aspergillus terreus, the acyltransferase mutant has higher activity. The mutant can be used for large-scale production of the statin compound simvastatin, and has the advantages of high conversion rate and high utilization ratio of raw materials.
Description
Technical field
The present invention relates to the synthetic field of medicine, be specifically related to the synthetic and synthetic with enzyme of Simvastatin, more specifically, relate toHave compared with wild type acyltransferase that increased activity, product tolerance improve, acyltransferase mutant that non-natural exists,The encode polynucleotides of described acyltransferase mutant, the host cell that comprises these type of polynucleotides, described acyltransferaseProduction method and use the method for the synthetic Simvastatin of acyltransferase mutant living things catalysis of the present invention.
Background technology
Simvastatin (simvastatin) is the hypolipidemic of Merck company development, and trade name Zocor, is LovastatinSemi-synthetic derivative, Lovastatin is by separating and obtain in Aspergillus terreus (Aspergillusterreus) zymotic fluid. Pungent cutting downThe pharmacological action in his spit of fland is to suppress HMG-CoA reductase (HMG-CoA in liver cell as competitive inhibitorReductase) activity, restriction HMG-CoA is to the conversion of methyldihydroxypentanoic acid, thus it is biosynthetic to reduce endogenous T-CHOLTotal amount. Compared with isodose Lovastatin (lovastatin), Simvastatin can more effectively reduce the total courage in serumSterol and LDL-C.
Produce at present the main flow process route that Simvastatin uses and proposed by Sleiteinger, by Verhoeven,Kumar etc. revise raising. This route, taking Lovastatin as starting material, experiences organic amine open loop, two hydroxyl protection, methylates, takes offThe processing steps such as protection, one-tenth ammonium salt, cyclization, obtain Simvastatin:
But this route exists reagent toxicity large, severe reaction conditions, side reaction is many, and the large grade of separation and purification of products difficulty is askedTopic, causes the production cost of current Simvastatin high, pollutes greatly. Therefore, be badly in need of that a kind of toxicity is low, efficiency is high, pollute few lifeProduct method. In recent years, along with the proposition of Green Chemistry concept and the pay attention to day by day to environmental protection, living things catalysis high-efficiency low-toxicity is lowThe feature of polluting has caused people's interest, and notice is turned to this field.
WO1994026920 has announced a kind of method of utilizing lipase and lipase to synthesize Simvastatin as catalyst, byCarry out regioselectivity esterification in needs, still must protect other hydroxyl, cause product yield to reduce.Also there is same problem in the method that WO2005040107 announces, efficiency is significantly improved not yet.
US2009191602 (A1) has reported the Lovastatin biological synthesis gene cluster of Aspergillus terreus, one of this gene cluster codingThe protein (SEQIDNo.1) of 46KDa, i.e. Lovastatin acyltransferase (LovD), can be by the carboxyl groups of acyl group thioestersRegioselectivity acyl group citrinin J acid C8 hydroxyl, the effective enzyme preparation for biosynthesis Simvastatin:
Although separation and purification clonal expression go out naturally occurring LovD at present, the LovD of wild type also exists a lotProblem, as lower in enzymatic activity, product inhibition etc., cause being difficult to realize industrial applications. US2010052038,US2010050253, CN201310315499.0, CN201310345811.0 etc. study this, obtained active raisingAcyltransferase mutant. But very important, still there is product/accessory substance in said mutation body under High Concentration SituationSuppress phenomenon, although take some measures, as used precipitating reagent to make enzymatic product separate out precipitation, add the adsorbents such as active carbonAdsorption reaction accessory substance mercaptan, alleviates the inhibition of product/accessory substance to enzyme, impels reaction to move to product direction, but above-mentionedMethod still can not thoroughly solve product/accessory substance and suppress problem, causes reaction conversion ratio conventionally in 95% left and right, has reduced raw materialWhen utilization rate, make troubles to downstream separation purifying. Therefore, utilize molecular biology method to change acyltransferaseMaking, in improving enzymatic activity, strengthen the tolerance of enzyme to product/accessory substance, remove product and suppress, is to promote acyl group to shiftEnzyme is produced the effective way of Simvastatin industrialization.
Summary of the invention
The present invention overcomes the deficiencies in the prior art, and a kind of new Lovastatin acyltransferase (LovD) mutant is provided,The encode gene of this mutant, and this mutant is in the purposes of preparing in Simvastatin.
The technical solution used in the present invention is that a kind of Lovastatin acyltransferase mutant comprises following processing: at ammoniaBase acid sequence is in the amino acid sequence of wild type acyltransferase of SEQIDNO.1, to insert, replace or 1 or many of disappearanceIndividual amino acid, or/and the wild type acyltransferase that is SEQIDNO.1 at amino acid sequence amino acid sequence oneIndividual or two ends add or delete one or more amino acid. Compared with using wild type acyltransferase, its acyl group shiftsEnzymatic activity strengthens 2-100 doubly.
In a specific embodiment of the present invention, the wild type that described mutant is SEQIDNO.1 at amino acid sequenceOn the basis of the amino acid sequence of acyltransferase, comprise following M1, V2, V11A, E28K, C111S, P125A, V159M,W174S, V194L, M243Q, K252R, T258S, H263A, M338L, M363L, I373V, S393N, any 1 in K307H to18 amino acid mutations; Each sudden change represents with triplet above: alphabet-numeric playing-letter, wherein numeral sudden change ammoniaThe position of base acid, the amino acid of the corresponding sudden change design of letter before numeral, the letter representation after numeral is used for replacing the front ammonia of numeralThe amino acid of base acid. The amino acid sequence of described mutant is SEQIDNO.4.
The present invention also provides its DNA sequence dna of a kind of encoding gene of the above Lovastatin acyltransferase mutant of encodingFor SEQIDNO.5. Its codon optimization be suitable at expression in escherichia coli. In some embodiments, multinuclearThuja acid comprises be optimized for the codon of expressing in the host cell of particular type. For various dissimilar microorganismsUse and the Preference of codon be known because it is specific amino acid whose excellent for the expression in these microorganismsThe codon of changing.
A kind of volume that comprises coding Lovastatin acyltransferase mutant is provided in another specific embodiment of the present inventionThe recombinant plasmid of code gene. Utilize this recombinant plasmid and Escherichia coli as host cell, build a kind of recombinant bacterium. Wherein large intestineBacillus is selected Escherichia coli E.coliDH5 α.
In embodiments of the invention, also provide a kind of Lovastatin acyltransferase mutant for catalysis citrinin J andThe purposes of the synthetic Simvastatin of alpha-alpha-dimethyl bytyry-S-methyl propionate reaction.
Come from the acyltransferase mutant of the wild type acyltransferase of Aspergillus terreus (Aspergillusterreus),Can make the hydrolysate citrinin J (MonacolinJ) of natural products Lovastatin be converted into Simvastatin. Described acylBased transferase mutant shows stronger catalytic activity compared with the wild type acyltransferase of SEQIDNO.1. Acyl group turnsThe polynucleotides that move enzyme mutant and this mutant of coding can use the normally used method preparation of those skilled in the art.Mutant can be by the vitro recombination of this enzyme that makes to encode, polynucleotides mutagenesis, DNA reorganization, fallibility PCR and orthogenesisThe acquisitions such as method.
LovD mutant of the present invention, when activity improves, has bright to the tolerance of product/accessory substance compared with wild typeAobvious improvement, has improved raw material availability, has reduced cost, has reduced energy consumption and environmental pollution.
Detailed description of the invention
The structure of embodiment 1 wild type and acyltransferase mutant expression vector
By the wild type acyltransferase from Aspergillus terreus (Aspergillusterreus), (amino acid sequence is SEQIDNO.1), the nucleotide sequence of wild type acyltransferase is optimized (obtaining SEQIDNO.2) (see Puigb ò P,GuzmánE,RomeuA,Garcia-VallvéS.OPTIMIZER:awebserverforoptimizingThecodonusageofDNAsequences.NucleicAcidsRes.2007). By the full base of PCR-basedBecause carrying out gene, synthesizes synthesis mode (PCR-basedgenesynthesismethod). By synthetic carrier segments utilizationFollowing primers F 1 and R1 carry out pcr amplification, and amplified production utilizes Not1 and Xho1 to carry out double digestion, and enzyme is cut product and is connected to expressionOn carrier, (obtain SEQIDNO.3), be transformed in Escherichia coli E.coliDH5 α and checking expression year by standard methodWhether body transforms successfully.
F1：5'CCGCTCGAGATGGGTTCTATCATCGACGCGGC3'；
R1：5'ATTTCGCCGGCGTTAACCCTGCTGGTACTGCGC3'。
Wild type acyltransferase is optimized to (obtaining SEQIDNO.4), the acyltransferase after optimizing is (prominentVariant) carry out sequence optimisation (obtaining SEQIDNO.5) according to e. coli codon Preference. By the full base of PCR-basedBecause carrying out gene, synthesizes synthesis mode. Utilize following primers F 2 and R2 to carry out pcr amplification, amplified production synthetic carrier segmentsUtilize Not1 and Xho1 to carry out double digestion, enzyme is cut product and is connected to (acquisition SEQIDNO.6) on expression vector, by standard sideMethod is transformed in Escherichia coli E.coliDH5 α and verifies whether expression vector transforms successfully.
F2：5'CCGCTCGAGATGGTTATGGGCTCTATCATCGA3'；
R2：5'ATTTCGCCGGCGTTAACCCTGCTGATACTGGGC3'。
The preparation of embodiment 2 acyltransferase mutant
That in picking embodiment 1, prepares contains in object expression vector plasmid transformation escherichia coli BL21, and picking list bacterium colony, connectsPlant in the culture medium after 10ml autoclaving: tryptone 10g/L, yeast extract 5g/L, sodium hydrogen phosphate 3.55G/L, potassium dihydrogen phosphate 3.4g/L, ammonium chloride 2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, six waterIron chloride 0.027g/L, glycerine 5g/L, glucose 0.8g/L, adds ampicillin to 50mg/L after sterilizing. 30 DEG C,250rpm incubated overnight. Get 1L triangular flask next day, be linked into the culture medium after 200ml autoclaving by the inoculative proportion of 1:20In: tryptone 10g/L, yeast extract 5g/L, sodium hydrogen phosphate 3.55g/L, potassium dihydrogen phosphate 3.4g/L, chlorinationAmmonium 2.68g/L, sodium sulphate 0.71g/L, epsom salt 0.493g/L, Iron trichloride hexahydrate 0.027g/L, glycerine 5g/L, PortugalGrape sugar 0.3g/L. After sterilizing, add ampicillin to 50mg/L. In 37 DEG C, be cultured to thalline OD5-6, at once by triangular flaskBe placed in 25 DEG C of shaking tables, 250rpm cultivates 1h. Add IPTG to final concentration 0.2mM, and in 37 DEG C, 250rpm continue to cultivate 3-5h。
After cultivation finishes, by nutrient solution, in 4 DEG C, under 6000g, centrifugal 20min finally obtains wet thallus 5.2g, collects thalline.Again use distilled water resuspended, under Ultrasonic Cell Disruptor, be crushed to clarification. After fragmentation, in 4 DEG C, centrifugal 30min under 12000g, collectsSupernatant, prepares freeze-dried powder with freeze drier after being chilled in advance-80 DEG C. Finally obtain thick enzyme freeze-dried powder 0.5g.
Catalyzing and synthesizing of embodiment 3 simvastatin ammonium salts
In 5mL centrifuge tube, add successively 1.5mL citrinin J solution (120mg/mL, pH10.0), 0.115mL α-Dimethyl butyrate acyl group-S-methyl propionate (DMB-S-MMP) (122mg, 1.05eq), 0.385mL acyltransferase mutantCorase meal (being obtained by embodiment 2) solution (7mg/mL), with being placed in 25 DEG C of water-baths, electromagnetic agitation, rotating speed 1200r/M, starts to react timing. HPLC monitors reaction process, conversion ratio > 99% after 32h.
Embodiment 4 utilizes acyltransferase mutant to prepare simvastatin ammonium salt and extracts product
In 500mL there-necked flask, add 0.1M, the ammonium chloride buffer 187mL of pH10.0,20g citrinin J(MonacolinJ), stir after with 25% ammoniacal liquor adjust pH10.0. Add again 13.55g(1.05eq) DMB-S-MMP andThe acyltransferase mutant corase meal that 0.3g embodiment 2 obtains, 25% ammoniacal liquor regulates after pH10.0, by there-necked flask as for waterIn bath, 250r/m mechanical agitation starts to react timing at 25 DEG C. Timing sampling HPLC monitors reaction process. Conversion ratio after 32h99.5%. By centrifugal reactant liquor 4000r/m 30min, collecting precipitation, final vacuum of the each washing of cold water and ethyl acetate is dry,Obtain simvastatin ammonium salt 24.58g, purity 99.1%.
Above the present invention is described in detail, its object is to allow the personage who is familiar with this art can understand thisThe content of invention is also implemented, and can not limit the scope of the invention with this, and the invention is not restricted to above-mentioned enforcementExample, the equivalence that all Spirit Essences according to the present invention are done changes or modifies, and all should be encompassed in protection scope of the present invention.
SEQUENCELISTING
<110>Chongqing Xinan Synthetic Pharmaceutical Co., Ltd
<120>a kind of Lovastatin acyltransferase mutant
<160>6
<210>1
<211>413
<212>PRT
<213>Aspergillus terreus (Aspergillusterreus)
<400>SEQIDNO.1
MetGlySerIleIleAspAlaAlaAlaAlaAlaAspProValValLeuMetGluThrAla
15101520
PheArgLysAlaValLysSerArgGlnIleProGlyAlaValIleMetAlaArgAspCys
25303540
SerGlyAsnLeuAsnTyrThrArgCysPheGlyAlaArgThrValArgArgAspGluCys
45505560
AsnGlnLeuProProLeuGlnValAspThrProCysArgLeuAlaSerAlaThrLysLeu
65707580
LeuThrThrIleMetAlaLeuGlnCysMetGluArgGlyLeuValAspLeuAspGluThr
859095100
ValAspArgLeuLeuProAspLeuSerAlaMetProValLeuGluGlyPheAspAspAla
105110115120
GlyAsnAlaArgLeuArgGluArgArgGlyLysIleThrLeuArgHisLeuLeuThrHis
125130135140
ThrSerGlyLeuSerTyrValPheLeuHisProLeuLeuArgGluTyrMetAlaGlnGly
145150155160
HisLeuGlnSerAlaGluLysPheGlyIleGlnSerArgLeuAlaProProAlaValAsn
165170175180
AspProGlyAlaGluTrpIleTyrGlyAlaAsnLeuAspTrpAlaGlyLysLeuValGlu
185190195200
ArgAlaThrGlyLeuAspLeuGluGlnTyrLeuGlnGluAsnIleCysAlaProLeuGly
205210215220
IleThrAspMetThrPheLysLeuGlnGlnArgProAspMetLeuAlaArgArgAlaAsp
225230235240
GlnThrHisArgAsnSerAlaAspGlyArgLeuArgTyrAspAspSerValTyrPheArg
245250255260
AlaAspGlyGluGluCysPheGlyGlyGlnGlyValPheSerGlyProGlySerTyrMet
265270275280
LysValLeuHisSerLeuLeuLysArgAspGlyLeuLeuLeuGlnProGlnThrValAsp
285290295300
LeuMetPheGlnProAlaLeuGluProArgLeuGluGluGlnMetAsnGlnHisMetAsp
305310315320
AlaSerProHisIleAsnTyrGlyGlyProMetProMetValLeuArgArgSerPheGly
325330335340
LeuGlyGlyIleIleAlaLeuGluAspLeuAspGlyGluAsnTrpArgArgLysGlySer
345350355360
LeuThrPheGlyGlyGlyProAsnIleValTrpGlnIleAspProLysAlaGlyLeuCys
365370375380
ThrLeuAlaPhePheGlnLeuGluProTrpAsnAspProValCysArgAspLeuThrArg
385390395400
ThrPheGluHisAlaIleTyrAlaGlnTyrGlnGlnGly
405410
<210>2
<211>1236
<212>DNA
<213>the parent acid sequence after optimization
<400>SEQIDNO.2
ggttctatcatcgacgcggcggcggcggcggacccggttgttctgatggaaaccgcgttc60
cgtaaagcggttaaatctcgtcagatcccgggtgcggttatcatggcgcgtgactgctct120
ggtaacctgaactacacccgttgcttcggtgcgcgtaccgttcgtcgtgacgaatgcaac18o
cagctgccgccgctgcaggttgacaccccgtgccgtctggcgtctgcgaccaaactgctg240
accaccatcatggcgctgcagtgcatggaacgtggtctggttgacctggacgaaaccgtt300
gaccgtctgctgccggacctgtctgcgatgccggttctggaaggtttcgacgacgcgggt360
aacgcgcgtctgcgtgaacgtcgtggtaaaatcaccctgcgtcacctgctgacccacacc420
tctggtctgtcttacgttttcctgcacccgctgctgcgtgaatacatggcgcagggtcac480
ctgcagtctgcggaaaaattcggtatccagtctcgtctggcgccgccggcggttaacgac540
ccgggtgcggaatggatctacggtgcgaacctggactgggcgggtaaactggttgaacgt600
gcgaccggtctggacctggaacagtacctgcaggaaaacatctgcgcgccgctgggtatc660
accgacatgaccttcaaactgcagcagcgtccggacatgctggcgcgtcgtgcggaccag720
acccaccgtaactctgcggacggtcgtctgcgttacgacgactctgtttacttccgtgcg780
gacggtgaagaatgcttcggtggtcagggtgttttctctggtccgggttcttacatgaaa840
gttctgcactctctgctgaaacgtgacggtctgctgctgcagccgcagaccgttgacctg900
atgttccagccggcgctggaaccgcgtctggaagaacagatgaaccagcacatggacgcg960
tctccgcacatcaactacggtggtccgatgccgatggttctgcgtcgttctttcggtctg1020
ggtggtatcatcgcgctggaagacctggacggtgaaaactggcgtcgtaaaggttctctg1080
accttcggtggtggtccgaacatcgtttggcagatcgacccgaaagcgggtctgtgcacc1140
ctggcgttcttccagctggaaccgtggaacgacccggtttgccgtgacctgacccgtacc1200
ttcgaacacgcgatctacgcgcagtaccagcagggt1236
<210>3
<211>5901
<212>DNA
<213>artificial sequence
<400>SEQIDNO.3
ctcgagatgggttctatcatcgacgcggcggcggcggcggacccggttgttctgatggaa60
accgcgttccgtaaagcggttaaatctcgtcagatcccgggtgcggttatcatggcgcgt120
gactgctctggtaacctgaactacacccgttgcttcggtgcgcgtaccgttcgtcgtgac180
gaatgcaaccagctgccgccgctgcaggttgacaccccgtgccgtctggcgtctgcgacc240
aaactgctgaccaccatcatggcgctgcagtgcatggaacgtggtctggttgacctggac300
gaaaccgttgaccgtctgctgccggacctgtctgcgatgccggttctggaaggtttcgac360
gacgcgggtaacgcgcgtctgcgtgaacgtcgtggtaaaatcaccctgcgtcacctgctg420
acccacacctctggtctgtcttacgttttcctgcacccgctgctgcgtgaatacatggcg480
cagggtcacctgcagtctgcggaaaaattcggtatccagtctcgtctggcgccgccggcg540
gttaacgacccgggtgcggaatggatctacggtgcgaacctggactgggcgggtaaactg600
gttgaacgtgcgaccggtctggacctggaacagtacctgcaggaaaacatctgcgcgccg660
ctgggtatcaccgacatgaccttcaaactgcagcagcgtccggacatgctggcgcgtcgt720
gcggaccagacccaccgtaactctgcggacggtcgtctgcgttacgacgactctgtttac780
ttccgtgcggacggtgaagaatgcttcggtggtcagggtgttttctctggtccgggttct840
tacatgaaagttctgcactctctgctgaaacgtgacggtctgctgctgcagccgcagacc900
gttgacctgatgttccagccggcgctggaaccgcgtctggaagaacagatgaaccagcac960
atggacgcgtctccgcacatcaactacggtggtccgatgccgatggttctgcgtcgttct1020
ttcggtctgggtggtatcatcgcgctggaagacctggacggtgaaaactggcgtcgtaaa1080
ggttctctgaccttcggtggtggtccgaacatcgtttggcagatcgacccgaaagcgggt1140
ctgtgcaccctggcgttcttccagctggaaccgtggaacgacccggtttgccgtgacctg1200
acccgtaccttcgaacacgcgatctacgcgcagtaccagcagggttaagcggccgcctag1260
gacccagctttcttgtacaaagtggtaagcttaattagctgagcttggactcctgttgat1320
agatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccg1380
ggcgttttttattggtgagaatccaagtaacaacaccatttaaatggagtggttacaaat1440
ggagtggttaattaaggctagcttggcgagattttcaggagctaaggaagctaaaatgga1500
gaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattt1560
tgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattac1620
ggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacat1680
tcttgcccgcctgatgaatgctcatccggaatttcgtatggcaatgaaagacggtgagct1740
ggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgtt1800
ttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgca1860
agatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatat1920
gtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaa1980
tatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaa2040
ggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcgg2100
cagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaattttt2160
ttaaggcagttattggtgcccttaaacgcctggggtaatgactctctagcttgaggcatc2220
aaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcgg2280
tgaacgctctcctgagtaggacaaatccgccctctagattacgtgcagtcgatgataagc2340
tgtcaaacatgagaattgtgcctaatgagtgagctaacttacattaattgcgttgcgctc2400
actgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacg2460
cgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgaga2520
cgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtcca2580
cgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataac2640
atgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcc2700
cggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcg2760
cagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcac2820
tccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgcc2880
agccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgattt2940
gctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggaga3000
aaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattag3060
tgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcc3120
cactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttc3180
gttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccg3240
cgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacg3300
actgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcg3360
ccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcggg3420
aaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttca3480
cattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttt3540
tgcaccattcgatggtgtcggaatttcgggcagcgttgggtcctggccacgggtgcgcat3600
gatctagagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagct3660
cccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcaggg3720
cgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatag3780
cggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccac3840
atgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttcc3900
gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct3960
cactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatg4020
tgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttc4080
cataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcga4140
aacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctct4200
cctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtg4260
gcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaag4320
ctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat4380
cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaac4440
aggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaac4500
tacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttc4560
ggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttt4620
tttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatc4680
ttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg4740
agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatca4800
atctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggca4860
cctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtag4920
ataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagac4980
ccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgc5040
agaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagct5100
agagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatc5160
gtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaagg5220
cgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatc5280
gttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataat5340
tctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaag5400
tcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggat5460
aataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcgggg5520
cgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgca5580
cccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacagga5640
aggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactc5700
ttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacata5760
tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtg5820
ccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatc5880
acgaggccctttcgtcttcac5901
<210>4
<211>415
<212>PRT
<213>protein sequence (mutant) after optimization
<400>SEQIDNO.4
MetValMetGlySerIleIleAspAlaAlaValAlaAlaAspProValValLeuMetGlu
15101520
ThrAlaPheArgLysAlaValGluSerArgGlnIleProGlyAlaValIleMetAlaArg
25303540
AspCysSerGlyAsnLeuAsnTyrThrArgCysPheGlyAlaArgThrValArgArgAsp
45505560
GluCysAsnGlnLeuProProLeuGlnValAspThrProCysArgLeuAlaSerAlaThr
65707580
LysLeuLeuThrThrIleMetAlaLeuGlnCysMetGluArgGlyLeuValAspLeuAsp
859095100
GluThrValAspArgLeuLeuProAspLeuCysAlaMetProValLeuGluGlyPheAsp
105110115120
AspAlaGlyAsnProArgLeuArgGluArgArgGlyLysIleThrLeuArgHisLeuLeu
125130135140
ThrHisThrSerGlyLeuSerTyrValPheLeuHisProLeuLeuArgGluTyrValAla
145150155160
GlnGlyHisLeuGlnSerAlaGluLysPheGlyIleGlnTrpArgLeuAlaProProAla
165170175180
ValAsnAspProGlyAlaGluTrpIleTyrGlyAlaAsnValAspTrpAlaGlyLysLeu
185190195200
ValGluArgAlaThrGlyLeuAspLeuGluGlnTyrLeuGlnGluAsnIleCysAlaPro
205210215220
LeuGlyIleThrAspMetThrPheLysLeuGlnGlnArgProAspMetLeuAlaArgArg
225230235240
AlaAspMetThrHisArgAsnSerAlaAspGlyLysLeuArgTyrAspAspThrValTyr
245250255260
PheArgHisAspGlyGluGluCysPheGlyGlyGlnGlyValPheSerGlyProGlySer
265270275280
TyrMetLysValLeuHisSerLeuLeuLysArgAspGlyLeuLeuLeuGlnProGlnThr
285290295300
ValAspLeuMetPheGlnProAlaLeuGluProArgLeuGluGluGlnMetAsnGlnHis
305310315320
MetAspAlaSerProHisIleAsnTyrGlyGlyProMetProMetValMetArgArgSer
325330335340
PheGlyLeuGlyGlyIleIleAlaLeuGluAspLeuAspGlyGluAsnTrpArgArgLys
345350355360
GlySerMetThrPheGlyGlyGlyProAsnIleIleTrpGlnIleAspProLysAlaGly
365370375380
LeuCysThrLeuAlaPhePheGlnLeuGluProTrpSerAspProValCysArgAspLeu
385390395400
ThrArgThrPheGluLysAlaIleTyrAlaGlnTyrGlnGlnGly
405410415
<210>5
<211>1242
<212>DNA
<213>DNA sequence dna of the encoding mutant body after optimization
<400>SEQIDNO.5
gttatgggctctatcatcgatgctgccgttgcagctgatccagttgtgctgatggaaacc60
gcctttcgtaaagctgtggaatctcgacagattccgggcgcagtgatcatggcacgcgat120
tgtagtggtaatctgaattatacccgttgctttggcgctcgaacggttcgtcgcgatgaa180
tgcaatcagttacctccattacaggttgatacaccttgtcgcttagcaagcgctaccaaa240
ctgctgacgacaatcatggccttacagtgcatggaacgcggcttagttgacttagatgaa300
acggtggatcgcctgttaccggatctgtgcgccatgcctgtgttagaaggctttgatgat360
gcgggtaatcctcgcttacgcgaacgccgcggcaaaatcaccctgcgtcatttgctgaca420
catacctcaggtctgagctatgtgtttctgcatccattattacgcgaatatgttgcccag480
ggtcatctccagtcggccgaaaaatttggtattcagtggcgcctggcaccaccggccgtg540
aatgatccgggtgccgagtggatctatggcgcagctgtcgattgggccggcaaactggtt600
gaacgtgcgaccggcctggacctggaacagtatctgcaagaaaatatttgtgctccactg660
ggcatcacggatatgacgtttaaattacagcagcgtccagatatgttagcccgccgcgcg720
gatatgacacatcgtaattctgcggatggtaaactgcgctatgatgataccgtgtatttt780
cgccatgatggtgaagaatgctttggtggtcagggcgtgttttcagggccgggctcttat840
atgaaagttctgcatagtctgttaaaacgtgatggcttattactgcaaccacagacggtt900
gacttgatgtttcagccagccctggaaccacgcttagaagaacagatgaatcagcacatg960
gatgcctctcctcatatcaattatggcggcccaatgccaatggttatgcgtcgtagtttt1020
ggtctgggtggcatcattgcactggaagatttggatggcgaaaattggcgtcgcaaaggc1080
tcaatgacctttggcggtggtcctaatatcatctggcagatcgatcctaaagcgggcctg1140
tgtacattagcgttcttccagctggaaccttggagcgatccggtttgtcgtgacctgacc1200
cgtacctttgaaaaagctatctatgcccagtatcagcagggt1242
<210>6
<211>5907
<212>DNA
<213>artificial sequence
<400>SEQIDNO.6
ctcgagatggttatgggctctatcatcgatgctgccgttgcagctgatccagttgtgctg60
atggaaaccgcctttcgtaaagctgtggaatctcgacagattccgggcqcagtgatcatg120
gcacgcgattgtagtggtaatctgaattatacccgttgctttggcgctcgaacggttcgt180
cgcgatgaatgcaatcagttacctccattacaggttgatacaccttgtcgcttagcaagc240
gctaccaaactgctgacgacaatcatggccttacagtgcatggaacgcggcttagttgac300
ttagatgaaacggtggatcgcctgttaccggatctgtgcgccatgcctgtgttagaaggc360
tttgatgatgcgggtaatcctcgcttacgcgaacgccgcggcaaaatcaccctgcgtcat420
ttgctgacacatacctcaggtctgagctatgtgtttctgcatccattattacgcgaatat480
gttgcccagggtcatctccagtcggccgaaaaatttggtattcagtggcgcctggcacca540
ccggccgtgaatgatccgggtgccgagtggatctatggcgcagctgtcgattgggccggc600
aaactggttgaacgtgcgaccggcctggacctggaacagtatctgcaagaaaatatttgt660
gctccactgggcatcacggatatgacgtttaaattacagcagcgtccagatatgttagcc720
cgccgcgcggatatgacacatcgtaattctgcggatggtaaactgcgctatgatgatacc780
gtgtattttcgccatgatggtgaagaatgctttggtggtcagggcgtgttttcagggccg840
ggctcttatatgaaagttctgcatagtctgttaaaacgtgatggcttattactgcaacca900
cagacggttgacttgatgtttcagccagccctggaaccacgcttagaagaacagatgaat960
cagcacatggatgcctctcctcatatcaattatggcggcgcaatgccaatggttatgcgt1020
cgtagttttggtctgggtggcatcattgcactggaagatttggatggcgaaaattggcgt1080
cgcaaaggctcaatgacctttggcggtggtcctaatatcatctggcagatcgatcctaaa1140
gcgggcctgtgtacattagcgttcttccagctggaaccttggagcgatccggtttgtcgt1200
gacctgacccgtacctttgaaaaagctatctatgcccagtatcagcagggttaagcggcc1260
gcctaggacccagctttcttgtacaaagtggtaagcttaattagctgagettggactcct1320
gttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttg1380
ccgccgggcgttttttattggtgagaatccaagtaacaacaccatttaaatggagtggtt1440
acaaatggagtggttaattaaggctagcttggcgagattttcaggagctaaggaagctaa1500
acaaatggagtggttaattaaggctagcttggcgagattttcaggagctaaggaagctaa1500
aatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaaga1560
acattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctgga1620
tattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttat1680
tcacattcttgcccgcctgatgaatgctcatccggaatttcgtatggcaatgaaagacgg1740
tgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactga1800
aacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatata1860
ttcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattga1920
gaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgt1980
ggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaagg2040
cgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttcca2100
tgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgta2160
atttttttaaggcagttattggtgcccttaaacgcctggggtaatgactctctagcttga2220
ggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtt2280
tgtcggtgaacgctctcctgagtaggacaaatccgccctctagattacgtgcagtcgatg2340
ataagctgtcaaacatgagaattgtgcctaatgagtgagctaacttacattaattgcgtt2400
gcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcgg2460
ccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcacca2520
gtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagc2580
ggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcggga2640
tataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgc2700
gcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaacca2760
gcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggaca2820
tggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatt2880
tatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcg2940
cgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcat3000
gggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaa3060
cattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatga3120
tcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgc3180
cgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaa3240
tcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatca3300
gcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccg3360
ccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcacca3420
cgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactg3480
gtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaa3540
aggttttgcaccattcgatggtgtcggaatttcgggcagcgttgggtcctggccacgggt3600
gcgcatgatctagagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacat3660
gcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccg3720
tcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtag3780
cgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtg3840
caccacatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgc3900
tcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggta3960
tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaag4020
aacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcg4080
tttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagagg4140
tggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg4200
cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggga4260
agcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc4320
tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggt4380
aactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccact4440
ggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg4500
cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtt4560
accttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt4620
ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcct4680
ttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttg4740
gtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttt4800
aaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt4860
gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtc4920
gtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccg4980
cgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggcc5040
gagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg5100
gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctaca5160
ggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacga5220
tcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcct5280
ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg5340
cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactca5400
accaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaata5460
cgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttct5520
tcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccact5580
cgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaa5640
acaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactc5700
atactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcgga5760
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccga5820
aaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaatagg5880
cgtatcacgaggccctttcgtcttcac5907
Claims (9)
1. a Lovastatin acyltransferase mutant, is characterized in that: described mutant comprises following processing: at amino acidSequence is to insert, replace or lack one or more ammonia in the amino acid sequence of wild type acyltransferase of SEQIDNO.1Base acid, or/and one of amino acid sequence of the wild type acyltransferase that is SEQIDNO.1 at amino acid sequence orTwo ends add or delete one or more amino acid.
2. a kind of Lovastatin acyltransferase mutant according to claim 1, is characterized in that: described mutant is at ammoniaBase acid sequence is on the basis of amino acid sequence of the wild type acyltransferase of SEQIDNO.1, comprises following M1, V2,V11A，E28K，C111S，P125A，V159M，W174S，V194L，M243Q，K252R，T258S，H263A，M338L，M363L，I373V, S393N, any 1 to 18 amino acid mutation in K307H.
3. according to a kind of Lovastatin acyltransferase mutant described in claim 1 or 2, it is characterized in that: described mutantAmino acid sequence be SEQIDNO.4.
4. the encoding gene of Lovastatin acyltransferase mutant described in the claim 1 or 2 or 3 of encoding.
5. encode the according to claim 3 encoding gene of Lovastatin acyltransferase mutant, is characterized in that: itsDNA sequence dna is SEQIDNO.5.
6. the restructuring of encoding gene that comprises Lovastatin acyltransferase mutant described in coding claim 1 or 2 or 3Plasmid.
7. a recombinant bacterium that produces Lovastatin acyltransferase mutant described in claim 1 or 2 or 3, is characterized in that:This bacterial classification comprises recombinant plasmid claimed in claim 6, and its host cell is Escherichia coli.
8. recombinant bacterium according to claim 7, is characterized in that: described Escherichia coli are selected Escherichia coli E.coliDH5 α.
Described in claim 1 Lovastatin acyltransferase mutant for catalysis citrinin J and alpha-alpha-dimethyl bytyry-The purposes of the synthetic Simvastatin of S-methyl propionate reaction.
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|---|---|---|---|
| CN201610062451.7A CN105586325A (en) | 2016-01-29 | 2016-01-29 | Lovastatin acyltransferase mutant |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610062451.7A CN105586325A (en) | 2016-01-29 | 2016-01-29 | Lovastatin acyltransferase mutant |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101490271A (en) * | 2006-05-24 | 2009-07-22 | 加利福尼亚大学董事会 | Methods and materials for making simvastatin and related compounds |
| CN102574896A (en) * | 2009-09-30 | 2012-07-11 | 科德克希思公司 | Variant lovd polypeptides and their uses |
| CN102695792A (en) * | 2009-10-08 | 2012-09-26 | 加利福尼亚大学董事会 | LovD mutants exhibiting improved properties towards simvastatin synthesis |
| CN104342416A (en) * | 2013-07-26 | 2015-02-11 | 石药集团中奇制药技术（石家庄）有限公司 | Lovastatin acyltransferase containing one or more point mutations |
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2016
- 2016-01-29 CN CN201610062451.7A patent/CN105586325A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101490271A (en) * | 2006-05-24 | 2009-07-22 | 加利福尼亚大学董事会 | Methods and materials for making simvastatin and related compounds |
| CN102574896A (en) * | 2009-09-30 | 2012-07-11 | 科德克希思公司 | Variant lovd polypeptides and their uses |
| CN102695792A (en) * | 2009-10-08 | 2012-09-26 | 加利福尼亚大学董事会 | LovD mutants exhibiting improved properties towards simvastatin synthesis |
| CN104342416A (en) * | 2013-07-26 | 2015-02-11 | 石药集团中奇制药技术（石家庄）有限公司 | Lovastatin acyltransferase containing one or more point mutations |
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| Title |
|---|
| PDB：4LCM_A: "Chain A,Simvastatin Synthase(lovd),From Aspergillus Terreus,Lovd9 Mutant(simh9014)", 《GENBANK》 * |
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Application publication date: 20160518 |