CN101942425A - Enzyme related to benzalacetone synthesis, coding gene and application thereof - Google Patents
Enzyme related to benzalacetone synthesis, coding gene and application thereof Download PDFInfo
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Abstract
本发明公开了一种与苯亚甲基丙酮合成相关的酶及其编码基因与应用。本发明提供的蛋白质，是如下1)或2)的蛋白质：1)由序列表中的序列4的氨基酸残基序列组成的蛋白质；2)将序列表中的序列4氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与苯亚甲基丙酮合成相关由1)衍生的蛋白质。本发明的实验证明，将本发明采用定点突变(C197L/G256L/S338I)技术对PcPKS2进行了突变研究，PcPKS2的三联体突变体命名为PcmBAS，结果显示，突变后的PcmBAS苯亚甲基丙酮合成能力较PcPKS2提高了7.52倍。The invention discloses an enzyme related to the synthesis of benzylidene acetone, its coding gene and application. The protein provided by the present invention is the protein of the following 1) or 2): 1) a protein consisting of the amino acid residue sequence of sequence 4 in the sequence listing; 2) the amino acid residue sequence of sequence 4 in the sequence listing is passed through one or A protein derived from 1) with substitution and/or deletion and/or addition of several amino acid residues and in relation to benzylideneacetone synthesis. Experiments of the present invention prove that the present invention adopts site-directed mutagenesis (C197L/G256L/S338I) technology to carry out mutation research to PcPKS2, and the triplet mutant of PcPKS2 is named as PcmBAS, and the results show that PcmBAS after mutation synthesizes benzylidene acetone The ability is 7.52 times higher than that of PcPKS2.
Description
Technical field
The present invention relates to a kind of and the synthetic involved enzyme of BENZALACETONE and encoding gene and application.
Background technology
Raspberry ketone, have another name called immature fruit of Juteleaf Raspberry ketone (raspberry ketone), chemistry 4-p-hydroxybenzene-2-butanone by name, (4-(4-hydroxyphenyl)-2-butanone), be a kind of a kind of safe edible spices that uses through the common approval of U.S. flavouring agent producer association (FEMA) and Council of Europe (COE) with quiet and tastefully laid out fruital note, its FEMA is numbered 2588, belong to that national customs is admitted, the new high-tech product of industry policy encourage growth, it is widely used in the synthetic of food, makeup and medicine intermediate.As Fructus Rubi real character fragrance matter, content is low and resource is very limited in the raspberry plant, and it is uneconomic extracting natural raspberry ketone from plant, and the raspberry ketone cost of natural extract is up to 20,000 dollars of per kilograms.
At occurring in nature, raspberry ketone extensively is present in the rubus raspberry.Raspberry, modern title raspberry is rubus, the Rosaceae, subshrub, little oar fruit.It is edible that raspberry can be cooked fruit, and its red ripe fruit claims the raspberry fruit, and mouthfeel perfume (or spice), sweet, sour can be eaten raw, is a kind of high-end fruit that has nutritive value and economic worth the America and Europe, and few well known in China, only there is a small amount of cultivation in the area northeastward, relatively more rare on the market.Raspberry has very high pharmaceutical use simultaneously; Its green fruit is made the traditional Chinese medicine raspberry through the process of preparing Chinese medicine, and documents such as Compendium of Material Medica records has " smell is sweet, and is flat, nontoxic ", and the supplementing the kidney to control the nocturnal urine yang invigorating functions that contracts is arranged.
Raspberry ketone is a kind of phenolic compound, its biosynthesizing derives from the polyketide biosynthetic pathway, BENZALACETONE synthase (Benzalacetone synthase (BAS)) (EC 2.3.1.-) catalysis 4-coumaric acyl coenzyme A and malonyl coenzyme A molecule generate BENZALACETONE (p-hydroxyphenylbut-3-ene-2-one) by step decarboxylation condensation reaction, and BENZALACETONE restores the direct down raspberry ketone that generates of catalysis of enzyme afterwards.BAS is vegetation type III polyketide synthase (PKS) superfamily member, has a series of benzene butane compounds of valuable pharmacological value and the C of derivative thereof at structure
6-C
4The molecular skeleton aspect has important function.BAS is the key enzyme and the rate-limiting enzyme of raspberry ketone biosynthetic pathway, is to utilize the biosynthetic target spot of genetic engineering technique molecular regulation raspberry ketone.BAS does not obtain separating in the raspberry plant always, but obtains separating in polygonum rheum palmatum (Rheum palmatum).
Summary of the invention
An object of the present invention is to provide and the synthetic involved enzyme of BENZALACETONE and encoding gene and application.
Protein provided by the invention, synthetic, called after PcmBAS is following 1) or 2) protein:
1) protein of forming by the amino acid residue sequence of the sequence in the sequence table 4;
2) with 4 amino acid residue sequences of the sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with BENZALACETONE by 1) deutero-protein.
Above-mentioned sequence 4 is made up of 389 amino-acid residues, the replacement of above-mentioned one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
Described proteinic encoding gene PcmBAS also is a scope of protection of the invention.
Described encoding gene is following 1)-5) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 3 in the sequence table;
2) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 4th to the 1167th Nucleotide;
3) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 1st to the 1167th Nucleotide;
4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization and the coding that limit synthesize the dna molecular shown in the relevant albumen with BENZALACETONE;
5) with 1) or 2) or 3) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have 99% homology at least and coding synthesizes the dna molecular shown in the relevant albumen with BENZALACETONE at least.
Above-mentioned sequence 3 is made up of 1167 Nucleotide, the sequence of OFR be sequence 3 from 5 ' terminal 1-1167 position nucleotide sequence.
Described stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS,, and wash film with this solution 65 ℃ of hybridization down.
The recombinant vectors, reorganization bacterium, transgenic cell line, expression cassette or the recombinant virus that contain above-mentioned encoding gene also are the scope of protection of the invention.
Described recombinant vectors is the recombinant vectors that obtains between the NdeI of above-mentioned encoding gene insertion carrier pET-30b (+) and SalI recognition site.
The above-mentioned encoding gene total length that increases or arbitrary segmental primer are to also being the scope of protection of the invention; Described primer is to as follows: a primer sequence is shown in sequence in the sequence table 5, and another primer sequence is shown in sequence in the sequence table 6.
The application of above-mentioned protein in synthetic BENZALACETONE, the application of above-mentioned encoding gene in synthetic BENZALACETONE all is the scope that the present invention will protect.Described synthetic BENZALACETONE is external synthetic BENZALACETONE.
Another object of the present invention provides a kind of method for preparing BENZALACETONE.
Method provided by the invention for being substrate with 4-coumaric acyl coenzyme A and malonyl coenzyme A, under the described proteinic katalysis of claim 1, obtains BENZALACETONE.
Of the present invention experimental results show that, adopt rite-directed mutagenesis (C197L/G256L/S338I) technology that PcPKS2 has been carried out mutation research the present invention, the triplet mutant called after PcmBAS of PcPKS2, the result shows that the PcmBAS BENZALACETONE synthesis capability after the sudden change has improved 7.52 times than PcPKS2.PcmBAS mutator gene and the biosynthesizing that is applied as raspberry ketone thereof provide direct otherwise effective technique and application foundation.
Description of drawings
Fig. 1 is the PcmBAS construction of prokaryotic expression vector
Fig. 2 is that the NdeI and the SalI double digestion of prokaryotic expression carrier identified
Fig. 3 is that BAS purifying SDS-PAGE analyzes
Fig. 4 is synthetic BENZALACETONE high performance liquid chromatography (HPLC) measurement result of BAS catalysis
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, PcPKS2 rite-directed mutagenesis
1.PcPKS2 rite-directed mutagenesis
Synthetic PcPKS2 (sequence 1 in the sequence table).The rite-directed mutagenesis of PcPKS2 (C197L/G256L/S338I) adopts QuikChange site-directed mutagenesis kit (available from Stratagene, Catalog #200518) kit method, concrete mutant primer following (mutational site marks with underscore): PcPKS2C197T/G256L/S338I (5 '-CATCGTTTGCTCGGAGATGACGGCAATT
ACATTCCGTGGGCCATCTGAAACAAAC-3 ', 5 '-GAATCCGATGGTGCAGTCGAG
TTACATTTGCTCGAATCTGGGCTTAGTTGCC-3 ' and 5 '-CAAGTGTTGAACGACTATGGAAACATG
ATTAGTGCTACTGTGTTTTTCATCATG-3 '), template is synthetic PcPKS2, carry out pcr amplification, the PCR product that obtains is sent to order-checking, and the result shows that this PCR product has the sequence of sequence 3 in the sequence table, the unnamed gene of this PCR product is PcmBAS, OFR is the 1-1170 position Nucleotide of sequence 3 in the sequence table, and PcmBAS encoded protein called after PcmBAS, this proteic aminoacid sequence are the sequence 4 in the sequence table.PcmBAS compares with PcPKS2 (sequence 2), the mutational site is: the 197th sports Leu (leucine) by Cys (halfcystine), the 256th sports Leu (leucine) by Gly (glycine), and the 338th sports Ile (Isoleucine) by Ser (Serine).PcmBAS compares with PcPKS2, and the mutational site is that the 583-585 position sports ACA by TGC, and the 760-762 position sports TTA by GGC, and 1006-1008 position TCA sports ATT.
2.PcmBAS heterogenous expression and protein purification
The ORF of PcmBAS cDNA uses the N-end and the terminal primer of C-that contain the specific limited restriction enzyme site to carry out pcr amplification.Sense primer is: 5 '-TATA
CATATGGCGGCTTCAATTGAGGAGATT-3 ' (sequence 5), underscore is represented the NdeI restriction enzyme site; Antisense primer is: 5 '-TATA
GTCGACCTAATGAATGATGGGCACGCTGCG-3 ' (sequence 6), underscore is represented the SalI restriction enzyme site.The PCR product (or artificial synthesized sequence 3) that obtains with step 1) is a template, carry out PCR, the fragment that obtains behind the PCR product process NdeI/SalI double digestion that obtains, insertion prokaryotic expression carrier pET-30b (+) (Novagen, Darmstadt is between NdeI Germany) and the recognition site of SalI, obtain recombinant vectors (Fig. 1 is the carrier structure synoptic diagram), recombinant vectors is cut evaluation with NdeI and SalI enzyme, result such as Fig. 2 (M, DL15000+2000 Marker; 1-3, positive plasmid.) shown in, as seen from the figure, obtain the dna fragmentation of about 1.2Kb.Enzyme is cut the correct reorganization positive plasmid order-checking of qualification result, this recombinant vectors is that the 4-1167 position Nucleotide of sequence 3 in the recognition site insertion sequence table of the NdeI of pET-30b (+) and SalI constitutes, should positive recombinant vectors called after pET-30b (+)-PcmBAS.
Change pET-30b (+)-PcmBAS over to E.coli BL21-Rosetta (DE3) (TransGen, Beijing, China) in, obtain the reorganization bacterium, enzyme was cut evaluation after the bacterium of will recombinating was extracted plasmid, obtained about 1.2Kb segmental reorganization bacterium called after BL21/pET-30b (+)-PcmBAS.
BL21/pET-30b (+)-PcmBAS is inserted 10ml contain kantlex (50 μ g ml
-1) and paraxin (34 μ g ml
-1) the LB substratum, 37 ℃ of overnight incubation are diluted 100 times next day and are inserted in the 200ml LB substratum in 37 ℃, 200rpm and continue to be cultured to OD
600During for 0.6-0.8, adding final concentration is the IPTG of 1mM, is cooled to 30 ℃, behind the 6h, by centrifugal collection thalline and be resuspended in 3ml 0.1M potassium phosphate buffer (pH 7.5), the broken thalline of ultrasonic wave on ice.Po Sui thalline homogenate on ice is in 4 ℃ 10, and centrifugal 10min under the 000g collects supernatant.
Supernatant passes through Ni
2+-agarose column (Novagen). with the 0.1M potassium phosphate buffer washing Ni that contains 0.5M NaCl and 40mM imidazoles (imidazole)
2+Behind-the agarose column three times, with the 0.1M potassium phosphate buffer washing wash-out that contains 400mM imidazole, collect the washing elutriant,, will wash elutriant again by PD-10 post (Amersham Pharmacia Biotech for the long-term recombinant protein activity that keeps, Uppsala, Sweden), with 0.1M Tris-HCl (pH 7.5) the displacement damping fluid that contains 10% (v/v) glycerine wash-out again, collect elutriant, obtain recombinant protein PcmBAS, be stored in-80 ℃.
The purification efficiency of recombinant protein detects with SDS-PAGE.
Adopting uses the same method imports PcPKS2 in the e. coli bl21, obtains reorganization bacterium BL21/pET-30b (+)-PcPKS2, and the bacterium of should recombinating again adopts aforesaid method fermentation, purifying to obtain recombinant protein PcPKS2.
Adopting uses the same method imports empty carrier pET-30b (+) in the e. coli bl21, obtains reorganization bacterium BL21/pET-30b (+), and the bacterium of should recombinating again adopts aforesaid method fermentation, purifying to obtain control group albumen.
Histidine six aggressiveness labels are constructed on the C-terminal of recombinant protein.Recombinant protein PcmBAS and PcPKS2 that above-mentioned purifying is obtained carry out the SDS-PAGE electrophoresis detection, are contrast with the control group albumen of above-mentioned acquisition, result such as Fig. 3 (M, molecular mass standard; 1, the PcmBAS purification result; 2, the PcPKS2 purification result) shown in, the result shows that the recombinant protein PcmBAS of purifying and PcPKS2 all form a single band on the position of 43kDa, this expection size with PcmBAS and PcPKS2 is consistent, and the protein purification success is described.The target protein band is not arranged in the control group albumen.
The functional study of embodiment 2, mutant
1, external enzymatic reaction and product analysis
The outer enzymatic reaction system of 250 μ l standard bodies contains 4-coumaric acyl coenzyme A (Beuerle T, Pichersky E (2002) the Enzymatic synthesis and purification of aromatic coenzymea esters.Anal Biochem 302:305-312 of 150 μ M; The public can obtain from Beijing Agricultural College.), the recombinant protein PcmBAS that obtains by embodiment 1 of 280 μ M malonyl coenzyme As (Sigma-Aldrich Catalog #M4263), 0.1M potassium phosphate buffer (pH 9.0) and 2.0 μ g, the pH value of reaction system is 9.0.Above-mentioned reaction system places 30 ℃ after following 30 minutes, adds final concentration and be 5% acetate (detection by product), uses the ethyl acetate extracting of 250 μ l afterwards and in 10, under the 000g centrifugal 10 minutes.After getting supernatant vacuum-drying, add the methanol aqueous solution of 50 μ l 50% (v/v).The recombinant protein PcPKS2 and the control group albumen that obtain with embodiment 1 are contrast.
The analysis of enzymatic preparation is used and is equipped with Kromosil C18 reversed-phase column (5 μ m, 250mm * 4.6mm; Macherey Nagel) Waters Alliance 2695 highly effective liquid phase chromatographic systems (HPLC) are finished.Moving phase is water (A) and methyl alcohol (B), and flow velocity is 0.6ml min
-1, use following gradient condition: 30%B 3 minutes, 30-70%B 27 minutes, 70-80%B 2 minutes, 80-95%B 3 minutes and 95%B 5 minutes.Detecting wavelength is respectively at following compound: naringenin is looked into youngster's ketone (naringenin chalcone), 289nm; BENZALACETONE (p-hydroxybenzalacetone), 323nm.With the corresponding standard product: naringenin cinnamophenone (Catalog #W530098 is available from Sigma-Aldrich company) and BENZALACETONE (Catalog#11960 is available from Sigma-Aldrich company), all cpds is carried out quantitatively.
For high performance liquid chromatography-mass spectroscopy, liquid phase separation is finished by Shimadzu LC-10ADvp HPLC system.Mass spectroscopy by Shimadzu LCMS-2010A mass spectrometer system finish (Shimadazu, Kyoto, Japan).The ShimadzuLC/ESIMS-2010 software system are finished data acquisition and processing (DAP).Liquid phase chromatogram condition is with aforementioned.The suitableeest mass spectrum condition is as follows: all m/z POP scopes are set in 120-350; The dry gas flow velocity, 1.5L min
-1The CDL temperature, 250 ℃; The block temperature, 200 ℃; Probe voltage+4.5kV.
Enzyme kinetics constant and catalytic efficiency (absolute activity) are enzymic activity indexs the most accurately (Kcat/Km).
The retention time of BENZALACETONE standard substance is 31.7 minutes, and the qualitative and quantitative analysis results of enzymatic preparation sees that shown in Figure 4 (A is the PcPKS2 catalytic result; B is the PcmBAS catalytic result), the result shows that the retention time that PcmBAS catalysis generates BENZALACETONE is 31.7 minutes, the retention time that PcPKS2 catalysis generates BENZALACETONE is 31.7 minutes.Control group albumen does not obtain BENZALACETONE, shows that it does not have enzymic activity.
Mass spectrometric detection result is 146.19 for the molecular weight of BENZALACETONE standard substance, the molecular weight that PcmBAS catalysis generates BENZALACETONE is 146.19, the molecular weight that PcPKS2 catalysis generates BENZALACETONE is 146.19, shows that the material that obtains is a BENZALACETONE.
The reorganization PcmBAS and 4-coumaric acyl coenzyme A and malonyl coenzyme A is hatched in the standard enzymatic reaction system jointly and enzymatic reaction system pH value is 9.0 o'clock, it is principal product that PcmBAS can generate BENZALACETONE efficiently, and the activity far above PcPKS2.
Proof: PcmBAS is the BENZALACETONE synthetic enzyme.
2, dynamic analysis
The mensuration of recombinant protein kinetic constant is under the situation of malonyl coenzyme A concentration saturated (concentration is 280 μ M/l), uses 4-coumaric acyl coenzyme A between its K
mThe 0.2-6.0 scope in 5 concentration values calculate acquisition.
250 μ l 0.1M potassium phosphate buffer reaction systems of above-mentioned standard are used in experiment, and each experiment repeats 3 times.The pH value of reaction system is 9.0, and above-mentioned reaction system places 30 ℃ after following 30 minutes, adds final concentration and be 5% acetate, uses the ethyl acetate extraction of 250 μ l afterwards, with organic phase in 10, under the 000g centrifugal 10 minutes.After getting supernatant vacuum-drying, add the methanol aqueous solution of 50 μ l 50% (v/v).
K
mAnd K
CatValue is drawn by the result of Lineweaver-Burke curve.
For synthesizing of BENZALACETONE, the kinetic constant of PcPKS2 is: K
m=46.7 μ m, K
Cat=0.24min
-1(4-coumaric acyl coenzyme A); The kinetic constant of PcmBAS is: K
m=35.7 μ m, K
Cat=1.38min
-1(4-coumaric acyl coenzyme A).Mutant PcmBAS (Kcat/Km) improves 7.52 times than the PcPKS2 catalytic efficiency, and PcmBAS mutator gene and the biosynthesizing that is applied as raspberry ketone thereof provide direct otherwise effective technique and application foundation.
Claims (9)
1. a protein is following 1) or 2) protein:
1) protein of forming by the amino acid residue sequence of the sequence in the sequence table 4;
2) with 4 amino acid residue sequences of the sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with BENZALACETONE by 1) deutero-protein.
2. the described proteinic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described encoding gene is following 1)-5) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 3 in the sequence table;
2) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 4th to the 1167th Nucleotide;
3) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 1st to the 1167th Nucleotide;
4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization and the coding that limit synthesize the dna molecular shown in the relevant albumen with BENZALACETONE;
5) with 1) or 2) or 3) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have 99% homology at least and coding synthesizes the dna molecular shown in the relevant albumen with BENZALACETONE at least.
4. the recombinant vectors, reorganization bacterium, transgenic cell line, expression cassette or the recombinant virus that contain claim 2 or 3 described encoding genes.
5. recombinant vectors according to claim 4 is characterized in that: described recombinant vectors is for inserting the recombinant vectors that obtains between the multiple clone site of carrier pET-30b (+) with claim 2 or 3 described encoding genes.
6. amplification claim 2 or 3 described encoding gene total lengths or arbitrary segmental primer are right; Described primer is to as follows: a primer sequence is shown in sequence in the sequence table 5, and another primer sequence is shown in sequence in the sequence table 6.
7. the application of the described protein of claim 1 in synthetic BENZALACETONE, or claim 2 or the application of 3 described encoding genes in synthetic BENZALACETONE.
8. application according to claim 7 is characterized in that: described synthetic BENZALACETONE is external synthetic BENZALACETONE.
9. a method for preparing BENZALACETONE is characterized in that: for being substrate with 4-coumaric acyl coenzyme A and malonyl coenzyme A, under the described proteinic katalysis of claim 1, obtain BENZALACETONE.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302482A (en) * | 2007-11-20 | 2008-11-12 | 大连理工大学 | A strain of marine Streptomyces S187 with broad-spectrum antibacterial activity |
| CN1633237B (en) * | 2002-02-19 | 2010-05-26 | 道农业科学公司 | Novel polyketide synthases producing SPINOSYN |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1633237B (en) * | 2002-02-19 | 2010-05-26 | 道农业科学公司 | Novel polyketide synthases producing SPINOSYN |
| CN101302482A (en) * | 2007-11-20 | 2008-11-12 | 大连理工大学 | A strain of marine Streptomyces S187 with broad-spectrum antibacterial activity |
Non-Patent Citations (4)
| Title |
|---|
| 《NCBI》 20090206 Ma LQ et al Polygonum cuspidatum type III polyketide synthase (PKS2) mRNA，complete cds，GenBank: EU305677，1417 bp mRNA linear 1-2 1-9 , 2 * |
| 《Planta》 20090228 Ma LQ et al A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum 457-469 1-9 第229卷, 第3期 2 * |
| 《中国植物学会七十五周年年会论文摘要汇编（1933-2008）》 20081231 马兰青等 虎杖(Polygonum cuspidatum Sieb. et Zucc)植物类型III聚酮合酶的分子克隆及功能研究 285-286 1-9 , 2 * |
| 《第八届全国药用植物及植物药学术研讨会论文集》 20091231 郭艳武等 虎杖白藜芦醇合酶基因的克隆及其功能鉴定 39 1-9 , 2 * |
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