CN101942425B - Enzyme related to benzalacetone synthesis, coding gene and application thereof - Google Patents

Abstract

The invention discloses an enzyme related to benzalacetone synthesis, a coding gene and an application thereof. The protein provided by the invention is a protein of 1) or 2), wherein 1) is a protein consisting of amino acid residual sequences shown in a sequence 4 in a sequence table; and 2) is a protein which is the amino acid sequence of the sequence 4 substituted and/or deleted and/or added with one or more amino acid residues, is related to benzalacetone synthesis, and derives from the 1). Experiments prove that the method performs mutation research on PcPKS2 by adopting directed mutagenesis (C197L/G256L/S338I) technology, and the triad mutant of the PcPKS2 is named to be PcmBAS; and results prove that the synthesizing capability of the mutated PcmBAS benzalacetone is improved by 7.52 times compared with PcPKS2.

Description

Enzyme related to the synthesis of benzylidene acetone and its coding gene and application

technical field

The invention relates to an enzyme related to the synthesis of benzylidene acetone, its coding gene and application.

Background technique

Raspberry ketone, also known as raspberry ketone (raspberry ketone), the chemical name is 4-p-hydroxyphenyl-2-butanone, (4-(4-hydroxyphenyl)-2-butanone), is a kind of food flavoring through the United States A safe edible spice with an elegant and fruity aroma jointly approved by the Manufacturers Association (FEMA) and the Council of Europe (COE). Its FEMA number is 2588. It is a high-tech product recognized by the national customs and encouraged by industrial policies. It is widely used in the synthesis of food, cosmetics and pharmaceutical intermediates. As a characteristic aroma substance of raspberry fruit, its content is low in raspberry plants and resources are very limited. It is uneconomical to extract natural raspberry ketones from plants, and the cost of naturally extracted raspberry ketones is as high as 20,000 US dollars per kilogram.

In nature, raspberry ketones are widely found in the Rubus genus raspberry. Raspberry, now known as raspberry, belongs to the genus Rubus, Rosaceae, semi-shrub, and small berry. Raspberry can be eaten as a fruit. Its red ripe fruit is called raspberry. It tastes fragrant, sweet, sour and can be eaten fresh. There is a small amount of cultivation in the Northeast, and it is relatively rare in the market. Raspberry has high medicinal value at the same time; its green fruit is processed into traditional Chinese medicine raspberry. It is recorded in "Compendium of Materia Medica" and other documents that it has "sweet smell, flat, non-toxic", beneficial to the kidney, strengthening essence, reducing urine and strengthening yang.

Raspberry ketone is a phenolic compound whose biosynthesis originates from the polyketide biosynthetic pathway, Benzalacetone synthase (BAS) (EC 2.3.1.-) catalyzes the conversion of 4-coumaroyl Coenzyme A and malonyl-CoA molecules undergo a one-step decarboxylation condensation reaction to generate benzylidene acetone (p-hydroxyphenylbut-3-ene-2-one), and then directly generate raspberry under the catalysis of benzylidene acetone ketone. BAS is a member of the plant type III polyketide synthase (PKS) superfamily, and plays an important role in the construction of a series of C ₆ -C ₄ molecular frameworks of phenylbutane compounds and their derivatives with important pharmacological value. BAS is the key enzyme and rate-limiting enzyme in the biosynthetic pathway of raspberry ketone, and is the target for molecular regulation of raspberry ketone biosynthesis by genetic engineering technology. BAS has not been isolated in raspberry plants, but has been isolated in Polygonaceae plants Rheumpalmatum.

Contents of the invention

An object of the present invention is to provide an enzyme related to the synthesis of benzylidene acetone, its coding gene and its application.

The protein provided by the present invention is artificially synthesized and named as PcmBAS, which 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) A protein derived from 1) that undergoes substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid residue sequence of Sequence 4 in the sequence listing and is related to the synthesis of benzylidene acetone.

The above-mentioned sequence 4 consists of 389 amino acid residues, and the above-mentioned substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein coding gene PcmBAS is also within the protection scope of the present invention.

The coding gene is the DNA molecule described in any one of the following 1)-5):

1) The DNA molecule shown in sequence 3 in the sequence listing;

2) The DNA molecule shown in sequence 3 from the 4th to the 1167th nucleotide at the 5' end in the sequence listing;

3) The DNA molecule shown in the sequence 3 from the 1st to the 1167th nucleotide at the 5' end in the sequence listing;

4) under stringent conditions, hybridize with the DNA sequence defined in 1) or 2) or 3) and encode the DNA molecule shown in the protein related to the synthesis of benzylidene acetone;

5) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of the DNA sequence defined in 1) or 2) or 3), A DNA molecule having at least 97%, at least 98%, or at least 99% homology and encoding a protein related to the synthesis of benzylidene acetone.

The above-mentioned sequence 3 consists of 1167 nucleotides, and the sequence of OFR is the 1-1167th nucleotide sequence from the 5' end of sequence 3.

The stringent conditions may be hybridization at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, and the solution is used to wash the membrane.

Recombinant vectors, recombinant bacteria, transgenic cell lines, expression cassettes or recombinant viruses containing the above-mentioned coding genes are also within the protection scope of the present invention.

The recombination vector is a recombination vector obtained by inserting the above coding gene into the NdeI and SalI recognition sites of the vector pET-30b(+).

The primer pair for amplifying the full length or any fragment of the above-mentioned coding gene is also within the protection scope of the present invention; the primer pair is as follows: one primer sequence is shown in sequence 5 in the sequence listing, and the other primer sequence is shown in the sequence listing sequence 6.

The application of the above-mentioned protein in the synthesis of benzylidene acetone and the application of the above-mentioned coding gene in the synthesis of benzylidene acetone are all within the protection scope of the present invention. The synthesis of benzylidene acetone is the synthesis of benzylidene acetone in vitro.

Another object of the present invention is to provide a method for preparing benzylidene acetone.

The method provided by the invention is to use 4-coumaroyl-CoA and malonyl-CoA as substrates to obtain benzylidene acetone under the catalysis of the protein described in claim 1.

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. The PcmBAS mutant gene and its application provide a direct and effective technical and application basis for the biosynthesis of raspberry ketone.

Description of drawings

Fig. 1 is the construction of PcmBAS prokaryotic expression vector

Figure 2 is the NdeI and SalI double enzyme digestion identification of the prokaryotic expression vector

Figure 3 is the BAS purification SDS-PAGE analysis

Fig. 4 is BAS catalytic synthesis benzylidene acetone high performance liquid chromatography (HPLC) measurement result

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the acquisition of PcPKS2 site-directed mutation

1. Site-directed mutation of PcPKS2

PcPKS2 (SEQ ID NO: 1 in the Sequence Listing) was artificially synthesized. The site-directed mutagenesis (C197L/G256L/S338I) of PcPKS2 adopts the QuikChange site-directed mutagenesis kit (purchased from Stratagene, Catalog #200518) kit method, and the specific mutation primers are as follows (mutation sites are underlined): PcPKS2C197T/G256L/S338I (5'-CATCGTTTGCTCGGAGATGACGGCAATT ACA TTCCGTGGGCCATCTGAAACAAAC-3', 5'-GAATCCGATGGTGCAGTCGAG TTA CATTTGCTCGAATCTGGGCTTAGTTGCC-3'and 5'-CAAGTGTTGAACGACTATGGAAACATG ATT AGTGCTACTGTGTTTTTCATCATG-3'), the template is sent to the PCR amplification product obtained by PCR, and the template is artificially synthesized PcPKS Sequencing, the results show that the PCR product has the sequence of sequence 3 in the sequence listing, the gene of the PCR product is named PcmBAS, OFR is the 1st-1170th nucleotide of sequence 3 in the sequence listing, and the protein encoded by PcmBAS is named PcmBAS , the amino acid sequence of the protein is sequence 4 in the sequence list. Compared with PcPKS2 (sequence 2), the mutation sites of PcmBAS are: the 197th position is mutated from Cys (cysteine) to Leu (leucine), and the 256th position is mutated from Gly (glycine) to Leu (leucine). ), the 338th position is mutated from Ser (serine) to Ile (isoleucine). Compared with PcPKS2, the mutation sites of PcmBAS were TGC to ACA at positions 583-585, GGC to TTA at positions 760-762, and TCA to ATT at positions 1006-1008.

2. PcmBAS heterologous expression and protein purification

The ORF of PcmBAS cDNA was PCR amplified using N-terminal and C-terminal primers containing specific restriction enzyme sites. The sense primer is: 5'-TATA CATATG GCGGCTTCAATTGAGGAGATT-3' (sequence 5), the underline indicates the NdeI restriction site; the antisense primer is: 5'-TATA GTCGAC CTAATGAATGATGGGCACGCTGCG-3' (sequence 6), the underline indicates the SalI restriction site location. Using the PCR product (or artificially synthesized sequence 3) obtained in step 1) as a template, PCR is carried out, and the obtained PCR product is inserted into the prokaryotic expression vector pET-30b (+) (Novagen, Between the recognition sites of NdeI and SalI in Darmstadt, Germany), the recombinant vector was obtained (Fig. 1 is a schematic diagram of the vector structure), and the recombinant vector was identified by digestion with NdeI and SalI, and the results were as shown in Figure 2 (M, DL15000+2000 Marker; 1 -3, positive plasmid.) Shown, find out from the figure, obtain the DNA fragment of about 1.2Kb. Sequence the recombinant positive plasmid with correct enzyme digestion and identification results. The recombinant vector is composed of the 4th-1167th nucleotides of sequence 3 inserted in the recognition site of pET-30b(+) NdeI and SalI. The positive recombinant vector was named pET-30b(+)-PcmBAS.

Transfer pET-30b(+)-PcmBAS into E.coli BL21-Rosetta(DE3) (TransGen, Beijing, China) to obtain recombinant bacteria, extract plasmids from the recombinant bacteria and identify them by enzyme digestion, and obtain a recombinant fragment of about 1.2Kb The strain was named BL21/pET-30b(+)-PcmBAS.

Insert BL21/pET-30b(+)-PcmBAS into 10ml of LB medium containing kanamycin (50μg ml ^-1 ) and chloramphenicol (34μg ml ^-1 ), culture at 37°C overnight, and dilute 100 times the next day Insert 200ml of LB medium at 37°C and 200rpm and continue to cultivate until the OD ₆₀₀ is 0.6-0.8, add IPTG with a final concentration of 1mM, cool down to 30°C, after 6h, collect the bacteria by centrifugation and resuspend in 3ml 0.1M Potassium phosphate buffer solution (pH 7.5), ultrasonically disrupted cells on ice. The homogenate of the crushed bacteria on ice was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was collected.

The supernatant was passed through a Ni ²⁺ -agarose column (Novagen). After washing the Ni ²⁺ -agarose column three times with 0.1M potassium phosphate buffer containing 0.5M NaCl and 40mM imidazole, wash it with 0.1M containing 400mM imidazole Potassium phosphate buffer was washed and eluted, and the washed eluate was collected. In order to maintain the activity of the recombinant protein for a long time, the washed eluate was passed through a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) with 10% (v/v ) glycerol with 0.1M Tris-HCl (pH 7.5) replacement buffer for re-elution, and the eluate was collected to obtain the recombinant protein PcmBAS, which was stored at -80°C.

The purification efficiency of the recombinant protein was detected by SDS-PAGE.

The same method was used to introduce PcPKS2 into Escherichia coli BL21 to obtain recombinant BL21/pET-30b(+)-PcPKS2, which was then fermented and purified by the above method to obtain recombinant protein PcPKS2.

Using the same method, the empty vector pET-30b(+) was introduced into Escherichia coli BL21 to obtain recombinant bacteria BL21/pET-30b(+), and then the recombinant bacteria were fermented and purified by the above method to obtain the control protein.

A histidine hexamer tag is built on the C-terminus of the recombinant protein. The recombinant proteins PcmBAS and PcPKS2 obtained by the above purification were detected by SDS-PAGE electrophoresis, and the control group protein obtained above was used as a contrast, and the results were shown in Figure 3 (M, molecular mass standard; 1, PcmBAS purification results; 2, PcPKS2 purification results) As shown, the results showed that the purified recombinant proteins PcmBAS and PcPKS2 both formed a single band at the position of 43kDa, which was consistent with the expected size of PcmBAS and PcPKS2, indicating that the protein was purified successfully. There was no target protein band in the control protein.

Embodiment 2, the functional research of mutant

1. In vitro enzymatic reaction and product analysis

250 μl standard in vitro enzymatic reaction system containing 150 μM 4-coumaroyl-CoA (Beuerle T, Pichersky E (2002) Enzymatic synthesis and purification of aromatic coenzymea esters. Anal Biochem 302: 305-312; the public can obtain from Beijing Agricultural University ), 280 μM malonyl-CoA (Sigma-Aldrich Catalog #M4263), 0.1M potassium phosphate buffer (pH 9.0) and 2.0 μg of the recombinant protein PcmBAS obtained in Example 1, the pH of the reaction system was 9.0. After the above reaction system was placed at 30° C. for 30 minutes, acetic acid with a final concentration of 5% (by-product detection) was added, followed by extraction with 250 μl of ethyl acetate and centrifugation at 10,000 g for 10 minutes. After the supernatant was vacuum-dried, 50 μl of 50% (v/v) aqueous methanol solution was added. The recombinant protein PcPKS2 obtained in Example 1 and the control protein were used as controls.

Analysis of enzymatic products was accomplished using a Waters Alliance 2695 high performance liquid chromatography system (HPLC) equipped with a Kromosil C18 reverse phase column (5 μm, 250 mm x 4.6 mm; Macherey Nagel). The mobile phase was water (A) and methanol (B), the flow rate was 0.6ml min ^-1 , and the following gradient conditions were used: 30% B for 3 minutes, 30-70% B for 27 minutes, 70-80% B for 2 minutes, 80- 95% B for 3 minutes and 95% B for 5 minutes. The detection wavelengths for the following compounds are respectively: naringenin chalcone (naringenin chalcone), 289 nm; benzylidene acetone (p-hydroxybenzalacetone), 323 nm. Various compounds were quantified with corresponding standard products: naringenin-chalcone (Catalog#W530098, purchased from Sigma-Aldrich Company) and benzylidene acetone (Catalog#11960, purchased from Sigma-Aldrich Company).

For high-performance liquid chromatography-mass spectrometry, liquid phase separation was performed by a Shimadzu LC-10ADvp HPLC system. Mass spectrometry was performed by Shimadzu LCMS-2010A mass spectrometry system (Shimadazu, Kyoto, Japan). ShimadzuLC/ESIMS-2010 software system completes data acquisition and processing. The liquid chromatography conditions are the same as above. The optimum mass spectrometry conditions are as follows: all m/z wave ranges are set at 120-350; dry gas flow rate, 1.5L min ^-1 ; CDL temperature, 250°C; block temperature, 200°C; probe voltage +4.5kV.

Enzyme kinetic constants and catalytic efficiency (absolute activity) (Kcat/Km) are the most accurate indicators of enzyme activity.

The retention time of benzylidene acetone standard substance is 31.7 minutes, and the qualitative and quantitative analysis result of enzymatic product is shown in Figure 4 (A is PcPKS2 catalysis result; B is PcmBAS catalysis result), the result shows, PcmBAS catalysis generates benzylidene The retention time of methyl acetone is 31.7 minutes, and the retention time of benzylidene acetone catalyzed by PcPKS2 is 31.7 minutes. The control protein did not yield benzylidene acetone, indicating no enzymatic activity.

Mass spectrometry detection result is that the molecular weight of benzylidene acetone standard substance is 146.19, and the molecular weight of benzylidene acetone catalyzed by PcmBAS is 146.19, and the molecular weight of benzylidene acetone catalyzed by PcPKS2 is 146.19, shows that the obtained substance is benzylidene methyl acetone.

When the recombinant PcmBAS is incubated with 4-coumaroyl-CoA and malonyl-CoA in a standard enzymatic reaction system and the pH of the enzymatic reaction system is 9.0, PcmBAS can efficiently generate benzylidene acetone as the main product , and the activity is much higher than that of PcPKS2.

Proof: PcmBAS is benzylidene acetone synthase.

2. Kinetic analysis

The kinetic constants of the recombinant protein were determined using 5 concentrations of 4-coumaroyl-CoA in the range of 0.2-6.0 of its K _m at a saturated concentration of malonyl-CoA (at a concentration of 280 μM/l). value is calculated.

The above-mentioned standard 250 μl 0.1M potassium phosphate buffer reaction system was used in the experiment, and each experiment was repeated 3 times. The pH value of the reaction system was 9.0. After the above reaction system was placed at 30° C. for 30 minutes, acetic acid with a final concentration of 5% was added, followed by extraction with 250 μl of ethyl acetate, and the organic phase was centrifuged at 10,000 g for 10 minutes. After the supernatant was vacuum-dried, 50 μl of 50% (v/v) aqueous methanol solution was added.

K _m and K _cat values were derived from the results of Lineweaver-Burke curves.

For the synthesis of benzylidene acetone, the kinetic constants of PcPKS2 are: K _m =46.7 μm, K _cat =0.24min ^-1 (4-coumaroyl-CoA); the kinetic constants of PcmBAS are: K _m =35.7 μm, K _cat =1.38 min ^-1 (4-coumaroyl-CoA). The catalytic efficiency of the mutant PcmBAS (Kcat/Km) is 7.52 times higher than that of PcPKS2. The PcmBAS mutant gene and its application provide a direct and effective technical and application basis for the biosynthesis of raspberry ketone.

Claims (8)

1. protein, the protein of forming by the amino acid residue sequence of the sequence in the sequence table 4.

2. the said proteinic encoding sox of claim 1.

3. encoding sox according to claim 2 is characterized in that: said encoding sox is the dna molecular shown in the sequence 3 in the sequence table.

4. the recombinant vectors, reorganization bacterium or the expression cassette that contain claim 2 or 3 said encoding soxs.

5. recombinant vectors according to claim 4 is characterized in that: said recombinant vectors is for inserting the recombinant vectors that obtains between the MCS of carrier pET-30b (+) with claim 2 or 3 said encoding soxs.

6. the application of the said protein of claim 1 in synthetic BENZALACETONE, or claim 2 or the application of 3 said encoding soxs in synthetic BENZALACETONE.

7. application according to claim 6 is characterized in that: said synthetic BENZALACETONE is external synthetic BENZALACETONE.

8. a method for preparing BENZALACETONE is characterized in that: for being substrate with 4-coumaric acyl coenzyme A and malonyl coenzyme A, under the said proteinic katalysis of claim 1, obtain BENZALACETONE.

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