JP2008048612A - Glycosyltransferase gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a polynucleotide encoding a glycosyltransferase, useful for producing a capsaicin glycoside, a glycosyltransferase encoded by the polynucleotide, a vector containing the polynucleotide, a transformant containing the vector, a recombinant glycosyltransferase produced by the transformant, to provide a method for producing a capsaicin glycoside using the transformant or the recombinant glycosyltransferase, to obtain a capsaicin glycoside obtained by the production method and a fragment of the polynucleotide. <P>SOLUTION: (a) The polynucleotide or (b) the polynucleotide encodes a protein having glycosyltransferase activity. (a) The polynucleotide comprises a specific base sequence or (b) the polynucleotide is hybridized with (i) a polynucleotide or (ii) a polynucleotide under a stringent condition. (i) The polynucleotide is composed of the specific base sequence (a) or (ii) the polynucleotide is composed of a base sequence complementary to the base sequence (a). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a glycosyltransferase-encoding polynucleotide, a glycosyltransferase encoded by the polynucleotide, a vector containing the polynucleotide, a transformant containing the vector, and a recombinant sugar transfer produced by the transformant. The present invention relates to a method for producing a capsaicin glycoside using an enzyme, the transformant or recombinant glycosyltransferase, a capsaicin glycoside obtained by the production method, and a fragment of the polynucleotide.

  Capsaicin is contained in capsicum in an amount of 10 to 20%, and is the most spicy component of spices. In addition, capsaicin is known to have various pharmacological activities such as an action of enhancing lipid metabolism, but it is extremely spicy and has low water solubility, so it is difficult to continuously take an effective amount of capsaicin.

Therefore, the use of capsaicin glycosides with weak pungency and water solubility has been studied, and various methods for producing capsaicin glycosides have been proposed (Patent Documents 1 to 4). However, capsaicin glycosides obtained using chemical synthesis methods or cultured cells have problems such as contamination of impurities and difficulties in isolating and purifying capsaicin glycosides. In this case, there was a problem that safety was not ensured. In addition, a gene encoding a glycosyltransferase involved in the production of capsaicin glycoside has not yet been found.
JP-A-7-82289 Japanese Patent Laid-Open No. 10-53512 JP 2003-310294 A JP 2004-217554 A

  The present invention relates to a glycosyltransferase-encoding polynucleotide, a glycosyltransferase encoded by the polynucleotide, a vector containing the polynucleotide, a transformant containing the vector, useful for the production of capsaicin glycosides, Recombinant glycosyltransferase produced by the transformant, method for producing capsaicin glycoside using the transformant or recombinant glycosyltransferase, capsaicin glycoside obtained by the method, and fragment of the polynucleotide I will provide a.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors succeeded in cloning a polynucleotide encoding the enzyme of the present invention from Phytolacca americana. Were determined. Furthermore, by constructing a vector containing the obtained polynucleotide and culturing a microorganism that holds this vector, it was found that the enzyme of the present invention can be produced efficiently and confirmed that it can be used for the synthesis of glycosides. The present invention has been completed.

Accordingly, the present invention provides the following:
1. A polynucleotide encoding a protein having glycosyltransferase activity,
The following polynucleotide (a) or (b):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1; or (b) a polynucleotide that hybridizes with any of the following (i) or (ii) under stringent conditions:
(I) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1; or (ii) a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1, A polypeptide having glycosyltransferase activity comprising:
(A) an amino acid sequence represented by SEQ ID NO: 2; or (b) a polyamino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 2, wherein one or several amino acids are substituted, deleted, inserted, or added. peptide,
3. A polynucleotide encoding the polypeptide according to 2 above,
4). A vector comprising the polynucleotide according to 1 or 3 above,
5. A transformant comprising the vector according to 4 above,
6). A recombinant glycosyltransferase produced by the transformant according to 5 above,
7). A method for producing a capsaicin glycoside comprising using the transformant according to 5 or the recombinant glycosyltransferase according to 6 above,
8). Capsaicin glycoside obtained by the production method according to 7 above,
9. A fragment comprising 15 or more consecutive nucleotide sequences of the polynucleotide according to 1 or 3 above,
About.

  The polynucleotide according to the present invention can be in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).

  The polynucleotide or oligonucleotide according to the present invention may contain a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).

  In the present invention, “stringent conditions” means that hybridization occurs only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. For example, hybridization at 42 ° C. and washing treatment at 42 ° C. with a buffer containing 1 × SSC (0.15 M NaCl, 0.015 M sodium citrate) and 0.1% SDS (Sodium dodecylsulfate) More preferred examples include hybridization at 65 ° C. and washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS. The hybridization can be performed by a conventionally known method such as the method described in “Molecular Cloning (Third Edition)” (J. Sambrook & D. W. Russell, Cold Spring Harbor Laboratory Press, 2001). Generally, the higher the temperature and the lower the salt concentration, the higher the stringency. In addition to the above temperature conditions, there are various factors that influence the stringency of hybridization. Those skilled in the art can combine various elements to obtain a string equivalent to the above-described hybridization stringency. You can realize the genie.

  In the present invention, “one or several amino acids are deleted, substituted, or added” can be deleted, substituted, or added by a known mutant polypeptide production method such as site-directed mutagenesis. A certain number (for example, 20 or less, preferably 10 or less, more preferably 7 or less, more preferably 5 or less, particularly preferably 3 or less) of amino acids are substituted, deleted, or added. means. Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, but is a product obtained by isolating and purifying a similar naturally occurring mutant polypeptide. It may be.

  Examples of a method for obtaining the polynucleotide or oligonucleotide according to the present invention include a method for isolating and cloning a DNA fragment containing the polynucleotide or oligonucleotide according to the present invention by a known technique. For example, a probe that specifically hybridizes with a part of the base sequence of the polynucleotide in the present invention can be prepared, and a genomic DNA library or cDNA library can be screened. Such a probe may be of any sequence and / or length as long as it specifically hybridizes to at least part of the base sequence of the polynucleotide of the present invention or its complementary sequence. Good.

  Examples of the method for obtaining the polynucleotide according to the present invention include a method using an amplification means such as PCR. For example, in the polynucleotide cDNA of the present invention, primers are prepared from the 5 ′ side and 3 ′ side sequences (or their complementary sequences), and genomic DNA (or cDNA) or the like is used as a template using these primers. By performing PCR or the like and amplifying the DNA region sandwiched between both primers, a large amount of DNA fragments containing the polynucleotide according to the present invention can be obtained.

  Examples of a source for obtaining the polynucleotide according to the present invention include Japanese pokeweed. In the examples described later, the polynucleotide according to the present invention is obtained from the pokeweed, but it is not limited thereto.

  The vector of the present invention can be used to express the polypeptide of the present invention. In order to confirm whether or not the polynucleotide according to the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell, various markers may be used. The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specific examples include bacteria such as Escherichia coli, yeast, plant cells and the like, but are not particularly limited.

  The method for introducing the expression vector into a host cell, that is, the transformation method is not particularly limited. Agrobacterium infection method, electroporation method (electroporation method), calcium phosphate method, protoplast method, lithium acetate method, etc. The conventionally known methods can be suitably used.

Cultivation of pokeweed cells As cells for cell culture, cells collected from any part of stems, leaves, and roots of pokeweed can be suitably used. Cultured cells can be prepared according to conventional methods. For example, the callus was induced from the leaves of the pokeweed moss under the Murashige sukuk medium (MS medium) to perform callus formation. Then, it culture | cultivated on 25 degreeC on light conditions.

  The pokeweed cells can be cultured with shaking at 25 ° C. under light irradiation using MS (Murashige-Skoog) medium.

Gene Extraction First, a gene is obtained from a culture obtained by culturing the above cells according to a conventional method. For this operation, a commercially available RNA extraction kit or DNA extraction kit can be used. When RNA is extracted, capsaicin can be added and cultured in advance to increase the expression level of the target gene. From the RNA extract, for example, cDNA can be obtained using RT-PCR.

Construction of cDNA Library The gene obtained can be inserted into a phage or plasmid vector to create a gene library. At this time, if necessary, the gene can be digested with an appropriate restriction enzyme and treated with a modifying enzyme such as phosphatase, or a linker or adapter can be added.

Cloning of target gene Total RNA was extracted from cultured pokeweed cultured cells, and fragments of the glycosyltransferase gene of pokeweed pokeweed by RT-PCR using primers designed from conserved amino acid sequences of heterologous glycosyltransferases and oligo dT primers Can be obtained. From the obtained gene fragment, a probe specific for the glycosyltransferase gene of Pythium can be designed. The target gene can be selected by hybridizing with the gene of the above cDNA library using this probe.

  By incorporating the thus cloned gene into a suitable vector, other prokaryotic or eukaryotic host cells can be transformed. Furthermore, genes can be expressed in the respective host cells by introducing appropriate promoters and sequences involved in expression into these vectors.

  Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. In order to express a gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences. Further, it is desirable that the vector has a sequence capable of conferring phenotypic (phenotypic) selectivity on transformed cells.

  For example, E. coli may be E. coli. E. coli K12 strain is often used, and pBR322 or pUC-type plasmid is generally used as a vector. However, the present invention is not limited to this, and any of various known strains and vectors can be used. As the promoter, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, bacteriophage-derived lambda (λ) PL promoter, polypeptide chain elongation factor in E. coli Tu (tufB) promoter and the like can be mentioned. As Bacillus subtilis, for example, 207-25 strain is preferable, and pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used as a vector, but is not limited thereto. is not. As a promoter, a regulatory sequence of the α-amylase gene of Bacillus subtilis is often used, and if necessary, a secretory expression outside the cell can also be achieved by connecting a DNA sequence encoding the signal peptide sequence of α-amylase. Become.

  Eukaryotic host cells include cells of vertebrates, insects, plants, yeasts, and the like. Examples of vertebrate cells include COS cells (Gluzman, Y. (1981) Cell 23,175- 182) or dihydrofolate reductase deficient strain of Chinese hamster ovary cells (CHO) (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220), etc. are often used. However, it is not limited to these.

As an expression vector for vertebrate cells, a vector having a promoter usually located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. You may have. Examples of the expression vector include, but are not limited to, pSV2dhfr (Subramani, S. et al. (1981) Mol. Cell. Biol. 1,854-864) having the SV40 early promoter.
As plants, Arabidopsis thaliana and cultured cells thereof are generally used. However, tobacco (Nicotiana tabacum), other horticultural plants, or cultured cells thereof may be used. The 35S promoter of cauliflower mosaic virus can be preferably used as the promoter for expression, and NOS of nopaline synthase can be preferably used as the terminator, but is not limited thereto.

  As eukaryotic microorganisms, yeasts are generally used, and among them, Saccharomyces genus yeasts such as Saccharomyces cerevisiae are preferred. Examples of expression vectors for eukaryotic microorganisms such as yeast include alcohol dehydrogenase gene promoters (Bennetzen, JLand Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) and acid phosphatase gene promoters. (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) and the like can be preferably used.

  The expression vector is prepared by DEAE-dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res. 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FLand van der Ed, AJ ( 1973) Virology 52,456-457) and electric pulse perforation (Neumann, E. et al. (1982) EMBO J. 1,841-845), etc., can be incorporated into host cells to obtain desired transformed cells Can do.

  The obtained transformant can be cultured according to a conventional method, and the enzyme of the present invention is produced intracellularly or extracellularly by the culture. As the medium used for the culture, various commonly used media can be appropriately selected depending on the employed host cells. For example, in the case of Escherichia coli, LB medium, YT medium, M9 medium, etc. can be used.

  As described above, the enzyme of the present invention produced inside or outside the transformant can be separated and purified by various known separation methods utilizing its physical properties and chemical properties. . Examples of the method include treatment with an ordinary protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchanger chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and the like. Examples of the various liquid chromatography, dialysis methods, combinations thereof, and the like. By the above method, the desired enzyme of the present invention can be easily produced on an industrial scale with high yield and high purity.

  The glycosyltransferase of the present invention that can be obtained by the above method has the following properties.

(A) Action Transfers a sugar activated by uridine nucleoside diphosphate (UDP) to a receptor.

(B) Specificity for sugar donors Acts on UDP-glucose and UDP-galactose but not on UDP-glucuronic acid.

(C) Specificity for sugar receptor m-hydroxybenzoic acid, salicyl alcohol, trans-p-coumaric acid, caffeic acid, daidzein, genistein, kaempferol, quercetin, apigenin, naringenin, 4,2 ′, 4 ′, 6'-tetrahydroxychalcone, aureusidin, esculetin, 8-nordihydrocapsaicin and betanidine can be accepted, but salicylic acid, p-hydroxybenzoic acid, hydroxyquinone and cyanidin cannot be accepted.

(D) Optimum reaction temperature 45 ° C

(E)
Temperature stability Stable at 40 ° C or less

(F) Optimum pH of reaction
6.5

(G) pH stability Stable at 7.0

(H) Influence of metal ions The influence of various metal ions on the enzyme of the present invention is shown below. Even when 1 mM EDTA was added to the assay system, almost no effect was observed on the activity of the enzyme of the present invention. Therefore, it is considered that the enzyme of the present invention does not require metal ions for its catalytic activity.

Metal ion (0.1 mM) Enzyme of the present invention Ca ²⁺ 34
Cd ²⁺ 9
Co ²⁺ 23
Cu ²⁺ 21
Mg ²⁺ 67
Mn ²⁺ 67
Ni ²⁺ 62
Sn ²⁺ 104
Zn ²⁺ −
EDTA (1 mM) 110
The numerical values are relative values with the control as 100.
No enzyme activity is observed with Fe ²⁺ , Fe ³⁺ , Hg ²⁺ and Zn ²⁺ .

(I) Influence of various inhibitors Inhibition of catalytic activity by DEPC which is a histidine residue modifying reagent is not observed. Further, inhibition of catalytic activity by PMSF, which is a serine residue modifying reagent, is not observed.

  Various glycosides can be produced using the transformant or a recombinant enzyme collected from the transformant. For example, the above transformant is cultured, the bacterial cells are collected from the culture, freeze-dried, and freeze-dried bacterial cells and UDP glucose are added to a buffer containing capsaicin and reacted to obtain a capsaicin glycoside. be able to.

  Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples.

Cultivation of Amaranthus cells Amaranthus cells were obtained from the Biochemistry Laboratory, Department of Basic Science, Okayama University, and subcultured every 4 weeks. About 20 g of Sphagnum pokeweed cells were added to 100 ml of the culture solution (MS medium), and cultured with shaking at 25 ° C. while irradiating 10,000 Lx light.

Acquirement of pokeweed glycosyltransferase (PaGT) gene fragment 20 mg capsaicin was added per 100 ml of the culture solution, and total RNA was extracted from pokeweed cells cultured for 3 days using RNeasy Plant Mini Kit (Qiagen). Was purified using DNase-Free kit. Using the obtained total RNA as a template, RT-PCR was performed using primers (LcGT1 (SEQ ID NO: 3) and oligo-dT) designed from the conserved amino acid sequence of glycosyltransferase. RT-PCR was performed using One Step RT-PCR Kit (Qiagen), and the composition of the reaction solution was in accordance with the manufacturer's instructions. The obtained PCR product was purified using PCR Purification Kit (Qiagen), and nested-PCR was performed. The composition of this reaction solution was RT-PCR product 1 μl, ExTaq buffer 2 ×, dNTPs 100 μM, LcGT2 primer (SEQ ID NO: 4) 0.2 μM, oligo dT 0.2 μM, Extaq polymerase (Takara) 2.5 U. The reaction conditions were initially 94 ° C for 30 seconds, then 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute for 30 cycles, and finally 72 ° C for 7 minutes. The amplified DNA fragment was TA cloned into pCR2.1-TOPO vector (TOPO TA cloning Kit, Invitrogen) and the nucleotide sequence was determined (DNA sequencer: Beckman Coulter CEQ 2000XL DNA Analysis System). A homology search was performed using the amino acid sequence deduced from the obtained base sequence.

  As a result of the homology, the homology of PaGT is 72% with UDP glucose: flavonoid-O-glucosyltransferase from Beta vulgaris, 70% with betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis, Dianthus UDP from caryophyllus: 67% with chalcononaringenin 2'-O-glucosyltransferase, 59% with phenylpropanoid glucosyltransferase 2 (U32644) from common tabacco, and from common tabacco It was 59% with phenylpropanoid glucosyltransferase 1 (AF346431).

Construction of cDNA library Poly (A) ⁺ -RNA was obtained from the pokeweed cells after addition of capsaicin using Straight A's mRNA Isolation System (Novagen). Using the obtained poly (A) ⁺ -RNA as a template, a double-stranded cDNA was prepared using ZAP-cDNA Synthesis Kit (STRATAGENE). The resulting double-stranded cDNA was purified using SizeSep 400 Spun Columns (Amersham Pharmacia Biotech) and cloned into Uni-ZAP XR insertion vector according to the prescription of ZAP-cDNA Synthesis Kit. This was packaged into λ phage using Gigapack III Gold Cloning Kit (STRATAGENE) to construct a cDNA phage library of P. argura cells.

CDNA cloning of PaGT Primer (forward primer: PaGT (F) (SEQ ID NO: 5), reverse primer: PCR21 (R) (SEQ ID NO: 6) designed from the pokeweed glycosyltransferase (PaGT) gene fragment obtained above. )) Was used to prepare digoxigenin (DIG) -labeled primers by PCR DIG Probe Synthesis Kit (Roche Biochemicals).

  The cDNA library was screened by plaque hybridization using the prepared DIG-labeled PaGT-specific probe. The resulting positive clone was subcloned into the pBluescript SK (−) phagemid vector according to the manufacturer's instructions (STRATAGENE). When the nucleotide sequence of the obtained clone (pBluescript-PaGT) was determined, the ORF length was 1455 bp (485 amino acids).

Construction of PaGT expression plasmid Since PaGT cDNA has a BamHI cleavage site inside, a primer having a BamHI end digested with BsaI was synthesized (forward primer: PaGT (BsaI-BamHI-F), SEQ ID NO: 7, reverse) Primer: PaGT (BsaI-BamHI-R), SEQ ID NO: 8). PCR was performed using the pBluescript-PaGT obtained above as a template. The composition of the reaction solution is pBluescript-PaGT 20ng, KOD-Plus Buffer 1x, dNTPs 0.2mM, PaGT (BsaI-BamHI-F) 0.3μM, PaGT (BsaI-BamHI-R) 0.3μM and KOD-Plus- (TOYOBO) It was 1U. The reaction conditions were 94 ° C. for 2 minutes, then 94 ° C. for 15 seconds, 51 ° C., 30 seconds and 68 ° C., 1.5 minutes for 30 cycles. The obtained PCR product was subcloned into the pCR4Blunt-TOPO vector using Zero Blunt TOPO PCR Cloning Kit For Sequencing (Invitrogen) according to the method recommended by the manufacturer. As a result, a plasmid (pCR4-PaGT) into which full-length PaGT was inserted was obtained. It was confirmed by the nucleotide sequence analysis that no mutation occurred in the PaGT gene. pCR4-PaGT was completely digested with BsaI, and the generated fragment of about 1.5 kb was recovered and subcloned into the BamHI site of the expression vector pQE-30 (QIAGEN) to construct pQE-30-PaGT. The obtained pQE-30-PaGT was used to transform E. coli XL1-Blue.

The obtained transformant (XL1-Blue) was shaken and cultured overnight in 10 ml of LB medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl) containing 60 μg / ml ampicillin. The whole culture broth was inoculated into 2 l of the medium having the same composition and cultured at 37 ° C. with shaking. When OD ₆₀₀ reached 0.5, isopropyl-β-thiogalactopyranoside (IPTG, final concentration 400 μM) was added, and the mixture was cultured with shaking at 20 ° C. overnight.

  The following operations were all performed at 4 ° C. The cultured transformant was collected by centrifugation (5,000 × g, 15 minutes), and a disruption buffer (100 mM potassium phosphate buffer: KPB, pH 7.2, 15 mM 2-mercaptoethanol: 2-ME) was used. In addition, well suspended. Subsequently, the microbial cells were pulverized using ultrasonic waves (1 minute × 10 times), and the precipitate was separated by centrifugation (8,000 × g, 25 minutes). Polyethyleneimine (final concentration 0.12%) was added to the supernatant, stirred well, and allowed to stand in an ice bath for 30 minutes. Thereafter, the supernatant was recovered as a crude enzyme solution by centrifugation (8,000 × g, 25 minutes).

  Subsequently, the crude enzyme solution was purified using HisTrap Kit (Amersham Pharmacia Biotech). The crude enzyme solution was applied to a HiTrap Chelating column equilibrated with Buffer K (100 mM KPB, 15 mM 2-ME, 10 mM imidazole), and the column was washed with Buffer K. Subsequently, the protein bound to the column was eluted with Buffer L (100 mM KPB, 15 mM 2-ME, 200 mM imidazole) (10 ml). This was concentrated to 1 ml using Amicon Ultra-15 30,000 MWCO (MILLIPORE).

Measurement of PaGT enzyme activity The composition of the reaction solution was 50 μM glucose receptor, 100 μM UDP glucose, 50 mM KPB pH 7.2, and the final solution volume was 100 μl. This reaction solution was preincubated with Dry Thermo Unit DTU (TAITEC) at 30 ° C. for 10 minutes, and an enzyme solution was added to start the reaction. After incubation at the same temperature for 1 hour, 150 μl of 2.5% TFA aqueous solution was added to stop the reaction. The reaction solution was centrifuged (15,000 rpm, 1 minute), and the reaction solution was analyzed using reverse phase high performance liquid chromatography (HPLC). Column was used COSMOSIL _{(TM) 5C 18 -MS-II (nakalai} tesque). The amount of enzyme used for the enzyme reaction was adjusted so that the amount of the reaction product was about 10% of the initial amount of the substrate.

  It was observed that PaGT was active against capsaicin, indicating that glycosylation of capsaicin by cultured pokeweed cultured cells was due to PaGT. PaGT showed activity against 8-nordihydrocapsaicin, betanidin, phenols, flavonoids and coumarins esculetin.

pH dependence The pH dependence of PaGT was evaluated by replacing the buffer in the reaction solution with an acetic acid-phosphate-sodium buffer (pH 4-10).

pH stability To 20 μl of the enzyme solution, the above 10 μl of 0.5 M buffer was added and incubated at 20 ° C. for 8 hours. Thereafter, 20 μl of 1M KPB (pH 7.2) was added, and the residual activity was measured by the method described in the above pH dependence to evaluate the pH stability of PaGT. As a result, the optimum reaction pH was 6.5 and was stable at pH 7.0.

Temperature dependence The temperature dependence of the PaGT activity was evaluated by performing the incubation temperature of the enzyme reaction at intervals of 5 ° C. from 20 to 55 ° C.

Temperature stability The enzyme solution was incubated at the temperature described above for 1 hour. The residual enzyme activity after the treatment was measured by the above method, and the temperature stability of PaGT was evaluated.

  As a result, the optimum reaction temperature of PaGT was 45 ° C, and it was stable at 40 ° C or less.

Influence of inhibitor The enzyme activity was measured with an assay system containing 0.1 mM of various metal ions, and the effect of each metal ion was examined by comparing with the enzyme activity of the assay system under the same conditions without metal ions. . Also measure enzyme activity in assay systems containing 1 mM uridine, UMP, UDP, UTP, diethylpyrocarbonate (DEPC), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA) to determine product inhibition It was. In the assay system for evaluating the influence of these inhibitors, HEPES-NaOH buffer (pH 7.2) was used in place of KPB as the buffer for the reaction solution.

As a result, PaGT activity was markedly inhibited by Fe ²⁺ , Fe ³⁺ , Hg ²⁺ or Zn ²⁺ . Relative activity is relative to the control, 34% ^{Ca 2+,} 9% for ^{Cd 2+,} 23% for ^{Co 2+,} 21% for ^{Cu 2+,} 67% for ^{Mg 2+,} 67% for ^{Mn 2+,} 62% for ^{Ni 2+, Sn 2+} It was 104%. Further, when 1 mM EDTA was added to the assay system for reaction, the enzyme activity was hardly affected. In addition, inhibition of PaGT activity by uridine, UMP, UDP, UTP, DEPC, PMSF was not observed.

Substrate specificity by HPLC Enzymatic reaction was carried out using UDP galactose and UDP glucuronic acid instead of UDP glucose. Further, salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, salicyl alcohol, hydroquinone, trans-p-coumaric acid, caffeic acid, daidzein, genistein, kaempferol, quercetin, apigenin, naringenin, 4 instead of betanidine , 2 ′, 4 ′, 6′-tetrahydroxychalcone, aureusidin, esculetin, and 8-nordihydrocapsaicin were used for the enzymatic reaction. The assay method followed the above method except that the substrate was different.

  PaGT is less active than UDP glucose (47% relative to UDP glucose), but UDP galactose was also recognized as a sugar donor.

Moreover, the activity with respect to each sugar receptor is shown below. In addition, a value is relative activity% when the activity with respect to caffeic acid is 100%.
Salicylic acid −
m-Hydroxybenzoic acid 27
p-hydroxybenzoic acid −
Salicyl alcohol 3.2
Hydroquinone −
trans-p-coumaric acid 19
Caffeic acid 100
Daidzein 66
Genistein 80
Kaempferol 37
Quercetin 80
Apigenin 83
Naringenin 59
4,2 ′, 4 ′, 6′-tetrahydroxychalcone 73
Aureushijin 91
Esculetin 65
8-Nordihydrocapsaicin 65
Betazinin 18

In the preparation of the DNA probe, the conditions for “hybridize under stringent conditions” in the base sequence of the present invention include, for example, hybridization at 42 ° C. and 1 × SSC (0.15 M NaCl, 0.015M sodium citrate), a washing treatment at 42 ° C. with a buffer containing 0.1% SDS (Sodium dodecylsulfate), hybridization at 65 ° C., and 0.1 × SSC, 0. A washing treatment at 65 ° C. with a buffer containing 1% SDS can be mentioned more preferably. In addition to the temperature conditions described above, there are various factors that affect the stringency of hybridization. Those skilled in the art can combine various elements to obtain a string equivalent to the above-described hybridization stringency. It is possible to realize a genie.

  According to the present invention, it is possible to efficiently produce a capsaicin glycoside that is less contaminated and safe for food applications.

  The enzyme of the present invention is useful for the production of a capsaicin glycoside capable of continuously ingesting an effective amount of capsaicin having various pharmacological activities such as lipid metabolism enhancing action.

Claims (9)

A polynucleotide encoding a protein having glycosyltransferase activity,
The following polynucleotide (a) or (b):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1; or (b) a polynucleotide that hybridizes with any of the following (i) or (ii) under stringent conditions:
(I) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1; or (ii) a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1. A polypeptide having glycosyltransferase activity comprising:
(A) an amino acid sequence represented by SEQ ID NO: 2; or (b) a polyamino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 2, wherein one or several amino acids are substituted, deleted, inserted, or added. peptide.   A polynucleotide encoding the polypeptide of claim 2.   A vector comprising the polynucleotide according to claim 1 or 3.   A transformant comprising the vector according to claim 4.   A recombinant glycosyltransferase produced by the transformant according to claim 5.   A method for producing a capsaicin glycoside, comprising using the transformant according to claim 5 or the recombinant glycosyltransferase according to claim 6.   A capsaicin glycoside obtained by the production method according to claim 7.   A fragment consisting of 15 or more consecutive nucleotide sequences of the polynucleotide according to claim 1 or 3.