WO2005052160A1 - L-sorbosone deshydrogenase, gene codant et utilisation de ceux-ci - Google Patents
L-sorbosone deshydrogenase, gene codant et utilisation de ceux-ci Download PDFInfo
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- WO2005052160A1 WO2005052160A1 PCT/CN2004/001356 CN2004001356W WO2005052160A1 WO 2005052160 A1 WO2005052160 A1 WO 2005052160A1 CN 2004001356 W CN2004001356 W CN 2004001356W WO 2005052160 A1 WO2005052160 A1 WO 2005052160A1
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- 108010053002 L-sorbosone dehydrogenase Proteins 0.000 title claims abstract 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 31
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Classifications
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
Definitions
- the invention relates to an L-sorbone dehydrogenase in the field of genetic engineering, a coding gene thereof, an expression method and application thereof. Background technique
- 2-keto-L-gulonic acid (2-KGA) is an important intermediate for the synthesis of ascorbic acid (vitamin C).
- vitamin C ascorbic acid
- NRRL B-21627 US Patent No. 5834231
- DSM4025 European patent EP9611500. 8
- L-sorbone oxidase found in IF03293 requires neither a coenzyme nor an electron acceptor.
- Makover et al. (Biotechnol. Bioeng, 17. 1485. 1975) also reported L-sorbone dehydrogenase (SNDH) activity It is present in Pseudomonas putida ATCC21812 and Gluconobacter suboxydans IF03293 and indicates that NAD or NADP is not its coenzyme.
- the molecular weight of SNDH discovered by Fujiwara et al. U.S. Patent No. 4,902,617) is 190,000 ⁇ 20,000 Daltons.
- 2-KGA is produced from sorbitol by 2-step fermentation with microorganisms, and then vitamin C is produced by methyl esterification.
- three kinds of microorganisms were used, that is, two kinds of microorganisms were involved in the conversion process from L-sorbose to 2-KGA, and one of them was an acid-producing bacterium that transformed L-sorbose to 2-KGA.
- Gluconobacter oxydans which has production value is usually slower to grow alone and has a lower conversion rate of 2-KGA.
- the object of the present invention is to provide an L-sorbone dehydrogenase and a gene encoding the same.
- the L-sorbone dehydrogenase provided by the present invention is a protein having the sequence of SEQ ID NTs: 2 amino acid residues in the Sequence Listing or has a sequence of amino acid residues of at least 80 with SEQ ID Na: 2 in the Sequence Listing.
- the SEQ ID No : 2 is derived from ketocoronic acid bacteria (eio ⁇ / Jo ⁇ e'M ⁇ A) WB0104 (CCTTCC No. M203094) and consists of 429 amino acid residues.
- the ketocoronic acid tKetogulonigeniwn sp.) WB0104 was deposited at the China Type Culture Collection (CCTCC) on November 24, 2003 under CCTCC No.M203094.
- the protein derived from SEQ ID Na: 2 having at least 80% homology with the amino acid residue sequence of SEQ ID Na: 2 in the Sequence Listing and having the same activity as SEQ ID No : 2 is preferably identical to SEQ ID Ns : 2
- the L-sorbone dehydrogenase may be a protein obtained by expressing a sequence having at least 80% homology with SEQ ID No : 1 in the sequence listing in a host bacterium Escherichia coli or Pichia pastoris.
- a gene encoding L-sorbone dehydrogenase,% rSNDH is one of the following nucleotide sequences:
- the SEQ ID Na: 1 sp.) WB0104 CCTCC No. M203094 consisting of 1290 bases.
- the open reading frame (0RF) of the gene is from base 1 to base 1290 at the 5 'end.
- Both the expression vector and the cell line containing the gene of the present invention belong to the protection scope of the present invention, and different expression vectors and cell lines (engineering bacteria) can be obtained by using existing molecular biology methods: for example, E. coli BL21 ( DE3), E. coli TOP10, or Pichia X-33.
- the above-mentioned engineered bacteria containing the gene of the present invention can also be fixed in different vectors by conventional methods to prepare immobilized cells.
- the L-sorbone dehydrogenase of the present invention can be immobilized on supports such as agarose, acrylamide, and sodium alginate, Preparation of immobilized enzyme.
- a second object of the present invention is to provide a method for expressing L-sorbone dehydrogenase.
- the method for expressing L-sorbone dehydrogenase provided by the present invention is a gene encoding an L-sorbone dehydrogenase obtained by amplifying genomic DNA of ketocoronic acid bacteria iKetogulonigenium sp. WB0104 CCTCC No. M20309 as a template
- the expression host bacteria were introduced to obtain positive clones, and the positive clones were cultured to express L-sorbone dehydrogenase.
- Ketogulonigenium sp.) WB0104 CCTCC No. M203094 genomic DNA can be extracted by conventional methods.
- a pair of primers for amplifying the L-sorbone dehydrogenase gene using ketogulonigeniim sp. WB0104 CCTCC No. M203094 as a template can be SEQ ID Na : 3 and SEQ ID Na : 4, SEQ ID Na in the sequence listing Na : 5 and SEQ ID Na : 6 or SEQ ID No : 7 and SEQ ID N 2: 8.
- SEQ ID No : 3 is composed of 26 bases
- SEQ ID Na : 4 is composed of 24 bases
- SEQ ID No : 5 is composed of 32 bases
- SEQ ID Ns: 6 is composed of 24 bases
- SEQ ID Na : 7 consists of 35 bases
- SEQ ID Na : 8 consists of 24 bases.
- the positive clone may be E. coli DH5 ⁇ , E.coli BL21 (DE3), E.coli TOP10, or Pichia X-33 containing an L-sorbone dehydrogenase-encoding gene.
- the L-sorbone dehydrogenase of the present invention does not depend on the coenzyme NAD, but the presence of coenzyme NAD can improve the conversion efficiency of the enzyme, the enzyme converts L-sorbone to 2-KGA in a range of 5. 0, the most suitable is 7.5.
- the whole genome sequence of NO. M203094 was determined by gene annotation, and the key enzyme gene related to the synthesis of 2-keto-L-gulonic acid-the L-sorbone dehydrogenase gene was predicted. Its DNA sequence has very low homology compared with all known L-sorbone dehydrogenase gene sequences.
- ⁇ Rhodobacter sphaeroides is 68% homologous, 53% homologous to P. syringae SNDH, 50% homologous to alfalfa rhizobium SNDH, and oxidized glucose with another vitamin C-producing bacterium
- the SNDH in Acidobacteria is essentially non-homologous.
- the protein (enzyme) produced by expressing this gene in different vectors and hosts by molecular biology methods can effectively convert L-sorbone to 2-keto-L-gulonic acid (2-KGA) in vitro No matter what form of SNDH (including body, secreted protein, fusion protein) has SNDH enzyme activity in vitro, at the same time, when the E.
- coli engineered bacteria carrying the rSNDH coding gene of the present invention is co-cultured with 2-KGA transforming bacteria
- the rSNDH of the present invention significantly improves the conversion rate of 2-KGA, and the rSNDH.
- Encoding gene of the present invention is a key enzyme gene in 2-KGA synthesis.
- the L-sorbone dehydrogenase gene of the present invention can be used to transform existing 2-KGA-producing bacteria and conduct targeted breeding to increase the yield of 2-KGA.
- the L-sorbone dehydrogenase gene of the invention or a mutant or fragment thereof is inserted into the genome of an existing 2-KGA-producing bacteria to enhance the activity of the L-sorbone dehydrogenase, or to express the present invention by molecular biological means
- the L-sorbone dehydrogenase gene is used to synthesize 2-KGA in vitro by using an immobilized enzyme method or an immobilized cell method, or a protein corresponding to the L-sorbone dehydrogenase gene of the present invention is used for the production of 2-KGA, Reduce the production cost of 2-KGA, increase the conversion rate of 2-KGA, reduce the environmental pollution, reduce the demand for energy during the production process, and improve the quality of the product.
- the invention has important industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
- Figure 1 shows the electrophoresis map of the PCR amplified product of the L-sorbone dehydrogenase gene.
- Figure 2 is an SDS-PAGE map of the expression product of the L-sorbone dehydrogenase gene in E. coli BL21 (DE3)
- Figure 3 is an SDS-PAGE chart of the expression product of L-sorbone dehydrogenase gene in E. coli TOP10.
- Figure 4 is an SDS-PAGE chart of the expression product of L-sorbone dehydrogenase gene in Pichia pastoris X-33.
- 5 is the rSNDH activity curve of the positive clone Escherichia coli T0P10 fusion expression
- Figure 7 shows the relationship between rSNDH enzyme activity and pH
- Figure 8 is an HPLC spectrum of rSNDH enzyme in the presence of coenzyme for 0 time
- Figure 9 shows the HPLC profile of rSNDH enzyme in the presence of coenzyme for 5 hours.
- Figure 10 shows the HPLC spectra of L-sorbone and 2-keto-L-gulonic acid standards.
- Figure 11 shows the HPLC spectrum of rSNDH enzyme in the absence of coenzyme for 5 hours.
- Fig. 12 is a mass spectrum of LSNsorbase to 2-KGA in the absence of coenzyme.
- Fig. 13 is a mass spectrum of a 2-KGA standard.
- TKetogulonigenium sp. WB0104 (CCTCC No. M203094) in the logarithmic growth phase was collected, and the culture supernatant was detected to contain 2-keto-L-gulonic acid by HPLC.
- the instrument used in this HPLC method and The reagents are: Two Waters 515 high-performance liquid pumps, Alltech 2000 ELSD; acetonitrile (HPLC grade); trifluoroacetic acid (analytical grade); Milli Q plus pure water.
- ELSD atomizing gas is N 2 , flow rate 2. 51 / min, drift tube temperature 100 ° C; chromatographic column is Thermo Quest APS2 4. 6mmi. D. X 25cm 5 ⁇ m; mobile phase: acetonitrile-0. 1% ⁇ Aqueous fluoroacetic acid (gradient) with a flow rate of 1.3 ml / min. The injection volume was 20ul. Controls: L-sorbose, 2-keto-L colic acid. Bacterial genomic DNA extraction method ( ⁇ Compiled Guide to the Experiment of Molecular Biology ⁇ Science Press. P.
- r-sorbone dehydrogenase gene r W sequence is predicted as SEQ ID No : 1, encoding a protein consisting of 429 amino acid residues. (SEQ ID No : 2), and its molecular weight is 45473. 41 Daltons.
- SEQ ID Na: 1 has very low homology compared to all known L-sorbone dehydrogenase gene sequences. After translation into protein, it has 68% homology with Rhodobacter sphaeroides (SNDH). It has 53% homology with P. syringae SNDH, 50% homology with S. meliloti SNDH, and basically no homology with SNDH in Gluconobacter oxidans, another vitamin C production bacterium.
- PET11B expression primers
- PCR was performed using genomic DNA of tKetogulonigenium sp. WB0104 (CCTCC No. M203094) as a template.
- the 50 ⁇ system contained a final concentration of 1.5 ol / L MgCl 2 and 0.2 ol / L. dNTPs, 0.2 ⁇ mol / L each of 5, primers and 3, primers, 10mraol / L Tris-HCl, 2u TaqDNA polymerase; PCR reaction conditions: 94 ° C for 5 minutes; and then performed 30 for the following conditions Cycle: 1 minute at 94 ° C, 1 minute at 55 ° C, 2 minutes at 72 ° C; 72 ⁇ 10 minutes.
- the PCR product electrophoresis results are shown in Figure 1.
- the molecular weight standard (marker) is 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 8000, 10000, and a, respectively.
- A is the PCR product of PET11B expression primer electrophoresis. result.
- the PCR product was recovered and purified by electrophoresis and ligated with pGEM-T-VECTOR using T 4 DNA ligase, and then transformed into DH5 a.
- the white spots were picked, and the plasmid was extracted with QIAGEN's MINI plasmid extraction column and verified by enzyme digestion to find the correct inserted pGEM- T-VECT0R- rSNDH plasmid, digested with NDEI / BAMHI, digested with agarose electrophoresis After receiving 1.3 kb, it was ligated with the expression vector PET11B, transformed into DH5 ⁇ , plated (containing AMP 100 ⁇ g / ml), picked clones and identified by enzyme digestion to obtain a positive clone plasmid PET11B-rSNDH.
- the two markers of molecular weight markers are 60,000 and 45,000 respectively; a is the control before induction, and b is the rSNDH expressed by PET11B after induction, and the arrow indicates the target band.
- the PBAD / T0P0 THIOFUSI0 expression system (Invitrogen) is a fast and efficient cloning and expression system. PCR products can be used directly for cloning without ligase treatment, and can be linked to the vector for expression in 5 minutes, and THI0RED0XIN is used. TRX thioredoxin is used as a fusion peptide to express a foreign protein, so that the foreign protein is expressed in a soluble active form. ⁇ Use PBAD promoter and arabinose as inducer.
- a PCR reaction was performed using genomic DNA of ketocoronic acid J (etogulonigeniwn sp.) WB0104 (CCTCC No. M203094) as a template.
- the 50 ⁇ system contained a final concentration of '1.5 ol / L MgCl 2) 0.2 mmol / L dNTPs, 0.2 ⁇ mol / L 5 'bow
- b is the electrophoresis result of the PCR product of the PBAD / THI0-TOP0 expression primer.
- the PCR product was directly connected to PBAD / THI0-T0P0 (for cloning process, refer to the INVIT0RGEN PBAD / T0P0-THI0FUSI0N expression kit), and then transformed into TOPIO.
- the INVITR0GEN Pichia picz ⁇ expression system was used to construct the expression vector piczaA / SND expressing rSNDH in Pichia pastoris.
- the PCR reaction conditions are as follows:
- Ketogulonigenium sp. WB0104 (CCTCC No. M203094) genomic DNA was used as a template for the PCR reaction, and then the PCR product was ligated to pGEM-T-VECTOR in accordance with conventional methods. After transformation, the white spot colonies were selected, and the plasmid was extracted and sequenced for identification. The correct pGEM-T-rSNDH and picz a A plasmid DNA identified by sequencing were treated with XHOI, respectively. The kit recovered approximately 1200bp and 3.3kb fragments, and ligated with T 4 DNA ligase. The ligated products were transformed into DH5 a competent bacteria.
- plasmid Coated on LB plate (containing ZE0CIN 25 ⁇ g / ml). The plasmid was extracted and identified by enzyme digestion. Select the recombinant plasmid picz a A-rSNDH with the correct ligation direction and insertion size, and use the QIAGEN MIDI plasmid extraction kit to extract the plasmid for transformation into Pichia pastoris.
- a single colony of the transformant was inoculated in 10 ⁇ l of sterile water, 5 ⁇ 1 of lysozyme (51) / ⁇ 1) was added, shaken at 30 ° C for 10 min, and placed in a liquid nitrogen phase for 1 min. After thawing at room temperature, it was directly used as a template.
- Primer a- Factor Hekou 3, A0X1 (Invitrigen easy select Pichia Expression Kit Manual) was used for PCR detection, and the L-sorbone dehydrogenase gene was detected.
- Transformant single colonies were inoculated in 50 ml BMGY medium and cultured at 30 ° C to A 6 . .
- nm «4.0 daily supplementation with methanol, ammonia, and induced expression for 6 days, the samples were centrifuged to collect the supernatant, and analyzed by SDS-PAGE. The results are shown in Figure 4, indicating that a specific band was detected at 45,000 Daltons. An expression product of the L-sorbone dehydrogenase gene was detected.
- the arrow points to rSNDH, and the molecular weight standards are 94000, 67000, 43000, 30000, and 20100.
- rSNDH L-sorbitan dehydrogenase
- the activity of rSNDH expressed by TOP10 fusion is shown in Figure 5, which indicates that the fusion rSNDH has enzymatic activity, and the enzymatic activity is 0.117 u / mg o, which is much higher than the crude enzyme activity reported in the literature (48.5 mu / mg).
- the unit of enzyme activity is defined as the amount of enzyme required to catalyze the reduction of luMol NAD per minute. Measurements have shown that this enzyme converts L-
- the pH range for the conversion of sorbone to 2 -KGA is 5.0-9. 0, of which the optimal action 13 ⁇ 4 is 7.5, as shown in FIG. 7.
- the rSNDH expressed by the fusion of the positive clone E. coli TOP10 was purified by Chelating-SFF chromatography, wherein the chromatography column: 1. 6 X 10cm, affinity ion: Ni 2+ , chromatography conditions: 50 mM PB buffer, pH 7. 0, Imidazole gradient.
- the purification result is shown in Fig. 6.
- the arrow indicates the purified rSNDH, the molecular weight is about 57000, and the molecular weight standards are 94000, 67000, 43000, 30000, 20100 in that order.
- the in vitro conversion test of L-sorbitan by rSNDH was performed according to the conventional method.
- the reaction system buffer: 50 mM PB, pH 8. 0, L-sorbone: 5 mg / ml, rSNDH enzyme: 0.08 mg / ral, NAD: 0 5mg / ml.
- the reaction was performed at 37 ° C for 5 hours, and the product 2-KGA was quantitatively determined by HPLC. The results are shown in Figures 8, 9, 10, and 11, indicating that rSNDH converts L-sorbone to 2-KGA in vitro, and the presence of coenzyme NAD is effective. Increase the conversion rate of 2-KGA.
- the standard concentration of 2-KGA is: 2mg / ml; the standard concentration of L-sorbone is: 2m g / ml; in the absence of coenzyme, the concentration of 2-KGA is: 0.86 m g / ml; In the presence of coenzyme, the production concentration of 2- KGA is: 1. 42 mg / ml. Transformation test in which no part in the reaction NAD verification LS- MS 2 -KGA production (electrospray ionization: ESI, electrospray voltage: 3.
- 0104 kinds of cultured ketocoronic acid bacteria and E. coli T0P10 with or without PBAD-rSNDH plasmid were mixed (10: 1) for a total of 3ml (containing L-sorbose 2.0%) inoculated into 20ml fermentation medium
- the final concentration of arabinose is 0.02%.
- Industrial applicability The present invention is based on Ketogulonigenium sp. WB0104 CCTCC.
- the genome-wide sequence of NO. M203094 was determined by gene annotation to predict the key enzyme gene related to 2-keto-L-gulonic acid synthesis-the L-sorbone dehydrogenase gene (irSNDH).
- the protein (enzyme) produced by expressing this gene in different vectors and hosts by molecular biology methods can effectively convert L-sorbone to 2-keto-L-gulonic acid (2-KGA) in vitro SNDH (including bodies, secreted proteins, and fusion proteins) expressed in any form has SNDH enzyme activity in vitro, and at the same time, when the engineered coli bacteria carrying the rSNDH-encoding gene of the present invention is co-cultured with 2-KGA transforming bacteria,
- the rSNDH of the present invention significantly improves the conversion rate of 2-KGA.
- the L-sorbone dehydrogenase gene of the present invention can be used to modify existing 2-KGA-producing bacteria and conduct targeted breeding to increase the yield of 2-KGA.
- the L-sorbone dehydrogenase gene of the present invention or the The mutant or fragment is inserted into the existing 2-KGA-producing bacteria genome to enhance the activity of L-sorbone dehydrogenase, or to express the L-sorbone dehydrogenase gene of the present invention by molecular biology means, and the immobilization Enzyme method or immobilized cell method to synthesize 2-KGA in vitro, or synthesize the protein corresponding to the L-sorbone dehydrogenase gene of the present invention for the production of 2-KGA, reduce the production cost of 2-KGA, and increase the 2-KGA
- the present invention has important industrial application prospects by reducing the environmental pollution, reducing the demand for energy during the production process, and improving the quality of products.
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US5352599A (en) * | 1988-01-14 | 1994-10-04 | Hoffmann-La Roche Inc. | Co-enzyme-independent L-sorbosone dehydrogenase of gluconobacter oxydans: isolation, characterization, and cloning and autologus expression of the gene |
US6197562B1 (en) * | 1993-03-08 | 2001-03-06 | Fujisawa Pharmaceutical Co., Ltd. | L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
CN1332249A (zh) * | 1994-02-25 | 2002-01-23 | 藤泽药品工业株式会社 | 2-酮-l-古洛糖酸的生产方法 |
WO2003008588A2 (fr) * | 2001-07-13 | 2003-01-30 | Basf Aktiengesellschaft | Procede de fabrication d'acide 2-ceto-l-gulonique et de vitamine c |
US20030087440A1 (en) * | 2000-04-05 | 2003-05-08 | Archer-Daniels-Midland Company | Ketogulonigenium endogenous plasmids |
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US5352599A (en) * | 1988-01-14 | 1994-10-04 | Hoffmann-La Roche Inc. | Co-enzyme-independent L-sorbosone dehydrogenase of gluconobacter oxydans: isolation, characterization, and cloning and autologus expression of the gene |
US6197562B1 (en) * | 1993-03-08 | 2001-03-06 | Fujisawa Pharmaceutical Co., Ltd. | L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
CN1332249A (zh) * | 1994-02-25 | 2002-01-23 | 藤泽药品工业株式会社 | 2-酮-l-古洛糖酸的生产方法 |
US20030087440A1 (en) * | 2000-04-05 | 2003-05-08 | Archer-Daniels-Midland Company | Ketogulonigenium endogenous plasmids |
WO2003008588A2 (fr) * | 2001-07-13 | 2003-01-30 | Basf Aktiengesellschaft | Procede de fabrication d'acide 2-ceto-l-gulonique et de vitamine c |
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