WO2005052160A1 - A L-sorbosone dehydrogenase as well as the encoding gene and the use thereof - Google Patents

Abstract

The present invention discloses a L-sorbosone dehydrogenase as well as the encoding gene and the use thereof. The L-sorbosone dehydrogenase provided by the invention is the protein with the amino acid residue sequence of SEQ ID NO:2 in sequence listing or the protein derived from SEQ ID NO:2, and sharing at least 80% homology with the amino acid residue sequence of SEQ ID NO:2 in sequence listing and having the same activity with SEQ ID NO:2. The gene encoding for the L-sorbosone dehydrogenase is one of the following nucleotide sequences: 1)SEQ ID NO:1 in sequence listing; 2) the polynucleotide encoding the protein sequence of SEQ ID NO:2 in sequence listing; 3)the DNA sequence sharing aboye 80% homology with the DNA sequence defined by SEQ ID NO:1 in sequence listing and encoding the saine functional protein. When the E. coli engineering strain carried the gene encoding for the L-sorbosone dehydrogenase of the invention is co-cultivated with the bacteria converting 2-KGA, the rSNDH of the invention can significantly improve the efficiency of conversion to 2-KGA .

Description

 L-sorbone dehydrogenase, coding gene and application thereof

 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). There are many known microorganisms that convert L-sorbitol / sorbose to 2-KGA. For example, NRRL B-21627 (US Patent No. 5834231) used by Stoddard et al., DSM4025 (European patent EP9611500. 8) mentioned by T. Hoshino et al., SCB329 et al. Mentioned by Yin Guanglin et al.

 There are many literatures on the use of microorganisms capable of converting L-sorbose to 2-KGA. U.S. Patent 3907.639 discloses belonging to the genus Acetobacter, Pseudomonas, Serratia, Staphylococcus, Aerobacter, Microorganisms of Alkali, Penicillium, Candida, and Gluconobacter have the above-mentioned transformation activity. Kitamura et al. (Europe J. Appl. Microbiol. 2. 1. 1975) reported in Gluconobacter oxydans

The enzymatic activity of 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. It consists of 4 subunits and is derived from Gluconobacter oxydans. Approximately 47,000 Daltons. At the gene level, only Yashimasa Saito et al. (Biotechnology and Bioengineering, Vol, 58, NOS APRIL 20 / May 5 1998) cloned the SNDH gene from Gluconobacter oxydans T-100, the SNDH 甶 498 Amino acid composition.

In contrast to the seven-step chemical reaction for the production of vitamin C by chemical synthesis, 2-KGA is produced from sorbitol by 2-step fermentation with microorganisms, and then vitamin C is produced by methyl esterification. But in the two-step fermentation, 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. Therefore, the current industrial production and application are based on the symbiosis of two bacteria, Π subtilis, Bacillus thuringiensis, Bacillus giant Acidogenic bacteria co-ferment. However, there are still many problems in actual production, such as low conversion rate and more complicated process control. SUMMARY OF THE INVENTION 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, named rSNDH, 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. A protein derived from SEQ ID Na: 2 with% homology and the same activity as SEQ ID No _: 2.

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 A protein derived from SEQ ID Na _: 2 having at least 90% homology and having the same activity as SEQ ID a: 2; it is particularly preferred to pass the amino acid residue sequence of SEQ ID No _: 2 through one or several amino acid residues A protein derived from SEQ ID Ns _: 2 with the substitution, deletion, or addition of a base and having the same activity as SEQ ID Na: 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:

1) SEQ ID Na: 1 in the sequence listing;

2) a polynucleotide encoding the protein sequence of SEQ ID No _: 2 in the sequence listing;

 3) It has a homology of more than 80% with the DNA sequence defined by SEQID No: 1 in the Sequence Listing, and encodes the same functional protein. Among them, it is preferably 90% of the DNA sequence defined by SEQID Na: 1 in the Sequence Listing. The above homology, and the DNA sequence encoding the same functional protein.

sp. )WB0104 CCTCC No. M203094, 由 1290个碱基组成， 该基因的开放阅读框架 (0RF)为自 5' 端第 1 到第 1290位碱基。 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.

Among them, 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.

Among them, 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, and SEQ ID Ns: 6 is composed of 24 bases, SEQ ID Na _: 7 consists of 35 bases, and SEQ ID Na _: 8 consists of 24 bases.

 The positive clone may be E. coli DH5α, E.coli BL21 (DE3), E.coli TOP10, or Pichia X-33 containing an L-sorbone dehydrogenase-encoding gene.

 0-9. 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.

In the present invention, Keto _g ulonigeniwn sp.) WB0104 CCTCC

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 6 Chelating-SFF chromatographic purification results of rSNDH expressed by the positive clone E. coli TOP10 fusion

 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.

 Example 1.Obtainment of L-sorbone dehydrogenase gene r0MW

 North China Pharmaceutical Group Co., Ltd. ketocoronic acid strain for vitamin C production

{Ketogulonigeniwn sp.) WB0104 CCTCC No. M203094, which is used in fermentation to convert L-sorbose to 2-keto-L-gulonic acid, which is an important precursor of vitamin C. 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 μ m; mobile phase: acetonitrile-0. 1% 三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. 39) was used to extract genomic DNA and determine the genome sequence according to the whole genome shotgun method (Fleischmann RD, Adams MD, et al. Science. 1995. 269 (5223): 496-512), and carried out a total of 35,000 reactions; the total length of the sequencing reached 18M, covering nearly 6 times of its chromosomal DNA. Then use PHRED-PHRAP, GLIMMER software to assemble annotations and perform 0RF prediction. According to the gene annotation results, the 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. The DNA sequence of 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.

 Example 2.Expression of L-sorbone dehydrogenase gene rfW

 1.PET-11B (Strategene) express rSNDH

 PET11B expression primers:

 5 'Primer: GACATATGAGCGTTCTGGCCAAATTC (SEQ ID Ns: 3)

3. Primer: CAGGATCCTTACGCAGCGGAAATC (SEQ ID Na _: 4)

PCR was performed using genomic DNA of tKetogulonigenium sp. WB0104 (CCTCC No. M203094) as a template. The 50 μΐ system contained a final concentration of 1.5 ol / L MgCl ₂ and 0.2 ol / L. dNTPs, 0.2 μmol / 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; 72Ό 10 minutes. 3Kb。 The PCR product electrophoresis results are shown in Figure 1. In the figure, 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 DH5α, plated (containing AMP 100 μg / ml), picked clones and identified by enzyme digestion to obtain a positive clone plasmid PET11B-rSNDH.

 Extract the plasmids from the positive clones (containing the plasmid PET11B-rSNDH) and transform them for expression with the host E. coli BL21 (DE3). Single colonies were inoculated into 5ml LB medium (containing AMP100 μg / ml) and cultured overnight at 37 ° C. Press 1% The inoculation amount was inoculated into 50ml LB medium (containing AMP100 μg / ml) and cultured to 0D = 0. 6-1, induced by IPTG (final concentration 1.0ramol / L), and continued to culture for 4 hours, and the cells were collected by centrifugation, The results of SDS-PAGE were shown in Fig. 2, which showed that a specific band was detected at 45,000 Daltons, indicating that the expression product of the L-sorbone dehydrogenase gene was detected. In the figure, 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.

 2.PBAD / THI0-T0P0 expresses SNDH

 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.釆 Use PBAD promoter and arabinose as inducer.

 PBAD / THI0-T0P0 expression primers:

5. Primer: GAGAATTCGATGAGCGTTCTGGCCAAATTCAC (SEQ ID Na _: 5) 3. Primer: GATTACGCAGCGGAAATCCGCCAG (SEQ ID Na: 6)

 PCR reaction conditions:

 94 ° C for 5 minutes, (94 ° C for 1 minute, 59 ° C for 1 minute, 72 ° C for 2 minutes) 30 cycles, 72 ° C for 10 minutes.

 PCR reaction system:

A PCR reaction was performed using genomic DNA of ketocoronic acid J (etogulonigeniwn sp.) WB0104 (CCTCC No. M203094) as a template. The 50 μΐ system contained a final concentration of '1.5 ol / L MgCl ₂₎ 0.2 mmol / L dNTPs, 0.2 μmol / L 5 'bow | each and 3, primers, lOmolol / LTris-HCl, 2uTaq DNA polymerase. 3Kb。 PCR product electrophoresis results shown in Figure 1, indicating that the PCR product size is 1.3Kb. In the figure, 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. Single colonies were inoculated into 5ml LB medium (containing AMP100 g / ml) and incubated at 37 ° C overnight Extraction with QIAGEN's MINI plasmid extraction column The plasmid was verified by enzyme digestion, and a plasmid that was correctly inserted into PBAD-rSNDH was found, and it was determined to be a positive clone.

 Pick a single colony of positive clones and inoculate it into 5ml LB medium (including P100 μg / ml) 37 Ό culture overnight, inoculate into 50ml LB medium (containing AMP100 μg / ml) at 1% inoculation volume and culture to 0D = 0.6- 1. Induced with arabinose (final concentration 0.02%), continued to culture for 4 hours, centrifuged to collect bacteria, and tested by SDS-PAGE. The results are shown in Figure 3, indicating that a specific band was detected at 57000 Daltons. This indicates that the expression product of the L-sorbone dehydrogenase gene was detected. In the figure, a is PBAD-rSNDH, and the molecular weight standards (marker) are 94000, 67000, 43000, and 30000 from large to small; the arrows indicate the bands of interest.

 3. Expression of SNDH in Pichia yeast

 (1) Introduction of L-sorbone dehydrogenase gene into yeast expression vector

 The INVITR0GEN Pichia picz α expression system was used to construct the expression vector piczaA / SND expressing rSNDH in Pichia pastoris.

 Design primers as follows:

5 'primer: CACTCGAGAAAAGA ATGAGCGTTCTGGCCAAATTC (SEQ ID Na _: 7) 3' primer: CACTCGAGTTACGCAGCGGAAATC (SEQ ID NQ: 8).

 The PCR reaction conditions are as follows:

 94 ° C for 3 minutes and then 30 cycles (94 ° C for 1 minute, 62 ° C for 1 minute, 72 ° C for 2 minutes), 72 ° C for 10 minutes.

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. Coated on LB plate (containing ZE0CIN 25 μg / 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.

 (2) Linearization of the recombinant plasmid

 20 μg picz a A-rSNDH was digested with Sac / single enzyme, and then expanded to 300 μ1 with water. An equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) was extracted once, and the supernatant was added. DNA was precipitated by 1/10 volume of 3M NaAc and 2.5 times volume of absolute ethanol, washed with 70% ethanol, dried under vacuum, and dissolved in 20 μ1 TE.

 (3) Preparation of competent yeast host cells

Inoculated Pichia expression hosts X- 33 in 5ml YPD medium, 3CTC shaken overnight, the next day to 500ml fresh YPD medium, 30 ° C with shaking to A _6. . ™ «1.3- 1 · 5, 4 ° C 5000rpm The heart was discarded for 5 minutes, and the supernatant was discarded, and the cells were washed and resuspended in 500 ml, 250 ml of ice-cold sterilized water and 20 ml of ice-cold 1M sorbitol, and the cells were resuspended in 1 ml of ice-cold 1M sorbitol to prepare competent cells.

 (4) Electric conversion

Mix 100 μl competent cells with 5- 10 μ _§ linearized recombinant plasmid, transfer to a 0.2 cm electric rotor, and ice-bath for 5 min. In a Bio-Rad Gene Pulser electro-rotator at 1500 V, 25 μΨ, 200 Ω, transform immediately and add immediately. 1ml sorbitol in 1ml ice bath was taken and spread on 100 μg of YPDS containing ZEOCIN (Invitrogen) 100 μg / ml selection plate and incubated at 30 ° C for 2 to 3 days. Transformants appeared.

 (5) Direct PCR detection of positive clones

 A single colony of the transformant was inoculated in 10 μl of sterile water, 5 μ1 of lysozyme (51) / μ1) 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.

 (6) Expression experiment

Transformant single colonies were inoculated in 50 ml BMGY medium and cultured at 30 ° C to A ₆ . . _{When 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. In the figure, the arrow points to rSNDH, and the molecular weight standards are 94000, 67000, 43000, 30000, and 20100.

 Example 3. Functional verification of L-sorbone dehydrogenase gene

 1. Using NAD as coenzyme, determination of enzyme activity

References (Agri. Biol. Chem. 54 (5) 121, 1218, 1990) and SugiSANA et al. (Agric, Biol, Chem, 55, 363-370, 1991), with L-sorbone as the substrate and AD as the electron Acceptor, and measured the light absorption value at a wavelength of 340 nm. The established method for determining the enzymatic activity of L-sorbone as a substrate and NAD as a coenzyme: The basic reaction solution was composed of L-sorbone 2.0 Omg, NAD 0. ½g, PB 50mM, pH7.0, at 25 Under the condition of a water bath of ° C, L-sorbitan dehydrogenase (rSNDH) crude enzyme expressed by the three expression systems in Example 2 was added separately 0.1 μg / ml 10 ul, total volume 820 ul, and positive clone yeast X- 33 was selected. Supernatant, positive clones E. coli BL21 (DE3) and TOP10 bacterial cells were broken to remove the supernatant, and the change rate of light absorption at 340 nm was measured. The results showed that rSNDH expressed in various forms was active. 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 1¾ is 7.5, as shown in FIG. 7.

 2. rSNDH in vitro transformation of L-sorbone

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. In the figure, 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. 0KV, electrospray interfaces drying gas: N _2, flow rate: 250L / hr, Desolvation temperature: 190 ° C, collision-induced dissociation voltage: 30V, ion source temperature: 120Ό), and the results are shown in Figs. 12 and 13, which demonstrate the characteristic 2-KGA absorption.

 Example 4.Co-cultivation of rSNDH-expressing host with ketocolic acid WB0104

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 Medium of fermentation bottle: L-sorbose 8.0%, yeast extract 0.2%, corn pulp 2.0%, urea 1.2%, K¾P0 ₄ 0.1%, MgS0 _4. 7Η ₂ 00. 5%。 01%, light calcium carbonate 0.5%. Incubate at 29 ° C and 200 rpm, measure the colic acid content at 24 h, sample the fermentation broth volume at about 40 h, measure the L-sorbose (anthrone method) and 2-keto-L-gulonic acid content (HPLC method ), Determine the fermentation end sampling time according to the L-sorbose content, and calculate the final conversion rate. The results are shown in Table 1, indicating that the expression of r ^ SMW significantly increased the conversion rate of 2-KGA.

Amount of colic acid (g / ml) * Fermentation volume (ml) * 0.928

 Conversion rate (%) = * 100%

L-sorbose (g) Table 1 Conversion of L-sorbose by ketocolic acid WB0104 and PBAD-rSNDH

Note: 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. For example, 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.

Claims

Rights request A L- sorbosone dehydrogenase, is having the sequence of SEQ ID _No: 2 in the Sequence Listing or a protein amino acid residue sequence in SEQ ID _No: 2 amino acid residue sequence having at least 80% homology And a protein derived from SEQ ID Na: 2 having the same activity as SEQ ID No _: 2.  2. The L-sorbone dehydrogenase according to claim 1, wherein the L-sorbone dehydrogenase is a protein having the amino acid sequence of SEQ ID Na: 2 in the sequence listing. 3. The L-sorbone dehydrogenase according to claim 1, characterized in that: said sequence has at least 80% homology with the amino acid residue sequence of SEQ ID Na: 2 in the sequence listing and has SEQ ID Ns : 2 The protein derived from SEQ ID No _: 2 with the same activity is a protein derived from SEQ ID Ne: 2 that has at least 90% homology with SEQ ID Na _: 2 and has the same activity as SEQ ID No .. 2.  4. The L-sorbone dehydrogenase according to claim 1, wherein the L-sorbone dehydrogenase is a sequence that will have at least 80% homology with SEQ ID Na: 1 in the sequence listing. The resulting protein is expressed in host bacteria E. coli or Pichia.  5. A gene encoding L-sorbone dehydrogenase, which is one of the following nucleotide sequences − 1) SEQ ID Na in the sequence list: 1; 2) a polynucleotide encoding the protein sequence of SEQ ID No _: 2 in the sequence listing; 3) A DNA sequence with more than 80% homology to the DNA sequence defined by SEQ ID No _: 1 in the Sequence Listing, and encoding the same functional protein.  6. An expression vector and a cell line containing the gene according to claim 5.  7. The expression vector and cell line according to claim 6, wherein the cell line is E. coli or Pichia yeast containing a gene encoding L-sorbone dehydrogenase.  8. The expression vector and cell line according to claim 7, wherein the cell line is an immobilized cell.  9. A method for expressing L-sorbone dehydrogenase, which comprises introducing a gene encoding L-sorbone dehydrogenase amplified by genomic DNA of ketocoronic acid bacteria iKetogulonigeniim sp. WB0104 CCTCC No. M203094 as a template The host bacteria were expressed to obtain positive clones, and the positive clones were cultured to express L-sorbone dehydrogenase.  10. The method according to claim 9, characterized in that: the ketocolic acid bacteria  (Ketogulonigenium sp.) WB0104 CCTCC No. M203094 Amplification of genomic DNA as a template The pair of primers of the L-sorbone dehydrogenase gene are SEQ ID No _: .3 and SEQ ID Na _: 4, SEQ ID No _: 5 and SEQ ID Na _: 6 or SEQ ID Na _: 7 and SEQ ID Ns: 8.  11. The method according to claim 9, characterized in that: the positive clones are E. coli DH5 ct, E. coli BL21 (DE3), E.coli TOP10, or Pichia X which contain an L-sorbone dehydrogenase gene. -33.  12. Use of the L-sorbone dehydrogenase according to any one of claims 1 to 4 in the process of converting L-sorbone to 2-KGA.  13. Use of a gene encoding L-sorbone dehydrogenase according to claim 5 in the process of converting L-sorbone to 2-KGA.  14. Use of an expression vector and a cell line containing a gene encoding an L-sorbone dehydrogenase according to any one of claims 6 to 8 in the process of converting L-sorbone to 2-KGA.  15. Use of the gene according to claim 5 in enhancing L-sorbone dehydrogenase activity.