CN114934027B - L-sorbose dehydrogenase mutant capable of improving 2-KLG yield - Google Patents
L-sorbose dehydrogenase mutant capable of improving 2-KLG yield Download PDFInfo
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Abstract
The invention discloses an L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG, belonging to the technical fields of genetic engineering and enzyme engineering. The invention takes the L-sorbose dehydrogenase from Gluconobacter oxydans as a starting sequence, and the L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG is obtained through site-directed mutagenesis. The 2-KLG production of mutants V368C, V368L, V S and V368T, respectively, reached 2.5g/L, 3.1g/L, 2.5g/L and 3.0g/L, respectively, 1.1, 1.4, 1.1 and 1.3 times that of the wild-type L-sorbose dehydrogenase strain, as determined by fermentation.
Description
Technical Field
The invention relates to an L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG, belonging to the technical fields of genetic engineering and enzyme engineering.
Background
Vitamin C, also known as ascorbic acid, is a vitamin essential to the human body. Is widely applied to the fields of food, beverage, pharmacy, cosmetics and feed. 2-keto-L-gulonic acid (2-KLG) is a direct precursor substance for the production of vitamin C. At present, the industrial production of 2-KLG utilizes a three-bacterium two-step fermentation method. Compared with one-step fermentation, the three-bacteria two-step fermentation method has the disadvantages of high energy consumption, high material consumption, difficult precise regulation and control of mixed bacteria fermentation, difficult breeding and the like. The search for a one-step fermentation to produce 2-KLG is a common goal of researchers. Since the production of 2-KLG from sorbitol involves only 3 enzymatic processes, D-sorbitol dehydrogenase, L-sorbose dehydrogenase and L-sorbosone dehydrogenase, respectively. Therefore, currently, researchers mostly conduct one-step bacterial research through metabolic engineering. The main method is to carry out heterologous expression on key enzyme genes in the 2-KLG synthesis path to produce 2-KLG, but the effect is not comparable with industrial production. Current studies indicate that L-sorbose dehydrogenase may be a key rate-limiting enzyme in the production of 2-KLG. NCBI alignment shows that L-sorbose dehydrogenase belongs to the glucose-methanol-choline oxidoreductase family. Such enzymes comprise two domains, the N-terminal domain belonging to the cofactor binding domain and the C-terminal domain belonging to the substrate binding domain. At present, less researches are carried out on the modification of the L-sorbose dehydrogenase, and the effective modification of the L-sorbose dehydrogenase can possibly improve the production strength of the 2-KLG.
Disclosure of Invention
The inventor obtains a strain of gluconobacter oxydans (Gluconobacter oxydans) WSH-004 which can directly convert D-sorbitol into 2-keto-L-gulonic acid (2-KLG) through screening, and identifies L-sorbose dehydrogenase and L-sorbosone dehydrogenase in the strain. The invention prepares the L-sorbose dehydrogenase mutant which can improve the yield of 2-KLG by means of genetic engineering and enzyme engineering.
The invention provides an L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG, which takes L-sorbose dehydrogenase derived from gluconobacter oxydans (Gluconobacter oxydans) WSH-004 as a starting sequence and mutates valine at position 368 into cysteine, leucine, serine or threonine.
In one embodiment, the amino acid sequence of the starting sequence is shown in SEQ ID NO. 1.
In one embodiment, the mutant is a valine (Val) to cysteine (Cys) at position 368, designated V368C, having the amino acid sequence shown in SEQ ID No. 2.
In one embodiment, the mutant is a valine (Val) to leucine (Leu) at position 368, designated V368L, and has the amino acid sequence shown in SEQ ID NO. 3.
In one embodiment, the mutant is a valine (Val) to serine (Ser) at position 368, designated V368S, having the amino acid sequence shown in SEQ ID No. 4.
In one embodiment, the mutant is a valine (Val) to threonine (Thr) at position 368, designated V368T, and has the amino acid sequence shown in SEQ ID No. 5.
The invention also provides genes encoding the mutants.
In one embodiment, the nucleotide sequence encoding mutant V368C is one in which codon 368 is replaced with UGC by GUG based on SEQ ID NO. 6.
In one embodiment, the nucleotide sequence encoding mutant V368L is based on SEQ ID NO.6, wherein codon 368 is replaced by GUG.
In one embodiment, the nucleotide sequence encoding mutant V368S is one in which codon 368 is replaced by UCG from GUG based on SEQ ID NO. 6.
In one embodiment, the nucleotide sequence encoding mutant V368T is based on SEQ ID NO.6 with the substitution of codon 368 for ACC by GUG.
The invention also provides a recombinant plasmid carrying the gene.
In one embodiment, the recombinant plasmid is a pMD 19-based starting plasmid.
The invention also provides a microbial cell expressing the L-sorbose dehydrogenase mutant or carrying the gene.
In one embodiment, the host cell of the microbial cell is a bacterial or fungal cell.
In one embodiment, the bacterium is a gram negative bacterium or a gram positive bacterium.
In one embodiment, the plasmid is pMD19-T.
In one embodiment, the microbial cells express the L-sorbose dehydrogenase, L-sorbose dehydrogenase mutant and key enzyme genes in the 2-KLG synthesis pathway using E.coli as a host and pMD19-T as an expression vector.
In one embodiment, the expression of the L-sorbose dehydrogenase gene and the L-sorbosone dehydrogenase gene is regulated with cspA promoter.
In one embodiment, the microbial cell hosts E.coli BL21 (DE 3).
The invention also provides application of the L-sorbose dehydrogenase mutant in producing 2-keto-L-gulonic acid.
In one embodiment, the use is to culture recombinant E.coli expressing the L-sorbose dehydrogenase mutant in LB medium at 30-37℃at 150-250 rpm to OD 600 Preparation of seed solution =3, transfer to sorbitol medium and fermentation at 30-37 ℃.
The invention also provides recombinant escherichia coli capable of producing 2-KLG, wherein the recombinant escherichia coli is provided with L-sorbose dehydrogenase and L-sorbosone dehydrogenase shown in any one of SEQ ID NO. 2-5.
In one embodiment, the gene for L-sorbosone dehydrogenase is shown in SEQ ID NO. 7.
In one embodiment, the recombinant E.coli is host BL21 (DE 3).
The invention also provides application of the microbial cells or the recombinant escherichia coli in the aspect of producing vitamin C or a precursor thereof.
The beneficial effects are that:
the invention aims at oxidizing L-sorbose dehydrogenase in gluconobacter (Gluconobacter oxydans) WSH-004, and modifies the amino acid sequence of the L-sorbose dehydrogenase through site-directed mutagenesis biotechnology to finally obtain 4L-sorbose dehydrogenase mutants V368C, V368L, V S and V368T with improved enzyme activity, and the 2-KLG yield reaches 2.5g/L, 3.1g/L, 2.5g/L and 3.0g/L which are 1.1, 1.4, 1.1 and 1.3 times of the strains expressing wild type L-sorbose dehydrogenase.
Drawings
FIG. 1 is a diagram showing construction of a site-directed mutagenesis-engineered L-sorbose dehydrogenase expression vector (pMD 19-cspA-SNDH-SDH).
FIG. 2 is a schematic representation of plasmid pMD19-cspA-SNDH-V368L in one embodiment of the invention.
FIG. 3 is a diagram showing the fermentation production of 2-KLG by high performance liquid chromatography detection of different strains.
FIG. 4 is a graph showing comparison of the production of 2-KLG by different strains.
Detailed Description
1. Culture medium and reagent:
LB medium: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride. The preparation of the solid culture medium also needs to be added with 18g/L agar powder.
Sorbitol medium: 10g/L of yeast powder and 50g/L of sorbitol. 18g/L agar powder was added to prepare a sorbitol solid medium.
Sorbitol medium: 10g/L of sorbose, 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride.
2. Cloning of the gene for L-sorbose dehydrogenase:
gluconobacter oxydans WSH-004 (CCTCC M2019481, strain disclosed in paper "High-Throughput Screening of a 2-Keto-L-Gulonic Acid-Producing Gluconobacter oxydans Strain Based on Related Dehydrogenases") was inoculated into sorbitol medium for cultivation. The bacterial cells were collected, the genome was extracted using Ezup-column bacterial genomic DNA extraction kit (purchased from Shanghai Biotechnology Co., ltd.), and L-sorbose dehydrogenase was amplified using PCR, and the amplification primers contained the homology arm sequences required for the ligation of plasmids. PCR was performed using 2X Phanta Max Master Mix (available from Nanjinozan Corp.). The PCR product was recovered using a SanPrep column type DNA gel recovery kit (available from Biotechnology (Shanghai) Co., ltd.).
3. Construction of L-sorbose dehydrogenase plasmid and expression of Gene:
1) The vector was amplified by PCR to linearize the vector and carry a homology arm sequence complementary to the amplified L-sorbose dehydrogenase gene.
2) The linearized vector and L-sorbose dehydrogenase were seamlessly ligated using the Information-Cloning kit (available from Nanjinopran Corp.) to construct a complete plasmid.
3) Transferring the constructed plasmid into target competence, coating the plasmid onto a plate containing corresponding antibiotics, and selecting positive clones for sequencing.
4. Fermenting the strain expressing the L-sorbose dehydrogenase and the mutant plasmid thereof:
1) The correct single colony was inoculated into LB medium and cultured overnight to prepare seed solution.
2) Seed solution was inoculated in an inoculum size of 5% to a sorbose medium, and after fermentation culture for 72 hours, the fermentation broth was collected.
3) The collected fermentation broth was centrifuged at 12000 Xg for 3 minutes, and the supernatant was collected for detection of 2-KLG production.
5. Detecting the yield of 2-KLG in the fermentation liquor by utilizing high performance liquid chromatography: the measurement was performed using high performance liquid chromatography. High performance liquid chromatography detection conditions: chromatographic column: aminex HPX-87H column (300 mm. Times.7.8 mm; bio-Rad, hercules, calif.); column temperature: 40 ℃; mobile phase: 5mM H 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 0.5mL/min, detected using a parallax detector.
The enzyme activity of the L-sorbose dehydrogenase and the mutant thereof is determined by a spectrophotometer capable of controlling temperature and monitoring absorbance in real time, a cuvette with a light path of 1.0cm is used, and the reaction system is as follows: substrate 25mM, phenazine Methosulfate (PMS) 2.5mM, sodium 2, 6-Dichloroindophenol (DCIP) 0.5mM, enzyme final concentration 0.04g/L, buffer 50mM Tris-HCl pH 7.0. The unit enzyme activity (U) is defined as the amount of enzyme required to reduce 1. Mu. Mol of DCIP in 1min at 37℃and pH 7.0.
Example 1: construction and expression of wild L-sorbose dehydrogenase gene clone and plasmid
(1) Construction of L-sorbose dehydrogenase expression vector:
from the deposited glycerol tubes, gluconobacter oxydans WSH-004 was inoculated into sorbitol medium, cultured at 37℃for 48h at 220rpm, centrifuged at 4000rpm, and the cells were collected, and Gluconobacter oxydans WSH-004 genome was extracted, and amplification of L-sorbose dehydrogenase gene (gene sequence shown in SEQ ID NO. 6) was performed using the primer pair F1/R1. And recovering the PCR product. Primer pair F1/R1 is as follows:
F1：GGATTTCGTAATGACGAGCGGTTTTGATTACATCGTTGTCG；
R1：ATCTGCAGAATTCTCAGGCGTTCCCCTGAATGAAATCCGC。
the production of the 2-KLG key gene L-sorbosone dehydrogenase (gene sequence shown in SEQ ID NO. 7) was amplified using the primer pair F2/R2 and the PCR product was recovered. Primer pair F2/R2 is as follows:
F2：AAAGGTAATACACTATGAATGTTGTCTCAAAGACTGTATCTTTACCG；
R2：AAACCGCTCGTCATTACGAAATCCAGTGCGAACGTTTG。
the pMD19 plasmid (pMD 19-cspA) carrying cspA promoter (SEQ ID NO. 8) was amplified linearly using primer pair F3/R3, (plasmid disclosed in High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase) and the PCR product was recovered. Primer pair F3/R3 is as follows:
F3：GGGGAACGCCTGAGAATTCTGCAGATATCCATCACACTGG；
R3：CAACATTCATAGTGTATTACCTTTAATAATTAAGTGTGCCTTTCGG。
the PCR reaction system is as follows: 25. Mu.L 2X Phanta Max Master Mix, 1. Mu.L forward primer (10. Mu. Mol. L) -1 ) 1. Mu.L of reverse primer (10. Mu. Mol.L) -1 ) 1 μl of template DNA was added with distilled water to 50 μl. The PCR amplification procedure of L-sorbose dehydrogenase and L-sorbosone dehydrogenase was set as follows: firstly, pre-denaturation at 95 ℃ for 3min; then enter 30 cycles: denaturation at 95℃for 30s, annealing at 56℃for 30s, and extension at 72℃for 1min; finally, the temperature is 72 ℃ for 5 minutes, and the temperature is kept at 4 ℃. The pMD19-cspA linearized vector PCR amplification procedure was set as follows: firstly, pre-denaturation at 95 ℃ for 3min; then 25 cycles are entered: denaturation at 95℃for 30s, annealing at 56℃for 30s, and extension at 72℃for 3min; finally, the mixture is extended for 10min at 72 ℃ and is kept at 4 ℃.
40ng of L-sorbose dehydrogenase and 40ng of the pcr product of L-sorbosone dehydrogenase were mixed with 20ng of pMD19-cspA linearization vector and ligated using the information-Cloning kit to construct plasmid pMD19-cspA-SNDH-SDH (FIG. 1). mu.L of pMD19-cspA-SNDH-SDH ligation product was transferred into E.coli BL21 (DE 3) competent, ice-bath for 30min, heat-shock at 42℃for 90s, ice-bath for 5min, 1ml of LB medium was added, culturing was carried out at 37℃and 220rpm for 45min, centrifugation was carried out at 3000rpm for 3min, the supernatant was removed, the bacterial body was resuspended in 100. Mu.L of LB medium, spread on LB plates containing 100mg/L ampicillin, and cultured overnight. The following day, positive clones were selected for sequencing to verify if the plasmid was correct. The strain with the correct sequence was designated as WT.
(2) Expression of L-sorbose dehydrogenase:
the correctly sequenced clones were transferred to 10ml LB containing 100mg/L ampicillin and cultured overnight to OD 600 =3, seed solution was prepared. The cultured seed solution was transferred to 25ml of sorbose medium containing 100mg/L ampicillin at 5% (OD after inoculation) 600 =0.153), cultured at 30 ℃ for 72 hours, and the fermentation broth was collected for product yield detection.
Example 2: preparation of L-sorbose dehydrogenase mutant
(1) Preparation of single mutations
Primers for introducing V368C, V368L, V S and V368T mutations were designed and synthesized respectively according to the pMD19-cspA-SNDH-SDH plasmid sequence constructed in example 1, site-directed mutagenesis was performed on the gene sequence of L-sorbose dehydrogenase, and the coding genes of the L-sorbose dehydrogenase mutants were confirmed to be correct by sequencing respectively; the vector carrying the mutant gene was introduced into E.coli BL21 (DE 3) for fermentation.
PCR amplification of the site-directed mutant encoding genes: the recombinant plasmid carrying the mutant gene is amplified by using the PCR technology and taking the expression vector pMD19-cspA-SNDH-SDH plasmid carrying the gene encoding the wild type L-sorbose dehydrogenase as a template.
The site-directed mutagenesis primer pair F4/R4 for introducing the V368C mutation is:
F4：GAGGCTGGGtgcACGTCCGTTCCCAAGGGAGCG (underline mutant base)
R4：TGGGAACGGACGTgcaCCCAGCCTCAGCCCCAGC (underline mutant base)
The site-directed mutagenesis primer pair F5/R5 for introducing the V368L mutation is:
F5：GAGGCTGGGcttACGTCCGTTCCCAAGGGAGCG (underlined mutant base))
R5：TGGGAACGGACGTaagCCCAGCCTCAGCCCCAGC (underline mutant base)
The site-directed mutagenesis primer pair F6/R6 for introducing the V368S mutation is:
F6：GAGGCTGGGtcgACGTCCGTTCCCAAGGGAGCG (underline mutant base)
R6：TGGGAACGGACGTcgaCCCAGCCTCAGCCCCAGC (underline mutant base)
The site-directed mutagenesis primer pair for introducing the V368T mutation is F7/R7:
F7：GAGGCTGGGaccACGTCCGTTCCCAAGGGAGCG (underline mutant base)
R7：TGGGAACGGACGTggtCCCAGCCTCAGCCCCAGC (underline mutant base)
The PCR system was the same as in example 1. The mutant plasmid PCR amplification procedure was set as follows: firstly, pre-denaturation at 95 ℃ for 3min; then 25 cycles are entered: denaturation at 95℃for 30s, annealing at 56℃for 30s, and extension at 72℃for 5min; finally, the mixture is extended for 10min at 72 ℃ and is kept at 4 ℃.
(2) Expression and validation of mutants
Verification of L-sorbose dehydrogenase mutant was performed according to the method of example 1, step (1). The strain expressing the mutant containing V368C was designated as M1, the strain expressing the mutant containing V368L (plasmid map see fig. 2) was designated as M2, the strain expressing the mutant containing V368S was designated as M3, and the strain expressing the mutant containing V368T was designated as M4. Culturing the recombinant strain in TB medium at 37deg.C to OD 600 The temperature is reduced to 20 ℃ and IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM/L is added for further culture for 20 hours, bacterial cells are collected by centrifugation, the enzyme is purified under the conditions of 37 ℃ and pH7.0 after the cells are broken, and the enzyme activity test is carried out on the purified wild type enzyme and mutant in vitro, so that the specific enzyme activity of the WT is 5400+/-400U/mg, and the specific enzyme activities of V368C, V368L, V S and V368T are 1.43, 1.83, 1.39 and 1.74 times of the WT respectively.
Example 3: l-sorbose dehydrogenase and mutant fermentation thereof for producing 2-KLG
The construction in example 1 and example 2 was followedStrains (WT, M1, M2, M3 and M4) were inoculated into LB supplemented with 100mg/L ampicillin, and cultured overnight at 37℃and 220rpm to OD 600 =3, seed solution was prepared. The next day, seed solution was inoculated in 5% in sorbitol medium and fermented at 30℃for 72 hours, the fermentation broth was collected, centrifuged at 12000 Xg for 3 minutes, the precipitate was discarded, and the supernatant was collected for detection of 2-KLG production.
The 2-KLG yield in the fermentation broth was determined using a high performance liquid chromatography parallax detector. Using 5mM H 2 SO 4 As a mobile phase, an Aminex HPX-87H column (300 mm. Times.7.8 mm; bio-Rad, hercules, calif.) was used at a flow rate of 0.5mL/min and a column temperature of 40℃for detection, the detection results are shown in FIG. 3. The results showed that the 2-KLG yield after 72 hours of fermentation of M1, M2, M3 and M4 reached 2.5g/L, 3.1g/L, 2.5g/L and 3.0g/L (OD 600 Up to 3.733, 3.796, 3.859 and 3.805), the 2-KLG yields were WT (2-KLG yield 2.2g/L, OD, respectively 600 Up to 3.799) 1.1, 1.4, 1.1 and 1.3 times (fig. 4).
Comparative example 1:
primers were designed according to the same strategy as example 2, and mutants V368D, V368E, V368F, V368G, V368K, V368M, V368P, V368Q, V R, V W, and V368Y were constructed, respectively. Mutants were prepared and expressed as in example 2, and fermented and tested as in example 3. The results are shown in Table 1.
TABLE 1 production of 2-KLG by different single mutants
| Mutant | 2-KLG(g/L) |
| V368D | 2.06297 |
| V368E | 0.85952 |
| V368F | 2.067019 |
| V368G | 0.677537 |
| V368K | 2.006918 |
| V368M | 2.175933 |
| V368P | 1.803614 |
| V368Q | 0.700799 |
| V368R | 1.535407 |
| V368W | 2.18846 |
| V368Y | 1.811543 |
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> L-sorbose dehydrogenase mutant capable of improving 2-KLG yield
<130> BAA220025A
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 531
<212> PRT
<213> Gluconobacter oxydans
<400> 1
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Val
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 2
<211> 531
<212> PRT
<213> artificial sequence
<400> 2
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Cys
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 3
<211> 531
<212> PRT
<213> artificial sequence
<400> 3
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Leu
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 4
<211> 531
<212> PRT
<213> artificial sequence
<400> 4
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Ser
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 5
<211> 531
<212> PRT
<213> artificial sequence
<400> 5
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Thr
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 6
<211> 1596
<212> DNA
<213> artificial sequence
<400> 6
atgacgagcg gttttgatta catcgttgtc ggtggcggtt cggctggctg tgttctcgca 60
gcccgccttt ccgaaaatcc ttccgtccgt gtctgcctca tcgaggcggg ccggcgggac 120
acgcatcccc tgatccacat gccggtcggt ttcgcgaaga tgaccacggg gccgcatacc 180
tgggatcttc tgacggagcc gcagaaacat gcgaacaacc gccagatccc ctatgtgcag 240
ggccggattc tgggcggcgg atcgtccatc aacgcggaag tcttcacgcg gggacaccct 300
tccgacttcg accgctgggc ggcggaaggt gcggatggct ggagcttccg ggatgtccag 360
aagtacttca tccgttccga aggcaatgcc gtgttttcgg gcacctggca tggcacgaac 420
gggccgctcg gggtgtccaa cctcgcggag ccgaacccga ccagccgtgc cttcgtgcag 480
agctgtcagg aaatggggct gccctacaac cctgacttca acggcgcatc gcaggaaggc 540
gcaggcatct atcagatgac gatccgcaac aaccggcgct gctcgacggc tgtggggtat 600
ctgcgtccgg ctctggggcg gaagaacctg acggttgtga cgcgggcgct ggtcctgaag 660
atcgtcttca acggaacgcg ggcgacgggc gtgcagtata tcgccaacgg caccctgaat 720
accgccgaag cgagccagga aatcgttgtg acggccggag cgatcggaac gccgaagctg 780
atgatgctgt cgggcgtcgg gcctgccgcg catcttcgcg aaaatggtat cccggtcgtg 840
caggatctgc cgggcgtggg cgagaacctt caggatcatt tcggtgtgga tatcgtagcc 900
gagctcaaga cggatgagag cttcgacaag taccggaaac tgcactggat gctgtgggca 960
ggtcttgaat ataccatgtt cagatccggt cccgttgcat ccaacgtggt tgagggcggc 1020
gcgttctggt actcggaccc gtcatcgggt gttcctgatc tccagttcca ttttcttgcg 1080
ggggctgggg ctgaggctgg ggtgacgtcc gttcccaagg gagcgtccgg gattacgctg 1140
aacagctatg tgctgcgtcc gaagtctcgt ggaactgtcc ggctgcgttc ggcagatcca 1200
agggtcaatc cgatggtcga tcccaatttc cttggagacc cggccgacct tgagacgtct 1260
gcggaaggtg tgcgcctgag ctacgagatg ttctcccagc cgtctttgca gaagcacatc 1320
cggaaaacct gtttctttag cggtaaacag ccgacgatgc agatgtatcg ggactatgcg 1380
cgggaacatg gccggacgtc ctatcatccg acatgcacct gcaagatggg tcgtgatgac 1440
atgtccgtcg tcgatccgcg tctgaaggtt catggccttg agggcatcag gatctgtgac 1500
agttcggtta tgccgtcgct gctcggttcc aacaccaatg ctgcgacgat catgatcagt 1560
gagcgggcag cggatttcat tcaggggaac gcctga 1596
<210> 7
<211> 1496
<212> DNA
<213> artificial sequence
<400> 7
atgaatgttg tctcaaagac tgtatcttta ccgttaaagc cgcgtgagtt cggattcttt 60
attgatggag aatggcgcgc aggtaaggat ttcttcgatc gttcctcgcc ggctcatgat 120
gttcccgtca cccgtattcc acgctgcacc cgtgaagacc ttgatgaggc agtcgctgct 180
gcacgtcgtg ctttcgagaa cggaagctgg gcgggtctgg cagccgcgga tcgtgcggcg 240
gttcttctga aagccgcggg ccttctgcgc gagcgccgtg atgacatcgc ttactgggaa 300
gttctcgaaa acgggaagcc catcagccag gcgaaaggtg agatcgatca ctgtatcgcc 360
tgtttcgaga tggcggccgg cgctgcgcgg atgctgcatg gtgatacgtt caacaatctg 420
ggcgaggggc tgtttggcat ggtcctgcgg gagcccatcg gtgtcgtcgg tctgattacg 480
ccgtggaact tcccgttcat gatcctgtgt gagcgggcgc ctttcattct cgcatccggc 540
tgcacgctgg tcgtcaagcc tgccgaagtc acgagtgcca cgacccttct tctggcagaa 600
atccttgccg atgccgggct gccgaagggt gtcttcaatg tcgtgacagg cacggggcgc 660
acggtcggtc aggccatgac cgagcatcag gatatcgaca tgctgtcctt cacgggctcc 720
acgggcgtcg gcaagtcctg tatccacgcg gcggctgaca gcaacctgaa gaaacttggc 780
ctcgaactgg gcggcaagaa cccgattgtc gtgttcgctg acagcaacct tgaggatgcg 840
gccgacgcgg tagccttcgg gatcagcttt aataccgggc agtgctgtgt gtcgtcgagc 900
cgcctgatcg tagagcggtc cgtggcggag aagttcgagc gcctcgtcgt ggcaaaaatg 960
gagaagatcc gcgttggtga tccgtttgat cccgaaacgc agattggcgc catcacgacg 1020
gaagcgcaga acaagaccat tctggactat atcgcgaaag gcaaggccga gggcgccaag 1080
ctgctctgtg gtggcgggat cgtcgatttc ggcaagggac agtatatcca gcccacgctt 1140
ttcacggatg tgaagccctc gatgggcatc gcgcgtgacg agatttttgg gccggttctg 1200
gcgtccttcc acttcgatac cgtcgatgag gcgatcgcga ttgccaatga cacggtttac 1260
ggcttggccg catcggtctg gagcaaggat atcgacaagg cgcttgccgt gacccgtcgt 1320
gttcgtgccg gccgcttctg ggtgaacacc atcatgagcg gtggtcccga gacgccgctg 1380
ggtggtttca agcagtcggg ctggggccgt gaggccggtc tgtacggcgt tgaggaatat 1440
acgcagatca aatctgtcca tatcgaaact ggcaaacgtt cgcactggat ttcgta 1496
<210> 8
<211> 265
<212> DNA
<213> artificial sequence
<400> 8
attgctgttt acggtcctga tgacaggacc gttttccaac ctattaatca taaatatgaa 60
aaataattgt tgcatcaccc gccaatgcgt ggcttaatgc acatcaacgg tttgacgtac 120
agaccattaa agcagtgtag taaggcaagt cccttcaaga gttatcgttg atacccctcg 180
tagtgcacat tcctttaacg cttcaaaatc tgtaaagcac gccatatcgc cgaaaggcac 240
acttaattat taaaggtaat acact 265
Claims (9)
- An L-sorbose dehydrogenase mutant characterized in that valine at position 368 is mutated into cysteine, leucine, serine or threonine by taking L-sorbose dehydrogenase with an amino acid sequence shown as SEQ ID NO.1 as a starting sequence.
- 2. A gene encoding the mutant of claim 1.
- 3. A recombinant plasmid carrying the gene of claim 2.
- 4. A microbial cell expressing the L-sorbose dehydrogenase mutant of claim 1, or carrying the gene of claim 2.
- 5. The microbial cell of claim 4, wherein the microbial cell is a bacterial or fungal cell.
- 6. A recombinant E.coli is characterized bypMD19-T is an expression vector, and expresses the L-sorbose dehydrogenase mutant as set forth in claim 1 and the L-sorbosone dehydrogenase with the encoding gene shown in SEQ ID NO. 7.
- 7. The recombinant E.coli according to claim 6, wherein the expression of the L-sorbose dehydrogenase mutant gene and the L-sorbosone dehydrogenase gene is regulated with cspA promoter.
- 8. Use of the L-sorbose dehydrogenase mutant of claim 1, or the recombinant escherichia coli of any one of claims 6-7, for the production of 2-keto-L-gulonic acid, vitamin C, or a product comprising 2-keto-L-gulonic acid.
- 9. A method for producing 2-keto-L-gulonic acid, which is characterized in that the recombinant escherichia coli of claim 6 or 7 is fermented in a medium containing sorbose at 30-37 ℃.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120350A (en) * | 1993-03-08 | 1996-04-10 | 藤泽药品工业株式会社 | Novel L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
| WO2010100236A1 (en) * | 2009-03-05 | 2010-09-10 | Dsm Ip Assets B.V. | Improved production of 2-keto-l-gulonic acid |
| CN111979259A (en) * | 2020-08-07 | 2020-11-24 | 江南大学 | Gluconobacter oxydans shuttle vector for high-efficiency gene expression |
| CN113913400A (en) * | 2021-11-26 | 2022-01-11 | 江南大学 | L-sorbosone dehydrogenase mutant with improved catalytic activity |
| CN114480236A (en) * | 2022-02-23 | 2022-05-13 | 江南大学 | Construction method of 2-KLG (bacillus gluconicum) production one-step strain chassis cell bank |
-
2022
- 2022-06-29 CN CN202210756716.9A patent/CN114934027B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120350A (en) * | 1993-03-08 | 1996-04-10 | 藤泽药品工业株式会社 | Novel L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
| WO2010100236A1 (en) * | 2009-03-05 | 2010-09-10 | Dsm Ip Assets B.V. | Improved production of 2-keto-l-gulonic acid |
| CN111979259A (en) * | 2020-08-07 | 2020-11-24 | 江南大学 | Gluconobacter oxydans shuttle vector for high-efficiency gene expression |
| CN113913400A (en) * | 2021-11-26 | 2022-01-11 | 江南大学 | L-sorbosone dehydrogenase mutant with improved catalytic activity |
| CN114480236A (en) * | 2022-02-23 | 2022-05-13 | 江南大学 | Construction method of 2-KLG (bacillus gluconicum) production one-step strain chassis cell bank |
Non-Patent Citations (1)
| Title |
|---|
| MULTISPECIES: GMC family oxidoreductase N-terminal domain-containing protein [Gluconobacter] NCBI Reference Sequence: WP_062447068.1.《NCBI》.2022,全文. * |
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|---|---|
| CN114934027A (en) | 2022-08-23 |
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