CN103184197B - Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof - Google Patents
Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof Download PDFInfo
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- CN103184197B CN103184197B CN201110457823.3A CN201110457823A CN103184197B CN 103184197 B CN103184197 B CN 103184197B CN 201110457823 A CN201110457823 A CN 201110457823A CN 103184197 B CN103184197 B CN 103184197B
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Abstract
The invention provides sorbose dehydrogenase and sorbosone dehydrogenase for gluconobacter oxydans CGMCC No. 1. 637, amino acid sequences of the sorbose dehydrogenase and sorbosone dehydrogenase are shown as SEQ ID No. 2 and SEQ ID No. 4. The invention also provides genes or gene clusters for coding the above proteins. SNDH-SDH gene clusters with self-controlling sequences are connected to broad host range plasmids pBBRiMCS2, and are introduced to the gluconobacter oxydans 621H through conjugative transfer, so that transformation from the sorbose to keto-gulonic acid can be realized.
Description
Technical field
The invention belongs to field of microbial biotechnology, be specifically related to sorbose dehydrogenase and sorbic ketone dehydrogenase gene and the proteins encoded thereof of gluconobacter oxydans 1.637.
Background technology
Gluconobacter oxydans (Gluconobacter oxydans) belongs to acetobacter section Gluconobacter, closely related with the mankind's productive life, all has wide practical use in industries such as food, beverage, medicine, chemical industry.
VC produces and generally adopts two-step fermentation at present, and the first step is converted into sorbose by gluconobacter oxydans catalytic substrate sorbyl alcohol, and second step is that sorbose is converted into ketone 2-KLG, is completed by big or small bacterium mixed fermentation.Two-step fermentation relates to fermenting twice, has three kinds of bacterium to participate in, and the cycle is long, and especially second step mixed fungus fermentation has increased difficulty to technology controlling and process, causes the waste of fermention medium nutritive ingredient and the energy, has increased environmental pollution, has improved production cost.If two-step fermentation can be integrated and completed by a kind of bacterium, be conducive to simplification of flowsheet, shorten fermentation time, reduce energy substrate consumption, thereby reduce production costs.
Summary of the invention
For addressing the above problem, the present invention aims to provide sorbose dehydrogenase and sorbic ketone dehydrogenase gene and application thereof.
For realizing the object of the invention, first the present invention provides, and the gluconobacter oxydans CGMCC sorbose dehydrogenase of No.1.637 is provided, and it is:
1) protein being formed by the aminoacid sequence shown in SEQ ID No.2, or
2) in the aminoacid sequence shown in SEQ ID No.2, be substituted, lack or add one or several amino acid and have same isoreactivity by 1) derivative protein.
The present invention also provides the gene of the sorbose dehydrogenase of the described gluconobacter oxydans CGMCC No.1.637 of coding.Preferably, its nucleotide sequence is as shown in SEQ ID No.1.
The present invention also provides the gluconobacter oxydans CGMCC sorbic ketone dehydrogenase of No.1.637, and it is:
1) protein being formed by the aminoacid sequence shown in SEQ ID No.4, or
2) in the aminoacid sequence shown in SEQ ID No.4, be substituted, lack or add one or several amino acid and have same isoreactivity by 1) derivative protein.
The present invention also provides the gene of the sorbic ketone dehydrogenase of the described gluconobacter oxydans of coding.Preferably, its nucleotide sequence is as shown in SEQ ID No.3.
Should be appreciated that those skilled in the art can, according to aminoacid sequence disclosed by the invention, not affect under its active prerequisite, replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.Therefore, the sorbose dehydrogenase of gluconobacter oxydans of the present invention also comprises by being substituted, lacking in the aminoacid sequence shown in SEQ ID No.2 or adding one or several amino acid and have the protein by the protein derived shown in SEQ ID No.2 of same isoreactivity.For example, by adding endways sequence label, as His-tag or Strep-tag and derivative protein.Equally, the sorbic ketone dehydrogenase of gluconobacter oxydans of the present invention also comprises by being substituted, lacking in the aminoacid sequence shown in SEQ ID No.4 or adding one or several amino acid and have the protein by the protein derived shown in SEQ ID NO.4 of same isoreactivity.For example, at nonactive section, for example, by adding endways sequence label, as His-tag or Strep-tag and derivative protein.
Preferably, the aminoacid sequence of the derived protein of the sorbose dehydrogenase of gluconobacter oxydans and the sorbic ketone dehydrogenase of gluconobacter oxydans can be more than 70% with the homology of the aminoacid sequence shown in SEQ ID No.2 and SEQ ID No.4 respectively, preferably more than 80%, more preferably more than 90%.
The present invention also provides the sorbose dehydrogenase gene that contains gluconobacter oxydans CGMCC No.1.637 and the gene cluster of sorbic ketone dehydrogenase gene, and its nucleotide sequence is as shown in SEQ ID No.5.Gene cluster shown in SEQ ID No.5, with the regulating and controlling sequence of self, comprises promotor original paper and the terminator element of himself.
Carrier, clone and Host Strains that what the present invention also provided contain aforementioned gene or gene cluster all belong to protection scope of the present invention.
In the present invention, the sorbose dehydrogenase SDH of gluconobacter oxydans CGMCC No.1.637 and operon of sorbic ketone dehydrogenase SNDH genomic constitution, there is the interval of 2 bases SNDH and SDH gene coding region.BLASTN comparison result shows, and gluconobacter oxydans T100 gene order homology is the highest, SNDH gene nucleotide series homology is that 74.6%, SDH gene nucleotide series homology is 72.4%.
Obtain the SNDH-SDH gene cluster of gluconobacter oxydans 1.637 by pcr amplification, this genomic cluster is connected into carrier, import in bacillus coli DH 5 alpha and express, DCIP method and active electrophoresis detected result show the activity of sorbose dehydrogenase SDH and sorbic ketone dehydrogenase SNDH.
SNDH-SDH gene cluster with autogenous control sequence is connected into broad host range plasmid pBBR1MCS2, shifts and import in gluconobacter oxydans 621H by combination, can realize the conversion of sorbyl alcohol to ketone 2-KLG.
Sorbose dehydrogenase of the present invention and sorbic ketone dehydrogenase gene and proteins encoded thereof will play an important role in the conversion of research sorbyl alcohol-sorbose-sorb ketone-one 2-KLG, VC ferment a step engineering strain structure.
Brief description of the drawings
Figure 1 shows that CGMCC No.1.637 genome detected result.Wherein, M: λ-HindIII DNA Marker; 1:CGMCC No.1.637 genomic dna.
Figure 2 shows that SDH gene and SNDH gene PCR product.Wherein, M:Trans plus II DNA marker; 1:SDH gene PCR product; 2:SNDH gene PCR product.
Figure 3 shows that SNDH-SDH gene cluster PCR product.Wherein, M:Trans plus II DNA marker; 1,2:SNDH-SDH PCR product.
Figure 4 shows that SDH and SNDH recombinant plasmid bacterium liquid PCR qualification.Wherein, M:Trans plus II DNA marker; 1-4:pMD 18T-SDH/DH5 α bacterium liquid PCR product; 5-7:pMD18T-SNDH/DH5 α bacterium liquid PCR product; 8:pMD18T/DH5 α bacterium liquid PCR product.
Figure 5 shows that pMD18T-SNDH-SDH/DH5 α bacterium liquid PCR qualification.M:Trans plus II DNA marker; 1-7:pMD18T-SDNHSDH/DH5 α bacterium liquid PCR product; 8:pMD18T/DH5 α bacterium liquid.
Figure 6 shows that pBBR1MCS2-SNDHSDH/DH5 α bacterium liquid PCR qualification.Wherein, M:Trans plus II DNA marker; 1～3,5～8:pBBR1MCS2-SNDHSDH/DH5 α bacterium liquid PCR product; 4:pBBR1MCS2/DH5 α bacterium liquid PCR product.
Figure 7 shows that the PCR qualification of pBBR1MCS2-SNDHSDH/621H bacterium liquid.Wherein, M:Trans plus II DNA marker; 1:pBBR1MCS2/621H bacterium liquid PCR product; 2-5:pBBR1MCS2-SNDHSDH/621H bacterium liquid PCR product.
Figure 8 shows that recombinant bacterium pBBR1MCS2-SNDHSDH/621H bacterium liquid carries out the result of thin-layer chromatography.Wherein, 1:pBBR1MCS2-SNDHSDH/621H bacterium liquid (sorbyl alcohol substrate, concentration is 10mg/mL); 2:pBBR1MCS2-SNDHSDH/621H (sorbyl alcohol substrate, concentration is 10mg/mL); 3:pBBR1MCS2/621H (sorbyl alcohol substrate, concentration is 10mg/mL); 4:621H (sorbyl alcohol substrate, concentration is 10mg/mL); 5: standard substance, sorbose/ketone 2-KLG (sorbose and ketone 2-KLG are respectively 2mg/mL).
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 extracts gluconobacter oxydans CGMCC No.1.637 genomic dna
(1) cultivating gluconobacter oxydans CGMCC No.1.637 (purchased from Institute of Micro-biology of the Chinese Academy of Sciences) is 2.0 left and right to OD value, 4 DEG C, the centrifugal 5min collection of 8000rpm 5mL bacterium liquid thalline.
(2) add 450 μ L TE, 50 μ L 10%SDS, 5 μ L Proteinase Ks, the vibration of 1800rpm vortex is fully resuspended.
(3) 80 DEG C of cracking 10min, let cool to room temperature, add 5 μ L RNaseA, put upside down and mix 10 times, 37 water-bath 2h.
(4) add 30 μ L 3M KAC, the 1800rpm 30s that vibrates, places 20min for-20 DEG C.
(5) 12000rpm, 4 DEG C of centrifugal 20min, draw supernatant in another centrifuge tube.
(6) add 5M NaCl 60 μ L, Virahol 300 μ L, put upside down and mix 20 times, place 20min for-20 DEG C.
(7) 12000rpm, 4 DEG C of centrifugal 20min, abandon supernatant.
(8) add 100 μ L 70% pre-cooled ethanol rinsing DNA precipitations, 12000rpm, 4 DEG C of centrifugal 10min, abandon supernatant.
(9) repeated washing once, is abandoned supernatant.Drying at room temperature.
(10) add 30 μ L TE to dissolve genomic dna precipitation ,-20 DEG C frozen for subsequent use.
Get 2 μ L samples and carry out 1% agarose gel electrophoresis detection, the about 50ng/ μ of sample concentration L, electrophorogram shows does not have obvious RNA band, meets pcr amplification template needs (Fig. 1).
Embodiment 2 increase gluconobacter oxydans 1.637 sorbose dehydrogenases and sorbic ketone dehydrogenase gene
The genomic dna of the CGMCC No.1.637 extracting taking embodiment 1 is template, uses primer shown in table 1, pcr amplification SDH respectively, SNDH gene and SNDH-SDH gene cluster, 1% agarose electrophoretic analysis (Fig. 2, Fig. 3).Cut glue and reclaim object fragment, connect pMD18T carrier, transform DH5 α, picking locus coeruleus bacterium colony is cultivated, bacterium liquid PCR qualification positive recombinant (Fig. 4, Fig. 5).Picking bacterium liquid PCR identifies positive strain, send Hua Da gene sequencing, preserves, respectively called after pMD18T-SDH, pMD 18T-SNDH, pMD 18T-SNDHSDH through clone's upgrading grain that order-checking qualification is correct.
Table 1PCR amplimer
| SDH-F： | 5′-ggccctcaaggtgacac-3′ | SEQ ID No.6 |
| SDH-R： | 5′-gagccttcagaacgtcatc-3′ | SEQ ID No.7 |
| SNDH-F： | 5′-agcaatatcccagagcagaag-3′ | SEQ ID No.8 |
| SNDH-R： | 5′-gcggtgtggtgacaagc-3′ | SEQ ID No.9 |
Embodiment 3DCIP and active electrophoresis detection of active
Centrifugal collection pMD18T-SNDHSDH/DH5 α thalline is through ultrasonic degradation, gets supernatant after centrifugal and carries out activity by DCIP method and Native electrophoresis and detect, and using sorbose and wood sugar as substrate, result shows SDH and SNDH activity (table 2) respectively.
Table 2DCIP and active electrophoresis detection of active result
Note: "+" table does not have activity, "-" table does not have activity.
Embodiment 4 builds pBBR1MCS2-SNDHSDH-1.637 plasmid
The pMD18T-SNDHSDH plasmid successfully constructing and pBBR1MCS2 (GenBank accession number U23751) plasmid vector are used respectively to Xba I/SalI double digestion, sepharose reclaims test kit and reclaims object fragment, 16 DEG C of connections (10 μ L system) are spent the night, transform DH5a, screening positive recombinant.Show the success of pBBR1MCS2-SNDHSDH plasmid construction through bacterium liquid PCR qualification (Fig. 6) and order-checking qualification.
The qualification of embodiment 5pBBR1MCS2-SNDHSDH/621H recombinant bacterium
The gluconobacter oxydans (ATCC 621H) (purchased from ATCC) (calling gluconobacter oxydans 621H in the following text) of activation is transferred in 5mL YBY (sorbyl alcohol 10g with the inoculum size of 1: 100, yeast extract paste 7g, calcium carbonate 3g, pH 4.8-5.0, adds dH
2o to 1000ml, 115 DEG C of sterilizing 20min), by the pRK2013/HB101 of activation, the pBBR1MCS2-SNDHSDH/DH5a of pBBR1MCS2/DH5a and structure transfers in 5mLLB substratum with the inoculum size of 1: 100 respectively, shaking culture is during to OD ≈ 0.6, centrifugal collection thalline separately, wash 3 times with 0.9%NaCl, with the resuspended thalline of 0.9%NaCl 500 μ L, by donor bacterium pBBR1MCS2/DH5a, pBBR1MCS2-SNDHSDH/DH5a respectively with auxiliary bacterium pRK2013/HB101, recipient bacterium 621H mixes according to the ratio of 1: 1: 2, getting mixed solution 100 μ L puts respectively on the filter membrane of YBY flat board, 30 DEG C of overnight incubation.On filter wash film, bacterium liquid, to YBY liquid nutrient medium, is got 200 μ L and is coated with respectively YBY flat board (kantlex Kan 50 μ g/ml), cultivates 24h for 30 DEG C, chooses single bacterium colony qualification.Show that through the bacterium liquid PCR of recombinant bacterium qualification result pBBR1MCS2-SNDHSDH plasmid successfully imports (Fig. 7) in 621H.
Embodiment 6 thin layer chromatographys detect the expression of pBBR1MCS2-SNDHSDH/621H recombinant bacterium
Get on 2 μ L recombinant bacterium pBBR1MCS2-SNDHSDH/621H bacterium liquid and check on thin layer plate, thin layer plate is placed in to chromatography cylinder inner equilibrium 20min.By n-propyl alcohol-water-1% phosphoric acid-formic acid volume ratio (400: 100: 10: 1) launch, to about Front distance thin layer plate upper limb 2cm, take out thin layer plate, dry up with blower in ventilation, with the colour developing of TCC solution spray.Result as shown in Figure 8, there is the sour point identical with standard substance in recombinant bacterium, this has illustrated that the SNDHSDH gene of gluconobacter oxydans CGMCC No.1.637 imports in gluconobacter oxydans 621H and has realized expression, has made 621H realize the conversion to ketone 2-KLG by sorbyl alcohol.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the sorbose dehydrogenase of gluconobacter oxydans CGMCC No.1.637, it is:
The protein being formed by the aminoacid sequence shown in SEQ ID No.2.
2. the gene of coding sorbose dehydrogenase claimed in claim 1.
3. sorbose dehydrogenase gene according to claim 2, is characterized in that, nucleotide sequence is as shown in SEQ ID No.1.
4. the sorbic ketone dehydrogenase of gluconobacter oxydans CGMCC No.1.637, it is:
The protein being formed by the aminoacid sequence shown in SEQ ID No.4.
5. coding sorbic ketone dehydrogenase gene claimed in claim 4.
6. sorbic ketone dehydrogenase gene according to claim 5, is characterized in that, nucleotide sequence is as shown in SEQ ID No.3.
7. the sorbose dehydrogenase gene of gluconobacter oxydans CGMCC No.1.637 and the gene cluster of sorbic ketone dehydrogenase gene, is characterized in that, nucleotide sequence is as shown in SEQ ID No.5.
8. contain the sorbose dehydrogenase gene described in claim 2 or 3 or contain the sorbic ketone dehydrogenase gene described in claim 5 or 6 or the carrier that contains gene cluster claimed in claim 7.
9. contain the Host Strains of carrier described in claim 8.
10. the sorbic ketone dehydrogenase gene described in sorbose dehydrogenase gene or the claim 5 or 6 described in claim 2 or 3 or gene cluster claimed in claim 7 application in ketone 2-KLG is produced.
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| CN112899211B (en) * | 2021-03-23 | 2022-10-11 | 江南大学 | Method for increasing yield of 2-KLG in gluconobacter oxydans |
| CN113913400B (en) * | 2021-11-26 | 2022-12-27 | 江南大学 | L-sorbosone dehydrogenase mutant with improved catalytic activity |
| CN114480237B (en) * | 2022-02-23 | 2023-10-03 | 江南大学 | Two-stage fermentation method for improving yield of 2-KLG in gluconobacter |
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| 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 |
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