CN102936576B - A kind of Xylitol genetic engineering bacterium and mixing thereof transform the method for producing Xylitol - Google Patents
A kind of Xylitol genetic engineering bacterium and mixing thereof transform the method for producing Xylitol Download PDFInfo
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Abstract
The present invention relates to a kind of method of Xylitol genetic engineering bacterium and mixing conversion production Xylitol thereof, relate to biological technical field.The present invention proposes to build a kind of Xylitol genetically engineered E.coli BL21-xdh06, this project bacterium passes through PCR method, xylitol dehydrogenase (xylitol dehydrogenase is obtained from gluconobacter oxydans (Gluconobacter oxydans), XDH) gene xdh, is cloned and is carried out stability and high efficiency expression at Host Strains E.coli BL21.The xylitol dehydrogenase that this genetic engineering bacterium is expressed, its enzymic activity improves 10 times, and utilize this genetic engineering bacterium and gluconobacter oxydans to mix static conversion D-R alcohol and produce Xylitol, the transformation efficiency of D-R alcohol brings up to 91.6% by 29%.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of structure of Xylitol genetic engineering bacterium and transform for mixing the method that Xylitol produced by D-R alcohol.
Background technology
Xylitol is a kind of five-carbon sugar, and its chemical name is 1,2,3,4, and 5-amylalcohol, molecular formula is C
5h
12o
5, be a kind of white crystals or crystalline powder, its solubleness, solution density are substantially identical with sucrose with physico-chemical properties such as refraction coefficients, can as the substitute of sucrose.Xylitol is a kind of natural sweet taste material having better nutritivity and be worth, and is also a kind of low-yield sweeting agent simultaneously.Xylitol is the intermediate of human body sugar metabolism, Regular Insulin is not consumed after edible, and Xylitol has special preventing decayed tooth function, can make the nutrition agent of diabetics, therapeutical agent and children's preventing decayed tooth food, Xylitol also has reduction blood sugar concentration, promotes the several functions such as calcium absorption simultaneously.
At present, Xylitol industrial production mainly adopts chemical acidification shortening method and utilizes microorganism direct fermentation hemicellulose hydrolysate to produce Xylitol, the raw material multi-source of these two kinds of methods is in the agricultural byproducts such as corn cob, bagasse being rich in pentosan, first these raw materials become the hydrolyzed solution containing a large amount of wood sugar after pyrohydrolysis, therefore there is the problems such as acid and alkali consumption is high, environmental pollution serious, complex process.The research of full Biological preparation Xylitol has caused the concern in the whole world, but does not also find the microorganism of direct fermentation glucose production Xylitol at occurring in nature.1969, it was Xylitol by conversion of glucose that Onishi and Suzuki first reported by three steps fermentations, first utilizes
osmophilic yeastbe D-R alcohol by conversion of glucose, recycling
acetic acid bacteriathe D-R alcohol of generation is oxidized to D-xylulose, and D-xylulose is reduced to Xylitol by a primary yeast afterwards, but its transformation efficiency is very low, and subsequent fermentations is complicated.
We utilize gluconobacter oxydans (
gluconobacter oxydans) be Xylitol by the redox of D-R alcohol, gluconobacter oxydans is purchased from Chinese microorganism strain preservation center, numbering: CGMCC1.110, this bacterium has D-R alcoholdehydrogenase (AraDH) and xylitol dehydrogenase (XDH) vigor simultaneously, D-R alcohol major part can be oxidized to xylulose, and the vigor of XDH is relatively low, the growing amount of Xylitol is just far smaller than the amount of D-xylulose, if product for the purpose of Xylitol, the vigor how improving XDH becomes the key of research.
Summary of the invention
The object of this invention is to provide the genetic engineering bacterium intestinal bacteria BL-xdh06 that a strain has high xylitol dehydrogenase enzyme activity
escherichia colibL-xdh06, is preserved in the China typical culture collection center CCTCC of the Wuhan University of Wuhan, China on June 7th, 2012, deposit number is CCTCC NO. M 2012207.
Another object of the present invention is to provide a kind of method that Xylitol produced by mixing static conversion D-R alcohol with high conversion.Utilize gluconobacter oxydans to mix the method for static conversion D-R alcohol production Xylitol, carry out according to following step:
(1) picking gluconobacter oxydans (
gluconobacter oxydans) inclined-plane bacterium colony in seed liquor substratum, 28-37 DEG C, 100-260 rpm shaking culture 8-20h.
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of volume ratio 1-10 %, 28-37 DEG C, 100-260 rpm shaking culture 48-96h.
(3) by the fermented liquid 6000-12000 rpm in (2), 4 DEG C of centrifugal 5-20 min, collect bacterium mud, wash, the resuspended thalline of cell transformation liquid that centrifugal rear use is appropriate with the potassium phosphate buffer of 0.1 mol/L pH 6.8;
(4) conversion fluid invert point is 28-37 DEG C, 100-260rpm transition 24-60h, 4h sampling, and 4h samples, and obtains Xylitol in conversion fluid.
Another object of the present invention is to provide a kind of method that Xylitol produced by mixing static conversion D-R alcohol with high conversion.Utilize the genetic engineering bacterium of structure and gluconobacter oxydans to mix static conversion D-R alcohol and produce Xylitol, the transformation efficiency of D-R alcohol brings up to 91.6 % by 29 %.
Utilize the genetic engineering bacterium of above-mentioned structure and gluconobacter oxydans to mix the method for static conversion D-R alcohol production Xylitol, carry out according to following step:
(1) picking gluconobacter oxydans (
gluconobacter oxydans) inclined-plane bacterium colony in seed liquor substratum, 28-37 DEG C, 100-260 rpm shaking culture 8-20h.
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of volume ratio 1-10 %, 28-37 DEG C, 100-260 rpm shaking culture 48-96h.
(3) by the fermented liquid 6000-12000 rpm in (2), 4 DEG C of centrifugal 5-20 min, collect bacterium mud, wash, the resuspended thalline of cell transformation liquid that centrifugal rear use is appropriate with the potassium phosphate buffer of 0.1 mol/L pH 6.8;
(4) by said gene engineering bacteria
e.colibL21-
xdh06be inoculated in LB-Amp(LB-penbritin) in substratum, 37 DEG C of shaking culture are spent the night, seed liquor is forwarded in fresh LB-Amp substratum with the inoculum size of 1-10 % by next day, 37 DEG C are cultured to thalline optical density value when being about 0.6-0.8, add IPTG(isopropyl-beta D-thio galactopyranoside) carry out abduction delivering about 5-15h to final concentration 0.5-1.2mmol/L.Get certain volume engineering bacteria liquid 6000-12000 rpm, 4 DEG C of centrifugal 5-20 min, collect bacterium mud, wash with the potassium phosphate buffer of 0.1 mol/L pH 6.8, thalline is stand-by.
(5) the resuspended thalline of the appropriate cell transformation liquid of thalline is obtained obtaining by centrifugal to (3) and (4).
(6) conversion fluid invert point is 28-37 DEG C, 100-260rpm transition 24-60h, 4h sampling, obtains Xylitol in conversion fluid.
Seed liquor substratum wherein described in step (1) is composed as follows: glucose 20-40.0 g/L, Tryptones 1-10.0 g/L, yeast extract paste 2-20.0 g/L; Gluconobacter oxydans slant medium: glucose 20-40.0 g/L, Tryptones 1-10.0 g/L, yeast extract paste 2-20.0 g/L, agar 10-20 g/L, all the other are water.
Enlarged culturing base wherein described in step (2) is composed as follows: glucose 20-40.0 g/L, yeast extract paste 2-20.0 g/L, Tryptones 1-10.0 g/L, D-R alcohol 5-20.0 g/L, calcium carbonate 10-20.0 g/L, all the other are water.
Cell transformation liquid wherein described in step (3) is composed as follows: D-R alcohol 20-40.0 g/L, calcium carbonate 10-30.0 g/L, ethanol 3-8%(V/V), all the other are water.
LB-Amp substratum wherein described in step (4) is composed as follows: tryptone 10g/L, and yeast extract 5g/L, NaCl 10g/L, Amp (penbritin) 50 μ g/L (final concentration), all the other are water.
Cell transformation liquid bag wherein described in step (5) is composed as follows: D-R alcohol 20-40.0 g/L, calcium carbonate 10-30.0 g/L, ethanol 3-8%(V/V), all the other are water.
Embodiment
Gluconobacter oxydans of the present invention (
gluconobacter oxydans) number purchased from Chinese microorganism strain preservation center: CGMCC1.110.Xylose dehydrogenase gene of the present invention be Christina Prus report sequence (
nature Biotechnology23(2), 195 – 200,2005, NC_006677.1).By the genetic engineering technique of routine xylose dehydrogenase gene imported intestinal bacteria obtain containing described xylose dehydrogenase gene (
xdh) intestinal bacteria.Genetic engineering bacterium of the present invention has higher xylitol dehydrogenase enzyme activity, and enzyme activity reaches 21U/mg, and its enzymic activity improves 10 times.Comprise the following steps:
1, cultivate gluconobacter oxydans, extract the STb gene of bacterial strain, go out object fragment by pcr amplification.
2, object fragment and expression vector are cut by enzyme and are connected construction recombination plasmid with T4 ligase enzyme, and recombinant plasmid is imported Host Strains
e.colibL21.
3, extract escherichia coli plasmid, carry out expression checking by PCR, double digestion and SDS-PAGE, prove that recombinant bacterium successfully constructs.
4, the xylitol dehydrogenase enzyme activity of genetic engineering bacterium is measured.Detailed process is shown in embodiment 1.
embodiment 1, there is the acquisition of the genetic engineering bacterium of xylitol dehydrogenase high enzymatic activity
According to feature NCBI delivering multiple clone site on the xylose dehydrogenase gene sequence of gluconobacter oxydans and expression vector pSE380, utilize bioinformatics software design and synthesis degenerated primer: P1:5 ＇ TA
cCATGGtTCACCACCATCACCATCATATGTCGAAGAAGTTTAAG3 ＇ (contains
ncoi restriction enzyme site); P2:5 ＇ TG
aAGCTTtCAACCGCCAGC AAT3 ＇ (contains
hindiII restriction enzyme site).
With gluconobacter oxydans genomic dna for template, pcr amplification goal gene.
PCR reaction parameter: denaturation: 95 DEG C of 2min; Sex change: 94 DEG C of 30sec; Renaturation: 55 DEG C of 30sec; Extend: 72 DEG C of 1min; Circulation: 35; Stop extending: 72 DEG C of 10min; Last 16 DEG C of insulations
PCR primer is detected by 1% agarose electrophoresis, and UNIQ-10 kits reclaims clip size and is about the PCR primer of 0.7-0.9kb for clonal expression.
PCR product and carrier pSE380 are used respectively
ncoi He
hindiII double digestion, uses T
4transformed competence colibacillus after DNA ligase enzyme connects
e.colibL21.
By PCR, digestion verification, a band should be there is by pcr amplification, PCR reaction parameter in recombinant plasmid:
Denaturation: 95 DEG C of 2min; Sex change: 94 DEG C of 30sec; Renaturation: 55 DEG C of 30sec; Extend: 72 DEG C of 1min; Circulation: 30; Stop extending: 72 DEG C of 10min; Last 16 DEG C of insulations.
Recombinant plasmid is used
hinda band should be there is during III single endonuclease digestion qualification, use
ncoi He
hindtwo bands should be there are during III double digestion qualification.Result shows: recombinant bacterium successfully constructs, called after intestinal bacteria BL-xdh06
escherichia colibL-xdh06, is preserved in the China typical culture collection center CCTCC of the Wuhan University of Wuhan, China on June 7th, 2012, deposit number is CCTCC NO. M 2012207.
Genetic engineering bacterium is inoculated in 3 ml LB-Amp substratum, and 37 DEG C of shaking culture are spent the night, and seed liquor is forwarded in fresh LB-Amp substratum with the inoculum size of 2 % by next day, and 37 DEG C are cultured to thalline optical density value (A
600) when being about 0.6, adding IPTG and carry out abduction delivering 10 about h to final concentration 1 mmol/L.Sds polyacrylamide gel electrophoresis is adopted to observe the expression of target protein.Result shows: foreign protein obtains high expression.LB-Amp substratum: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Amp 50 μ g/L (final concentration).
Genetic engineering bacterium is inoculated in 3 ml LB-Amp substratum, and 37 DEG C of shaking culture are spent the night, and seed liquor is forwarded in fresh LB-Amp substratum with the inoculum size of 2 % by next day, and 37 DEG C are cultured to thalline optical density value (A
600) when being about 0.6, adding IPTG and carry out abduction delivering 10 about h to final concentration 1 mmol/L.Get appropriate bacterium liquid 8000 r/min, 4 DEG C of centrifugal 10 min, collect bacterium mud, wash twice with the potassium phosphate buffer of 0.1 mol/L pH 6.8, the resuspended thalline of potassium phosphate buffer that centrifugal rear use is appropriate, ultrasonication in ice bath (surpasses 3 s and stops 3 s, all times 10 min, 200 w).Afterwards in 10000 rpm, 4 DEG C of centrifugal 10 min, supernatant is crude enzyme liquid.
Crude enzyme liquid is utilized to measure the enzyme activity of xylitol dehydrogenase, result shows: recombinant bacterium has the xylitol dehydrogenase enzyme activity of gene clone donor bacterium gluconobacter oxydans, enzyme activity is 21 U/ml, and the xylitol dehydrogenase enzyme activity of recombinant bacterium is about 10 times of gluconobacter oxydans.Obtain the genetic engineering bacterium with xylitol dehydrogenase high enzymatic activity.
measuring method:
Xylitol dehydrogenase is a kind of oxydo-reductase, and its catalytic activity is reversible, and xylulose is converted into Xylitol for its reduction reaction, needs coenzyme NAD H, and Xylitol is converted into xylulose for its oxidizing reaction, needs coenzyme NAD
+.React as follows:
By unit of measure's time endoenzyme catalystic converter system at the increment of 340 nm place absorbancys thus calculate xylitol dehydrogenase enzyme live.Reaction system is respectively 50 mmol/ L NADH solution, 0.4 mol/L xylulose solution, 0.1mol/ L pH5.0 sodium acetate buffer.The volume of each solution is: 1.8 mL damping fluids, 0.03 mL NAD
+/ NADH solution, 0.05 mL Xylitol/xylulose solution, 0.03 mL enzyme sample.
Enzyme lives unit definition when being 25 DEG C, and per minute reduces the NAD of 1 μm of ol
+required enzyme amount.
embodiment 2, prepare Xylitol
(1) first glucose oxidation and bacillus is cultivated in seed liquor substratum, picking 2-3 epoxidation gluconobacter sp inclined-plane bacterium colony in 10 ml seed liquor substratum, 28 DEG C, 100 rpm shaking culture 8h.Seed liquor substratum comprises: glucose 20.0 g/L, Tryptones 1.0 g/L, yeast extract paste 2.0 g/L; Gluconobacter oxydans slant medium: glucose 20 g/L, Tryptones 1 g/L, yeast extract paste 2 g/L, agar 10 g/L.
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of 1 %, 28 DEG C, 100 rpm shaking culture 48h.Enlarged culturing base comprises: glucose 20.0 g/L, yeast extract paste 2.0 g/L, Tryptones 1.0 g/L, D-R alcohol 5.0 g/L, calcium carbonate 10.0 g/L.
(3) by fermented liquid 6000 rpm in (2), 4 DEG C of centrifugal 5 min, collect bacterium mud, wash twice, the resuspended thalline of cell transformation liquid that centrifugal rear use is appropriate with the potassium phosphate buffer of 0.1 mol/L pH 6.8.Cell transformation liquid comprises: D-R alcohol 20 g/L, calcium carbonate 10 g/L, ethanol 3%(V/V).
(4) conversion fluid invert point is 28 DEG C, 100 rpm transition 24h, and interval 4h samples, and detect conversion fluid composition with high performance liquid chromatography, the transformation efficiency that D-R alcohol is converted into Xylitol is up to 29 %.
embodiment 3, prepare Xylitol
(1) first glucose oxidation and bacillus is cultivated in seed liquor substratum, picking 2-3 epoxidation gluconobacter sp inclined-plane bacterium colony in 10 ml seed liquor substratum, 37 DEG C, 260 rpm shaking culture 20 h.Seed liquor substratum comprises: glucose 40.0 g/L, Tryptones 10.0 g/L, yeast extract paste 20.0 g/L.
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of 10 %, 37 DEG C, 260 rpm shaking culture 96 hours.Enlarged culturing base comprises: glucose 40.0 g/L, yeast extract paste 20.0 g/L, Tryptones 10.0 g/L, D-R alcohol 20.0 g/L, calcium carbonate 20.0 g/L; Gluconobacter oxydans slant medium: glucose 40.0 g/L, Tryptones 10.0 g/L, yeast extract paste 20.0 g/L, agar 20 g/L.
(3) by fermented liquid 12000 rpm in (2), 4 DEG C of centrifugal 20 min, collect bacterium mud, wash twice, the resuspended thalline of cell transformation liquid that centrifugal rear use is appropriate with the potassium phosphate buffer of 0.1 mol/L pH 6.8.Cell transformation liquid comprises: D-R alcohol 40 g/L, calcium carbonate 30 g/L, ethanol 8%(V/V)
(4) conversion fluid invert point is 37 DEG C, 260 rpm transition 60h, and interval 4h samples, and detect conversion fluid composition with high performance liquid chromatography, the transformation efficiency that D-R alcohol is converted into Xylitol is up to 29 %.
embodiment 4, mixing conversion prepare Xylitol
(1) first glucose oxidation and bacillus is cultivated in seed liquor substratum, picking 2-3 epoxidation gluconobacter sp inclined-plane bacterium colony in 10 ml seed liquor substratum, 28 DEG C, 100 rpm shaking culture 8h.Seed liquor substratum comprises: glucose 20.0 g/L, Tryptones 1.0 g/L, yeast extract paste 2.0 g/L.
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of 1 %, 28 DEG C, 100 rpm shaking culture 48h.Enlarged culturing base comprises: glucose 20.0 g/L, yeast extract paste 2.0 g/L, Tryptones 1.0 g/L, D-R alcohol 5.0 g/L, calcium carbonate 10.0 g/L.
(3) genetic engineering bacterium is inoculated in LB-Amp substratum, 37 DEG C of shaking culture are spent the night, next day with 1% inoculum size seed liquor is forwarded in fresh LB-Amp substratum, 37 DEG C are cultured to thalline optical density value when being about 0.6, add IPTG and carry out abduction delivering 5 about h to final concentration 0.5 mmol/L.Get certain volume engineering bacteria liquid 6000 rpm, 4 DEG C of centrifugal 5 min, collect bacterium mud, wash twice with the potassium phosphate buffer of 0.1 mol/L pH 6.8, thalline is stand-by.LB-Amp substratum: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Amp 50 μ g/L (final concentration).
(4) by fermented liquid 6000 rpm in (2), 4 DEG C of centrifugal 5min, collect bacterium mud, wash twice with the potassium phosphate buffer of 0.1 mol/L pH 6.8, thalline is stand-by.
(5) the resuspended thalline of the appropriate cell transformation liquid of thalline is obtained obtaining by centrifugal to (3) and (4).Cell transformation liquid comprises: D-R alcohol 20 g/L, calcium carbonate 10 g/L, ethanol 3%(V/V).
(6) conversion fluid invert point is 28 DEG C, 100 rpm transition 24h, and interval 4h samples, and detect conversion fluid composition with high performance liquid chromatography, the transformation efficiency that D-R alcohol is converted into Xylitol is up to 91.6%.
embodiment 5, mixing conversion prepare Xylitol
(1) first glucose oxidation and bacillus is cultivated in seed liquor substratum, picking 2-3 epoxidation gluconobacter sp inclined-plane bacterium colony in 10 ml seed liquor substratum, 37 DEG C, 260 rpm shaking culture 20h.Seed liquor substratum comprises: glucose 40.0 g/L, Tryptones 10.0 g/L, yeast extract paste 20.0 g/L.
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of 10 %, 37 DEG C, 260 rpm shaking culture 96h.Enlarged culturing base comprises: glucose 40.0 g/L, yeast extract paste 20.0 g/L, Tryptones 10.0 g/L, D-R alcohol 20.0 g/L, calcium carbonate 20.0 g/L.
(3) genetic engineering bacterium is inoculated in LB-Amp substratum, 37 DEG C of shaking culture are spent the night, next day with 10% inoculum size seed liquor is forwarded in fresh LB-Amp substratum, 37 DEG C are cultured to thalline optical density value when being about 0.8, add IPTG and carry out abduction delivering 15 about h to final concentration 1.2 mmol/L.Get certain volume engineering bacteria liquid 12000 rpm, 4 DEG C of centrifugal 20 min, collect bacterium mud, wash twice with the potassium phosphate buffer of 0.1 mol/L pH 6.8, thalline is stand-by.LB-Amp substratum: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Amp 50 μ g/L (final concentration).
(4) by fermented liquid 12000 rpm in (2), 4 DEG C of centrifugal 20min, collect bacterium mud, wash twice with the potassium phosphate buffer of 0.1 mol/L pH 6.8, thalline is stand-by.
(5) the resuspended thalline of the appropriate cell transformation liquid of thalline is obtained obtaining by centrifugal to (3) and (4).Cell transformation liquid comprises: D-R alcohol 40 g/L, calcium carbonate 30 g/L, ethanol 8%(V/V).
(6) conversion fluid invert point is 37 DEG C, 260 rpm transition 60h, and interval 4h samples, and detect conversion fluid composition with high performance liquid chromatography, the transformation efficiency that D-R alcohol is converted into Xylitol is up to 91.6%.
SEQUENCE LISTING
<110> Jiangsu University
<120> Xylitol genetic engineering bacterium and mixing thereof transform the method for producing Xylitol
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<170> PatentIn version 3.3
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<213> artificial sequence
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TACCATGGTT CACCACCATC ACCATCATAT GTCGAAGAAG TTTAAG
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TGAAGCTTTC AACCGCCAGC AAT
Claims (2)
1. a recombination bacillus coli (
escherichia coli) BL-xdh06, deposit number is CCTCC NO. M 2012207.
2. utilize recombination bacillus coli BL-xdh06 described in claim 1 and gluconobacter oxydans (
gluconobacter oxydans) mix the method that Xylitol produced by static conversion D-R alcohol, it is characterized in that carrying out according to following step:
(1) picking gluconobacter oxydans inclined-plane bacterium colony is in seed liquor substratum, 28-37 DEG C, 100-260 rpm shaking culture 8-20h;
(2) seed liquor is inoculated into enlarged culturing base with the inoculum size of volume ratio 1-10 %, 28-37 DEG C, 100-260 rpm shaking culture 48-96h;
(3) by the fermented liquid 6000-12000 rpm in (2), 4 DEG C of centrifugal 5-20 min, collect bacterium mud, wash, the resuspended thalline of cell transformation liquid that centrifugal rear use is appropriate with the potassium phosphate buffer of 0.1 mol/L pH 6.8;
(4) recombination bacillus coli BL-xdh06 is inoculated in LB-ampicillin medium, 37 DEG C of shaking culture are spent the night, seed liquor is forwarded in fresh LB-Amp substratum with the inoculum size of 1-10 % by next day, 37 DEG C are cultured to thalline optical density value when being 0.6-0.8, add isopropyl-beta D-thio galactopyranoside and carry out abduction delivering 5-15h to final concentration 0.5-1.2mmol/L;
Get the bacterium liquid 6000-12000 rpm after certain volume induction, 4 DEG C of centrifugal 5-20 min, collect bacterium mud, wash with the potassium phosphate buffer of 0.1 mol/L pH 6.8, thalline is stand-by;
(5) the resuspended thalline of the appropriate cell transformation liquid of thalline is obtained obtaining by centrifugal to (3) and (4);
(6) conversion fluid invert point is 28-37 DEG C, 100-260rpm transition 24-60h, 4h sampling, obtains Xylitol in conversion fluid;
Seed liquor substratum wherein described in step (1) is composed as follows: glucose 20-40.0 g/L, Tryptones 1-10.0 g/L, yeast extract paste 2-20.0 g/L; Gluconobacter oxydans slant medium: glucose 20-40.0 g/L, Tryptones 1-10.0 g/L, yeast extract paste 2-20.0 g/L, agar 10-20 g/L, all the other are water;
Enlarged culturing base wherein described in step (2) is composed as follows: glucose 20-40.0 g/L, yeast extract paste 2-20.0 g/L, Tryptones 1-10.0 g/L, D-R alcohol 5-20.0 g/L, calcium carbonate 10-20.0 g/L, and all the other are water;
Cell transformation liquid wherein described in step (3) is composed as follows: D-R alcohol 20-40.0 g/L, calcium carbonate 10-30.0 g/L, ethanol 3-8%(V/V), all the other are water;
LB-Amp substratum wherein described in step (4) is composed as follows: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, penbritin final concentration 50 μ g/L, and all the other are water;
Cell transformation liquid wherein described in step (5) is composed as follows: D-R alcohol 20-40.0 g/L, calcium carbonate 10-30.0 g/L, ethanol 3-8%(V/V), all the other are water.
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| CN102250820A (en) * | 2010-06-03 | 2011-11-23 | 南京工业大学 | Genetic engineering bacteria of gluconobacter oxydans and construction methods thereof |
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| CN102250820A (en) * | 2010-06-03 | 2011-11-23 | 南京工业大学 | Genetic engineering bacteria of gluconobacter oxydans and construction methods thereof |
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| Gluconobacter oxydans木糖醇脱氢酶基因的克隆表达及木糖醇的转化分析;沈晓波 等;《中国生物工程杂志》;20091231;第29卷(第12期);摘要、第55页左栏第4段-第56页左栏第7段 * |
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