CN109097314A - A method of the full cell of Gluconobacter oxvdans is prepared by carbon source of glycerol - Google Patents
A method of the full cell of Gluconobacter oxvdans is prepared by carbon source of glycerol Download PDFInfo
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Abstract
The invention discloses a kind of methods for preparing the full cell of Gluconobacter oxvdans as carbon source using glycerol, it is that genetic engineering transformation is carried out to wild type Gluconobacter oxvdans, it can be using glycerol as carbon source for growth, recombination Gluconobacter oxvdans Gluconobacter oxydans Δ GOX1068 Δ GOX0854 is made, then obtained recombination Gluconobacter oxvdans are inoculated in glycerol minimal medium, are obtained after 30 ± 1 DEG C of shaking table shaken cultivations.Experiment confirms, full cell preparation process is easy in method of the invention, the whole-cell biocatalyst of preparation is suitable with the Gluconobacter oxvdans original bacteria Gluconobacter oxydans 621H whole-cell biocatalyst catalytic efficiency prepared in sorb alkoxide complex culture medium, and biocatalyst preparation cost is lower, has good application value.
Description
Technical field
The present invention relates to a kind of preparation methods of the full cell of Gluconobacter oxvdans, more particularly to one kind is using glycerol as carbon
The method that source prepares the full cell of Gluconobacter oxvdans.
Background technique
Gluconobacter oxvdans are Gram-negative, stringent aerobic acidophil, belong to Acetobacteraceae gluconobacter sp
Belong to (Gupta A et al., J.Mol.Microbiol.Biotechnol., 2001,3:445-456).Gluconobacter oxydans bar
Bacterium can a large amount of alcohols of incomplete oxidation and carbohydrate, periplasmic space of many compounds in Gluconobacter oxvdans
Middle envelope combination dehydrogenase is aoxidized in a manner of chemistry-, site-, three-dimensional-selectivity for corresponding ketone, aldehyde and organic acid
(Keliang G et al.,Appl.Microbiol.Biotechnol.,2006,70:135-139；Deppenmeier U et
al.,J.Mol.Microbiol.Biotechnol.,2002,16:69-80).In addition, Gluconobacter oxvdans have hyperoxia
The characteristic of rate and low biomass becomes preferred biocatalyst (the De Muynck C of a variety of biotechnology production processes
et al.,Crit.Rev.Biotechnol.,2007,27:147-171).At present the full cell of Gluconobacter oxvdans by
It is widely used in the production of various industrial products, such as vitamin C, dihydroxyacetone (DHA), xylonic and gluconic acid etc.
(Pappenberger G et al.,Adv.Biochem.Eng.Biotechnol.,2014,143:143-188；Mishra R
et al.,Biotechnol.Adv.,2008,26:293-303；Toivari M H et al.,
Appl.Microbiol.Biotechnol.,2012,96:1-8；Silberbach M et al.,
Appl.Microbiol.Biotechnol.,2003,62:92-98)。
The nutritional need of Gluconobacter oxvdans is higher, usually need to be in answering containing higher concentration sorbierite and yeast powder
It is cultivated in miscellaneous culture medium, culture medium cost seriously limits application of the full cell of Gluconobacter oxvdans in bioconversion.
In order to solve this problem, many people have carried out the research using other low price carbon source culture Gluconobacter oxvdans.It grinds
The person of studying carefully knocks out the glucose dehydrogenase in Gluconobacter oxvdans 621H, and then the knockout bacterium is using glucose as carbon source yeast
Powder is that nitrogen source carries out metabolism evolution, the results showed that evolved with the metabolism of low concentration glucose culture knock out the full cell of bacterium with highly concentrated
The catalytic efficiency of the full cell of wild mushroom of the sorbierite culture of degree it is identical (Zhu K et al., Mol.Biotechnol., 2011,
49:56-64).Kiefler et al. supplements tricarboxylic acid cycle in Gluconobacter oxvdans completely, and eliminates Pyruvate production
The approach of acetaldehyde and acetic acid, biomass (Kiefler I when improving using glucose as carbon source culture Gluconobacter oxvdans
et al.,Appl.Microbiol.Biotechnol.,2017,101:5453-5467).But prepare the full cell of above-mentioned bacterial strains
Cost it is still higher, and full cell preparation efficiency is lower, therefore there is an urgent need to find a kind of cheap carbon source efficiently to prepare
Gluconobacter oxvdans biocatalyst.
Glycerol is inevitable by-product in bio-fuel production, higher and cheap (the Gao C et of yield
al.,Green Chem.,2015,17:804-807).It is entirely thin that retrieval discovery using glycerol as carbon source prepares Gluconobacter oxvdans
The method of born of the same parents has not been reported.
Summary of the invention
For preparing in the prior art, the full cell cost of Gluconobacter oxvdans is expensive, it is difficult to expand its industrial application
Deficiency, the problem to be solved in the present invention is to provide a kind of sides that the full cell of Gluconobacter oxvdans is prepared using glycerol as carbon source
Method.
The method of the present invention for preparing the full cell of Gluconobacter oxvdans as carbon source using glycerol, step is:
(1) genetic engineering transformation is carried out to wild type Gluconobacter oxvdans, can be made using glycerol as carbon source for growth
Gluconobacter oxvdans must be recombinated；
(2) obtained recombination Gluconobacter oxvdans are crossed to containing mass volume ratio is 1.5~1.8% agar and to contain
On the sorb alkoxide complex culture medium flat plate of 50 μ g/mL Cefoxitins, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours；
(3) under sterile conditions, the single colonie on step (2) plate is picked them separately with sterile toothpick, be then inoculated with
Into the sorb alkoxide complex culture medium containing 50 μ g/mL Cefoxitins of 5mL, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours,
Obtain recombination Gluconobacter oxvdans first order seed；
(4) under sterile conditions, the resulting first order seed bacterium solution of step (3) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the sorb alkoxide complex culture medium of 50mL, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, obtain recombination oxidation
Gluconobacter suboxydans secondary seed bacterium solution；
(5) aseptically, the recombination Gluconobacter oxvdans secondary seed bacterium solution 8,000 step (4) obtained
± 500 revs/min are centrifuged 2~5 minutes, with 0.85% brine thallus 2~3 times, then again hang thallus and play life
Recombination Gluconobacter oxvdans bacterium solution is obtained in reason salt water, by bacterium solution final concentration OD600nmRecombination is aoxidized for 0.1 inoculum concentration
Gluconobacter suboxydans are inoculated into 100mL glycerol minimal medium, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, obtain
Gluconobacter oxvdans culture；
(6) by step (5) culture obtain 6,000 ± 500 revs/min of Gluconobacter oxvdans culture centrifugation 10~
15 minutes；And with pH 7.4, phosphate buffer washing thalline 2~3 times of 1/15M, the phosphoric acid then is played by cell is outstanding
In salt buffer, make the final concentration OD of somatic cells600nmIt is 30~50, that is, obtains the Gluconobacter oxydans for being used for biocatalyst
The full cell bacterium solution of bacillus；
Wherein:
The formula of sorb alkoxide complex culture medium described in above-mentioned steps (2)~(3) is: sorbierite 73g/L, yeast powder
18.4g/L, (NH4)2SO41.5g/L, KH2PO41.5g/L, MgSO4·7H2O 0.47g/L；121 DEG C sterilize 20 minutes；
The formula of glycerol minimal medium described in above-mentioned steps (5) is: (NH4)2SO42g/L, KH2PO42.2g/L
Na2HPO40.2g/L, MgSO4·7H2O 0.2g/L, metal ion solution (50 ×) 20mL, 121 DEG C sterilize 20 minutes；Glycerol
2g/L, 121 DEG C individually sterilize 20 minutes；Vitamin (100 ×) solution 10mL, filtration sterilization；Wherein, metal ion solution (50
×) formula are as follows: nitro triacetic acid 0.25g/L, EDTA 1.5g/L, FeSO4·7H2O 0.55g/L, ZnSO4·7H2O
0.45g/L, CoCl2·6H2O 0.03g/L, MnCl2·4H2O 0.1g/L, CuSO4·5H2O 0.03g/L, CaCl2·2H2O
0.45g/L, NaMoO4·2H2O 0.004g/L, H3BO30.05g/L, KI 0.01g/L；The formula of vitamin (100 ×) solution
Are as follows: calcium pantothenate 0.05g/L, niacin 0.04g/L, p-aminobenzoic acid 0.04g/L, glutamine 20g/L.
In the above-mentioned method for preparing the full cell of Gluconobacter oxvdans using glycerol as carbon source: the recombination oxidation obtained
Gluconobacter suboxydans are preferably to be named as the genetic engineering bacterium of Gluconobacter oxydans Δ GOX1068 Δ GOX0854,
G.oxydans Δ GOX1068 Δ GOX0854 is with wild type Gluconobacter oxvdans G.oxydans 621H (DSM
No.2343) it is starting strain, knocks out the alcohol dehydrogenase GOX1068 that film combines and the polyol dehydrogenase GOX0854 system that film combines
?.
Wherein: the Gluconobacter oxydans Δ GOX1068 Δ GOX0854 genetic engineering bacterium is gram-negative
Property bacterium, stringent aerobic growth, optimum culturing temperature be 30 ± 1 DEG C, can be in the sorb alkoxide complex containing 50 μ g/mL Cefoxitins
It is grown on culture medium or glycerol minimal medium.
The full cell of Gluconobacter oxvdans of the method for the invention preparation is raw in catalysis xylose as biocatalyst
Produce the application in xylonic.
Wherein, specific application method is:
Using the full cell of the Gluconobacter oxvdans of preparation as biocatalyst, make final concentration of cells in catalyst system
OD600nmIt is 10~20, under 30 DEG C, the stringent aerobic condition of pH 7.0~7.5, converts the xylose that concentration is 5~60g/L；150
Rev/min oscillation 3~30 hours, obtain the conversion fluid containing xylonic；By resulting conversion fluid, with 13,000 ± 500 revs/min from
The heart 5~8 minutes, biocatalyst be added is removed, draws after supernatant is diluted to the multiple suitable for test and carries out efficient liquid phase
Chromatography measures the concentration of catalysate xylonic.
The measuring method of above-mentioned catalysate xylonic is:
The model of high performance liquid chromatograph used is Agilent 1100Hewlett-Packard, is equipped with UV detector
(210nm) and Bio-Rad Aminex HPX-87H analytical column (300 × 7.8mm), 55 DEG C of column temperature, mobile phase 10mM H2SO4, stream
Fast 0.4mL/min, 5 μ L of sampling volume.
The invention discloses a kind of methods for preparing the full cell of Gluconobacter oxvdans as carbon source low cost using glycerol, first
Genetic engineering transformation was carried out to wild type Gluconobacter oxvdans before this, allowing to glycerol is carbon source for growth, and gene is made
The full cell of engineering bacteria Gluconobacter oxvdans, the engineering bacteria knocked out film combination alcohol dehydrogenase GOX1068 and film combine
Polyol dehydrogenase GOX0854.The full cell of the engineering bacteria prepared in this way is catalyzed xylose and obtains xylose as biocatalyst
For acid, its catalytic efficiency is verified.As the result is shown: the whole-cell catalytic process is shown in attached drawing 2, G.oxydans Δ GOX1068 Δ
GOX0854 whole-cell catalytic 30 hours, xylonic 61.6g/L is generated, the yield of xylose to xylonic is 1.05g/g.
The outstanding feature and beneficial effect that the present invention has are:
(1) present invention with cheap glycerol minimal medium instead of traditional expensive sorb alkoxide complex culture medium,
The preparation cost for greatly reducing Gluconobacter oxvdans biocatalyst expands Gluconobacter oxvdans biocatalysis
Application of the agent on conversion technology.
(2) the alcohol dehydrogenase GOX1068 and film that the present invention has knocked out film combination in G.oxydans 621H are combined polynary
Alcohol dehydrogenase GOX0854, when using glycerol as the carbon source culture recombinant bacterium, the generation of glyceric acid and dihydroxyacetone (DHA) is blocked,
The glycerol of consumption is only used to support thallus own growth, thus improves the accumulation of Gluconobacter oxvdans biomass.
(3) after knocking out GOX1068 and GOX0854, still there is function known to six kinds on the cell membrane of G.oxydans 621H
The dehydrogenase of energy plays a role, and the present invention demonstrates the catalytic action of one of dehydrogenase glucose dehydrogenase, uses
G.oxydans Δ GOX1068 Δ GOX0854 whole-cell catalytic xylose production xylonic, the yield of xylonic reach 61.6g/L,
The yield of xylose to xylonic is 1.05g/g.Based on the dehydrogenase that other films combine, which is urged
Agent is also applied in the production of other compounds.
(4) the full cell of complete G.oxydans Δ GOX1068 Δ GOX0854 is directly used to be given birth to as biocatalyst
Object conversion, reaction condition is mild, easy to operate.And biocatalyst can be removed with centrifugal process, subsequent products separation and Extraction
Simply.
Detailed description of the invention
Fig. 1 G.oxydans 621H and G.oxydans Δ GOX1068 Δ GOX0854 whole-cell catalytic xylose consume column
Figure
Wherein, 1: the full cell of G.oxydans 621H prepared using sorbierite as carbon source；2: being prepared by carbon source of sorbierite
The full cell of G.oxydans Δ GOX1068 Δ GOX0854；3: the full cell of G.oxydans 621H prepared using glycerol as carbon source；
4: the full cell of G.oxydans Δ GOX1068 Δ GOX0854 prepared using glycerol as carbon source.
Fig. 2 G.oxydans Δ GOX1068 Δ GOX0854 whole-cell catalytic xylose production xylonic conditional curve.
Specific embodiment
The original strain Gluconobacter oxydans 621H purchase that the present invention and embodiment are related to is in the micro- life of Germany
Object Culture Collection Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen,
Braunschweig, Germany), deposit number be DSM No.2343, the bacterial strain application examples see document ( C et
al.,Appl.Microbiol.Biotechnol.,2007,76:553-559；Zhang M et al.,
J.Ind.Microbiol.Biotechnol.,2013,40:379-388)。
Embodiment 1: gene relevant to glycerol metabolism product is knocked out
(1) knockout for the alcohol dehydrogenase large subunit GOX1068 that film combines
The alcohol dehydrogenase large subunit GOX1068 sequence length that film combines is 2274 bases, nucleotide sequence such as SEQ
Shown in ID NO.1.
The bacterial strain that the present invention constructs is to carry out genetic modification on the basis of G.oxydans 621H original bacteria.
The genomic DNA of bacterial strain G.oxydans 621H is prepared using conventional method, which can refer to scientific publication
The method of bacterial genomes prepared in a small amount in " the fine works molecular biology experiment guide " that society publishes, extracts G.oxydans
The genomic DNA of 621H；Using G.oxydans 621H genome as template, using primer " GOX1068up.f and
The upstream and downstream of GOX1068up.r " and " GOX1068down.f and GOX1068down.r " PCR amplification GOX1068 encoding gene are same
Source arm.Then GOX1068up.f and GOX1068down.r primer is utilized, is carried out by template of upstream and downstream homology arm obtained
Recombinant PCR, obtains the truncated segment of GOX1068, and both ends include EcoRI and BamHI restriction enzyme site.
EcoRI and BamHI is used to carry out double enzymes respectively the truncated segment of GOX1068 and suicide plasmid pK18mobsacB
It cuts, digestion products use T after glue recycles4DNA ligase connection, obtains and knocks out plasmid pK18mobsacB- Δ GOX1068.
Primer sequence is as follows:
GOX1068up.f:5’-CCAGAATTCGATGACTTCTGGTCTACTGAC-3’(EcoRI)
GOX1068up.r:5’-GAGCAGAACGTCTGAGTTGGTCGTGGCGTA-3’
GOX1068down.f:5’-TACGCCACGACCAACTCAGACGTTCTGCTC-3’
GOX1068down.r:5’-TAAGGATCCTCAGGGGTGATCCGCGGTCG’(BamHI)
To be containing the Escherichia coli Escherichia coli DH5 α for knocking out plasmid pK18mobsacB- Δ GOX1068
F+strain, to contain the Escherichia coli Escherichia coli HB101 of helper plasmid pRK2013 as supplementary strain, with
G.oxydans621H as F-strain, using sorb alkoxide complex medium agar sugar plate add 50 μ g/mL kanamycins,
50 μ g/mL Cefoxitins and 0.1% acetic acid (v/v) screen homologous single-crossover bacterial strain, and wherein acetic acid is for removing Escherichia coli
Growth.
Using the sorb alkoxide complex medium agar sugar plate screening homologous double-crossover containing 10% sucrose, that is, knock out
The bacterial strain of GOX1068 gene.
Using GOX1068up.f and GOX1068down.r as primer, with previous step in the sorb alkoxide complex containing 10% sucrose
The genome of the transformant grown on medium agar sugar plate is template, carries out PCR amplification.GOX1068 is only obtained to amplification
The bacterial strain of truncated segment further expands 16rDNA segment using primer 2 8S and 1492R using its genome as template, through surveying
Blast is carried out after sequence to compare to be Gluconobacter oxvdans, i.e., successfully constructs Gluconobacter oxvdans knock-out bacterial strain Δ
GOX1068。
(2) knockout for the polyol dehydrogenase subunit GOX0854 that film combines
The polyol dehydrogenase subunit GOX0854 sequence length that film combines is 2232 bases, nucleotide sequence such as SEQ
Shown in ID NO.2.
Knockout step of the knockout step of gene GOX0854 referring to GOX1068 in the present embodiment step (1), primer sequence
It is as follows:
GOX0854up.f:5’-CCAGAATTCGATGCGCAGATCCCATCTTCT-3’(EcoRI)
GOX0854up.r:5’-GCCCCACGGACCATTCTGGCACTCTGCTGC-3’
GOX0854down.f:5’-GCAGCAGAGTGCCAGAATGGTCCGTGGGGC-3’
GOX0854down.r:5’-TATAAGCTTTCAGCCCTTGTGATCAGGCA’(HindIII)
The recombination Gluconobacter oxvdans bacterial strain for having knocked out two genes that will finally obtain, the bacterium are named as
Gluconobacter oxydansΔGOX1068ΔGOX0854。
Experiment confirms: above-mentioned Gluconobacter oxydans Δ GOX1068 Δ GOX0854 genetic engineering bacterium is that leather is blue
Family name's negative bacterium, stringent aerobic growth, optimum culturing temperature are 30 ± 1 DEG C, can be in the sorbierite containing 50 μ g/mL Cefoxitins
It is grown on complicated culture medium or glycerol minimal medium.
The preparation of 2: four kinds of thallus whole-cell catalysts of embodiment
(1) by bacterial strain Gluconobacter oxvdans G.oxydans 621H and G.oxydans Δ GOX1068 Δ GOX0854
Being crossed to respectively containing mass volume ratio is 1.5~1.8% agar and the sorb alkoxide complex culture medium for containing 50 μ g/mL Cefoxitins
On plate, G.oxydans 621H in 30 ± 1 DEG C shaking table shaken cultivation 24 ± 1 hours, G.oxydans Δ GOX1068 Δ
GOX0854 in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours；
(2) first order seed: under sterile conditions, picking them separately the single colonie on step (1) plate with sterile toothpick,
Then be inoculated into respectively in the sorb alkoxide complex culture medium containing 50 μ g/mL Cefoxitins of 5mL, G.oxydans 621H in 30 ±
1 DEG C shaking table shaken cultivation 24 ± 1 hours, G.oxydans Δ GOX1068 Δ GOX0854 in 30 ± 1 DEG C of shaking table shaken cultivations 36 ±
1 hour；
(3) secondary seed: aseptically, the inoculum concentration that step (2) resulting bacterium solution is 2~4% with volume ratio is taken
It is inoculated into the sorb alkoxide complex culture medium of 50mL respectively, G.oxydans 621H is small in 30 ± 1 DEG C of shaking table shaken cultivations 24 ± 1
When, G.oxydans Δ GOX1068 Δ GOX0854 in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours；
(4) shaking flask culture: aseptically, the culture for two kinds of bacterium that step (3) is obtained respectively with 8,000 ±
500 revs/min are centrifuged 2 minutes, are distinguished washing thalline 2 times with 0.85% physiological saline, then respectively hang thallus and play physiology
G.oxydans 621H bacterium solution and recombination Gluconobacter oxvdans G.oxydans Δ GOX1068 Δ GOX0854 are obtained in salt water
Bacterium solution；
By bacterium solution final concentration OD600nmG.oxydans 621H bacterium solution is inoculated into 100mL sorb respectively for 0.1 inoculum concentration
In alkoxide complex culture medium and 100mL glycerol minimal medium, in 30 ± 1 DEG C shaking table shaken cultivation about 36 ± 1 hours；
By bacterium solution final concentration OD600nmG.oxydans Δ GOX1068 Δ GOX0854 bacterium solution is connect respectively for 0.1 inoculum concentration
Kind is into 100mL sorb alkoxide complex culture medium and 100mL glycerol minimal medium, in 30 ± 1 DEG C of shaking table shaken cultivations about 36
± 1 hour；
Wherein: sorb alkoxide complex culture medium prescription described in above-mentioned steps (1)~(4) is: sorbierite 73g/L；Yeast
Powder 18.4g/L；(NH4)2SO41.5g/L, KH2PO41.5g/L, MgSO4·7H2O 0.47g/L；121 DEG C sterilize 20 minutes.
Glycerol minimal medium formula is: (NH4)2SO42g/L, KH2PO42.2g/L, Na2HPO40.2g/L,
MgSO4·7H2O 0.2g/L, metal ion solution (50 ×) 20mL, 121 DEG C sterilize 20 minutes；Glycerol 2g/L, 121 DEG C individually go out
Bacterium 20 minutes；Vitamin (100 ×) solution 10mL, filtration sterilization.Wherein, metal ion solution (50 ×) is formulated are as follows: nitrotrimethylolmethane
Acetic acid 0.25g/L, EDTA 1.5g/L, FeSO4·7H2O 0.55g/L, ZnSO4·7H2O 0.45g/L, CoCl2·6H2O
0.03g/L, MnCl2·4H2O 0.1g/L, CuSO4·5H2O 0.03g/L, CaCl2·2H2O 0.45g/L, NaMoO4·2H2O
0.004g/L, H3BO30.05g/L, KI 0.01g/L；Vitamin (100 ×) solution formula are as follows: calcium pantothenate 0.05g/L, niacin
0.04g/L, p-aminobenzoic acid 0.04g/L, glutamine 20g/L.
(5) thallus is collected: by the four kinds of cultures cultivated in (4) two kinds of culture mediums of step respectively with 6,000 ± 500
Rev/min centrifugation 10 minutes；And distinguished washing thalline 2~3 times with the phosphate buffer of pH 7.4,1/15M, then by cell
It hangs and is played in the phosphate buffer respectively, and make the final concentration (OD of cell respectively600nm) it is 30~50, that is, obtain two kinds of trainings
Four kinds of full cell bacterium solutions for biocatalyst in base source are supported, for use.
Embodiment 3: the comparison of the catalytic effect for four kinds of biocatalysts that embodiment 2 obtains
Four kinds of full cell bacterium solutions for obtaining two kinds of culture medium sources using embodiment 2 make catalyst system as biocatalyst
Middle final concentration of cells (OD600nm) it is 13, under 30 DEG C, the stringent aerobic condition of pH 7.0~7.5, conversion concentration is about 7.8g/L's
Xylose；150 revs/min of shaking bath oscillations；It is catalyzed the consumption of 6 hours sample detection xyloses and the generation of xylonic；By gained
Conversion fluid, be centrifuged 5 minutes with 13,000 ± 500 revs/min, remove biocatalyst be added, draw supernatant carry out it is efficient
The concentration of liquid-phase chromatographic analysis measurement xylose.
As the result is shown: the xylose consumption process is shown in attached drawing 1, reacts 6 hours, is prepared using sorbierite as carbon source
It G.oxydans621H, G.oxydans Δ GOX1068 Δ GOX0854 full cell and is prepared using glycerol as carbon source
Xylose can be catalyzed completely by the full cell of G.oxydans Δ GOX1068 Δ GOX0854, the oxidation Portugal prepared using glycerol as carbon source
The catalysis of the catalytic levels of the full cell of grape saccharic acid bacillus and the full cell of Gluconobacter oxvdans prepared using sorbierite as carbon source
It is on close level.
Embodiment 4: the G.oxydans Δ GOX1068 Δ GOX0854 whole-cell catalytic xylose life prepared using glycerol as carbon source
Produce the application of xylonic
Referring to embodiment 1, using wild type Gluconobacter oxvdans G.oxydans 621H as starting strain, film knot is knocked out
Recombination Gluconobacter oxvdans, name is made in the polyol dehydrogenase GOX0854 that the alcohol dehydrogenase GOX1068 and film of conjunction are combined
For Gluconobacter oxydans Δ GOX1068 Δ GOX0854.
(1) by obtained recombination Gluconobacter oxvdans Gluconobacter oxydans Δ GOX1068 Δ GOX0854
Being crossed to containing mass volume ratio is 1.5~1.8% agar and the sorb alkoxide complex culture medium flat plate for containing 50 μ g/mL Cefoxitins
On, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours；
(2) under sterile conditions, the single colonie on step (1) plate is picked them separately with sterile toothpick, be then inoculated with
Into the sorb alkoxide complex culture medium containing 50 μ g/mL Cefoxitins of 5mL, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours,
Obtain recombination Gluconobacter oxvdans first order seed；
(3) under sterile conditions, the resulting first order seed bacterium solution of step (2) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the sorb alkoxide complex culture medium of 50mL, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, obtain recombination oxidation
Gluconobacter suboxydans secondary seed bacterium solution；
(4) aseptically, the recombination Gluconobacter oxvdans secondary seed bacterium solution 8,000 step (3) obtained
± 500 revs/min are centrifuged 2~5 minutes, with 0.85% brine thallus 2~3 times, then again hang thallus and play life
Recombination Gluconobacter oxvdans bacterium solution is obtained in reason salt water, by bacterium solution final concentration OD600nmRecombination is aoxidized for 0.1 inoculum concentration
Gluconobacter suboxydans are inoculated into 100mL glycerol minimal medium, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, obtain
Gluconobacter oxvdans culture；
(5) by step (4) culture obtain 6,000 ± 500 revs/min of Gluconobacter oxvdans culture centrifugation 10~
15 minutes；And with pH 7.4, phosphate buffer washing thalline 2~3 times of 1/15M, the phosphoric acid then is played by cell is outstanding
In salt buffer, make the final concentration OD of somatic cells600nmIt is 30~50, that is, obtains the Gluconobacter oxydans for being used for biocatalyst
The full cell bacterium solution of bacillus Gluconobacter oxydans Δ GOX1068 Δ GOX0854；
Wherein:
The formula of above-mentioned sorb alkoxide complex culture medium is: sorbierite 73g/L, yeast powder 18.4g/L, (NH4)2SO4 1.5g/
L, KH2PO41.5g/L, MgSO4·7H2O 0.47g/L；121 DEG C sterilize 20 minutes；
The formula of above-mentioned glycerol minimal medium is: (NH4)2SO42g/L, KH2PO42.2g/L, Na2HPO4 0.2g/
L, MgSO4·7H2O 0.2g/L, metal ion solution (50 ×) 20mL, 121 DEG C sterilize 20 minutes；Glycerol 2g/L, 121 DEG C individually
Sterilizing 20 minutes；Vitamin (100 ×) solution 10mL, filtration sterilization；Wherein, the formula of metal ion solution (50 ×) are as follows: nitre
Base triacetic acid 0.25g/L, EDTA 1.5g/L, FeSO4·7H2O 0.55g/L, ZnSO4·7H2O 0.45g/L, CoCl2·6H2O
0.03g/L, MnCl2·4H2O 0.1g/L, CuSO4·5H2O 0.03g/L, CaCl2·2H2O 0.45g/L, NaMoO4·2H2O
0.004g/L, H3BO30.05g/L, KI 0.01g/L；The formula of vitamin (100 ×) solution are as follows: calcium pantothenate 0.05g/L, niacin
0.04g/L, p-aminobenzoic acid 0.04g/L, glutamine 20g/L.
With the full cell of Gluconobacter oxvdans Gluconobacter oxydans Δ GOX1068 Δ GOX0854 of preparation
As biocatalyst, make final concentration of cells OD in catalyst system600nmFor 20 biocatalyst, at 30 DEG C, pH 7.0~7.5
Under stringent aerobic condition, the xylose that concentration is 60g/L is converted；150 revs/min of shaking bath oscillations；Therebetween, it samples within every 3 hours
The consumption of xylose and the generation of xylonic are detected, and is 7.0~7.5 with the pH that 6M NaOH adjusts catalyst system, substrate xylose consumption
Stop catalysis when to the greatest extent；It by resulting conversion fluid, is centrifuged 5 minutes with 13,000 ± 500 revs/min, removes biocatalysis be added
The concentration that supernatant dilution suitable multiple carries out efficient liquid phase chromatographic analysis measurement whole-cell catalytic product xylonic is drawn in agent.
As the result is shown: the whole-cell catalytic process is shown in that attached drawing 2, the full cell of G.oxydans Δ GOX1068 Δ GOX0854 are urged
Change 30 hours, generate xylonic 61.6g/L, the yield of xylose to xylonic is 1.05g/g.
Sequence table
<110>Shandong University
<120>a kind of method that the full cell of Gluconobacter oxvdans is prepared as carbon source using glycerol
<141> 2018-07-20
<160> 2
<170> SIPO SequenceListing 1.0
<210> 1
<211> 2274
<212> DNA
<213>the alcohol dehydrogenase large subunit GOX1068 sequence that film combines
<400> 1
tcaggggtga tccgcggtcg gaacatcggc cgtggactgc gggacattga ggaactgatc 60
gttcggaacg ccagttgagt tttcacgtgc cttgacttcg tcgtcgtagg attcgtttgc 120
gcgcttcacg atgaagttgc ggatatcctc gatctgctca ggcgtcatgg acgtgtcgaa 180
gcggtccatg ccgtaagccg tcagagcacc gcgaccaacg agcttgtaga agctttccct 240
tccgcgcgga gcaccggacc agcgaaggtc aggcagaacg ccaccggaga tcgcgttgtc 300
gccatggcag gccgagcaga acgtctgata catgaagtat ccgtccttct ggcgggcctc 360
gtcataggtc ggaaccggct tgacgggggt gaagcccagc tcgttcttcg gcggcaggct 420
gtccttgccg tcaagcgaga aggcgatcac gcgggagtgg ttgaccgtcc agcccgaggt 480
gcgggcaacg ccgccgtaca ggaacggata gatgccaccc cagcccactt caaccgcgac 540
atactgcttg ccgttggccg tataggtcac cggcggagcg atgatcgcgc tctgtgcggg 600
gaaggaatag agatcgttgc cgttcgtggc gtcgtaggcg tggaattcgc cgtttgccag 660
accctggaag atcacgttac cggccgtggc cagaagacca ccgttccacg gacccttgtg 720
gttgatcgtg aaggccggag ccatcttttc cggatcccag gcaaccgtcc agcccttcag 780
gaccttcagg aagtccttct tggcggccac atgctccgga tcattgtcgt cgaagagacc 840
gatcttgttc atgtcgaggc cgaggttcca ggcgtcatgg tacgccttga agccgccttt 900
ctggccatcg tacaggagcg ggatctgctg tgccgggatg tagaccagct tcgtcttcgg 960
gctgtaggcc atggcggcga agttatggcc accaagctca cccgggatgc cgagccaggg 1020
cttgcccgtc agggtccaca gggcgtcagg cacgtagttc ggacgacccg taaccggatc 1080
caggccgttg gcccagttct cgtaggtgta gggcttgccg gtgatgaact taccggtctt 1140
tgcatcgatg atgtagaaga agccgttctt cggagcatgc acgatgacgt ggcgcatctc 1200
gccgttgacc ggcatgtcga gcgtcatgat ctgctggacg gaggtgtaat cccactcgtc 1260
catcggcgtt tcctggaagt gccagacata cttgccggtg tcaggattga tcgcgacgat 1320
gctgcccagg aagaggttgt cgcccttgcc ttcggagcgg aacttgtagt tccacggcga 1380
gccgttgccg acgcccaggt agacgaggtc cgtcaccggg tcatagacga gcgaatccca 1440
gacggtaccg ccgccgccct gctgcttcca ggcgccgttc ttgccccagg tcgggtaggc 1500
cttggacatc aggatgtcgt cggaagctgc gccgtccggc ttgttctcgg ggttcggaac 1560
ggtgaagaag cgccagtcga gcttgccggt ttccgcgtcg aaggcagaga cgaagccgcg 1620
ggcaccgaat tctgcaccac cgttgccgat caggaccttg cccttggcga tacggggggc 1680
accgtcaacc gtgtaggacc gctggtggcc gagctgggcc tccttgggga tcgtgtagac 1740
gctccagacc agcttgccgg tcttggcgtc gagtgcgatc agacggccgt cgaacgtgcc 1800
gaaatagacc ttgccgttcc agtaggcggc gccacggctg accgtgtcac agcagccacg 1860
gtcggcgatg ttgcccggaa ccttcggatc gtaggaccac aggagcttgc cggtcgctgc 1920
atccagcgcc ttcatcttgc tccagttggt cgtggcgtac atgacgccgt tgacgatcag 1980
gggagtgcct tcctggccgc ggttcgtgtc cagatcatag tgccaggcga gcttcagctt 2040
gccgacattc tccgtattga tctggtcgag cgggctgtag cgctgctcgg aataggaacg 2100
gccgtaggac agccagtccc cgggatggcc gccgttatcg gagctcgtga ttgctgtgcc 2160
ggtatcttcc tgcgcaaaag cgacaggtac agctgctgaa aatgccagcg cggcagcgca 2220
gctgagaagg cgctttttcg taaccttgat cggcgtcagt agaccagaag tcat 2274
<210> 2
<211> 2232
<212> DNA
<213>the polyol dehydrogenase subunit GOX0854 sequence that film combines
<400> 2
tcagcccttg tgatcaggca gtgcgtaaac caccagctga tcgctgaccg gcgtcatcat 60
gaagtgatga ccacccgcca tgatggcgac gtactgatga ccattggctt cgtaggtcat 120
cgggttagcc tgaccgccgc ccggcaggac cgcgctccag accaccttgc cggtgtgctc 180
gtcgatggca cggatctggt tatccgtagc tgccgcgatg aacaccacgc caccggccgt 240
cacgaccgag ccaccattat tcggcgtacc gatttcccag ggaagaccgg tcggcaggcc 300
ccacggacca ttggcgcggg ccgttcccag cgggtgctgc cacagcacct tctggccgtg 360
cttcatgtcg atggccgtga tcatgccgta gggcgggcgg ttgcacatca tacccgtgta 420
ctgatcccag aacggggtca cgacgatacc gtaaggcgtg ccgtccatgg cgccgttacc 480
ttcggcgcca ccgccacccg gcttgtagtt cgggtcatcg atcggcatca ggccaagttc 540
gtcggccttc ttgcgggtta cgagctggtc gtacatcggg gtgatgttcc agttcgcaat 600
caggatgccg ctctgcgggt cataggacac ggaaccccag tcgctgccgc cgttatagcc 660
cggatactcg atccagggct tgtcgacgct cggtggcgtg aactcacccg tatagttcgc 720
acggcggaac ttgatacggc agaagagctg gtcgatgggg gacatgcccc acatatccgt 780
ctctttcaga tccggcacgc gcagagccgg cattcccgtg gaccagggct gcgtcggcga 840
acgcggatcg cccgggatca cgcccggcga cggagcggga cgctcttcga ccggcaggat 900
cggcttgccg tcacgacggt cgagcacgaa ggtctggcca cgcttggtcg gcatgatgag 960
tgcgggaaca ggctgaccat cctggccggg catgtccatg agggtggcct gcgagccgat 1020
gtcatagtcc cagacgtcct tgtgaacggt ctggaagacc cagcgcggcg aacccgtctt 1080
tacgtcaagc gcgacaactg cggacgagac cttgttttct tcagggctac gcagggcact 1140
gtagtaatcg gaagccgagt tgccggtcgg gacgtagacg aggcccagcg cattgtcgcc 1200
ggtcatcgca gcccaggagt tcggcgtacc acggctgtaa tggttgttgc cggtcggctg 1260
gctgtgatcg ttggggcggt tcacgtccca ggcccacagg aacttgccgc tctcggcatc 1320
atagccacgg atcacacccg acggagccca gcggcgctga ccgtcgagaa cttcgtggtt 1380
gaccacaacc acaccgttca cgaccggcgg cggcgtcgtc atggagacga agccggggac 1440
ggattcgcca agaccctgca tcaggttgac ctggccgcca ttgccgaagc cttcgcacag 1500
attgccggtc gcggcatcaa ccgcgatcag gcgcatgtcg agcgtgcctt caaggatacg 1560
gttatggcag ggctggcctt cgggcacctg agacgacgtg aaatacgtca cgcccttgca 1620
cgctgcggtg taggggatgg cttcgtattt ctcgttgatg ttgtgacgcc agatctcctt 1680
acccgtcgcc gggtcgatct tcatgatgtc gttctgtgcc gagcacatgt agaggccgtc 1740
acccaccttg atcggggtgg tttcggcagc ccatttgttc gtctggcccg gacgcggata 1800
gctaccggtg tgatagacga aagcgacctt gagctgatcg gcgttctgcg gcgtgatctc 1860
ggacagcggc gaatagcgca tctgactgtc gtcatggccg taggcggccc agtctccgga 1920
ggcggggctg gcactctgct gcggggccat ctgcggaacg tcgttcggat ccatggaccc 1980
gatatccggc aggttggccg cgttcacacc cggagcctgg ggcgcatagg gggacgggcc 2040
ggaaaagcgg ctcttcgcgg ccggcgtgtt ctcggtcggc tcgaaggaat tgccgctgcc 2100
attgccgggg cccggcacgg aggaggtcgg cgagccaccg ctgcctgcgg gggcgaactg 2160
ggcattggca gccagcggtg cgcaggccag cgtggcacag gcaacggtgg cgagaagatg 2220
ggatctgcgc at 2232
Claims (5)
1. a kind of method for preparing the full cell of Gluconobacter oxvdans as carbon source using glycerol, step is:
(1) genetic engineering transformation is carried out to wild type Gluconobacter oxvdans, weight can be made using glycerol as carbon source for growth
Group Gluconobacter oxvdans；
(2) obtained recombination Gluconobacter oxvdans are crossed to containing mass volume ratio for 1.5~1.8% agar and containing 50 μ
On the sorb alkoxide complex culture medium flat plate of g/mL Cefoxitin, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours；
(3) under sterile conditions, the single colonie on step (2) plate is picked them separately with sterile toothpick, is then seeded into 5mL
The sorb alkoxide complex culture medium containing 50 μ g/mL Cefoxitins in, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, weighed
Group Gluconobacter oxvdans first order seed；
(4) under sterile conditions, the resulting first order seed bacterium solution of step (3) is taken, is connect with the inoculum concentration that volume ratio is 2~4%
Kind into the sorb alkoxide complex culture medium of 50mL, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, obtain recombination oxidation grape
Saccharic acid bacillus secondary seed bacterium solution；
(5) aseptically, the recombination Gluconobacter oxvdans secondary seed bacterium solution 8,000 ± 500 step (4) obtained
Revs/min centrifugation 2~5 minutes, with 0.85% brine thallus 2~3 times, physiological saline then was played by thallus is outstanding again
In obtain recombination Gluconobacter oxvdans bacterium solution, by bacterium solution final concentration OD600nmOxidizing glucose will be recombinated for 0.1 inoculum concentration
Acidfast bacilli is inoculated into 100mL glycerol minimal medium, in 30 ± 1 DEG C shaking table shaken cultivation 36 ± 1 hours, obtain oxidation Portugal
Grape saccharic acid alphacterium culture；
(6) 6,000 ± 500 revs/min of Gluconobacter oxvdans culture for obtaining step (5) culture are centrifuged 10~15 points
Clock；And with pH 7.4, phosphate buffer washing thalline 2~3 times of 1/15M, then plays cell is outstanding the phosphate and delay
In fliud flushing, make the final concentration OD of somatic cells600nmIt is 30~50, that is, obtains the Gluconobacter oxvdans for being used for biocatalyst
Full cell bacterium solution；
Wherein:
The formula of sorb alkoxide complex culture medium described in above-mentioned steps (2)~(3) is: sorbierite 73g/L, yeast powder 18.4g/L,
(NH4)2SO41.5g/L, KH2PO41.5g/L, MgSO4·7H2O 0.47g/L；121 DEG C sterilize 20 minutes；
The formula of glycerol minimal medium described in above-mentioned steps (5) is: (NH4)2SO42g/L, KH2PO42.2g/L
Na2HPO40.2g/L, MgSO4·7H2O 0.2g/L, metal ion solution (50 ×) 20mL, 121 DEG C sterilize 20 minutes；Glycerol
2g/L, 121 DEG C individually sterilize 20 minutes；Vitamin (100 ×) solution 10mL, filtration sterilization；Wherein, metal ion solution (50
×) formula are as follows: nitro triacetic acid 0.25g/L, EDTA 1.5g/L, FeSO4·7H2O 0.55g/L, ZnSO4·7H2O
0.45g/L, CoCl2·6H2O 0.03g/L, MnCl2·4H2O 0.1g/L, CuSO4·5H2O 0.03g/L, CaCl2·2H2O
0.45g/L, NaMoO4·2H2O 0.004g/L, H3BO30.05g/L, KI 0.01g/L；The formula of vitamin (100 ×) solution
Are as follows: calcium pantothenate 0.05g/L, niacin 0.04g/L, p-aminobenzoic acid 0.04g/L, glutamine 20g/L.
2. the method for preparing the full cell of Gluconobacter oxvdans as carbon source using glycerol according to claim 1, feature exist
In: the recombination Gluconobacter oxvdans obtained are to be named as Gluconobacter oxydans Δ GOX1068 Δ
The genetic engineering bacterium of GOX0854, G.oxydans Δ GOX1068 Δ GOX0854 is with wild type Gluconobacter oxvdans
G.oxydans 621H is starting strain, knocks out the alcohol dehydrogenase GOX1068 that film combines and the polyol dehydrogenase that film combines
GOX0854 is made.
3. the method for preparing the full cell of Gluconobacter oxvdans as carbon source using glycerol according to claim 2, feature exist
In: the Gluconobacter oxydans Δ GOX1068 Δ GOX0854 genetic engineering bacterium is Gram-negative bacteria, stringent good
Oxide growth, cultivation temperature are 30 ± 1 DEG C, can the sorb alkoxide complex culture medium containing 50 μ g/mL Cefoxitins or glycerol without
It is grown on machine salt culture medium.
4. the full cell of Gluconobacter oxvdans of method preparation as claimed in claim 1 or 2 is as biocatalyst in catalysis wood
Application in sugar production xylonic.
5. application according to claim 4, which is characterized in that application method is:
Using the full cell of the Gluconobacter oxvdans of preparation as biocatalyst, make final concentration of cells OD in catalyst system600nm
It is 10~20, under 30 DEG C, the stringent aerobic condition of pH 7.0~7.5, converts the xylose that concentration is 5~60g/L；150 revs/min
Oscillation 3~30 hours, obtains the conversion fluid containing xylonic；By resulting conversion fluid, with 13,000 ± 500 revs/min of centrifugations 5~8
Minute, biocatalyst be added is removed, draws after supernatant is diluted to the multiple suitable for test and carries out high performance liquid chromatography point
Analysis measures the concentration of catalysate xylonic.
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