CN110016455A - A kind of conversion bacterial strain catalyzing and synthesizing Miglitol intermediate and method - Google Patents
A kind of conversion bacterial strain catalyzing and synthesizing Miglitol intermediate and method Download PDFInfo
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Abstract
The invention discloses a kind of conversion bacterial strain for catalyzing and synthesizing Miglitol intermediate and methods, after the conversion bacterial strain is by the inverted recombinant expression plasmid pBBR-sldAB of starting strain ZJB16009, it is obtained again through screening, it is named as gluconobacter oxydans (Gluconobacter oxydans), strain pBBR-sldAB, deposit number is CCTCC No.M 2019033, its N- hydroxyethyl glucosamine dehydrogenase activity has a distinct increment compared with starting strain ZJB16009, and space-time yield is about 2 times of starting strain ZJB16009, show gluconobacter oxydans pBBR-sldAB of the present invention potential using value with higher in the catalyzing and synthesizing of miglitol key intermediate 6NSL.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of conversion for catalyzing and synthesizing Miglitol intermediate
Bacterial strain and method.
Background technique
Miglitol [1- (2- ethoxy) -2- (methylol) -3,4,5- piperidines triols, miglitol] is glucose knot
Structure analog (as shown in formula I) is a kind of alpha-glycosidase inhibitor class antidiabetic drug of Bayer Bitterfeld GmbH (Bayer) company exploitation
Object has high-affinity to amylopsin and alpha-glucosidase, is able to suppress the hydrolysis of disaccharides, polysaccharide and glycoconjugate, prolongs
The absorption of slow glucose and other monosaccharide, hypoglycemic effect is obvious, and toxic side effect is significantly lower than sulfonephthalein arteries and veins class and double flesh class drugs,
Through one of the critical treatment drug for becoming treatment type-2 diabetes mellitus.
Patent US4246345, US4806650, US5401645 etc. are disclosed using N- hydroxyethyl glucosamine as substrate
The process route of student's object combined method synthesis Miglitol is the main technological route of current synthesis Miglitol, and the substrate is by Portugal
Grape sugar and ethanol amine are synthesized through a step chemistry hydrogenation process, and it is right to introduce gluconobacter oxydans (Gluconobacter oxydans)
Substrate N- hydroxyethyl glucosamine carries out the selective asymmetric oxidation of 4- hydroxyl, and the intermediate product direct-coupling chemistry of acquisition adds
Hydrogen Cyclization Miglitol.The more traditional full chemistry synthesis of the technique has synthesis route short, and at low cost, yield is higher
Advantage.Wherein, gluconobacter oxydans asymmetry selective oxidation is the critical limitation step of the route.
Although research has the bacterial strain for producing Miglitol intermediate, the especially phase of Gluconobacter oxydans source bacterial strain both at home and abroad
Report is closed, such as patent application CN101302549A, CN105968042A, CN104693109A etc., but due to gluconobacter oxydans
The biomass of fermentation unit is few, and the vigor of wild strain N- hydroxyethyl glucosamine dehydrogenase is relatively weak, to substrate N- hydroxyl second
The tolerance of base gucosamine is poor.Catalysis process is slower, and concentration of substrate and conversion ratio are lower, as used in CN101029321A
N- hydroxyethyl glucosamine concentration in 40g/L, limit the production level of Miglitol, need to establish high sorbitol dehydrogenase
The bacterial strain of enzyme activity.In addition, the cell fixation or multiple resting cell by gluconobacter oxydans repeatedly apply strategy
(CN101270378A) yield of unit cell can be improved to a certain extent, but resting cell is to substrate N- ethoxy Portugal
The specific enzyme activity power of the selection dehydrogenation of grapes glucosamine does not significantly improve, single batch substrate feed intake 50g/L and it is following (Hu, ZC., Bu,
JL.,Wang,RY.et al.Appl Biochem Biotechnol(2018).https://doi.org/10.1007/
s12010-018-2924-y).Patent CN108441491A is by establishing miglitol key intermediate 6- deoxidation -6- amino (N-
Ethoxy)-α-L- furans sorbose (6NSL) fast quantitative measurement method for detecting, combined mutagenesis screening obtains the catalysis of high vigor and closes
At Miglitol intermediate 6NSL bacterial strain ZJB16009, concentration of substrate is promoted to 60-80/L, if it is dense to further increase substrate
Degree, can significantly reduce the yield of product 6NSL.Early period document report -6 amino of intermediate 6- deoxidation (N- ethoxy)-α-L- furan
Mutter sorbose (6NSL) isomerization and its stability can significantly affect Miglitol ultimate yield (J.Org.Chem.2009,
74,1766–1769).Therefore, gluconobacter oxydans are improved to the dehydrogenase activity and full cell of substrate N- hydroxyethyl glucosamine
The space-time yield of synthesis key intermediate 6NSL is to promote the Key Strategy of Miglitol bio-conversion process.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provides one kind and catalyzes and synthesizes the production of Miglitol intermediate space-time
The higher conversion bacterial strain of rate, for being catalyzed N- hydroxyethyl glucosamine synthesis -6 amino of 6- deoxidation (N- ethoxy)-α-L- furans
Sorbose.
A kind of conversion bacterial strain catalyzing and synthesizing Miglitol intermediate, is named as gluconobacter oxydans (Gluconobacter
Oxydans), strain pBBR-sldAB, deposit number are CCTCC No.M 2019033.The oxygen that the bacterial strain is obtained early period by inventor
Changing gluconobacter suboxydans, (bacterial strain ZJB16009 (deposit number CCTCC No.M201703), is detailed in Publication No. CN108441491A
Patent application), after an inverted recombinant expression plasmid pBBR-sldAB, obtained by screening, wherein recombinantly expressing matter
The construction method of grain pBB-sldAB are as follows: by the big small subunit of the sorbitol dehydrogenase of Gluconobacter oxvdans cell membrane localization
The coded sequence of SldA (protein sequence accession number ABP65281.1) and SldB (protein sequence accession number ABP65282.1)
(No. GenBank is EF493853.1) is inserted into promoter sequence (nucleotide sequence is as shown in SEQ ID No.1) and one
A terminator sequence (nucleotide sequence is as shown in SEQ ID No.2) is intermediate, then using restriction site XhoI and
EcoRI is inserted into extensive host expression vector pBBR1MCS-5, and building obtains pBBR-sldAB recombinant expression plasmid.Bacterial strain
PBBR-sldAB compared with original strain ZJB16009 for, catalyze and synthesize Miglitol intermediate (catalysis N- hydroxyethyl glucosamine
Synthesize -6 amino of 6- deoxidation (N- ethoxy)-α-L- furans sorbose) when have higher space-time yield.
Invention further provides the conversion bacterial strains to synthesize -6 amino (N- of 6- deoxidation in catalysis N- hydroxyethyl glucosamine
Ethoxy) application in-α-L- furans sorbose.
The present invention also provides a kind of methods for catalyzing and synthesizing Miglitol intermediate using the conversion bacterial strain.
The method, comprising the following steps:
(1) it by the conversion bacterial strain enzymatic production, and collects the thallus after fermentation and obtains resting cell；
(2) product Miglitol is obtained using resting cell catalysis substrate N- hydroxyethyl glucosamine prepared by step (1)
Intermediate: -6 amino of 6- deoxidation (N- ethoxy)-α-L- furans sorbose.
Wherein, in step (1) preparation method of resting cell the following steps are included: the conversion bacterial strain is inoculated into culture
Fermented and cultured is carried out in base, is then collected thallus and is obtained the resting cell, wherein the culture medium includes: D-glucitol
50g/L, yeast extract powder 25g/L, KH2PO45g/L, K2HPO45g/L, solvent are water.
Preferably, the culture medium further includes sodium glutamate, isoleucine, serine and arginine, and each amino acid makes
It is respectively 0.5~5g/L with concentration.
It is furthermore preferred that sodium glutamate 2g/L, isoleucine, serine and each 1g/L of arginine.
Preferably, catalysis reaction is formed using system in step (2) are as follows: 60~120g/ of substrate N- hydroxyethyl glucosamine
L, for the resting cell of step (1) preparation with wet thallus 40~80g/L of poidometer, remaining is water.
It is furthermore preferred that catalysis reaction is formed using system in step (2) are as follows: substrate N- hydroxyethyl glucosamine 60g/L, seven
Water magnesium sulfate 5g/L, for the resting cell of step (1) preparation with wet thallus poidometer 60g/L, remaining is water, and adjusting pH is 5.0.
Gluconobacter oxydans pBBR-sldAB of the present invention is by the inverted recombinant expression plasmid pBBR- of starting strain ZJB16009
After sldAB, then through screening acquisition, N- hydroxyethyl glucosamine dehydrogenase activity has larger mention compared with starting strain ZJB16009
It rises, and space-time yield is about 2 times of starting strain ZJB16009, it is shown that gluconobacter oxydans pBBR-sldAB of the present invention is in rice
Potential using value with higher in the catalyzing and synthesizing of lattice column alcohol key intermediate 6NSL.
Detailed description of the invention
Fig. 1 is that external source adds PQQ to the influence result figure of N- hydroxyethyl glucosamine dehydrogenase activity.
Fig. 2 is that external source adds sodium glutamate to the influence result figure of N- hydroxyethyl glucosamine dehydrogenase activity.
Fig. 3 is that external source adds isoleucine to the influence result figure of N- hydroxyethyl glucosamine dehydrogenase activity.
Fig. 4 is that external source adds serine to the influence result figure of N- hydroxyethyl glucosamine dehydrogenase activity.
Fig. 5 is that external source adds arginine to the influence result figure of N- hydroxyethyl glucosamine dehydrogenase activity.
Specific embodiment
Embodiment 1
Gluconobacter oxydans recombinant bacterium Gluconobacter oxydans pBBR-sldAB (CCTCC No.M
2019033) building.
(1) molecule construction of recombinant expression plasmid pBBR-sldAB:
By the big small subunit SldA of the sorbitol dehydrogenase of Gluconobacter oxvdans cell membrane localization, (protein sequence is logged in
Number ABP65281.1) and the coded sequence of SldB (protein sequence accession number ABP65282.1) (No. GenBank is
EF493853.1), the substitution of one of them or several nucleotide can cause the substitution of corresponding amino acid residue, to influence N-
The function of hydroxyethyl glucosamine dehydrogenase.The coded sequence is inserted into SEQ ID No.1 (promoter sequence) and SEQ ID
No.2 (terminator sequence) is intermediate, is inserted into extensive host expression vector using restriction site XhoI and EcoRI
In pBBR1MCS-5, building obtains pBBR-sldAB recombinant expression plasmid.
(2) (bacterial strain ZJB16009 (deposit number CCTCC No.M201703), is detailed in Publication No. to Gluconobacter oxvdans
The patent application of CN108441491A) electricity turns the preparation of competent cell:
1) saved from 4 DEG C it is flat pull upper picking single colonie and be inoculated in the test tube of the MP culture medium equipped with 5mL, and in 30 DEG C
Preculture, until OD600For 0.4-0.6 range；
2) take the preculture bacterium solution of 1mL, access equipped with 50mL EP culture medium (mannitol 80g/L, yeast powder 15g/L,
MgSO4·7H2O 2.5g/L, glycerol 0.5g/L, CaCl2In 500mL triangular flask 1.5g/L), 30 DEG C, 200rpm cultivates to bacterium
Body OD600For 0.5-0.6 range；
3) triangular flask equipped with bacterium solution is placed in 20min on ice；
4) 4 DEG C, 10min is centrifuged under the conditions of 8000rpm, remove supernatant；
5) the 10% sterile glycerite of 30mL pre-cooling, sufficiently resuspension thallus is added；
6) step 4,5 are repeated twice；
7) 10% glycerite of 1mL pre-cooling, abundant suspension thalline is added, and is dispensed into the 1.5mL EP pipe of oneself sterilizing
In, every 100 μ L of pipe is placed in -80 DEG C of refrigerators and saves backup.
(3) Gluconobacter oxvdans is electroporated:
1) it takes the competent cell of above-mentioned preparation to melt on ice, plasmid (pBBR-sldAB recombinant expression plasmid) 5 μ is added
L is mixed gently, and stands 30min；
2) it is transferred to the electric revolving cup of pre-cooling, 2000V, 200 Ω, 25 μ F carry out electricity and turn；
3) electric shock terminates the 400 μ L of MP culture medium that pre-cooling is added at once, mixed liquor removal is transferred in 1.5mLEP, 30
DEG C, 200rpm cultivates 4-6h；
4) 200 μ L coated plates are taken, the sorbierite for embracing western fourth sodium and 25ng/mL gentamicin sulphate containing 50ng/mL is coated on
On solid plate, 30 DEG C are grown to resistant clones after culture 2-4 days；
5) 4 days are cultivated to visible single colonie for 30 DEG C；
6) bacterium colony PCR is verified, sequence verification, primer sequence are as follows:
Upstream primer: 5 '-CTGGAGGCTTTCACCAAATGCCGAATACTTATGGC-3 '；
Downstream primer: 5 '-TCAGCCCCCGCTTTCTCAGCCCTTGTGATCAGG-3 '；
(4) screening of Gluconobacter oxvdans recon:
The correct positive recombinant of verifying is selected, using quick colour-developing method to the N- hydroxyethyl glucosamine in recon
Dehydrogenase activity carries out primary dcreening operation, obtains the recon Gluconobacter oxydans pBBR-sldAB of one plant of higher vigor,
It is named as gluconobacter oxydans (Gluconobacter oxydans), strain pBBR-sldAB, is preserved on January 8th, 2019
Positioned at the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC No.M 2019033.
Embodiment 2
Gluconobacter oxydans Gluconobacter oxydans pBBR-sldAB (CCTCC No.M 2019033) with lure
The N- hydroxyethyl glucosamine dehydrogenase activity for becoming bacterial strain ZJB16009 (CCTCC No.M201703) compares.
(1) preparation of gluconobacter oxydans resting cell: by starting strain gluconobacter oxydans mutagenic strain ZJB16009
And the recombinant bacterial strain Gluconobacter oxydans pBBR-sldAB that the present invention constructs is inoculated into equipped with 40mL respectively
In the 250mL triangular flask of seed culture fluid, the group of seed culture fluid becomes D-glucitol 50g/L, yeast extract powder 25g/L,
KH2PO45g/L, K2HPO45g/L, solvent are water, and pH value is natural (refer to and do not need to adjust pH again).28 DEG C, 235rpm cultivates 48h,
Seed liquor is obtained, 10000rpm, 10min are centrifuged, abandons supernatant, clear water washs one time, and supernatant is abandoned in centrifugation, is precipitated as being subsequently used for turning
The resting cell of change, resting cell measurement biomass, PQQ supernatant secretion content and N- hydroxyethyl glucosamine dehydrogenation obtained
Enzyme activity.Wherein, N- hydroxyethyl glucosamine Dehydrogenase activtity unit of force (U) is defined as under the conditions of 15 DEG C, catalysis substrate per minute
N- hydroxyethyl glucosamine generates the enzyme amount of 1.0 μm of oL Miglitol intermediate 6NSL.N- hydroxyethyl glucosamine dehydrogenase
Rate activity is that the resting cell N- hydroxyethyl glucosamine dehydrogenase activity of unit biomass is defined as contained by every gram of biomass
Enzyme activity unit number (U/g).
Enzyme activity (U/mL)=X × 10 × 103/(M×T)
X:6NSL (g/L)
10: reaction volume (mL)
103: unit conversion
The molal weight (g/mol) of M:6NSL
T: reaction time (h).
The comparison of 1 N- hydroxyethyl glucosamine dehydrogenase activity of table
Analyzed from result above, the N- hydroxyethyl glucosamine dehydrogenase activity of recombinant cell (strain pBBR-sldAB) compared with
Starting strain ZJB16009 has a distinct increment, but highest bioaccumulation amount has decline slightly, the ratio consumed from sorbierite
Also it can show that the metabolism D-glucitol consumption ratio of recombinant bacterium is obviously improved.
Embodiment 3
Investigate influence of the different aminoacids to fermented and cultured gluconobacter oxydans synthesis sorbitol dehydrogenase.
As it can be seen that although the N- hydroxyethyl glucosamine dehydrogenase activity of recombinant bacterium has is obviously improved from embodiment 2,
It is that the content of extracellular coenzyme PQQ is significantly reduced (23.32vs.39.93mg/L) compared with starting strain, prompts limited PQQ
Content may inhibit the raising of N- hydroxyethyl glucosamine dehydrogenase activity vigor in cell.In order to verify the hypothesis, pass through
The PQQ that various concentration is added in recombinant bacterium culture supernatant measures corresponding dehydrogenase activity.It can be seen from figure 1 that with outer
The raising of source PQQ content, corresponding N- hydroxyethyl glucosamine dehydrogenase activity vigor are also significantly increased, it was demonstrated that in recombinant bacterium
The rate-limiting step for becoming the realization of N- hydroxyethyl glucosamine dehydrogenase activity of PQQ, the most suitable addition of external source in strain
Concentration is 60ng/mL.In order to further promote the PQQ in recombinant bacterium to synthesize, consider to synthesize precusor amino acids from external source addition PQQ
Promote its synthesis capability in recombinant bacterium Gluconobacter oxydans pBBR-sldAB.High spot reviews in the present embodiment
The addition of different exogenous amino acids is to the vigor of recombinant bacterium N- hydroxyethyl glucosamine dehydrogenase and the influence of PQQ content.It is different
The fermentative medium formula of amino acid are as follows: D-glucitol 50g/L, yeast extract 20g/L, KH2PO4 5g/L、K2HPO4 5g/
L, 0.5~5g/L (sodium glutamate, isoleucine, serine and arginine), solvent are deionized water, and pH is natural；Similarity condition
Under, not add any amino acid as control.
Embodiment 1 is recombinated 2019033 seed liquor of gluconobacter oxydans CCTCC No.M to be seeded to volumetric concentration 2%
Fermentation medium, for 24 hours, fermentation liquid centrifugation, gained thallus is for detecting N- ethoxy grape for fermented and cultured at 28 DEG C, 150rpm
The content of osamine dehydrogenase activity and coenzyme PQQ.
N- hydroxyethyl glucosamine dehydrogenase activity detects operating process: take fermentation liquid 20mL to be centrifuged 10000rpm,
10min abandons supernatant, washs one time, and supernatant is abandoned in centrifugation, prepares buffer 100mM PBS, then by 20g/LN- ethoxy glucose
Amine and 5g/L epsom salt are dissolved in buffer, adjust pH to 6.0 again, establish the reaction system of 10mL, 28 DEG C of reaction 30min,
It samples 1mL centrifugation and carries out conversion ratio and product 6NSL yield that liquid phase tests and analyzes N- hydroxyethyl glucosamine；Liquid phase detector bar
Part are as follows: mobile phase is methanol-water (2: 98), 4mM sodium heptanesulfonate+10mM K2HPO4, it is 3.5 with phosphoric acid tune pH value；Flow velocity is
0.5mL/min, sample volume 20μL；30 DEG C of column temperature, time 20min；Calculate product 6NSL yield.
The independent addition pair of PQQ precusor amino acids (sodium glutamate, isoleucine, serine and arginine) is primarily looked at
The influence of N- hydroxyethyl glucosamine dehydrogenase activity.From shown in Fig. 2-5, four kinds of amino acid add 0.5~2g/L respectively can
Enough raisings for improving N- ethoxy dehydrogenase activity to a certain extent, most suitable addition concentration is respectively sodium glutamate (2g/
L), isoleucine (1g/L), serine (1g/L) and arginine (1g/L), corresponding N- hydroxyethyl glucosamine Dehydrogenase activtity
Power is able to ascend 1~20% or so.
The addition of amino acid has further been investigated to the synthesis of coenzyme PQQ and N- hydroxyethyl glucosamine dehydrogenase activity
It influences, and under optimum concentration, investigates the shadow of the joint addition to N- hydroxyethyl glucosamine dehydrogenase activity of four kinds of amino acid
It rings, the results are shown in Table 2:
The addition of 2 additional amino acid of table is synthesized to PQQ and the promotion of N- hydroxyethyl glucosamine dehydrogenase activity
Interpretation of result: as shown in table 2, different types of amino acid is added into culture medium, after shake flask fermentation culture for 24 hours,
Compared to group is not added, unit volume N- hydroxyethyl glucosamine dehydrogenase activity has to be changed to some extent.Wherein, when
Non- PQQ synthesis precusor amino acids are added, such as when proline (1g/L), the vigor of N- hydroxyethyl glucosamine dehydrogenase does not almost have
It changes；And when adding the synthesis precusor amino acids of coenzyme PQQ, such as 2g/L sodium glutamate, 1g/L isoleucine, 1g/L ammonia
Acid and when 1g/L arginine, compared to blank control group, recombinant bacterial strain Gluconobacter oxydans pBBR-sldAB's
The content of coenzyme PQQ is significantly increased in culture supernatant；Corresponding, the vigor of N- hydroxyethyl glucosamine dehydrogenase is also significant
It improves, and positive correlation is presented in the raising ratio of the two.It is worth noting that, after four kinds of precusor amino acids are added simultaneously, PQQ
Exocytosis content reach 44.43 ± 1.78mg/L of peak, the vigor of N- hydroxyethyl glucosamine dehydrogenase also reaches most
676.5 ± 25.6U/L of high level, the vigor is suitable with the N- ethoxy enzyme activity that the PQQ of external source addition 60ng/mL is promoted, but
It is to effectively reduce fermentation costs.The above results show that by before the synthesis of additionally adding coenzyme PQQ in conventional medium
Body can promote biosynthesis and exocytosis of the recombinant bacterial strain Gluconobacter oxydans pBBR-sldAB to PQQ,
To further improve the vigor of N- hydroxyethyl glucosamine dehydrogenase, the dehydrogenase activity of unit volume is compared with control medium
Improve 20% or more.
Embodiment 4
G.oxydans pBBR-sldAB resting cell catalyzes and synthesizes 6NSL under different concentration of substrate.
Transformation system: having flask with indentation 50mL/500mL, prepares N- hydroxyethyl glucosamine and (is first adjusted to meta-acid with dense HCl
Condition, then being adjusted to 5.0, N- hydroxyethyl glucosamine concentration with 2M NaOH is respectively 60,80,100 and 120g/L；MgSO4·
7H2O final concentration 5g/L adds the wet bacterium of resting cell that G.oxydanspBBR-sldAB fermented and cultured obtains in 3 method of embodiment
The additive amount of body, wet thallus is 60g/L, is constituted 50mL reaction system by reaction medium of deionized water.
Experiment condition: temperature: 15 DEG C；Revolving speed: 220rpm；Every 12h 2M NaOH alkali tune, maintains the pH value of conversion fluid
4.5~5.0.
Sampling: every 12h sampling, centrifuging and taking supernatant, liquid phase test sample yield, thallus microscopy.
Interpretation of result: the substrate transformation rate and efficiency of pcr product under the conditions of different concentration of substrate have been investigated, as a result such as 3 institute of table
Show, under different concentration of substrate, the substrate transformation rate reaches 97% or more；Efficiency of pcr product is analyzed, substrate 60g/L, 80g/L,
Efficiency of pcr product can achieve 90% or so when 100g/L, and when concentration of substrate is promoted to 120g/L, efficiency of pcr product is significantly reduced, and be
71.17%.Comprehensively consider, most suitable concentration of substrate selects 100g/L, yield 88.04%.
Resting cell catalyzes and synthesizes 6NSL in flask system under the different concentration of substrate of table 3
Embodiment 5
Influence of the different G.oxydans pBBR-sldAB resting cell throwing amounts to 6NSL is catalyzed and synthesized.
Transformation system: having flask with indentation 50mL/500mL, prepares N- hydroxyethyl glucosamine and (is first adjusted to meta-acid with dense HCl
Condition, then being adjusted to 5.0, N- hydroxyethyl glucosamine concentration with 2M NaOH is respectively 100g/L；MgSO4·7H2O final concentration 5g/
L, according in 3 method of embodiment G.oxydans pBBR-sldAB fermented and cultured obtain resting cell wet thallus, wet thallus
Additive amount is 40,60,80g/L, is constituted 50mL reaction system by reaction medium of deionized water.
Experiment condition: temperature: 15 DEG C；Revolving speed: 220rpm；Every 12h 2M NaOH alkali tune, maintains the pH value of conversion fluid
4.5~5.0.
Sampling: every 12h sampling, centrifuging and taking supernatant, liquid phase test sample yield, thallus microscopy.
Interpretation of result: under the conditions of having investigated 100g/L concentration of substrate, the additive amount of different catalysts is timely to reaction process
The influence of empty yield is significant.The results are shown in Table 4, with the raising of catalyst amount, the reaction time needed for 100g/L substrate
Significant to shorten, when cell throwing amount is more than or equal to 60g/L wet cell concentration, the substrate transformation rate reaches 90% or more；Efficiency of pcr product
88% or more, when cell throwing amount is 80g/L, 6NSL cumulative concentration reaches 92.4g/L, space-time yield 110.88gproduct
L-1d-1.Overall cost considers that optimal cell throwing amount is 60g wet cell/L.
Influence of the 4 different catalysts throwing amount of table to 6NSL space-time yield is catalyzed and synthesized
Embodiment 6
Recombinant bacterial strain Gluconobacter oxydans pBBR-sldAB and gluconobacter oxydans mutagenic strain
ZJB16009 catalyzes and synthesizes Miglitol intermediate 6NSL ability and compares.
(1) preparation of gluconobacter oxydans resting cell: by starting strain gluconobacter oxydans mutagenic strain ZJB16009
And recombinant bacterial strain Gluconobacter oxydans pBBR-sldAB carries out enzymatic production (with embodiment 2), and 28 DEG C, 235rpm
After fermented and cultured 18h, the fermentation liquid 20mL containing wet thallus is obtained, is centrifuged 10000rpm, 10min, abandons supernatant, clear water washing one
Time, supernatant is abandoned in centrifugation, is precipitated as being subsequently used for the resting cell of conversion.
(2) foundation and catalysis of catalysis substrate N- hydroxyethyl glucosamine synthesis Miglitol intermediate 6NSL transformation system
Process compares:
Reaction system: substrate N- hydroxyethyl glucosamine 100g/L, resting cell wet thallus concentration be 60g/L, with go from
Sub- water is reaction medium, and saturation HCl adjusts pH to 5.0, establishes 50mL reaction system, rear for 24 hours whole in 15 DEG C of progress conversion reactions
It only reacts, detects the cumulative concentration of product 6NSL, liquid phase testing conditions are same
Embodiment 1.
The key parameter of 5 two plants of bacterium catalysis substrate N- hydroxyethyl glucosamine synthesis 6NSL of table compares
As shown in table 5, under 100g/L concentration of substrate, recombinant bacterial strain G.oxydans pBBR-sldAB catalyzes and synthesizes 6NSL
Efficiency be significantly higher than starting strain ZJB-16009.After reaction for 24 hours, the conversion of concentration of substrate 100g/L can be realized substantially, and
And the yield of product 6NSL is significantly higher than control strain (86.8% and 71.4%), and the transformation period shortens 12h.Further meter
Space-time yield is calculated, the space-time yield of recombinant bacterium is about 2 times of control strain, it is shown that recombinant bacterium G.oxydans pBBR-sldAB
Potential using value in the catalyzing and synthesizing of miglitol key intermediate 6NSL.
Sequence table
<110>Zhejiang Polytechnical University
Zhejiang Medicine Co
<120>a kind of conversion bacterial strain for catalyzing and synthesizing Miglitol intermediate and method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 133
<212> DNA
<213>gluconobacter oxydans (Gluconobacter oxydans)
<400> 1
tgaaagcggc tggcgcgtgc tggcggggct gtaacagaac tgcaaaaccg gaacgctgag 60
aaagttggta acatcccact ttttcagggt ataacgcctt ccgcggactt tcagttctgg 120
aggctttcac caa 133
<210> 2
<211> 115
<212> DNA
<213>gluconobacter oxydans (Gluconobacter oxydans)
<400> 2
gaaagcgggg gctgaggccg ccgggtgatg gaaggggtgg ttttcgtgct gcaaatggca 60
cggagaccgc ccctttgtcg tttccggggc cgttttttcc atttttcggg tggga 115
<210> 3
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctggaggctt tcaccaaatg ccgaatactt atggc 35
<210> 4
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tcagcccccg ctttctcagc ccttgtgatc agg 33
Claims (9)
1. a kind of conversion bacterial strain for catalyzing and synthesizing Miglitol intermediate, which is characterized in that be named as gluconobacter oxydans
(Gluconobacter oxydans), strain pBBR-sldAB, deposit number are CCTCC No.M2019033.
2. conversion bacterial strain synthesizes -6 amino of 6- deoxidation (N- hydroxyl second in catalysis N- hydroxyethyl glucosamine as described in claim 1
Base) application in-α-L- furans sorbose.
3. a kind of use the method that conversion bacterial strain catalyzes and synthesizes Miglitol intermediate as described in claim 1.
4. method as claimed in claim 3, which comprises the following steps:
(1) bacterial strain enzymatic production will be converted as described in claim 1, and be collected the thallus after fermentation and obtained resting cell；
(2) it is obtained among product Miglitol using resting cell catalysis substrate N- hydroxyethyl glucosamine prepared by step (1)
Body: -6 amino of 6- deoxidation (N- ethoxy)-α-L- furans sorbose.
5. method as claimed in claim 4, which is characterized in that the preparation method of resting cell includes following step in step (1)
It is rapid: conversion bacterial strain as described in claim 1 being inoculated into culture medium and carries out fermented and cultured, it is described quiet then to collect thallus acquisition
Cease cell, wherein the culture medium includes: D-glucitol 50g/L, yeast extract powder 25g/L, KH2PO45g/L, K2HPO4
5g/L, solvent are water.
6. method as claimed in claim 5, which is characterized in that the culture medium further includes sodium glutamate, isoleucine, silk ammonia
Acid and arginine, the use concentration of each amino acid is respectively 0.5~5g/L.
7. method as claimed in claim 6, which is characterized in that sodium glutamate 2g/L, isoleucine, serine and arginine are each
1g/L。
8. method as claimed in claim 4, which is characterized in that catalysis reaction is formed using system in step (2) are as follows: substrate N-
60~120g/L of hydroxyethyl glucosamine, with wet thallus 40~80g/L of poidometer, remaining is the resting cell of step (1) preparation
Water.
9. method according to claim 8, which is characterized in that catalysis reaction is formed using system in step (2) are as follows: substrate N-
Hydroxyethyl glucosamine 100g/L, epsom salt 5g/L, the resting cell of step (1) preparation with wet thallus poidometer 60g/L,
Remaining is water, and adjusting pH is 5.0.
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|---|---|---|---|---|
| CN112592879A (en) * | 2020-12-31 | 2021-04-02 | 浙江工业大学 | Recombinant Gluconobacter oxydans engineering bacterium and application thereof in synthesizing miglitol intermediate |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456665A (en) * | 2018-02-01 | 2018-08-28 | 浙江工业大学 | A method of promoting gluconobacter oxydans synthesis sorbitol dehydrogenase and coenzyme pyrroloquinoline quinone |
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2019
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456665A (en) * | 2018-02-01 | 2018-08-28 | 浙江工业大学 | A method of promoting gluconobacter oxydans synthesis sorbitol dehydrogenase and coenzyme pyrroloquinoline quinone |
Non-Patent Citations (2)
| Title |
|---|
| XIA KE等: "Biosynthesis of miglitol intermediate 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose by an improved d-sorbitol dehydrogenase from Gluconobacter oxydans", 《3 BIOTECH》 * |
| XUE-PENG YANG等: "Membrane-Bound Pyrroloquinoline Quinone-Dependent Dehydrogenase in Gluconobacter oxydans M5, Responsible for Production of 6-(2-Hydroxyethyl) Amino-6-Deoxy-L-Sorbose", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592879A (en) * | 2020-12-31 | 2021-04-02 | 浙江工业大学 | Recombinant Gluconobacter oxydans engineering bacterium and application thereof in synthesizing miglitol intermediate |
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