CN108949658A - A kind of whole-cell catalyst and its preparation method and application increasing permeability - Google Patents
A kind of whole-cell catalyst and its preparation method and application increasing permeability Download PDFInfo
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- CN108949658A CN108949658A CN201810685948.3A CN201810685948A CN108949658A CN 108949658 A CN108949658 A CN 108949658A CN 201810685948 A CN201810685948 A CN 201810685948A CN 108949658 A CN108949658 A CN 108949658A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
A kind of whole-cell catalyst and its preparation method and application increasing permeability.The coli somatic of the expression rhamnosidase of fermented and cultured is resuspended using phosphate buffer, obtains whole-cell catalyst, adds ionic liquid or eutectic solvent is placed in shaking bath and is reacted；Ionic liquid or eutectic solvent of the total volume 1% ~ 20%, 30 ~ 60 DEG C of reaction temperature, 180 rpm of revolving speed；Handle 0.5 ~ 3 h of time；Thallus is centrifuged and collected after processing, washes residual ion liquid/eutectic solvent using phosphate buffer, has both obtained the whole-cell catalyst that permeability increases.The present invention improves the permeability of whole-cell catalyst using ionic liquid/eutectic solvent, improves enzyme-to-substrate contact probability intracellular.
Description
Technical field
The invention belongs to biocatalysis technology fields, and in particular to a kind of whole-cell catalyst and its system for increasing permeability
Preparation Method and application.
Background technique
Whole-cell catalytic avoids the separation and purification process of pure enzyme, so as to reduce production cost, and cell and production
Object can be easily separated to realize the recycling and reuse (Renewable Energy, 2016,85:1002-1010) of enzyme.In addition,
It is more stable (Algal Research, 2017,28:16-23) by the endocellular enzyme specific ionization enzyme of full cytoprotection.However, due to complete
There are cell wall/film in cell catalyst, the combination of the active site of substrate and enzyme is restricted, and product is difficult to flow out in time thin
It is extracellular, lead to the reduction of the yield of Product inhibiton and target product.Therefore, it is necessary to take measures to increase connecing between enzyme-to-substrate
Touching, to improve the efficiency of whole-cell catalytic.Currently, most widely used method has following two: 1) utilizing cell surface exhibition
Show that destination protein gene is anchored on cell surface by technology, enzyme-to-substrate directly can combine (Applied in cell surface
Biochemistry and Biotechnology, 2017,1:1-23), carry out catalytic process；2) by physics, chemistry and
Molecular biology method improves cell permeability to increase the mass-transfer efficiency of intraor extracellular to enhance the contact of enzyme-to-substrate
(Journal of Biotechnology,2017,248:9-14).Although first method can increase enzyme to a certain extent
Contact with substrate, but the screening and other complexity for the molecular size and target protein for needing to consider to be demonstrated destination protein
Molecular biology processes.Therefore more scholars tend to second method.Cell permeability is improved, small molecule and certain big is allowed
Molecule is freely through cell without destroying multicellular organism and can be to avoid complicated operating process.
Ionic liquid and eutectic solvent are the promotors for receiving significant attention catalytic process in recent years.Ionic liquid has
It is environmental-friendly, there is low saturated vapour pressure, low flammability, hydrophobicity and good thermodynamic stability (Carbohydrate
Polymers, 2015,118:150-155) etc. characteristics.There is research as solvent, to pass through fat using ionic liquid [EMIM] [BF4]
Enzymatic synthesis indoles tetrahydro chromene, and higher yield (77-98%) (Catalysts, 2017,7:185-195) is obtained, from
Since eutectic solvent is found, the fields such as separation process, functional material, chemical reaction, electrochemistry are had been used for.Since its is low
Cost, hypotoxicity and biodegradable characteristic and show good application prospect, ionic liquid has with eutectic solvent
Closely similar physicochemical properties become atom utilization close to 100% green solvent.
Summary of the invention
The technical issues of solution: the present invention is asked for the correlation of whole-cell catalyst intraor extracellular mass transfer and its ameliorative way
Topic a kind of whole-cell catalyst and preparation method thereof for increasing permeability and is answered using recombinant Bacillus coli cells as research object
With being capable of increasing cell permeability, enhance intraor extracellular mass transfer, glucosides enzymatic rutin intracellular is utilized to synthesize rare natural products
Method.
Technical solution: a kind of preparation method for the whole-cell catalyst increasing permeability, step are as follows: utilize phosphate buffer
Be resuspended fermented and cultured expression rhamnosidase coli somatic, obtain whole-cell catalyst, add ionic liquid or
Eutectic solvent is placed in shaking bath and is reacted；Ionic liquid or eutectic solvent of the total volume 1%~20%, reaction temperature
30~60 DEG C of degree, revolving speed 180rpm；Handle 0.5~3h of time；Thallus is centrifuged and collected after processing, is cleaned using phosphate buffer
Fall residual ion liquid/eutectic solvent, both obtains the whole-cell catalyst that permeability increases.
Above-mentioned ionic liquid is 1- butyl -3- methylimidazole bis-trifluoromethylsulfoandimide salt, 1- butyl -3- methylimidazole six
Fluorophosphate, 1- butyl -3- methyl imidazolium tetrafluoroborate, 1- hexyl -3- methylimidazole hexafluorophosphate, 1- ethyl -3- first
Base limidazolium hexafluorophosphate；Above-mentioned eutectic solvent is the chlorination gallbladder of choline chloride-urea of mass ratio 1:2, mass ratio 1:2
Alkali-glycerol, choline chloride-acetamide of mass ratio 1:2, mass ratio 1:2 choline chloride-ethylene glycol, mass ratio 1:1 chlorination
Choline-malonic acid.
Above-mentioned Escherichia coli are escherichia coli (Escherichia coli).
Whole-cell catalyst made from above-mentioned preparation method.
Application of the above-mentioned whole-cell catalyst in bioconversion rutin.
The specific steps of application are as follows: whole-cell catalyst is mixed with rutin, carries out rutin bioconversion, reaction temperature is
Reaction temperature is 30~60 DEG C；Reaction pH is pH 4.5~7.0；Substrate rutin concentration is 0.02~2g/L；Reaction time is 0.5
~6h.
The utility model has the advantages that the present invention improves the permeability of whole-cell catalyst using ionic liquid/eutectic solvent, born of the same parents are improved
Interior enzyme-to-substrate contact probability.Since the presence of cell wall/film has seriously affected enhancing intraor extracellular mass-transfer efficiency, make enzyme and bottom
The combination of object is seriously obstructed, while substrate can not flow out extracellularly in time, easily cause Product inhibiton, using ionic liquid/low
Congruent melting solvent increases the permeability of cell, helps to solve the above problems, to expand the application range of whole-cell catalyst.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The recombinant Bacillus coli cells method of preparation expression rhamnosidase in the embodiment of the present invention are as follows: be inoculated in containing ammonia
In the LB liquid medium of parasiticin resistance (50 μ g/mL) (3~5mL), 37 DEG C of shaken cultivations grow to exponential phase.According to
2% bacterium amount that connects is transferred to (500mL), 22 DEG C of constant temperature in the LB liquid medium containing amicillin resistance (50 μ g/mL)
Shaken cultivation is to OD600When reaching 0.6~0.8,400 μM of isopropyl-β-D-thiogalactosides (IPTG) are added, temperature is dropped
16~20h is expressed to 17 DEG C of progress low temperature inductions.The bacterium solution that inducing expression terminates 8000rpm at 4 DEG C is centrifuged 5min, collects bacterium
Body；Whole-cell catalyst is both obtained twice with pH7.2~7.4PBS buffer solution for cleaning thallus.
It is high performance liquid chromatography that the measuring method of detection rutin and isoquercitrin qualitative, quantitative is used in the embodiment of the present invention
Method, condition are as follows: H&E Pump P3000A constant flow pump, UV-VIS detector, column model Alltima C18, length are
25mm, internal diameter are 5 μm, maintain 30 DEG C using HPLC-UV separation and measurement rutin and isoquercitrin, column temperature.Mobile phase is second
Nitrile: (0.02%) phosphoric acid (volume ratio 20:80), flow velocity 1.0mlmin-1, Detection wavelength 360nm.Sample volume is 20 μ L.Sample introduction
Before, sample passes through 0.45 μm of filter filtering.
Wherein, rutin conversion ratio and isoquercitrin yield calculation method are as follows:
Embodiment 1
This example demonstrates that preparing what catalyst was urged with eutectic solvent choline chloride-Urea treatment E. coli whole cell
Process.
Express the coli somatic cell and greening choline-urea quality ratio 1:20 of rhamnosidase, the chlorination gallbladder
Alkali: the mass ratio of urea is 1:2, is dissolved in 10mL citrate-phosphate disodium hydrogen buffer (pH=5.0), mixture is placed in water
Shaking table is bathed, 30 DEG C, 180rpm handles 0.5h.After 10min is centrifuged at high speed freezing centrifuge 6000rpm, 4 DEG C, collect
Thallus.Thallus is collected again after washing 2 times with phosphate buffer (pH=7.4), prepares whole-cell catalyst.
Embodiment 2
This example demonstrates that preparing what catalyst was urged with eutectic solvent choline chloride-Urea treatment E. coli whole cell
Process.
Express the coli somatic cell and greening choline-urea quality ratio 1:4 of rhamnosidase, the chlorination gallbladder
Alkali: the mass ratio of urea is 1:2, is dissolved in 10mL citrate-phosphate disodium hydrogen buffer (pH=5.0), mixture is placed in water
Shaking table is bathed, 60 DEG C, 180rpm handles 3h.After be centrifuged 10min at high speed freezing centrifuge 6000rpm, 4 DEG C, collect bacterium
Body.Thallus is collected again after washing 2 times with phosphate buffer (pH=7.4), prepares whole-cell catalyst.
Embodiment 3
This example demonstrates that preparing what catalyst was urged with eutectic solvent choline chloride-Urea treatment E. coli whole cell
Process.
Express the coli somatic cell and greening choline-urea quality ratio 1:10 of rhamnosidase, the chlorination gallbladder
Alkali: the mass ratio of urea is 1:2, is dissolved in 10mL citrate-phosphate disodium hydrogen buffer (pH=5.0), mixture is placed in water
Shaking table is bathed, 45 DEG C, 180rpm handles 1.5h.After 10min is centrifuged at high speed freezing centrifuge 6000rpm, 4 DEG C, collect
Thallus.Thallus is collected again after washing 2 times with phosphate buffer (pH=7.4), prepares whole-cell catalyst.
Embodiment 4
This example demonstrates that be urged through choline chloride-Urea treatment Bacillus coli cells as catalyst in conventional reactor
Change the process that rutin generates isoquercitrin.
Using according to whole-cell catalyst prepared by embodiment 1 (being dissolved in pH=7.4 phosphate buffer) and substrate rutin
(being dissolved in pH=5.0 citrate-phosphate disodium hydrogen) 20:1 in mass ratio takes above-mentioned solution 3mL in 10mL centrifuge tube respectively, will be from
Heart pipe is placed in 30 DEG C of shaking baths, reaction time 2h, interval 30min sampling.Every 200 μ L of sub-sampling, sample be placed in 1.5mL from
Heart pipe 10000rpm is centrifuged 2min, collects supernatant 150uL, carries out liquid chromatographic detection, the conversion of rutin after methanol dilution is added
Rate is 40 ± 2.3%, and the yield of isoquercitrin is 29 ± 1.2%.
Embodiment 5
This example demonstrates that be urged through choline chloride-Urea treatment Bacillus coli cells as catalyst in conventional reactor
Change the process that rutin generates isoquercitrin.
Using according to whole-cell catalyst prepared by embodiment 2 (being dissolved in pH=7.4 phosphate buffer) and substrate rutin
(being dissolved in pH=5.0 citrate-phosphate disodium hydrogen) 1:1 in mass ratio takes above-mentioned solution 3mL in 10mL centrifuge tube respectively, will be from
Heart pipe is placed in 45 DEG C of shaking baths, reaction time 6h, interval 30min sampling.Every sub-sampling 200uL, sample be placed in 1.5mL from
Heart pipe 10000rpm is centrifuged 2min, collects supernatant 150uL, carries out liquid chromatographic detection, the conversion of rutin after methanol dilution is added
Rate is 96.65 ± 1.9%, and the yield of isoquercitrin is 93.03 ± 3.3%.
Embodiment 6
This example demonstrates that be urged through choline chloride-Urea treatment Bacillus coli cells as catalyst in conventional reactor
Change the process that rutin generates isoquercitrin.
Using according to whole-cell catalyst prepared by embodiment 3 (being dissolved in pH=7.4 phosphate buffer) and substrate rutin
(being dissolved in pH=5.0 citrate-phosphate disodium hydrogen) 1:10 in mass ratio takes above-mentioned solution 3mL in 10mL centrifuge tube respectively, will be from
Heart pipe is placed in 60 DEG C of shaking baths, reaction time 3h, interval 30min sampling.Every sub-sampling 200uL, sample be placed in 1.5mL from
Heart pipe 10000rpm is centrifuged 2min, collects supernatant 150uL, carries out liquid chromatographic detection, the conversion of rutin after methanol dilution is added
Rate is 26 ± 2.2%, and the yield of isoquercitrin is 18 ± 1.6%.
Claims (6)
1. a kind of preparation method for the whole-cell catalyst for increasing permeability, it is characterised in that step are as follows: utilize phosphate buffer
Be resuspended fermented and cultured expression rhamnosidase coli somatic, obtain whole-cell catalyst, add ionic liquid or
Eutectic solvent is placed in shaking bath and is reacted；Ionic liquid or eutectic solvent of the total volume 1% ~ 20%, reaction temperature
30 ~ 60 DEG C, 180 rpm of revolving speed；Handle 0.5 ~ 3 h of time；Thallus is centrifuged and collected after processing, is washed using phosphate buffer
Residual ion liquid/eutectic solvent had both obtained the whole-cell catalyst that permeability increases.
2. a kind of preparation method for the whole-cell catalyst for increasing permeability according to claim 1, it is characterised in that described
Ionic liquid is 1- butyl -3- methylimidazole bis-trifluoromethylsulfoandimide salt, 1- butyl -3- methylimidazole hexafluorophosphate, 1-
Butyl -3- methyl imidazolium tetrafluoroborate, 1- hexyl -3- methylimidazole hexafluorophosphate, 1- ethyl-3-methylimidazole hexafluoro phosphorus
Hydrochlorate；The eutectic solvent is choline chloride-glycerol, the mass ratio of choline chloride-urea of mass ratio 1:2, mass ratio 1:2
Choline chloride-acetamide of 1:2, choline chloride-ethylene glycol of mass ratio 1:2, mass ratio 1:1 choline chloride-malonic acid.
3. a kind of preparation method for the whole-cell catalyst for increasing permeability according to claim 1, it is characterised in that described
Escherichia coli be escherichia coli (Escherichia coli).
4. whole-cell catalyst made from any preparation method of claim 1 ~ 3.
5. application of the whole-cell catalyst described in claim 4 in bioconversion rutin.
6. application according to claim 5, it is characterised in that step are as follows: mix whole-cell catalyst with rutin, carry out
Rutin bioconversion, reaction temperature are that reaction temperature is 30~60 DEG C；Reaction pH is pH 4.5~7.0；Substrate rutin concentration is
0.02~2 g/L；Reaction time is 0.5~6 h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643592A (en) * | 2019-10-29 | 2020-01-03 | 南京工业大学 | Method for modifying whole cells by cholate-metal ion composite and application thereof |
| CN111500661A (en) * | 2020-04-23 | 2020-08-07 | 江南大学 | Method for simultaneously preparing L-rhamnose and isoquercetin |
| CN113201565A (en) * | 2021-05-27 | 2021-08-03 | 中国科学院广州能源研究所 | Method for improving catalytic efficiency of whole-cell preparation of D-psicose based on eutectic solvent |
| CN113684235A (en) * | 2021-08-19 | 2021-11-23 | 江苏科技大学 | Method for converting mulberry red pigment component by double aqueous phase whole cell catalysis |
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| CN106119319A (en) * | 2016-08-25 | 2016-11-16 | 江苏科技大学 | Recombinant alpha L rhamnoside enzyme extract is catalyzed the method for directionally hydrolyzing flavonoid glycoside in micro passage reaction |
| CN107287223A (en) * | 2017-06-20 | 2017-10-24 | 江苏科技大学 | α L rhamnosides enzyme genes and its application |
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2018
- 2018-06-28 CN CN201810685948.3A patent/CN108949658A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119319A (en) * | 2016-08-25 | 2016-11-16 | 江苏科技大学 | Recombinant alpha L rhamnoside enzyme extract is catalyzed the method for directionally hydrolyzing flavonoid glycoside in micro passage reaction |
| CN107287223A (en) * | 2017-06-20 | 2017-10-24 | 江苏科技大学 | α L rhamnosides enzyme genes and its application |
Non-Patent Citations (1)
| Title |
|---|
| FAN ZHANG ET.AL.，: "Ionic liquids/deep eutectic solvents enhance isoquercitrin production by the conversion of rutin extracted from grapefruit peel", 《HTTP://UEST.NTUA.GR/NAXOS2018/PROCEEDINGS/PDF/NAXOS2018_ZHANG(E)_ETAL.PDF》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643592A (en) * | 2019-10-29 | 2020-01-03 | 南京工业大学 | Method for modifying whole cells by cholate-metal ion composite and application thereof |
| CN111500661A (en) * | 2020-04-23 | 2020-08-07 | 江南大学 | Method for simultaneously preparing L-rhamnose and isoquercetin |
| CN113201565A (en) * | 2021-05-27 | 2021-08-03 | 中国科学院广州能源研究所 | Method for improving catalytic efficiency of whole-cell preparation of D-psicose based on eutectic solvent |
| CN113684235A (en) * | 2021-08-19 | 2021-11-23 | 江苏科技大学 | Method for converting mulberry red pigment component by double aqueous phase whole cell catalysis |
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Application publication date: 20181207 |