CN109554359A - The method of immobilization embedded microorganism and its preparation contain microbial immobilization bead and its application - Google Patents
The method of immobilization embedded microorganism and its preparation contain microbial immobilization bead and its application Download PDFInfo
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Abstract
The present invention provides a kind of method of immobilization embedded microorganism, it is related to immobilization embedded technical field, comprising the following steps: poly-vinyl alcohol solution is mixed with the bacterium solution of microorganism, 1~72h is freezed at -85~-75 DEG C, balling-up is cut after defrosting, is obtained containing microbial immobilization bead.Immobilization embedded method of the present invention passes through to be handled under low temperature (- 85~-75 DEG C), improves the immobilized spherule intensity using polyvinyl alcohol as carrier material；The method of the invention improves the stability of immobilized spherule, and microorganism is embedded in support material internal, provides stable environment for the catalysis reaction of microorganism and its enzyme, completely cuts off the interference of external high concentration and high ph-values condition.What immobilization embedded method of the present invention was prepared reacts non-breakable in the high concentration salt solutions of pH neutral containing microbial immobilization bead, can be repeatedly circulated, effectively increase the efficiency of biocatalysis synthetic reaction, reduce cost.
Description
Technical field
The present invention relates to the methods and its preparation of immobilization embedded technical field more particularly to immobilization embedded microorganism
Contain microbial immobilization bead and its application.
Background technique
R-3- quinuclidinol is that important intermediate of many anticholinergic agents, such as Suo Linaxin, Revatropate etc. all contain
There is the newest anticholinergic agent of R-3- quinuclidinol structure, has well for the treatment urinary incontinence and chronic obstructive pulmonary disease (COPD)
Curative effect.The synthesis of R-3- quinuclidinol has chemical method and two kinds of bioanalysis.Chemical method synthesizes R-3- quinuclidinol, can be residual in product
Metallic catalyst is stayed, compared with chemical method, it is more that bioanalysis has that reaction condition is mild, high conversion rate, stereoselectivity are strong etc.
Kind advantage, therefore bioanalysis is developed to synthesize the direction that R-3- quinuclidinol is industrialized production.
Inventor's early-stage study has found one plant of rhodothece rubra, is named as Rhodotorula mucilaginosa X15
(culture presevation number are as follows: CGMCC No:3664) can catalyze and synthesize R-3- quinuclidinol (patent are as follows: ZL by substrate of quinuclidone
201010197502.X)；Inventor also construct glucose dehydrogenase and carbonyl reductase coexpression engineering bacteria BW (full cell is urged
Agent), R-3- quinuclidinol is catalyzed and synthesized by substrate of quininone hydrochlorate, quinuclidone conversion ratio reaches 100%, product R-3- Kui
The optical purity of peaceful alcohol reaches 99% (Zhenhua Jia, Hong Ma, Yali Huang et al.Production of (R)-
3-quinuclidinol by a whole-cell biocatalyst with high efficiency.Biocatalysis
andBiotransformation,2017,36:4,316-323).But the stability of carbonyl reductase and catalytic activity exist
Defect, use environment requires stringent and easy loss of activity, and immobilization technology is the common method for solving this predicament.
Immobilization is fixed on enzyme (cell) on support carrier, can be improved the stability of enzyme, be widened application range and drop
Low cost, embedded immobilization microbial technology are the carrier skies that free microorganism is positioned to restriction with means chemically or physically
Between field, and it is made to keep activity and the method recycled.The method of this immobilization bacterium can purify and keep Black Liquor with Efficient Bacteria
Kind, therefore have many advantages, such as that treatment effeciency is high, reaction is easily controllable, strain is high-purity efficiently, biological concentration is high.
There are many carrier of immobilization, mainly have sodium alginate (CA), agar, carrageenan, polyvinyl alcohol (PVA),
Polypropylene phthalein is hungry, poly- alum, silica gel, light-hardening resin etc..Compare all carriers, finds polyvinyl alcohol and sodium alginate gel machine
Tool intensity and mass-transfer performance are preferable, and to biological nontoxic, and resistance to Biodegradable is good, are that more appropriate immobilized cell carries
Body (comparison [J] of the fragrant several fixed cell carriers of Jiang Yuhong, Huang Xia, Yu Yu, environmental science, 2001,14 (2)).Utilize sea
Alginates method and Polyvinyl alcohol method method embed engineering bacteria, and catalyze and synthesize R-3- quinuclidinol with the engineering bacteria after embedding, due to
The reaction solution of the reaction needs high salt concentration, and reacting liquid pH value is 7, so that the bead of these methods embedding is during the reaction
It is easy to be crushed, and in normal conditions (when salinity is lower, reaction solution is acid), alginate method and Polyvinyl alcohol method side
The immobilized spherule of method preparation is more stable.
Summary of the invention
For the present invention in order to overcome in existing biocatalysis embedding reaction, immobilized spherule intensity is small, service life is short, easy
The defect of water absorption and swelling, the present invention provide a kind of immobilization embedded to solve the problems of immobilization embedded microbial inoculum
The method of microorganism can be suitable for highly concentrated solution and/or neutral pH reaction condition；According to immobilization embedded side of the present invention
The microbial immobilized bead that method is prepared not only can effectively catalyze synthesis, but also reduce the broken of embedding bead.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of methods of immobilization embedded microorganism, comprising the following steps:
Poly-vinyl alcohol solution is mixed with the bacterium solution of microorganism, 1~72h is freezed at -85~-75 DEG C, is cut after defrosting
Balling-up is obtained containing microbial immobilization bead.
Preferably, the poly-vinyl alcohol solution concentration is 0.07~0.13mg/g.
Preferably, the preparation method of the poly-vinyl alcohol solution includes: to mix polyvinyl alcohol with water, is added after heating for dissolving
Enter DMSO to make it dissolve, it is cooling, obtain poly-vinyl alcohol solution；The mass ratio of the polyvinyl alcohol, water and DMSO is 1:7~10:
0.1~2.
Preferably, the bacterial concentration of the microorganism is 0.2~1.0g/ml.
Preferably, the microorganism includes rhodothece rubra X15 or engineering bacteria；It is general that the rhodothece rubra X15 is deposited in China
Logical Microbiological Culture Collection administrative center, deposit number are CGMCC No:3664.
Preferably, when the microorganism is engineering bacteria, the preparation method of the bacterium solution of the engineering bacteria the following steps are included:
(1) engineering bacteria is inoculated in LB culture medium and is activated；
(2) single colonie of engineering bacteria is inoculated in LB culture medium after picking activation, and 32~40 DEG C of cultures 12~for 24 hours, it is planted
Sub- culture solution；
(3) seed culture fluid is seeded in LB liquid medium, culture to OD600Value adds IPTG when being 0.6~0.8
Induction, centrifuging and taking precipitating, obtains thallus；
(4) thallus that step (3) obtains is redissolved with phosphate buffer, obtains the bacterium solution of engineering bacteria, the engineering bacteria
Bacterium solution OD600Value is 0.3~0.6.
Preferably, the volume ratio of the bacterium solution of poly-vinyl alcohol solution and microorganism is 1~100:1.
Preferably, the thaw point is 28~35 DEG C.
Contain microbial immobilization bead the present invention provides a kind of preparation of above-mentioned technical proposal the method.
The present invention also provides synthesizing containing microbial immobilization bead in biocatalysis described in above-mentioned technical proposal
Application in drug or pharmaceutical intermediate.
Compared with prior art, beneficial effects of the present invention:
(1) microorganism is fixed on inside immobilized spherule by the method for the invention, so that microorganism be prevented to be lost, passes through
The carrier material of outside cladding provides a metastable environment for microorganism, and catalyzing enzyme is made to play optimal efficacy.
(2) melt the fixed embedding cell of method using the low temperature-heat through the invention, can enhance containing microorganism
Stability of the immobilized spherule under high salt concentration and/or condition of neutral pH, improves bead intensity and service life, Jin Erke
To realize, repeatedly circulation is catalyzed and synthesized, and significantly improves catalysis reaction efficiency, provides new thinking to be immobilization embedded.
(3) present invention is used as fixation support only with polyvinyl alcohol (PVA), and PVA passes through low-temperature treatment (- 85~-75
DEG C) after can effectively improve the intensity of bead, overcome immobilized spherule that conventional immobilization embedded method obtains because of different pH value
Reaction solution and broken or strength reduction defect.The present invention contains the strong of microbial immobilization bead as obtained by improving
Degree, makes the reaction solution of the high salt concentration and/or neutral pH in its appropriate biological catalytic synthesis, reduces the fortune of microorganism catalysis
Row cost has more significant economic benefit and environmental benefit.
(4) microbial immobilization embedding method of the present invention can strengthen various high-concentration biological processing reactions, utilize
Immobilization embedded method preparation of the present invention contains microbial immobilization bead, effectively reduces reaction condition to microorganism
It influences, catalyzes and synthesizes drug for biology or pharmaceutical intermediate provides new thinking and opens up new approach, while being microorganism
Development and application in complex environment provide broader prospect.
Detailed description of the invention
Fig. 1 engineering bacteria immobilized spherule is catalyzed the ability of quinuclidone conversion R-3- quinuclidinol；
Fig. 2 saccharomycete immobilized spherule is catalyzed the ability of quinuclidone conversion R-3- quinuclidinol；
Influence of Fig. 3 reaction solution to different immobilized spherules；
The ability of Fig. 4 different immobilized spherule catalysis quinuclidone conversion R-3- quinuclidinols；
The ability of Fig. 5 immobilized spherule continuous catalysis quinuclidone conversion R-3- quinuclidinol.
Specific embodiment
The present invention provides a kind of methods of immobilization embedded microorganism, comprising the following steps:
The bacterium solution of poly-vinyl alcohol solution and microorganism is sufficiently mixed, the mixture solidified, by the mixture of solidification
1~72h is freezed at -85~-75 DEG C, is cut balling-up after defrosting, is obtained containing microbial immobilization bead.
Using polyvinyl alcohol as immobilization embedded carrier, the concentration of the poly-vinyl alcohol solution is preferably the present invention
0.07~0.13mg/g, more preferably 0.09~0.11mg/g.
In the present invention, the preparation method of the poly-vinyl alcohol solution preferably includes: to mix polyvinyl alcohol with water, is added
DMSO is added after heat of solution to make it dissolve, it is cooling, obtain poly-vinyl alcohol solution；The mass ratio of the polyvinyl alcohol, water and DMSO
For 1:7~10:0.1~2.
In the present invention, the mass ratio of the polyvinyl alcohol, water and DMSO are preferably 1:7~10:0.1~2, more preferably
1:8~9:0.4~1；Most preferably 1:0.8:0.5.In the present invention, the effect of the DMSO is to improve polyvinyl alcohol and exist
Dissolution in water.Heating finally obtains uniform gather also for its dissolution is accelerated after the present invention mixes polyvinyl alcohol with water
Glycohol solution.
In the present invention, the microorganism refers to the various microorganisms for biocatalysis synthesis.Using of the present invention
Method immobilizes embedding, is able to solve immobilized spherule and carries out biocatalysis conjunction in pH neutral, high concentration salt solutions
The problem of being easily broken at reaction.In the present invention, the microorganism includes bacterium and saccharomycete.In specific implementation of the invention
In example, the microorganism preferably uses rhodothece rubra X15 or glucose dehydrogenase and carbonyl reductase to co-express engineering bacteria.This
Invention the method is not limited in embedding for being catalyzed the microorganism of (R) -3- quinuclidinol synthesis, can be also used for immobilization packet
Bury other microorganisms for facing the microorganism similar problems synthesized with catalysis (R) -3- quinuclidinol.In the present invention, described dark red
Yeast X15, is deposited in China General Microbiological culture presevation administrative center, and deposit number is CGMCC No:3664.
In the present invention, the bacterial concentration of the microorganism is preferably 0.2~1.0g/ml, more preferably 0.4~0.6g/
Ml, most preferably 0.45g/ml.
In the present invention, when the bacterium solution that the concentration of the poly-vinyl alcohol solution is preferably 0.07~0.13mg/g, microorganism
When concentration is preferably 0.2~1.0g/ml, the bacterium solution volume of the polyvinyl alcohol and microorganism is more excellent than being preferably 1~100:1
It is selected as 2~20:1, most preferably 5:1.
In the present invention, to resulting mixed solution after the mixing of the bacterium solution of poly-vinyl alcohol solution and microorganism carry out low temperature (-
85~-75 DEG C) processing can significantly improve immobilized spherule intensity obtained, and overcome that immobilized spherule is easily broken asks
Topic.Currently preferred, the cryogenic temperature is preferably -80 DEG C；The cooling time is preferably 16~36h, more preferably
24h。
In the present invention, the microorganism can derive from commercial goods or voluntarily prepare.In the present invention, when described micro-
When biology is saccharomycete, bacterium solution is prepared according to the cultural method of saccharomycete；In a specific embodiment of the present invention, microorganism is preferred
For rhodothece rubra X15, the preparation method of bacterium solution is referring to Chinese patent ZL 201010197502.X.
Currently preferred, when the microorganism is engineering bacteria, voluntarily preparation method includes the bacterium solution of the engineering bacteria
Following steps:
(1) engineering bacteria is inoculated in LB culture medium and is activated；
(2) single colonie of engineering bacteria is inoculated in LB culture medium after picking activation, and 32~40 DEG C of cultures 12~for 24 hours, it is planted
Sub- culture solution；
(3) seed culture fluid is seeded in LB liquid medium, culture to OD600Value adds IPTG when being 0.6~0.8
Induction, centrifuging and taking precipitating, obtains thallus；
(4) thallus that step (3) obtains is redissolved with phosphate buffer, obtains the bacterium solution of engineering bacteria, the engineering bacteria
Bacterium solution OD600Value is 0.3~0.6.
The engineering bacteria strain of refrigeration is inoculated in LB culture medium and activates by the present invention, currently preferred, the work
Changing temperature is preferably 32~38 DEG C, and more preferably 37 DEG C；The activation time is preferably 12~for 24 hours, more preferably 14~20h.
Engineering bacteria strain inoculum concentration when the present invention is to the activation is not particularly limited, and is inoculated with according to conventional method in that art, than
Such as with oese picking.
After the activation, the single colonie of engineering bacteria is inoculated in LB culture medium after picking activation of the present invention, and 32~40
DEG C culture 12~for 24 hours, obtain seed culture fluid.In the present invention, the cultivation temperature is preferably 36~38 DEG C, more preferably 37
℃；The incubation time is preferably 14~20h, more preferably 16~18h.
After obtaining seed culture fluid, seed culture fluid is seeded in LB liquid medium by the present invention, culture to OD600Value
IPTG induction is added when being 0.6~0.8, centrifuging and taking precipitating obtains thallus.In the present invention, the inoculum concentration of the seed liquor is excellent
It is selected as the 10% of LB liquid medium volume.It is currently preferred, add IPTG into fluid nutrient medium final concentration of 0.1~
0.8mM, more preferably 0.6mM.The present invention is to make engineering bacterium expression albumen using the purpose of IPTG induction, the egg of inducing expression
White ginseng and biocatalytic reaction.In the present invention, the temperature when IPTG is induced is preferably 25~35 DEG C, more preferably 30
℃.In the present invention, the time of the IPTG induction is preferably 6~12h, more preferably 8~10h.In the present invention, it is described from
The rate of the heart is preferably 5000~8000rpm, more preferably 6000rpm；The time of the centrifugation is preferably 15~30min, more
Preferably 20min.It is currently preferred, sediment fraction is taken after centrifugation, is washed 3~5 times, is obtained with 1 times of phosphate buffer solution
To thallus.
After obtaining thallus, the present invention is redissolved with phosphate buffer, obtains the bacterium solution of engineering bacteria, the bacterium solution of the engineering bacteria
OD600Value is 0.3~0.6, more preferably 0.4~0.5.
It is currently preferred to thaw at 28~35 DEG C after freezing；The present invention is to the thawing mode without spy
It is different to limit, using manner known in the art, such as water-bath or microwave treatment.The present invention after thawing cuts resulting material
Ball is cut into get the immobilized spherule containing engineering bacteria, the engineering bacteria is embedded in the inside of PVA Carrier.In this hair
In bright, the immobilized spherule diameter being prepared is preferably 1~10mm, more preferably 2~8nm.
The present invention also provides the consolidating containing microorganism that immobilization embedded method described in above-mentioned technical proposal is prepared
Surely change bead.In the present invention, the viable count containing in microbial immobilization bead is preferably 104~109Cfu/g,
More preferably 106cfu/g.It is described to be hardly damaged containing microbial immobilization bead, it is suitable for high salt concentration, pH neutral item
The biocatalysis synthetic reaction of part, can be repeatedly circulated in above-mentioned reaction, and production can also be reduced by improving catalytic reaction efficiency
Cost.
One kind is prepared the present invention provides above-mentioned technical proposal the method and contains microbial immobilization bead, it can
It is suitable for the biocatalysis synthetic reaction of high salt concentration and/or neutral pH conditions.
The present invention also provides synthesizing containing microbial immobilization bead in biocatalysis described in above-mentioned technical proposal
Application in drug or pharmaceutical intermediate.
In a specific embodiment of the present invention, contain microbial immobilization bead using the method for the invention building
It can be applied in the reaction of biocatalysis synthesis R-3- quinuclidinol.
It is currently preferred, utilize the R- of the present invention that biocatalysis synthesis is carried out containing microbial immobilization bead
The method of 3- quinuclidinol the following steps are included:
A, it prepares and contains 8~12g/L of sodium dihydrogen phosphate, 20~30g/L of disodium hydrogen phosphate, 250~500g/L of glucose, Kui
It is peaceful 1~500g/L's of ketone and coenzyme (100mM NAD+) 0.05~0.2mL/L, pH value is adjusted to 7.0, obtains reaction solution；
B, into reaction solution, addition contains microbial immobilization bead, and conversion reaction is carried out at 28~32 DEG C, and conversion is anti-
PH value maintains 7.0 during answering.
It in the present invention, preferably include: sodium dihydrogen phosphate 10g/L, disodium hydrogen phosphate 27g/L, Portugal in the reaction solution
Grape sugar 400g/L, quinuclidone 250g/L and coenzyme (100mM NAD+) 0.1mL.
In the present invention, the volume of the reaction solution and the mass ratio containing microbial immobilization bead (weight in wet base) are excellent
It is selected as 1L:35~60g, more preferably 1L:45g.In conversion reaction of the present invention, the reaction solution is entered containing micro-
Biology immobilized spherule inside with mmp reaction therein, occur catalysis reaction, can effectively avoid it is disposable, connect on a large scale
Salinity is higher for touching, the neutral reaction solution of pH and cause enzyme to inactivate, improve the stability of immobilized spherule and recyclable
Utilization ability.
Biocatalysis synthesis R-3- quinuclidinol reaction is carried out containing microbial immobilization bead using prepared by the present invention
When, R-3- quinuclidinol is converted by quinuclidone completely, and be not crushed containing microbial immobilization bead；And use ability
The immobilized spherule (immobilized spherule 2, immobilized spherule 3) of the two kinds of conventional methods in domain preparation, converts R-3- Kui for quinuclidone
The efficiency of peaceful alcohol is respectively 54% and 61%, and both immobilized spherules have respectively in the reaction system it is different degrees of
Broken, 2 percentage of damage of immobilized spherule is 67%, and 3 percentage of damage of immobilized spherule is 45%.
Meanwhile the embodiment of the present invention is also shown, can be carried out using provided by the invention containing microbial immobilization bead
Catalysis is repeatedly recycled, can only carry out the primary microbial immobilization bead that contains reacted that is catalyzed with routine has marked improvement, is
The cost for further decreasing industrial production R-3- quinuclidinol provides technical foundation.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Whole-cell catalyst is constructed according to the record of the specification word section Example of Chinese patent CN106282135A
(engineering bacteria), the whole-cell catalyst have the ability that 3- quinuclidone is catalyzed and synthesized to (R) -3- quinuclidinol.
The engineering bacteria streak inoculation that -70 DEG C are saved is in LB solid medium, and 37 DEG C of culture 12h, be restored activity
Engineering bacteria；One single colonie of the engineering bacteria of picking activity recovery is inoculated in LB liquid medium, and 37 DEG C of culture 12h are obtained
Seed culture fluid.
By seed culture fluid, it is seeded in LB liquid medium according to the inoculum concentration of fluid nutrient medium 10%.Temperature
37 DEG C, 250rpm stirring under conditions of expand culture, when its culture solution OD600 be 0.6~0.8 when, be added IPTG to train
Final concentration of 0.6mM in nutrient solution is induced: inducing temperature is 30 DEG C, and induction time is 8 hours.After induction, by institute
It obtains culture solution and is centrifuged 20min at 6000rpm, collect thallus, after being washed 3 times using 1 times of phosphate buffer, with phosphoric acid buffer
Thallus is resuspended in liquid, is configured to OD600It is spare for 0.5 engineering bacterium suspension.The bacteria concentration of the engineering bacterium suspension is
0.45g/ml。
The preparation of PVA solution: the mass ratio according to PVA, water, DMSO is that 1:8:0.5 weighs raw material, by polyvinyl alcohol
(PVA) it is added in cold water, heating dissolves PVA, and DMSO is then added, and makes it dissolve uniformly, placement is cooled to room temperature.
Engineering bacterium suspension is mixed with PVA according to volume ratio 1:5, by mixed solution quick-frozen 24 hours at -80 DEG C, is taken
30 DEG C of defrostings after out, wash 1 time, are then cut into bead, obtain the immobilized spherule containing engineering bacteria.The present invention is prepared
The immobilized spherule containing engineering bacteria in containing enzyme activity about: glucose dehydrogenase 5 × 104Ug, carbonyl reductase 3.5 × 104U/g。
Embodiment 2
The immobilized spherule containing engineering bacteria and engineering bacteria free cell of this Experimental Comparison embodiment 1 preparation carry out
Performance when biocatalysis synthetic reaction
Catalytic synthesis is carried out according to optimum reaction condition:
Prepare reaction solution: i.e. sodium dihydrogen phosphate 10g/L, disodium hydrogen phosphate 27g/L, glucose 400g/L, quinuclidone 250g/
L, coenzyme (100mM NAD+) 0.1mL, pH value is adjusted to 7.0, obtains reaction solution.
Reaction solution is divided into two groups:
(1) test group, the immobilized spherule 33g containing engineering bacteria that prepared by addition embodiment 1 in every liter of reaction solution are (fixed
Change cell)；
(2) control group, according to method preparation engineering bacterium (cell catalyst) suspension shown in embodiment 1, every liter of reaction
Engineering bacterium suspension 45g (engineering bacteria free cell) is added in liquid.
Test group and the control group pH in 30 DEG C of progress conversion reactions, reaction process maintain pH7.0, and record catalysis is anti-
Conversion ratio between seasonable with quinuclidone.
Experimental result as shown in Figure 1, test group (engineering bacteria immobilized cell) and control group (engineering bacteria free cell) all
R-3- quinuclidinol can be catalyzed and synthesized by substrate of quinuclidone well, catalysis reaction can be completed within 7 hours, and each
The transformation efficiency of the quinuclidone of period is essentially identical, illustrates that engineering bacteria immobilized cell can be good at being catalyzed quinuclidone in this way
It is converted into R-3- quinuclidinol.And using the engineering bacteria immobilized spherule of the method for the invention preparation not because reaction system is sent out
Now it is crushed.
Embodiment 3
According to the record Rhodotorula of the specification word section Example of Chinese patent ZL 201010197502.X
Mucilaginosa X15 (culture presevation number are as follows: CGMCC No:3664), which, which has, catalyzes and synthesizes (R)-for 3- quinuclidone
The ability of 3- quinuclidinol.
It cultivates to obtain rhodothece rubra suspension, bacteria concentration according to the record of the Chinese patent ZL 201010197502.X
For 0.45g/ml.
The preparation of PVA solution: the mass ratio according to PVA, water, DMSO is that 1:8:0.5 weighs raw material, by polyvinyl alcohol
(PVA) it is added in cold water, heating dissolves PVA, and DMSO is then added, and makes it dissolve uniformly, placement is cooled to room temperature.
Rhodothece rubra X15 suspension is mixed with PVA according to volume ratio 1:5, mixed solution is quick-frozen 24 small at -80 DEG C
When, 30 DEG C of defrostings after taking-up wash 1 time, are then cut into bead, obtain the immobilized spherule containing rhodothece rubra X15.This hair
In the bright immobilized spherule containing rhodothece rubra X15 being prepared about containing enzyme activity: glucose dehydrogenase 5 × 104Ug, carbonyl is also
Protoenzyme 3.5 × 104U/g。
This Experimental Comparison embodiment 3 above-mentioned prepared immobilized spherule containing rhodothece rubra X15 and dark red ferment
Female X15 free cell carries out performance when biocatalysis synthetic reaction.
Catalytic synthesis is carried out according to optimum reaction condition in embodiment 2.
Reaction solution is divided into two groups:
(1) test group, the above-mentioned prepared immobilization containing rhodothece rubra X15 of addition embodiment 3 in every liter of reaction solution
Bead 33g (immobilized cell)；
(2) control group prepares rhodothece rubra X15 (cell catalyst) suspension according to method shown in embodiment 3, and every liter
Rhodothece rubra X15 suspension 45g (rhodothece rubra X15 free cell) is added in reaction solution.
Test group and the control group pH in 30 DEG C of progress conversion reactions, reaction process maintain pH7.0, and record catalysis is anti-
Conversion ratio between seasonable with quinuclidone.
Experimental result is as shown in Fig. 2, test group (immobilized spherule of rhodothece rubra X15) and control group (rhodothece rubra X15
Free cell) R-3- quinuclidinol can be catalyzed and synthesized by substrate of quinuclidone well, it is anti-that catalysis can be completed within 8 hours
It answers, and the transformation efficiency of the quinuclidone of each period is essentially identical, illustrates the immobilized spherule energy of rhodothece rubra X15 in this way
Enough quinuclidones of catalysis well are converted into R-3- quinuclidinol.And contain rhodothece rubra using the method for the invention preparation
The immobilized spherule of X15 is not because reaction system discovery is broken.
Embodiment 4
Catalytic synthesis is carried out according to optimum reaction condition:
1, reaction solution: i.e. sodium dihydrogen phosphate 10g/L, disodium hydrogen phosphate 27g/L, glucose 400g/L, quinuclidone is prepared
250g/L, coenzyme (100mM NAD+) 0.1mL, pH value is adjusted to 7.0, obtains reaction solution.
2, immobilized spherule 1: preparing the immobilized spherule containing engineering bacteria according to method shown in embodiment 1, is denoted as solid
Surely change bead 1；
Immobilized spherule 2: after engineering bacteria (cell catalyst) suspension is prepared according to 1 the method for embodiment, with
The sodium alginate of mass fraction 2.5% is embedding medium, and the calcium chloride of mass fraction 4% is fixative to engineering bacteria (cell catalysis
Agent) suspension embedded, embedding method are as follows: fixative is added dropwise to bacteria concentration as quality using the rate of addition of 2.0mL/min
In the engineering bacterium suspension of score 20%, the gel particle after fixative is added dropwise places 1.5h, then is immersed in 4 DEG C, mass fraction
To be crosslinked 4h in 4% calcium chloride solution, immobilized spherule 2 is obtained；
Immobilized spherule 3: after engineering bacterium suspension is prepared according to 1 the method for embodiment, with mass fraction 10%
PVA be embedding medium, the calcium chloride of mass fraction 5% is that fixative embeds engineering bacterium suspension, embedding method are as follows: with
Fixative is added dropwise in the engineering bacterium suspension that bacteria concentration is mass fraction 20% by the rate of addition of 2mL/min, is added dropwise and is fixed
Gel particle after agent places 1.5h, then is immersed in 4 DEG C, is crosslinked 5h in the calcium chloride solution that mass fraction is 5%, is fixed
Change bead 3.
3, respectively according to each 33g of immobilized spherule 1~3 for adding above-mentioned preparation in every liter of reaction solution, gained reaction solution is equal
PH maintains pH7.0 in 30 DEG C of progress conversion reactions, reaction process, and it is broken to count immobilized spherule 1~3 in catalytic reaction process
Broken situation and the conversion capability to quinuclidone.
As a result as shown in Figure 3,4, it is converted into R-3- quinuclidinol using various forms of immobilized cells catalysis quinuclidone, urged
Change reaction and proceed to 7h, immobilized spherule 1 converts R-3- quinuclidinol for quinuclidone completely and without broken；Immobilized spherule
2 and immobilized spherule 3 by the efficiency that quinuclidone is converted into R-3- quinuclidinol be respectively 54% and 61%, and both immobilizations
Bead has different degrees of broken respectively in the reaction system, and 2 percentage of damage of immobilized spherule is 67%, and immobilized spherule 3 is broken
Broken rate is 45%.This explanation has stronger stability using the immobilized spherule that PVA is prepared at low temperature.
Embodiment 5
This test recycles ability containing microbial immobilization bead to preparation and verifies.
Urge for the first time according to the immobilized spherule containing engineering bacteria that method shown in embodiment 2 prepares embodiment 1
Change reaction, after the quinuclidone in first time catalysis reaction is fully converted to R-3- quinuclidinol, by the immobilization containing engineering bacteria
After bead is using phosphate buffer solution cleaning, it is added in new reaction solution, is catalyzed again according to method shown in embodiment 2
Reaction.The immobilized spherule containing engineering bacteria of preparation is recycled in the manner described above, with detection present invention preparation
The ability and efficiency that are repeatedly catalyzed containing microbial immobilization bead.
Test result is as shown in figure 5, the immobilized spherule containing engineering bacteria is fully converted to R-3- Kui in catalysis quinuclidone
During peaceful alcohol, continuous several times catalysis all has very strong catalytic capability, in first 6 times of continuous catalysis, catalytic capability base
Originally it remains unchanged, so that quinuclidone is fully converted to R-3- quinuclidinol in a short time, but as the immobilization containing engineering bacteria is small
The number that ball uses is excessive, and catalytic capability is weakened；But give the regular hour, the immobilized spherule containing engineering bacteria is same
Quinuclidone can be fully converted to R-3- quinuclidinol by sample.
The immobilized spherule containing rhodothece rubra X15 for preparing embodiment 3 also according to method shown in embodiment 3 carries out
Catalysis reaction, the reaction process are fully converted to R-3- Kui with engineering bacteria immobilized spherule described in embodiment 4 catalysis quinuclidone
Peaceful alcohol reaction.Test result is as shown in figure 5, the immobilized spherule containing rhodothece rubra X15 is fully converted in catalysis quinuclidone
During R-3- quinuclidinol, continuous several times catalysis all has very strong catalytic capability, in first 8 times of continuous catalysis, catalysis
Ability is held essentially constant, and quinuclidone is made to be fully converted to R-3- quinuclidinol in a short time.
These explanations are prepared by the present invention containing microbial immobilization bead to there is repeatedly catalysis quinuclidone to convert completely
For the ability of R-3- quinuclidinol, technical foundation is provided to further decrease the cost of industrial production R-3- quinuclidinol.
Embodiment 6
Catalytic capability containing microbial immobilization bead under the substrate of various concentration of this test to preparation.
1, the immobilized spherule containing engineering bacteria is prepared according to method shown in embodiment 1；According to shown in embodiment 3
The immobilized spherule containing rhodothece rubra X15 is prepared in method；
2, reaction solution: sodium dihydrogen phosphate 10g/L, disodium hydrogen phosphate 27g/L, glucose 400g/L, quinuclidone, coenzyme is prepared
(100mM NAD+) 0.1mL, adjust pH value to 7.0, wherein the concentration of quinuclidone be respectively 250g/L, 350g/L, 400g/L,
450g/L, 500g/L, 550g/L and 600g/L obtain the different reaction solution of concentration of substrate.
3, respectively into the different reaction solution of concentration of substrate, contain engineering according to add above-mentioned preparation in every liter of reaction solution
The immobilized spherule of bacterium and each 33g of immobilized spherule containing rhodothece rubra X15, gained reaction solution carry out converting at 30 DEG C anti-
It answers, pH maintains pH7.0 in reaction process, counts in catalytic reaction process and respectively converts completely containing microbial immobilization bead
The time of quinuclidone.
The results are shown in Table 1, as the catalysis of the increase of substrate quinuclidone concentration, the immobilized spherule containing engineering bacteria is anti-
The time used is answered to increase, but when concentration of substrate is higher than 500g/L, quinuclidinol cannot be fully converted to R-3- quinuclidinol, work as bottom
When object concentration is 550g/L and 600g/L, after catalysis reaction has carried out 72 hours, quinuclidinol cannot all be fully converted to R-3- Kui
Peaceful alcohol, immobilized spherule enzyme activity completely disappears at this time.Immobilized spherule containing rhodothece rubra X15 obtains same result.
This illustrates the influence of immobilized spherule catalytic capability and vigor by concentration of substrate.
1 concentration of substrate of table is to the influence containing the catalysis reaction of microbial immobilization bead
As seen from the above embodiment, the present invention provides it is a kind of be not easily broken in biocatalytic reaction contain microorganism
Immobilized spherule preparation method, and be prepared containing microbial immobilization bead catalysis respond it is strong, can be more
Secondary recycling effectively reduces production cost, improves growth efficiency.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method of immobilization embedded microorganism, comprising the following steps:
Poly-vinyl alcohol solution is mixed with the bacterium solution of microorganism, 1~72h is freezed at -85~-75 DEG C, cuts balling-up after defrosting,
It obtains containing microbial immobilization bead.
2. the method according to claim 1, wherein the poly-vinyl alcohol solution concentration is 0.07~0.13mg/
g。
3. according to the method described in claim 2, it is characterized in that, the preparation method of the poly-vinyl alcohol solution includes:
Polyvinyl alcohol is mixed with water, DMSO is added after heating for dissolving and makes it dissolve, it is cooling, obtain poly-vinyl alcohol solution；It is described
The mass ratio of polyvinyl alcohol, water and DMSO is 1:7~10:0.1~2.
4. the method according to claim 1, wherein the bacterial concentration of the microorganism is 0.2~1.0g/ml.
5. according to claim 1 or 4 the methods, which is characterized in that the microorganism includes rhodothece rubra X15 or engineering bacteria；
The rhodothece rubra X15 is deposited in China General Microbiological culture presevation administrative center, and deposit number is CGMCC No:3664.
6. the method according to any one of claim 5, which is characterized in that when the microorganism is engineering bacteria, institute
State the preparation method of the bacterium solution of engineering bacteria the following steps are included:
(1) engineering bacteria is inoculated in LB culture medium and is activated；
(2) single colonie of engineering bacteria is inoculated in LB culture medium after picking activation, and 32~40 DEG C of cultures 12~for 24 hours, obtain seed training
Nutrient solution；
(3) seed culture fluid is seeded in LB liquid medium and is cultivated, culture to OD600Value is added when being 0.6~0.8
IPTG induction, centrifuging and taking precipitating, obtains thallus；
(4) thallus that step (3) obtains is redissolved with phosphate buffer, obtains the bacterium solution of engineering bacteria, the bacterium of the engineering bacteria
The OD of liquid600Value is 0.3~0.6.
7. the method according to any one of claim 2,3 and 4, which is characterized in that poly-vinyl alcohol solution and microorganism
Bacterium solution volume ratio be 1~100:1.
8. the method according to claim 1, wherein the thaw point is 28~35 DEG C.
9. a kind of preparation of any one of claim 1~8 the method contains microbial immobilization bead.
10. it is according to any one of claims 8 containing microbial immobilization bead in biocatalysis synthetic drug or pharmaceutical intermediate
In application.
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| CN111593044A (en) * | 2019-02-20 | 2020-08-28 | 成都医学院 | Rhodotorula mucilaginosa immobilized cell and application thereof |
| CN112457307A (en) * | 2020-12-15 | 2021-03-09 | 河北省科学院生物研究所 | Extraction method of R-quininol |
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| CN101864370A (en) * | 2010-06-11 | 2010-10-20 | 河北省科学院生物研究所 | Yeast strain converting quinuclidone into R-3-quinuclidinol and conversion method thereof |
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Cited By (3)
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| CN111593044A (en) * | 2019-02-20 | 2020-08-28 | 成都医学院 | Rhodotorula mucilaginosa immobilized cell and application thereof |
| CN112457307A (en) * | 2020-12-15 | 2021-03-09 | 河北省科学院生物研究所 | Extraction method of R-quininol |
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