CN108841816A - Organic-inorganic composite immobilized cell and its preparation method and application - Google Patents
Organic-inorganic composite immobilized cell and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of preparation methods of Organic-inorganic composite immobilized cell, include the following steps：It is mixed again with inorganic material after the bacteria suspension of nitrilase producing strains is mixed with organic material aqueous solution, carries out cell fixation in 2-5 DEG C of immobilization liquid, obtain Organic-inorganic composite immobilized cell；The concentration of bacteria suspension is 10-40mg/mL；Organic material is sodium alginate and polyvinyl alcohol.The present invention also provides a kind of biological synthesis methods of niacin：The Organic-inorganic composite immobilized cell of above-mentioned preparation is mixed with substrate nicotinonitrile, by mixed liquor in pH=7.5-8.5, temperature reacts 30-60min under the conditions of being 20-55 DEG C, prepares niacin by multiple batches of reaction.Process for fixation provided by the present invention is easy to operate, at low cost, immobilized cell high mechanical strength, mass-transfer performance is good, thermal stability is high, reusable rate is high, and beneficial thinking is provided for industrial application.
Description
Technical field
The present invention relates to industrial biotechnology field more particularly to a kind of Organic-inorganic composite immobilized cell and its systems
Preparation Method and application.
Background technique
Nitrilase contains a catalytic triads (Glu-Lys-Cys), being capable of step catalysis nitrile compounds generation phase
The carboxylic acid and ammonia answered.But nitrile compounds have certain cytotoxicity mostly, can inhibit nitrilase enzyme activity, and then lead to cell
Denaturation, cracking.Free cell catalyst in the conversion process can be directly by the toxic effect from nitrile substrate, and cell is solid
Surely the reaction stability, thermal stability, storage-stable, operational stability of cell catalyst can not only be improved by changing technology, and
Mitigate cell to a certain extent by the toxic effect of substrate, and is conducive to the reaction continuous processing of carboxylic acids product and subsequent
The techniques such as isolate and purify.Immobilized cell technology theoretically have the advantages that it is very more, but at present it is most of research and
Experiment all only rests on laboratory stage and level, and is introduced into industrialized production not successfully, maximum bottleneck it
One be exactly particle mechanical strength it is inadequate.Currently, scholar both domestic and external puts into sight on complex carrier, such as SA/SiO2It is multiple
Heshui gel is conducive to the two performance advantage complementation, makes up mutual shortcoming in conjunction with organic carrier and inorganic carrier.
Immobilized cell technique is interior in a limited space by the way that either physically or chemically the cells are fixed, but cell is still
The technology that keeps activity and can be recycled.The affine nontoxicity of high-molecular organic material in immobilization material, but there is also machines
The disadvantages of tool intensity is low, mass-transfer performance is inadequate can be efficiently modified this deficiency by adding inorganic material.It is fixed improving cell
In terms of changing bead intensity, some researches show that add suitable active carbon, CaCO3、Ca(OH)2Powder or SiO2, can not only determine
To the specific gravity for adjusting immobilized spherule, the compressive strength of immobilized spherule also can be improved.Chotaniy et al. is in PVA immobilization
The inorganic material such as silica sand are added in journey to increase the mechanical strength, settleability and permeability of immobilized spherule.Also some researches show that
The calcium silicates that 2-30% is added in PVA immobilization material can significantly improve the stability of immobilized spherule, enhancing cell is lived
Property.In terms of the transmission characteristic for improving cell fixation bead.According to it has been reported that addition CaCO3Immobilization particle can be improved
Transmission characteristic, CaCO3On the one hand it can slowly be utilized by microorganism, another aspect CaCO3In the immobilization process of slant acidity condition
In can gradually decompose generate CO2, so that immobilized spherule internal voids increase, this is conducive to the proliferation and immobilized spherule of cell
Internal aeration, water flowing and the diffusion of reaction substrate transmitting.Microvoid structure inside immobilized spherule is solid to cell
Surely change performance and mass transfer reaction is crucial, square chapter equality people is with CaCO3Change with pyrolusite powder is inorganic filling material
Into porous polymer carrier performance, modified carrier is more conducive to the immobilization of cell as the result is shown.Liu Shu text et al. was once
Add bentonite, CaCO3And SiO2The mechanical strength and mass transfer ability for improving immobilized spherule, through analyzing in immobilization material
Add CaCO3The mechanical stability of immobilization particle can be significantly improved.
But inorganic material up to now, is added in organic immobilized cell, and it is raw to be applied to nitrilase
Object converts there is not yet any document report.Early period of the invention has carried out the compound immobilization of sodium alginate-PVA to mut-D3,
Mut-D3 cell after immobilization greatly promotes substrate tolerance, how to further increase tolerance of the immobilized cell to substrate
Property, it also needs further to study.
Summary of the invention
In order to solve the above technical problems, the object of the present invention is to provide a kind of Organic-inorganic composite immobilized cell and its
Preparation method and application, the immobilized cell after adding inorganic material are produced in mechanical strength, mass transfer ability, thermal stability and substrate
It is promoted in terms of object tolerance significantly, when being applied to niacin production, the yield of niacin is significantly improved.
The first purpose of the invention is to provide a kind of preparation methods of Organic-inorganic composite immobilized cell, including with
Lower step：
Compositing organic material solution is obtained after the bacteria suspension of nitrilase producing strains and organic material aqueous solution are mixed, then
Compositing organic material solution is mixed with inorganic material, carries out cell fixation in 2-5 DEG C of immobilization liquid, obtain it is organic-
Inorganic compounding immobilized cell；Wherein, the concentration of bacteria suspension is 10-40mg/mL；Organic material is sodium alginate, polyvinyl alcohol
(PVA) and one or more of chitosan, the concentration of organic material aqueous solution are 2%-12%, organic material aqueous solution and bacterium
The volume ratio of suspension is 5:1-1:5, inorganic material accounts for the 0.2%-6.0% of the mass fraction of compositing organic material solution.
Further, bacteria suspension is prepared by the following method：
The nitrilase that fermentation is obtained generates fermented liquid, and thalline were collected by centrifugation, is washed using sodium phosphate buffer, and
Thallus is resuspended in the buffer, bacteria suspension is made.
Further, nitrilase producing strains are nitrilase producing strains pseudomonas putida mutant mut-D3.mut-
The deposit number CGMCC No.14276 of D3 bacterial strain, preservation date on June 26th, 2017, for application No. is 201710666935.7
Chinese patent disclosed in.
Further, inorganic material SiO2、CaCO3One or more of with bentonite.
Further, SiO2Account for the 1.0-3.0% of the mass fraction of compositing organic material solution；CaCO3Account for compound organic material
Expect the 0.2-1.0% of the mass fraction of solution；Bentonite accounts for the 2-6% of the mass fraction of compositing organic material solution.
Further, organic material is sodium alginate and polyvinyl alcohol, and the mass ratio of sodium alginate and polyvinyl alcohol is 2-
7:1-3。
Further, sodium alginate, polyvinyl alcohol and inorganic material are mixed with bacteria suspension by the following method：
By the PVA aqueous solution of sodium alginate aqueous solution and 4-12% that mass concentration is 2-7% according to 9:1 volume ratio is mixed
Close, then with bacteria suspension 1:1 (volume ratio) mixing, then inorganic material is added thereto, it stirs evenly.
Further, immobilization liquid is 0.1-1.5%CaCl2Saturation boric acid solution.
Further, the immobilization time is 1-10h.
Second object of the present invention is to be claimed a kind of to fix using Organic-inorganic composite prepared by the above method
Change cell.
Further, Organic-inorganic composite immobilized cell prepared by method of the invention, in 25-55 DEG C of temperature model
Enclose it is interior show excellent stability to thermal stimulus, half-life period 33-272h, than the immobilized cell for being free of inorganic material
Thermal stability improve 1.2-1.65 times.Meanwhile the storage-stable of Organic-inorganic composite immobilized cell is good, at 4 DEG C
When storing 40 days, Organic-inorganic composite immobilized cell of the invention remains to retain 72% catalytic activity, is higher than without inorganic
The immobilized cell (60%) of material.
Third object of the present invention is to provide a kind of biological synthesis methods of niacin, include the following steps：
Above-mentioned Organic-inorganic composite immobilized cell is mixed with substrate nicotinonitrile, by mixed liquor in pH=7.5-
8.5, temperature reacts 30-60min under the conditions of being 20-55 DEG C and adds next batch substrate after a batch is completed in conversion, until turning
Rate is lower than 70%, obtains niacin.
Further, the concentration of substrate nicotinonitrile is 150mM-550mM in mixed liquor.Preferably, concentration is
250mM。
Further, the mass ratio of Organic-inorganic composite immobilized cell and substrate nicotinonitrile is 2:1-1:3.
Organic-inorganic composite immobilized cell prepared by the present invention further enhances high concentration substrate tolerance, can answer
Efficient Conversion for niacin synthesizes, and yield improves 21% compared to the immobilized cell for not adding inorganic material.
The latent of the performance boosts such as immobilized cell mechanical strength, substrate tolerance and stability is made for addition inorganic material
In reason, key factor is in following aspect：Nontoxicity that high-molecular organic material is affine, but there is also mechanical strengths low, mass transfer performances
Can be inadequate the disadvantages of, can be efficiently modified this deficiency by adding inorganic material.In terms of improving cell fixation bead intensity,
Add suitable active carbon, CaCO3、Ca(OH)2Powder or SiO2, not only can may be used also with the specific gravity of orientation adjustment immobilized spherule
Improve the compressive strength of immobilized spherule.In terms of the transmission characteristic for improving cell fixation bead, CaCO is added3It can improve solid
Surely change the transmission characteristic of particle, CaCO3On the one hand it can slowly be utilized by microorganism, another aspect CaCO3In consolidating for slant acidity condition
It can gradually be decomposed during fixedization and generate CO2, so that immobilized spherule internal voids increase, this is conducive to the proliferation of cell and consolidates
Surely change aeration, water flowing and the diffusion of the reaction substrate transmitting inside bead.The knot of electron microscope analysis immobilized cell structure
Fruit also shows, and the immobilized spherule internal voids structure after adding inorganic material obviously becomes larger loose.This is conducive to improve every
A hole inner cell quantity increases aeration, water flowing and the resistance to mass tranfer for reducing reaction.In addition, after addition inorganic material
The mechanical strength of immobilized cell is promoted so that immobilized cell is stronger to extraneous adaptability, and immobilized cell is not easy to crack
Solution, accordingly extends the service life and stability of cell.Meanwhile inorganic and organic materials are combined for microbial cell inside
Micromorphology also has different degrees of influence, promotes cell to extraneous adverse environment and harsh reaction condition tolerance.
According to the above aspect of the present invention, the present invention has at least the following advantages：
The present invention provides a kind of promotion of mechanical strength, resistance to mass tranfer reduction, improved thermo stability, repeatability are high and right
The Organic-inorganic composite immobilized cell and preparation method thereof that substrate tolerance is further promoted, using method system of the invention
Standby immobilized cell can convert the nicotinonitrile of 14 batches, and accumulation obtains 418g/L niacin, inorganic compared to not adding
The niacin output increased 21% of the immobilized cell of material.
Process for fixation provided by the present invention is easy to operate, at low cost, immobilized cell high mechanical strength, mass-transfer performance
Good, thermal stability height and reusable rate height, beneficial thinking is provided for industrial application.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Fig. 1 is influence knot of the single inorganic material of the present invention to prepared Organic-inorganic composite immobilized cell performance
Fruit；
Fig. 2 is influence knot of the composite inorganic material of the present invention to prepared Organic-inorganic composite immobilized cell performance
Fruit；
Fig. 3 is the SEM test chart of different Organic-inorganic composite immobilized cells；
Fig. 4 is the influence and its thermal stability and storage of temperature, pH to Organic-inorganic composite immobilized cell performance of the present invention
Hide stability test result；
Fig. 5 is that concentration of substrate influences Organic-inorganic composite immobilized cell catalysis activity of the present invention and fixation cell
The conversion ratio test result of niacin in born of the same parents' batch conversion process.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
The preparation of 1 bacteria suspension of embodiment
The mutant bacteria mut-D3 for producing nitrilase is inoculated in 50mL fluid nutrient medium, in 30 DEG C of shaken cultivation 20-36h,
The present embodiment is preferably for 24 hours, as seed liquor.30 DEG C of oscillation trainings in fresh fermentation medium are forwarded to the inoculum concentration of 1-5%
Support 30-45h.In the present embodiment, preferably 1% inoculum concentration, incubation time 40h.8000rpm is centrifuged 10min and goes under the conditions of 4 DEG C
Supernatant collects thallus.It is washed 2-3 times using the sodium phosphate buffer of 100mM, pH7.2, the present embodiment is preferably 2 times, and will
The thallus is resuspended in the buffer, and the bacteria suspension of 20mg/mL is made, and it is spare to be placed in 4 DEG C of refrigerators.
The preparation of 2 Organic-inorganic composite immobilized cell of embodiment
This implementation provides a kind of preparation method of Organic-inorganic composite immobilized cell, fixes to sodium alginate-PVA
Change in cell and adds inorganic material SiO respectively2、CaCO3, bentonite, to investigate single inorganic material to immobilized cell performance
It influences, the specific method is as follows：
By mass concentration be 2.5% sodium alginate aqueous solution and mass concentration be 8% PVA aqueous solution according to 9:1
Ratio (volume ratio) mixing, as organic fixed material.The bacteria suspension that will be prepared in above-mentioned organic fixed material and embodiment 1
1:1 mixing (volume ratio), obtains compositing organic material solution.It is added respectively into compositing organic material solution again different inorganic
Material：SiO2(1.0%, 1.5%, 2.0%, 2.5%, 3.0%), CaCO3(0.2%, 0.4%, 0.6%, 0.8%, 1.0%),
Bentonite (2%, 3%, 4%, 5%, 6%), stirs evenly.Inorganic material added by above, representated by the numerical value in bracket
Meaning be that inorganic material accounts for the mass ratio of compositing organic material solution, such as 1% inorganic material refers to compound organic in 100g
1g inorganic material is added in material solution.
Mixed liquor is slowly dropped into the immobilization liquid (0.6%CaCl of 4 DEG C of pre-coolings with syringe2Saturation boric acid solution)
In, the immobilization time is 5h, and operation carries out in ice bath.Immobilized cell is washed with sodium phosphate buffer (100mM, pH7.2)
It washs 3 times, obtains Organic-inorganic composite immobilized cell, and measure its catalytic capability and mechanical strength.
As seen from Figure 1 with SiO2Concentration is continuously increased, and the mechanical strength of Organic-inorganic composite immobilized cell mentions
It rises obviously, when concentration is 2.0%, mechanical strength reaches maximum, 21% is improved, and remain enzyme activity and have dropped 21-32%；
With CaCO3Concentration is continuously increased, and the residual enzyme activity and mechanical strength of Organic-inorganic composite immobilized cell, which are in, first to be increased
Downward trend afterwards, residual enzyme activity reach maximum when concentration is 0.8%, only lose 10% enzyme activity, and mechanical strength is promoted
It is not obvious (4-6%)；The increase of bentonite concentration makes the residual enzyme activity of immobilized spherule and mechanical strength be constantly in decline
Trend.In summary experimental result, SiO2It can significantly improve the mechanical strength of immobilized spherule, and CaCO3Be conducive to residual enzyme
It is living, so following embodiment of the present invention, which uses, is added to seaweed for both materials simultaneously in order to obtain both effects simultaneously
In sour sodium-PVA immobilized cell, it is desirably to obtain high mechanical strength and remains the high immobilized cell of enzyme activity.
The preparation of 3 Organic-inorganic composite immobilized cell of embodiment
This implementation provides a kind of preparation method of Organic-inorganic composite immobilized cell, fixes to sodium alginate-PVA
Change the inorganic material SiO for adding various concentration in cell simultaneously2、CaCO3, to investigate composite inorganic material to immobilized cell
It influences, the specific method is as follows：
By mass concentration be 2.5% sodium alginate aqueous solution and mass concentration be 8% PVA aqueous solution according to 9:1
Ratio (volume ratio) mixing, as organic fixed material.The bacteria suspension that will be prepared in above-mentioned organic fixed material and embodiment 1
1:1 mixing (volume ratio), obtains compositing organic material solution.1.5%SiO is separately added into compositing organic material solution again2+
0.6%CaCO3, 1.5%SiO2+ 0.8%CaCO3, 1.5%SiO2+ 1.0%CaCO3, 2.0%SiO2+ 0.6%CaCO3, 2.0%
SiO2+ 0.8%CaCO3, 2.0%SiO2+ 1.0%CaCO3, 2.5%SiO2+ 0.6%CaCO3, 2.5%SiO2+ 0.8%CaCO3Or
2.5%SiO2+ 1.0%CaCO3, stir evenly.Inorganic material added by above, meaning representated by the numerical value in bracket are
Inorganic material accounts for the mass ratio of compositing organic material solution, and such as 1% inorganic material refers in 100g compositing organic material solution
Middle addition 1g inorganic material.
Mixed liquor is slowly dropped into the immobilization liquid (0.6%CaCl of 4 DEG C of pre-coolings with syringe2Saturation boric acid solution)
In, the immobilization time is 5h, and operation carries out in ice bath.Immobilized cell is washed with sodium phosphate buffer (100mM, pH7.2)
It washs 3 times, obtains Organic-inorganic composite immobilized cell, and measure its catalytic capability and mechanical strength.
As seen from Figure 2, with SiO2Concentration improves, and the mechanical strength of Organic-inorganic composite immobilized cell is overall
In the trend of raising, this meets existing document SiO2Pressure-bearing center can be provided for immobilization particle and improves the specific gravity of immobilized spherule
With the report of intensity.High mechanical strength illustrates that the ability of immobilization particle reply external environment collision is strong, the repeatable benefit of particle
It is also high with rate.And with CaCO3Concentration improves, and catalysis activity is fallen after rising instead.CaCO3Addition can improve immobilization particle
Permeability, the exchange capacity of cell and external substance is also better, but permeability is excessively high, and mechanical strength cannot be guaranteed.
So the two indexs should be comprehensively considered in material selection and find optimum combination.It is added simultaneously in immobilized spherule
SiO2And CaCO3, it is also beneficial to form CaOSiO2, to improve the activity and stability of immobilized spherule.With mechanical strength
It is index with opposite enzyme activity, adds 2.0%SiO in sodium alginate-PVA immobilization material2And 0.6%CaCO3To be prepared with
Machine-inorganic compounding immobilized cell preferred plan.
Sodium alginate-PVA immobilized spherule prepared by inorganic material and addition 2.0% is not added respectively in Fig. 3 A, B
SiO2And 0.6%CaCO3The SEM characterization result of prepared Organic-inorganic composite immobilized spherule adds as seen from Figure 3
Add 2.0%SiO2And 0.6%CaCO3Organic-inorganic composite immobilized cell internal voids structure afterwards obviously becomes larger loose
(Fig. 3 B), this is conducive to improve each hole inner cell quantity, while increasing aeration, water flowing and the mass transfer for reducing reaction
Resistance.
In addition, taking addition 2.0%SiO2And 0.6%CaCO3Organic-inorganic composite immobilized cell afterwards, respectively pH6,
6.5, after reacting different time under the conditions of 7,7.5,8,8.5,9, separately sampled measurement catalysis activity is inorganic to adding to measure pH
The influence of the sodium alginate-PVA immobilized cell catalytic activity of material.
Separately take addition 2.0%SiO2And 0.6%CaCO3Organic-inorganic composite immobilized cell afterwards is respectively in 25 DEG C, 30
DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C when measure enzyme activity, with measuring temperature to addition inorganic material sodium alginate-
The influence of PVA immobilized cell catalytic activity.
Separately take addition 2.0%SiO2And 0.6%CaCO3Organic-inorganic composite immobilized cell afterwards, respectively at 30 DEG C, 40
DEG C, 45 DEG C, 50 DEG C of water-baths heat preservation, period sampling measuring catalysis activity investigates the situation of change of catalysis activity during warm bath, directly
Until it is down to less than half.Refer to the opposite enzyme activity for calculating each point in time sampling detection as 100% using initial catalyst vigor,
And mapped with time and Ln (RA), partly declining for immobilized cell catalysis activity at each temperature is calculated using the slope of matched curve
Phase, with half-life period t1/2As temperature stability measurement index, the sodium alginate-PVA immobilized cell of addition inorganic material is investigated
Thermal stability influence.
Separately take addition 2.0%SiO2And 0.6%CaCO3Organic-inorganic composite immobilized cell afterwards is stored in 4 DEG C of ice
In case, respectively in 10d (day), 20d, 10d, 30d, 40d, 50d, 60d sample detection catalysis activity, to investigate immobilized cell
Storage-stable.
As a result as shown in figure 4, as seen from Figure 4, the sodium alginate-PVA immobilized cell after adding inorganic material is most
Suitable reaction pH (Fig. 4 A) and temperature (Fig. 4 B) are respectively 8.0 and 45 DEG C, such as the immobilized cell for not adding inorganic material.
But under the conditions of slant acidity and meta-alkalescence, the sodium alginate-PVA immobilized cell that inorganic material is added, which has, preferably adapts to energy
Power and catalysis activity.And in lower than 30 DEG C, higher than 45 DEG C temperature ranges, the sodium alginate-PVA immobilization of inorganic material is added
Cell shows superior stability to thermal stimulus.
Sodium alginate-PVA the immobilized cell of inorganic material is added in 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C of half-life period
(t1/2) it is respectively 271.48h, 150.92h, 92.92h, 33.12h (Fig. 4 C).And sodium alginate-PVA immobilized cell partly declines
Phase (t1/2) it is respectively 192.54h, 111.79h, 76.17h, 20.32h.Addition inorganic material makes sodium alginate-PVA fixation cell
Born of the same parents' thermal stability is further enhanced, and has been respectively increased 1.41 times, 1.35 times, 1.22 times, 1.63 times.
It can be seen that by Fig. 4 D, the storage-stable of the sodium alginate-PVA immobilized cell of inorganic material be added in storage
Between be 10-50d when have small size raising.When 4 DEG C of refrigerators are stored 40 days, the sodium alginate-PVA fixation cell of inorganic material is added
Born of the same parents remain to retain 72% catalytic activity, and the sodium alginate-PVA immobilized cell without addition is only 60% or so.
In addition, using nicotinonitrile as substrate, concentration is respectively：150,250,350,450,550mmol/L utilize this
Sodium alginate-PVA immobilized cell after the addition inorganic material of invention carries out catalyzed conversion to it, is turned with studying it to batch
Changing influences.To the addition 2.0%SiO prepared2And 0.6%CaCO3Every batch of in obtained Organic-inorganic composite immobilized cell
The secondary nicotinonitrile for being separately added into final concentration of 150,250,350,450 or 550mmol/L, Organic-inorganic composite immobilization
The mass ratio of cell and substrate nicotinonitrile is 2:1-1:3, and in 30 DEG C of oscillating reactions 180min, niacin is detected using HPLC
Production rate, wherein HPLC condition is instrument：Wear peace Ultimate 3000 series HPLC, chromatographic column：Atlantis dC18(5μ
M, 4.6 × 150mm；Waters, USA), mobile phase：Acetonitrile：Water=3:2 (containing 0.02% trifluoroacetic acid), flow velocity：0.5mL/
Min, Detection wavelength：268nm, column temperature：30 DEG C, sample volume：10μL.
The substrate tolerance of immobilized cell is further promoted it can be seen from Fig. 5 A.When initial nicotinonitrile concentration
When within the scope of 150mM-550mM, catalysis of the sodium alginate-PVA immobilized cell of inorganic material to nicotinonitrile is added
Vigor gets a promotion compared to sodium alginate-PVA immobilized cell, especially when concentration of substrate is promoted to 350mM, is added inorganic
Sodium alginate-PVA the immobilized cell of material needs 60min that can convert substrate completely, and sodium alginate-PVA immobilization
Cell needs 80min.And when concentration of substrate is promoted to 450mM and 550mM, the sodium alginate-PVA that inorganic material is added is fixed
Change transformation rate and improves 5%-15%.In view of maximum conversion efficiency, therefore 250mM is the concentration of substrate that most preferably feeds intake.
Embodiment 4 uses immobilized cell batch Synthesis niacin
To the addition 2.0%SiO prepared2And 0.6%CaCO3Every batch of in obtained Organic-inorganic composite immobilized cell
The secondary nicotinonitrile that final concentration of 250mM is added, and in 30 DEG C of oscillating reactions 60min, until conversion ratio is lower than 70%.Have
The mass ratio of machine-inorganic compounding immobilized cell and substrate nicotinonitrile is 2:1-1:3.According to the condition in embodiment 3, use
HPLC detects niacin and nicotinonitrile content.It can be converted it can be seen from Fig. 5 B using immobilized spherule prepared by the present invention
The nicotinonitrile of 14 batches, accumulation obtains 418g/L niacin, compared to the immobilized cell niacin for not adding inorganic material
21% (the immobilized cell cumulative production for not adding inorganic material is only 346g/L) of output increased.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of Organic-inorganic composite immobilized cell, which is characterized in that include the following steps：
After the bacteria suspension of nitrilase producing strains and organic material aqueous solution are mixed, compositing organic material solution is obtained, then
It is mixed again with inorganic material, carries out cell fixation in 2-5 DEG C of immobilization liquid, obtained the Organic-inorganic composite and fix
Change cell；Wherein, the concentration of the bacteria suspension is 10-40mg/mL；The organic material is sodium alginate, polyvinyl alcohol and shell
One or more of glycan, the concentration of organic material aqueous solution are 2%-12%, the body of organic material aqueous solution and bacteria suspension
Product is than being 5:1-1:5, the inorganic material accounts for the 0.2%-6.0% of the mass fraction of the compositing organic material solution.
2. preparation method according to claim 1, it is characterised in that：The nitrilase producing strains are nitrilase generation
Bacterium mutant mut-D3.
3. preparation method according to claim 1, it is characterised in that：The inorganic material is SiO2、CaCO3And bentonite
One or more of.
4. preparation method according to claim 1, it is characterised in that：The organic material is sodium alginate and polyethylene
The mass ratio of alcohol, the sodium alginate and polyvinyl alcohol is 2-7:1-3.
5. preparation method according to claim 1, it is characterised in that：The immobilization liquid is 0.1-1.5%CaCl2It is full
And boric acid solution.
6. preparation method according to claim 1, it is characterised in that：The immobilization time is 1-10h.
7. Organic-inorganic composite immobilized cell prepared by a kind of method of any of claims 1-6.
8. a kind of biological synthesis method of niacin, which is characterized in that include the following steps：
Organic-inorganic composite immobilized cell as claimed in claim 7 is mixed with substrate nicotinonitrile, by mixed liquor in pH
=7.5-8.5, temperature react 30-60min under the conditions of being 20-55 DEG C, obtain the niacin.
9. the biological synthesis method of niacin according to claim 8, it is characterised in that：Substrate 3- cyano in the mixed liquor
The concentration of pyridine is 150mM-550mM.
10. the biological synthesis method of niacin according to claim 8, it is characterised in that：The Organic-inorganic composite is fixed
The mass ratio for changing cell and substrate nicotinonitrile is 2:1-1:3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480361A (en) * | 2021-12-27 | 2022-05-13 | 安徽泰格生物科技有限公司 | Immobilized bacteria and preparation method of nicotinamide |
| CN115627268A (en) * | 2022-10-31 | 2023-01-20 | 东北农业大学 | Method for extracting eleutheroside B by combination of immobilized enzyme and high pressure |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460188A (en) * | 2017-08-07 | 2017-12-12 | 江南大学 | A kind of compound immobilization method and its application of nitrilase producing strains mutant |
-
2018
- 2018-08-03 CN CN201810877199.4A patent/CN108841816B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460188A (en) * | 2017-08-07 | 2017-12-12 | 江南大学 | A kind of compound immobilization method and its application of nitrilase producing strains mutant |
Non-Patent Citations (2)
| Title |
|---|
| 吴芳芳等: "聚乙烯醇包埋 Trichosporon sp．X1 处理苯酚废水", 《科学技术与工程》 * |
| 孙晓红等: "有机-无机复合载体固定化酵母生物催化剂生产燃料乙醇的研究", 《第十一届全国青年催化会议论文集》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480361A (en) * | 2021-12-27 | 2022-05-13 | 安徽泰格生物科技有限公司 | Immobilized bacteria and preparation method of nicotinamide |
| CN114480361B (en) * | 2021-12-27 | 2024-05-10 | 安徽泰格生物科技有限公司 | Preparation method of immobilized thalli and nicotinamide |
| CN115627268A (en) * | 2022-10-31 | 2023-01-20 | 东北农业大学 | Method for extracting eleutheroside B by combination of immobilized enzyme and high pressure |
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