CN101265453B - Beer yeast strain containing CMP kinase and CDP kinase and application - Google Patents
Beer yeast strain containing CMP kinase and CDP kinase and application Download PDFInfo
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- CN101265453B CN101265453B CN2008100239036A CN200810023903A CN101265453B CN 101265453 B CN101265453 B CN 101265453B CN 2008100239036 A CN2008100239036 A CN 2008100239036A CN 200810023903 A CN200810023903 A CN 200810023903A CN 101265453 B CN101265453 B CN 101265453B
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Abstract
The invention relates to a method for screening Saccharomyces cerevisiae with high-activity CMP kinase and CDP kinase, and the use of the Saccharomyces cerevisiae in the production of cytidine triphosphate, and belongs to the technology field of biological engineering. The Saccharomyces cerevisiae strains is preserved in the China General Microbiological Culture Collection Center (CGMCC) with accession number of CGMCC NO.2448. The Saccharomyces cerevisiae strains with high-activity CMP kinase and CDP kinase are screened by means of nitrosoguanidine (NTG) mutagenesis. In the production of the cytidine triphosphate, the Saccharomyces cerevisiae strains are used to be prepared into immobilized cells of various grain diameters with carrageenan as carriers by different controlled stirring speeds realized through embedding. Compared with manual cutting, the screening method of the invention has the advantages of small cell leakage, regular grains with high strength, low labor intensity, simple grain-producing procedure, no complicated equipment and instrument, and is suitable for industrialized production. Furthermore, bean oil used in the invention is safe and nontoxic solvent, suitable for various requirements and easy to recycle.
Description
Technical field
The present invention relates to a kind of screening and the application of this saccharomyces cerevisiae in producing cytidine triphosphate(CTP) that contains high vigor CMP kinases and the kinase whose saccharomyces cerevisiae of CDP, belong to technical field of bioengineering.
Background technology
Cytidine triphosphate(CTP) (is called for short: CTP) be a kind of normal composition in the body cell, it participates in the anabolism of nucleic acid and phospholipid in vivo, promote proteinic synthetic, and provide energy, can also regulate and promote the synthetic of neurocyte, neurogliocyte and cells of vascular wall membranous structure and make up, can resist the neural cell injury that causes by excitatory amino acid, free radical, thereby play anti-angiogenic hardened effect.And the main effect of cytidine monophosphate kinases (CMP kinase) and cytidine diphosphate(CDP) kinases (CDP kinase) is to be responsible for activating UTP-ammonia ligase (CTP synthetase), the activation UTP-ammonia ligase can reduce cytidine monophosphate, and (be called for short: CMP) level makes CMP synthesize CTP.At present existing manufacturer production CTP injection formulation is mainly used in neural system and cardiovascular disease that the multiple reason of treatment causes, clinically as cerebrovascular accident and sequela thereof; Dysfunction after cerebral concussion, traumatic stupor, the craniocerebral operations; Neurosis; Cerebrovascular sclerosis, senile dementia; Peripheral nerve injury; Children's cerebral dysgenesis etc.(Ishige Kazuya, 2002) subtilis jofC coding CMP kinases, URA6 coding CMP kinases (Shinya Kaneko, 1998) in the cereuisiae fermentum.MssA genes encoding CMP kinases is confirmed by people such as Fricke in the intestinal bacteria, though the CMP kinases is not that intestinal bacteria institute is essential, the CMP kinases provides under the phosphoryl situation at ATP, and reversible catalysis CMP transforms CDP, and then generates CTP:
ATP·Mg
2++CMP→ADP·Mg
2++CDP
ATP·Mg
2++CDP→ADP·Mg
2++CTP
The synthetic process of CTP mainly comprises chemical method, biological synthesis process, photosynthetic phosphoric acid method etc.Because the substrate price, very few to the research of CTP suitability for industrialized production both at home and abroad, the enterprise that really produces CTP in China does not have basically, but find certainly to can be used as after the medicine, the market requirement increases year by year, and along with the decline year by year of CMP price, prospect of production is very good.CTP the eighties in last century domestic production method mainly all be CM P to be transformed by isolating intravital self enzyme of yeast system, the production technique comparative maturity, but this technology can not be isolated all enzymes in the glycolysis-system fully, enzyme deactivation rate height, transformation efficiency is low, (Ethan SSimon, 1990).People such as Ying Guoqing (2004) utilize the free cereuisiae fermentum that CMP is synthesized CTP, and the transformation efficiency of CTP can maintain about 80%, and people (1997) such as Zhan Gu word synthesize CTP by the photophosphorylation reaction with CMP with spinach chloroplast, and transformation efficiency is 85%.
Summary of the invention
The objective of the invention is to disclose a kind of high vigor CMP kinases and kinase whose saccharomyces cerevisiae of CDP of containing.
Another object of the present invention provides the method for this saccharomyces cerevisiae of screening.
A further object of the present invention is to disclose the application of this saccharomyces cerevisiae in producing cytidine triphosphate(CTP).
The present invention reaches above purpose by the following technical programs: a kind of CMP of containing kinases and the kinase whose saccharomyces cerevisiae of CDP (Saccharomyces cerevisiae) bacterial strain, bacterium colony is an oyster white, glossy, smooth, neat in edge, it is preserved in Chinese common micro-organisms culture presevation administrative center on April 10th, 2008, and preserving number is: CGMCC NO.2448.
The step of the screening method of described bacterial strain comprises:
(1) makes the yeast suspension, yeast is put into the shaking table shaking culture be in the centrifugal recovery thalline of logarithmic growth after date after the substratum activation, add phosphate buffered saline buffer and make bacteria suspension to cell;
(2) above-mentioned bacteria suspension is mixed by 1: 1 volume ratio with mutagenic compound, 30 ℃ after water bath with thermostatic control 10-50 minute, mixed solution is diluted to 10
-3Doubly-10
-6Back spread plate obtains resistant strain;
(3) above-mentioned resistant strain was cultivated 24-36 hour through shaking table, detected and filter out with high performance liquid chromatography and contain high vigor CMP kinases and the kinase whose Yeast strain of beer of CDP; Wherein, the temperature of shaking table is 26-36 ℃, and rotating speed is 160-300rpm, and the mutagenic compound in the described step (2) are nitrosoguanidines of 2mg/ml; Substratum in the described flat board contains 300 μ g/ml cytidine triphosphate(CTP) analog cytidine diphosphate(CDP)-thanomins, and described resistant strain is cytidine diphosphate(CDP)-thanomin resistant mutant strain.
The application of cereuisiae fermentum bacteria strain of the present invention in producing cytidine triphosphate(CTP) comprises: utilizing entrapping method is to add after carrier is made immobilized cell with described bacterial classification in the reaction system of synthetic cytidine triphosphate(CTP) with carrageenin-konjac polysaccharide gum blend, utilize the method separation and purification reaction solution of ion-exchange separation and purification again, last crystallization obtains cytidine triphosphate(CTP).Its concrete steps are:
(1) carrageenin sodium salt and Rhizoma amorphophalli powder are dissolved in the deionized water, are incubated 40～70 minutes after the steam heating to 121 ℃, pour into and naturally cool to 40 ℃ in the reactor, the glue insulation, wherein the weight ratio of k-carrageenin sodium salt and Rhizoma amorphophalli powder is 2: 1;
(2) make sterilised yeast suspension with stirring in the physiological saline adding washing cereuisiae fermentum wet thallus well, 35 ℃ of insulations, wherein the weight ratio of physiological saline and cereuisiae fermentum wet thallus is 1: 4;
(3) sterilised yeast suspension in the step (2) is poured in the glue of step (1), stirred 20～40 minutes down, make it mixing and obtain mixture in 40 ℃;
(4) add solvent in the reactor that band stirs and be preheated to 40 ℃, pour the mixture that step (3) obtains into reactor, stir 20～40 minutes things to be mixed and form particle, filter, wherein stirring velocity is 50-150rpm;
(5) particle after the middle filtration of step (4) was soaked in Klorvess Liquid 30～90 minutes, filter, soaked 30～90 minutes with glutaraldehyde again, behind the most particle surface moisture of screen cloth drop, pack to refrigerator cold-storage.Wherein the solvent that adopts in the step (4) is a soya-bean oil, and described immobilized cell is spherical.Adopt HZ-1 ion exchange resin to carry out ion-exchange chromatography in the step of the method separation and purification reaction solution of described ion-exchange separation and purification, and per hour carry out gradient elution with the 0.2-0.5 column volume with the 1.0mol/L sodium chloride solution, obtain the cytidine triphosphate(CTP) elutriant.With elutriant concentrating under reduced pressure under 50～70 ℃ of water-baths, add 95% ethanol of 3 times of volumes in the described crystalline step, suction filtration after the refrigerator standing over night obtains cytidine triphosphate(CTP) with filtration cakes torrefaction.
It is that carrier generates the spheroidal particle immobilized cell automatically with the carrageenin that the present invention adopts entrapping method, can prepare the spheroidal particle of various particle diameters easily, compares with manual cut, the cell seepage is few, regular particles, intensity height, labour intensity is little, and the granulation program is simple, need not complex apparatus and instrument.Its advantage is that employed solvent is a soya-bean oil, and safety non-toxic obtains conveniently, is fit to various requirement, and is easy to reclaim; This process stabilizing, easy and simple to handle, the product aftertreatment is simple, is easy to suitability for industrialized production.
Use the synthetic CTP of ball shape fixation yeast particles catalyzed reaction of the present invention, the length of holding time, and can make things convenient for the solid-liquid sharp separation, reduce the CTP that is generated and further degrade; Spheroidal particle can use more than ten batches repeatedly and not reduce the CTP transformation efficiency, and its reaction solution is limpid, need not be further complicated particle reclaimer operation, reaction batch is apparently higher than free yeast and manual cut particle; Reaction solution separates through ion-exchange, and gained CTP strong solution purity reaches more than 94%, and yield is all more than 80% on the post; And the CTP ion exchange liquid purity difference of free yeast reaction solution gained, yield is low; After the crystallization, because CTP strong solution purity height, the gained crystal structure is easy, and crystal purity reaches more than 97%, and crystalline content reaches more than 94%, and apparently higher than the particle reaction of free yeast and manual cut, the CTP transformation efficiency can reach 97%.
Description of drawings
The present invention is further illustrated below in conjunction with accompanying drawing.
Fig. 1 is a Yeast strain of beer of the present invention.
Fig. 2 is the ball shape fixation particulate reaction repeated synoptic diagram of the present invention's preparation, among the figure :-◆-expression spheroidal particle;-zero-expression free yeast;-▲-expression manual cut.
Fig. 3 is the HPLC collection of illustrative plates of CTP standard substance.
Fig. 4 is the HPLC collection of illustrative plates of CTP sample.
Embodiment
Material and instrument: commercially available cereuisiae fermentum, shake-flask culture base, slant medium, nitrosoguanidine (are called for short: NTG) solution, substrate solution, shaking table, 20L stirred autoclave, 100L stirred autoclave, high performance liquid chromatograph.
The preparation of shake-flask culture base: 40g glucose, 15g ammonium sulfate, 9g potassium primary phosphate, 3g dipotassium hydrogen phosphate, 3g magnesium sulfate heptahydrate, 0.03g iron vitriol, 3g yeast extract paste, 3g corn steep liquor, be dissolved in the 1000ml distilled water, pH 4.0-6.5,115 ℃ of sterilization 20min.
The preparation of slant medium: 40g glucose, 15g ammonium sulfate, 9g potassium primary phosphate, 3g dipotassium hydrogen phosphate, 3g magnesium sulfate heptahydrate, 0.03g iron vitriol, 3g yeast extract paste, 3g corn steep liquor, 20 agar are dissolved in the 1000ml distilled water pH 4.0-6.5.
Embodiment one
(1) commercially available cereuisiae fermentum that will be to be screened changes shaking table over to and cultivates after the slant medium activation, 30 ℃ of shaking table temperature, after rotating speed 200rpm cultivates 24h, getting the 1ml nutrient solution again transfers in the shake-flask culture base, shaking table is cultured to logarithmic phase, centrifugal recovery thalline, the phosphate buffered saline buffer of adding 5ml, pH 7.0 is made bacteria suspension.
(2) take by weighing the NTG of 20mg, behind acetone solution, add distilled water and be settled to 10ml, be the NTG solution of 2mg/ml, 1ml bacteria suspension and 1ml nitrosoguanidine (NTG) solution are mixed, behind the vibration mixing, at 30 ℃ of thermostat water bath water-bath 30min, then mixed solution is diluted to 10
-3Stop mutagenesis.Mutagenesis is diluted to 10 with stop buffer after stopping again
-5, 10
-6The back coating is added with cytidine triphosphate(CTP) analog cytidine diphosphate(CDP)-thanomin (300 μ g/ml) flat board, filters out cytidine diphosphate(CDP)-thanomin resistant mutant strain.
(3) resistant mutant strain that filters out is inserted in the slant medium, cultivate 24h as the slant activation seed for 30 ℃.Bottle is shaken in the access of slant activation seed, and 30 ℃, the 200rpm shaking table is cultured to logarithmic phase, centrifugal recovery thalline, and with this bacterial strain switching slant medium, cultivate after 24 hours to 4 ℃ of refrigerators preservations for 30 ℃.In 25g yeast amount, add the 20ml substrate solution, comprising CMP 60mmol/L, glucose 150mmol/L, phosphate buffered saline buffer (sodium salt) 250mmol/L, magnesium chloride 8mmol/L, pH is 6.0,35 ℃ of temperature, by adding the 0.15ml tween 80, ATP concentration is 3mmol/L, reaction times 20h, under this reaction system, filter out and contain high vigor CMP kinases, the kinase whose cereuisiae fermentum bacteria strain of CDP, see Fig. 1, and detect with high performance liquid chromatography.Wherein, the chromatographic condition of high performance liquid chromatography is: chromatographic column: the Lichrospher C of Chinese nation
18Post (250mm * 4.6mm i.d., 5 μ m); Moving phase: methyl alcohol 6 ‰ (volume fraction), phosphate aqueous solution (regulating pH value to 6.6) (volume ratio is 11: 89) with triethylamine; Flow velocity 1.0mL/min; Detect wavelength 271nm; Column temperature is a room temperature; Sampling volume 20 μ L.
Embodiment two
(1) take by weighing 200g kappa-carrageenan sodium salt, 100g makes with extra care Rhizoma amorphophalli powder, is dissolved in the 5L deionized water, leads to be steam heated to 121 ℃, behind the insulation 60min, pours in the reactor of 20L band stirring, and cold naturally promptly to 40 ℃, the glue insulation is standby.
(2) take by weighing the good cereuisiae fermentum wet thallus 4kg of washing and add 1kg physiological saline, stir, 35 ℃ of insulations are standby.
(3) in the sterilised yeast suspension impouring glue with good heat insulation, stir in 40 ℃ and to make that to mix the back standby half an hour.
(4) in the reactor that the 100L band stirs, add the 60L soybean oil, be preheated to 40 ℃, control stirring velocity 125rpm, the mixture of yeast and glue is poured in the soybean oil, stirred and treat shaping particles half an hour, filter, leave standstill after soya-bean oil reclaims, discard behind lower floor's moisture content reusable.
(5) particle after filtering in the step (4) was soaked one hour with 3% Klorvess Liquid, filter, soaked one hour with 1% glutaraldehyde again,, pack that to put refrigerator cold-storage standby with the moisture content of the most particle surface of screen cloth drop.
Present embodiment can be controlled the spheroidal particle that mixing speed prepares various particle diameters as required, and mixing speed and spherical particle diameters relation see Table 1
Table 1
| Batch | Mixing speed (r/min) | Spherical particle diameters (mm) * |
| 1 | 50 | 5-6 |
| 2 | 75 | 4-5 |
| 3 | 100 | 3-4 |
| 4 | 125 | 2-3 |
[0043]
| 5 | 150 | 1-2 |
| 6 | 200 | 0.5-1 |
Wherein, * represents that the diameter particle weight that requires is not less than 90% than last total particle weight,
As shown in Table 1, this method can prepare the spheroidal particle of particle diameter 1-5mm easily, need not complex apparatus and operational condition.
Prilling process of the present invention and manual cut method form particulate and relatively see Table 2
Table 2
| Batch | Operating method | Processing mode * | OD 660 | Fragment |
| 1 | Granulation (sphere | 200r/min | 11.3 | Do not have |
| 2 | Manual cut (3-5mm square) | 200r/min | 201 | Obviously |
* the ratio of representing particle and physiological saline is 1: 1.
The particle that prilling process of the present invention as shown in Table 2 makes is evenly spherical, the intensity height, and Premeabilisation of cells is few.
Embodiment three
In the reaction system of the synthetic CTP of fixed yeast, except that generating a large amount of CTP, also there are impurity such as unreacted CMP and by product CDP, phosphoric acid salt, for CTP is separated from reaction solution, adopt HZ-1 ion exchange resin to carry out ion-exchange chromatography, and use 0.4mol/L sodium-chlor earlier, per hour carry out gradient elution with the 1.0mol/L sodium chloride solution again with the 0.2-0.5 column volume.With elutriant concentrating under reduced pressure under 50～60 ℃ of water-baths, add 95% ethanol of 3 times of volumes, in the refrigerator standing over night, suction filtration, filtration cakes torrefaction, finished product detects with high performance liquid chromatography, and chromatographic condition is identical with embodiment one.Fixed yeast is seen Fig. 2 to the successive reaction situation of CTP, and as seen from the figure: applying immobilized spheroidal particle can successive reaction more than ten batches, and reaction solution is limpid, does not have the tangible cell phenomenon of being left out; And use amorphous dice with the quadrat method manual cut, though can successive reaction, after the 4th, reaction conversion ratio obviously reduces, and the yeast seepage is serious in the reaction solution, the solution muddiness; Free yeast can only react once, and reaction conversion ratio significantly reduces later on for the second time.The detected result that obtains CTP in the present embodiment is seen Fig. 3, Fig. 4.Ion exchange method and crystallization method are the laboratory ordinary method, repeat no more herein.The CTP yield sees Table 3.The crystallization data see Table 4
Table 3
| Batch | Applied sample amount (Kg) | Elution amount (Kg) | Elutriant purity (%)) | Yield (%) |
| 071103 | 1.06 | 0.86 | 94.4 | 81.1 |
| 071114 | 1.24 | 1.03 | 96.1 | 83.1 |
| 071122 | 1.13 | 0.93 | 95.1 | 82 |
| 071202 | 1.31 | 1.11 | 95.6 | 84.3 |
| 071210 | 1.17 | 0.96 | 94.1 | 82.1 |
| 071020* | 1.31 | 0.85 | 82.2 | 65.1 |
| 071024** | 1.28 | 0.93 | 86.7 | 72.6 |
| 071204** | 1.04 | 0.73 | 84.2 | 70.0 |
Wherein, * represents the free cell reaction, and * * represents the reaction of manual cut particulate catalytic;
Table 4
| Batch | Total amount (Kg) | Crystal amount (Kg) | Crystal purity (HPLC, %)) | Crystalline content (UV, %) | Yield (%) |
| 071103 | 0.86 | 0.77 | 97.3 | 94.2 | 90 |
[0058]
| 071114 | 1.03 | 0.94 | 97.8 | 96.1 | 91.1 |
| 071122 | 0.93 | 0.84 | 97.3 | 94.9 | 90.5 |
| 071202 | 1.11 | 1.01 | 97.7 | 96.3 | 90.8 |
| 071210 | 0.96 | 0.86 | 97.2 | 94.1 | 90.1 |
| 071020* | 0.85 | 0.68 | 84.6 | 85.9 | 80 |
| 071024** | 0.93 | 0.78 | 90.9 | 91.6 | 84.1 |
| 071204** | 0.73 | 60.1 | 90.3 | 91.4 | 83.4 |
Wherein, * represents the free cell reaction, and * * represents the reaction of manual cut particulate catalytic;
Result and discussion
As shown in Table 3, use the catalytic reaction of spheroidal particle, CMP and CDP foreign matter content are few, ion-exchange yield height, and elutriant purity is very high; And the reaction of employing free cell or manual cut particulate catalytic, yield is low, and elutriant purity has only about 85%.
As shown in Table 4, use the catalytic reaction of spheroidal particle, crystallization yield can be more than 90%, and crystalline purity meets the requirement of country fully up to more than 94%; And the reaction of employing free cell, the crystal yield is low, and the crystallisation process difficulty has more oily matter to occur; The reaction of manual cut particulate catalytic though crystallization is not difficult, because of the elutriant moderate purity is original just not high, causes crystalline purity low, needs periodic crystallisation just can reach more than 90%.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (6)
1. one kind contains CMP kinases and the kinase whose saccharomyces cerevisiae of CDP (Saccharomycescerevisiae) bacterial strain, and its preserving number in Chinese common micro-organisms culture presevation administrative center is: CGMCCNO.2448.
2. according to the application of the described bacterial strain of claim 1 in producing cytidine triphosphate(CTP), it is characterized in that: at first utilizing entrapping method is to add in the reaction system of synthetic cytidine triphosphate(CTP) after carrier is made immobilized cell with described bacterial classification with carrageenin-konjac polysaccharide gum blend, utilize the method separation and purification reaction solution of ion-exchange separation and purification again, crystallization obtains cytidine triphosphate(CTP).
3. according to the application of the described bacterial strain of claim 2 in producing cytidine triphosphate(CTP), it is characterized in that: described entrapping method is that the concrete steps that carrier is made immobilized cell with described bacterial classification are with carrageenin-konjac polysaccharide gum blend:
(1) carrageenin sodium salt and Rhizoma amorphophalli powder are dissolved in the deionized water, are incubated 40～70 minutes after the steam heating to 121 ℃, pour into and naturally cool to 40 ℃ in the reactor, the glue insulation, wherein the weight ratio of k-carrageenin sodium salt and Rhizoma amorphophalli powder is 2: 1;
(2) make sterilised yeast suspension with stirring in the physiological saline adding washing cereuisiae fermentum wet thallus well in addition, 35 ℃ of insulations, wherein the weight ratio of physiological saline and cereuisiae fermentum wet thallus is 1: 4;
(3) sterilised yeast suspension in the step (2) is poured in the glue of step (1), stirred 20～40 minutes down, make it mixing and obtain mixture in 40 ℃;
(4) add solvent in the reactor that band stirs and be preheated to 40 ℃, pour the mixture that step (3) obtains into reactor that band stirs, stir 20～40 minutes things to be mixed and form particle, filter, wherein stirring velocity is 50-150rpm;
(5) particle after the middle filtration of step (4) was soaked in Klorvess Liquid 30～90 minutes, filter, soaked 30～90 minutes with glutaraldehyde again, behind the most particle surface moisture of screen cloth drop, pack to refrigerator cold-storage.
4. according to the application of the described bacterial strain of claim 3 in producing cytidine triphosphate(CTP), it is characterized in that: the solvent that adopts in the described step (4) is a soya-bean oil, and described immobilized cell is spherical.
5. according to the application of the described bacterial strain of claim 2 in producing cytidine triphosphate(CTP), it is characterized in that: adopt HZ-1 ion exchange resin to carry out ion-exchange chromatography in the step of the method separation and purification reaction solution of described ion-exchange separation and purification, and per hour carry out gradient elution with the 0.2-0.5 column volume with 0.4～1.0mol/L sodium chloride solution, obtain the cytidine triphosphate(CTP) elutriant.
6. according to the application of the described bacterial strain of claim 5 in producing cytidine triphosphate(CTP), it is characterized in that: in the described crystalline step with elutriant concentrating under reduced pressure under 50～70 ℃ of water-baths, add 95% ethanol of 3 times of volumes, suction filtration after the refrigerator standing over night obtains cytidine triphosphate(CTP) with filtration cakes torrefaction.
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| CN102199644B (en) * | 2011-04-15 | 2014-12-17 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
| CN105039188A (en) * | 2015-08-28 | 2015-11-11 | 广东海纳川生物科技股份有限公司 | Recombinant Pichia pastoris culture medium, and culture method and application thereof |
| CN113881737B (en) * | 2021-05-20 | 2024-03-01 | 江南大学 | Method for producing CMP-sialic acid on large scale by coupling fermentation of genetically engineered bacteria and yeast |
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| CN1234444A (en) * | 1999-05-17 | 1999-11-10 | 华东理工大学 | Microorganism carrier fixed with glue compounded with carragheen and konjak polysaccharide and use thereof |
| CN1800381A (en) * | 2005-12-21 | 2006-07-12 | 上海秋之友生物科技有限公司 | Preparation method of carrageeenen immobilized microbe cell spherical granule and its uses |
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| CN1234444A (en) * | 1999-05-17 | 1999-11-10 | 华东理工大学 | Microorganism carrier fixed with glue compounded with carragheen and konjak polysaccharide and use thereof |
| CN1800381A (en) * | 2005-12-21 | 2006-07-12 | 上海秋之友生物科技有限公司 | Preparation method of carrageeenen immobilized microbe cell spherical granule and its uses |
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