CN1970747A - Process for preparing spherical immobilized cell and enzyme particulate - Google Patents
Process for preparing spherical immobilized cell and enzyme particulate Download PDFInfo
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- CN1970747A CN1970747A CN 200510123838 CN200510123838A CN1970747A CN 1970747 A CN1970747 A CN 1970747A CN 200510123838 CN200510123838 CN 200510123838 CN 200510123838 A CN200510123838 A CN 200510123838A CN 1970747 A CN1970747 A CN 1970747A
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Abstract
The invention discloses a making method of fixed biological catalyst, which comprises the following steps: adopting nontoxic polyvinyl alcohol composite material as carrier; allocating mixed soluble gel; adding microbe cell or enzyme according to proportion; blending evenly to form ball-shaped particle in the polyvalent metal ion solution; freezing particle; placing in the low-density hole-forming agent to obtain needed fixed cell or enzyme particle.
Description
The present invention relates to a kind of preparation method of immobilized biocatalyst, more precisely a kind of preparation method of immobilized cell/enzyme.
Adopt entrapping method to prepare the used carrier of immobilized cell/enzyme two classes are arranged.One class is a natural macromolecular material, as gelatin, agar and alginate calcium etc., because its physical strength is generally all relatively poor, is difficult to be applicable in the bio-reactor, especially medium-term and long-term ground of fluid bed type bio-reactor continuous operation.Another kind of is synthesized polymer material, as polyacrylamide etc., often toxic to thalline or enzyme mainly due to monomer whose, perhaps condition is harsh relatively in preparation process, be easy to damage the activity of enzyme, and generally also be difficult to make spheroidal particle, to adapt to the operation of fluidized bed bio reactor.In recent years, many patent documentations have been reported with polyvinyl alcohol (PVA) as carrier, with the polyvinyl alcohol aqueous sol-gelization, improve forming technique and make it to become sphere, improve the gelled particles physical strength, carrier gel metallization processes condition and minimizing enzyme activity loss aspect have obtained remarkable achievement when relaxing the microorganism cells embedding.Specially permit clear 61-100193A as Japan and adopt the boric acid manufacturing process to make the polyvinyl alcohol aqueous sol-gelization, made spheric immobilized cell/enzyme particle, and physical strength is good.But this process has used pair cell/enzyme to have toxic boric acid, therefore the activity of damaging cells/enzyme certainly will be not suitable for the immobilization that PH is the microorganism cells/enzyme of acid range.Chinese patent literature CN1076488 report improves this borate method recently, adopted the method for the two-stage shaping of boric acid one phosphoric acid, shortened the duration of contact of cell/enzyme and boric acid, but do not broken away from boric acid, and used phosphate concn is also higher, about 3～20%.In addition, the adjusting of relevant porosity in all not mentioned immobilized microorganism catalyst preparation process in the patent documentation of being delivered up to now, make it to adapt to the requirement of different big minicell/enzymes and different substrates, cause prepared immobilized biocatalyst permeability good, resistance to mass transfer is little, the physical strength height.
The object of the present invention is to provide a kind of preparation method of ball shape fixation cell/enzyme particle.Immobilized cell/the enzyme of Using such method preparation, spheroidal particle with rule, flexible and toughness, the physical strength height, wear-resisting, there is abundant reticulated structure its inside, and the porosity size can be adjusted, it is good to be used in the biochemical reaction process permeability, and resistance to mass transfer is little, immobilized cell/enzyme activity height.
In order to achieve the above object, the present invention is realized by following technical proposals.Adopt non-toxic polyethylene alcohol, alginate calcium, silicon sol matrix material, be made into mixing water colloidal sol, and add microorganism cells/enzyme in proportion, in polyvalent metal ion solution, begin to take shape the spheroidal particle of rule behind the uniform mixing, particle again through freezing and part wind more than 2 times in process, and then place the pore-creating agent of lower concentration, just can obtain required immobilized cell/enzyme particle after placing certain hour.Its technical characterictic is in the above-mentioned preparation process: 1. the non-toxic polyethylene alcohol complex carrier that uses, it is by the made mixture gel of polyvinyl alcohol, alginate calcium and silicon sol, their weight percentage in the water-sol are: polyvinyl alcohol 6～15%, alginate calcium is 0.5～1.5%, and silicon sol is 0.3～5.0%; 2. employed polyvalent metal ion solution is CaCl
2The aqueous solution, its concentration are 0.1～0.5M; 3. phosphoric acid or phosphoric acid salt that employed pore-creating agent is a lower concentration, as Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC or corresponding sylvite etc., its concentration is 0.03～0.55% (weight).
Specific implementation process of the present invention is: after at first polyvinyl alcohol, alginate calcium and silicon sol being made mixing water colloidal sol according to aforementioned proportion, and autoclaving sterilization.Add the microorganism cells wet thallus then, its content is generally 1～10% (weight), fully is mixed, and the mixing water colloidal sol that will contain this microorganism cells is more tentatively made the spheroidal particle of rule in containing the aqueous solution of polyvalent metal ion.The granulation mode can adopt centrifugal granulation or multi-nozzle pressure type to granulate, and the laboratory can be adopted peristaltic pump to drip and be granulated.The polyvalent metal ion aqueous solution can be CaCl
2The aqueous solution, its concentration is generally 0.1～0.5M.Just the immobilized cell particle of tool spheroidal particle is at CaCl
2Need in the aqueous solution to keep 2～6 hours, it is air-dry at room temperature to thaw subsequently, remove part moisture content, so freezing, thaw and part air-dry more than twice, again with after the physiological saline washing, be dipped in the buffered soln of the phosphoric acid that contains 0.03～0.55% (weight) or phosphoric acid salt (as Sodium phosphate dibasic), make it further to form required immobilized cell particle with certain porosity.
Make a general survey of the forming process of immobilized cell particle, the principal element that influences its size of particles is the composition and the viscosity of mixing water colloidal sol, and the size of nozzle and colloidal sol are in the spouting velocity at nozzle place.And influence the aperture of particle and the factor of porosity is, polyvinyl alcohol sodium alginate and the silicon sol content in the water-sol, the concentration of phosphate anion and the gel particles residence time in this substratum or damping fluid in substratum or the damping fluid, also has the air-dry degree of dehydration of gel etc.These factors also directly have influence on the intensity of immobilized cell particle.
With the prepared immobilized cell/enzyme particle of the present invention, outward appearance is the spheroidal particle of rule, general 1.5～the 3.0mm of its particle diameter, flexible, physical strength good, wear-resistant, anti-extrusion, its inside has the reticulated structure of enriching, and helps adhering to and growth of cell, when carrying out biochemical reaction, permeability is good, and resistance to mass transfer is little.The CO that in anaerobic fermentation process, produces
2Very easily discharge.Be used for successive fluidized-bed or fixed-bed reactor, the immobilized cell particle does not have hydro-planing, has guaranteed the reactor normal operation, and in use has enzyme height alive and long characteristics of life-span.
The fixed yeast cell particle that will make with aforesaid method places and contains 0.04% phosphoric acid substratum and carry out cell proliferation, and approximately through 28 hours, yeast concentration reaches (2～4) * 10 in it
9Individual cells/ml particle, its particle diameter is about 3mm, is placed on then in the reactive system of a two-stage injection circulation moving-burden bed reactor and a multi-stage fixed-bed reactors in series, be that raw material carries out zymamsis with molasses.This fixed yeast cell does not have hydro-planing in reactor, flow state is good, the physical strength height.When substrate sugar concentration is 16%, be 5.01 o'clock when empty, its end of a period ethanol concn is 8.5% (volume), and sugared transformation efficiency is 94.4%, is 93.4% to sugared yield, and throughput is 48.07 gram alcohol/(catalyzer hour).This fixed yeast cell reaches more than 3 months work-ing life.
The invention will be further described with embodiment below.
Embodiment one:
Polyvinyl alcohol 40 grams (polymerization degree is 1750 ± 50, and alcoholysis degree is greater than 97%);
Sodium alginate 5 grams;
40 milliliters of silicon sol (concentration is 28%);
400 milliliters in water;
Yeast wet thallus 10 grams;
1 liter of calcium chloride water (0.3M).
Polyvinyl alcohol, sodium alginate, silicon sol and water are made mixing water colloidal sol in 85 ℃～95 ℃ following thorough mixing, the horizontal high voltage sterilization of going forward side by side.Treat that this water-sol is cooled to about 30 ℃, adding has been cultivated and through the yeast wet thallus behind the high speed centrifugation, thorough mixing is even, splashes in the calcium chloride water for preparing in advance by peristaltic pump then, become 3～4mm spheroidal particle, and in this calcium chloride solution, slowly stirred 2 hours.The elimination calcium chloride solution, after physiological saline washing once, move into icehouse, under-25 ℃ of conditions freezing 10 hours, take out again and under 28～30 ℃, thaw air-dry 8 hours, freezing so repeatedly and air-dry three times, be placed on again in the liquid nutrient medium that contains 0.04% (weight) phosphoric acid under 28 ℃ and carry out cell proliferation,, just make required fixed yeast cell particle through 28 hours, its particle diameter is 3mm, and yeast concentration is up to (2～4) * 10 in this fixed yeast spheroidal particle
9Individual cells/ml particle, and flexible and toughness, wear-resisting resistance to compression, it combines synthetic macromolecule and the natural polymer advantage as fixation support.
Embodiment two:
Polyvinyl alcohol 9 grams (polymerization degree is 1750 ± 50, and alcoholysis degree is greater than 97%);
Sodium alginate 1.2 grams;
90 milliliters in water;
Arthrobacter simplex wet thallus 5 grams;
200 milliliters of calcium chloride waters (0.3M concentration).
Preparation immobilization Arthrobacter particle method is with embodiment one.Prepared immobilization Arthrobacter particle is placed the substratum that contains 0.5% potassium primary phosphate, temperature is 32 ℃, and the time is 48 hours, just makes required immobilization Arthrobacter spherical catalyst, use it in the process of cortisone acetate deoxidation system prednisoni acetas its Δ
1.2The vigor of desaturase can improve 3.8 times.Carry out shake flat experiment, when concentration of substrate was 10mg/ml, the reaction times only needed 13 hours just can reach 98% transformation efficiency, and when concentration of substrate was 20mg/ml, the reaction times also only just can reach 97% transformation efficiency in 17 hours.
Claims (1)
1. the preparation method of a ball shape fixation cell/enzyme particle, it is after being mixed into the water-sol with polyvinyl alcohol composite material and water, add microorganism cells/enzyme in proportion, in binder, make tool spheroidal particle colloid just behind the uniform mixing, colloidal particle again through freezing more than 2 times, thaw with the part air drying process after, place pore-creating agent just can obtain immobilized cell/enzyme particle that outward appearance is spherical in shape, inside is porous network structure again.The technical characterictic of this method is:
A. polyvinyl alcohol composite material is combined by polyvinyl alcohol, alginate calcium and silicon sol, and their weight percents in the water-sol are respectively 6～15%, and 0.5～1.5% and 0.3～5.0%;
B. binder is the CaCl of polyvalent metal ion
2The aqueous solution, its concentration are 0.1～0.5M;
C. pore-creating agent is phosphoric acid, phosphoric acid salt (as Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC etc.) or the corresponding sylvite of lower concentration, and as potassium primary phosphate and dipotassium hydrogen phosphate etc., its concentration is 0.03～0.55% (weight).
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| CN 200510123838 CN1970747A (en) | 2005-11-23 | 2005-11-23 | Process for preparing spherical immobilized cell and enzyme particulate |
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| CN 200510123838 CN1970747A (en) | 2005-11-23 | 2005-11-23 | Process for preparing spherical immobilized cell and enzyme particulate |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102030539A (en) * | 2010-09-29 | 2011-04-27 | 桂劲宁 | Method for preparing solid composite particles |
| CN106399290A (en) * | 2016-10-08 | 2017-02-15 | 甘琦 | Method of utilizing polysaccharide plant gum to prepare embedded microorganisms |
| CN108642032A (en) * | 2018-07-02 | 2018-10-12 | 深圳市长隆科技有限公司 | Embedded immobilization microbial carrier and preparation method thereof and sewage water treatment method |
| CN110304802A (en) * | 2019-06-25 | 2019-10-08 | 西安建筑科技大学 | A kind of preparation method of masking material, application and septic-tank device |
-
2005
- 2005-11-23 CN CN 200510123838 patent/CN1970747A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102030539A (en) * | 2010-09-29 | 2011-04-27 | 桂劲宁 | Method for preparing solid composite particles |
| CN106399290A (en) * | 2016-10-08 | 2017-02-15 | 甘琦 | Method of utilizing polysaccharide plant gum to prepare embedded microorganisms |
| CN106399290B (en) * | 2016-10-08 | 2019-09-13 | 上海明奥环保科技有限公司 | A method of imbedded microbe is prepared using polysaccharide vegetable glue |
| CN108642032A (en) * | 2018-07-02 | 2018-10-12 | 深圳市长隆科技有限公司 | Embedded immobilization microbial carrier and preparation method thereof and sewage water treatment method |
| CN110304802A (en) * | 2019-06-25 | 2019-10-08 | 西安建筑科技大学 | A kind of preparation method of masking material, application and septic-tank device |
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