CN1059759A - The preparation method of complex solidifying enzyme - Google Patents
The preparation method of complex solidifying enzyme Download PDFInfo
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- CN1059759A CN1059759A CN 90108304 CN90108304A CN1059759A CN 1059759 A CN1059759 A CN 1059759A CN 90108304 CN90108304 CN 90108304 CN 90108304 A CN90108304 A CN 90108304A CN 1059759 A CN1059759 A CN 1059759A
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- enzyme
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Abstract
The present invention is a kind of method for preparing complex solidifying enzyme, comprises the steps:
(1) preparation conjugation immobilized enzyme: will be attached to polyamino compound and the reactive material of amine that has pendant amine groups on the porous support by handling---plant dual-function compound, as the amine reactive group phase reaction in the glutaraldehyde, the reaction of the free amino of its another amine reactive group and enzyme or enzyme, and link with it and to constitute the conjugation immobilized enzyme.
(2) conjugation immobilized enzyme and the microorganism cells that produces this enzyme are embedded in one or more embedding mediums jointly, constitute complex solidifying enzyme.
Description
The present invention proposes and illustrates in detail the preparation method of " complex solidifying enzyme " (Complex Immobi-lized Enzymes), belongs to bioengineering field.
At medicine, food and the increasingly extensive enzyme of industrial purposes, its essence belongs to protein, and is normally water miscible, and generally be unsettled.Because it can only be disposable that enzyme is used in solution, causes all inconvenience in industrial production.So invented the various methodologies of immobilized enzyme.The process for fixation of various enzymes and the existing in the literature a large amount of records of process for fixation of producing the microorganism cells of enzyme.Now the relevant background material that relevant background material is particularly related to the glucose isomerase aspect is done a general introduction.
Takasaki(Gao Qi) etc. nineteen sixty-five, Tsumura(Jin Cun), find to be fit to industrial glucose isomerase respectively and produced bacterial classification-dead color product look streptomycete (Strepto-myces phaeochromogenes) and streptomyces albus (Streptomyces albus).Echinopanax japonicum pine company produced enzyme process isomery syrup in 1966.Fixed glucose isomerase industrialization in 1974 success promotes the immobilized biocatalyst researchdevelopment.China all did research to bacterial classification, fermentation, the immobilization technology of production glucose isomerases such as rose-red streptomycete 336, thermophilic actinomycete M1033, actinoplanes since 1970.National immobilized biocatalyst academic discussion papers in 1988 have been summarized the progress of each side such as immobilized enzyme.
The invention provides a kind of method for preparing complex solidifying enzyme, it comprises following content:
(1) preparation conjugation immobilized enzyme, its step is as follows:
(a) porous support contacts with having pendant amine groups polyamino compound solution, and polyamine is attached on the porous support;
(b) wash with deionized water, the polyamine that excludes solvent and disperse wherein not adhere to, contact with the reactive material of porous support of having handled and amine then, this solution is a kind of multifunctional aldehyde, a kind of multi-functional Organohalogen compounds, a kind of polyfunctional acid anhydride or a kind of multi-functional azo-compound, one of them amine reactive group and pendant amine groups are reacted, and staying another amine reactive group can further react;
(c), exclude solvent and any being dissolved in wherein and the reactive material of unreacted amine with the deionized water washing.The porous support of having handled contacts with at least a enzyme solution, with amido and the unreacted amine reaction-ity group reaction that causes enzyme, by the form immobilized enzyme of covalent linkage.
Porous support comprises among the present invention:
(1) granular diatomaceous earth (Granular diatomaceous earth),
(2) cenosphere (Cenosphere),
(3) chitin (Chitin).
The physicochemical property of granular diatomaceous earth are as follows:
Physical properties:
Granular size: 120-180 order
Density: 575 kilograms/cubic metre
Shape: sphere
Crushing strength: 17.6-18.6 kg/cm
Color: white to shallow brown
Average surface area: 40 meters squared per gram
Average pore volume: 0.16 milliliter/gram
Mean pore size: 10-40 micron
Chemical ingredients (%) and character:
SiO
2：90.0
AL
2O
3：6.5
Fe
2O
3：2.3
CaO：0.2
MgO：0.3
Other oxide compound: 0.3
Water content: be less than 1.0
PH value of solution: 6.5-7.0
Structure: mainly be amorphous diatom soil.
This porous particle diatomite is because chemically stable, rigidity and cheap physically, thereby it can be as a kind of porous inorganic carrier.
The physico-chemical property of cenosphere is as follows:
1, outward appearance: garden sphere
2, granularity: particle diameter is 1～300 micron
3, unit weight: 250～400 kilograms/cubic metre
4, proportion: 0.5～0.75 gram/cubic centimetre
Chemical ingredients (%):
SiO
251.32～65.98
Al
2O
322.62～39.86
Fe
2O
32.18～8.73
CaO 0.49～3.35
MgO 0.86～1.96
The polyamine that is suitable among the present invention comprises: polyethylene diamines, polymine, polymethylene diamines, polyhexamethylene diamines, poly-tetren, polyethylene polyamine.Through the porous support of polyamine treatment, then handle with the reactive material of amine.
The reactive material of amine is glutaraldehyde, Toluene-2,4-diisocyanate, 4-diisothiocyanic acid salt, diazo p-diaminodiphenyl.The concentration of the reactive material of amine is that every liter of solution contains the 1-10 gram.The water-soluble respectively and organic solvent of the reactive material of reactive material of water-soluble amine and non-water-soluble amine is applied to porous support again.Organic solvent comprises: methyl alcohol, ethanol, propyl alcohol, Virahol, acetone, methyl ether, ether, propyl ether, isopropyl ether.The reaction certain hour, so that the reactive material of amine is from the polyamine free amino of deriving, the porous support of having handled, with the deionized water washing several times.
Use the present invention, earlier with deionized water washing porous support until totally, vacuum exhaust then.With polyamine solution, be mixed with finite concentration, as being added to polyethylene polyamine on the porous support that has washed, porous support and polyethylene polyamine aqueous solution volume ratio value are about 1: 5, the polyethylene polyamine concentration of aqueous solution is about 0.1%-0.5%, under 20-30 ℃ of slight the stirring, effective reaction time 2-5 hour, the porous support that excessive polyethylene polyamine solution with water washing had been handled was got rid of.If make linking agent with glutaraldehyde, its volume and polyethylene polyamine solution are approximate, and promptly porous support and glutaraldehyde solution volume ratio are 1: 5, and glutaraldehyde concentration is 0.1%～1.0%, is advisable with 0.25-0.7% concentration especially.Employing contains the glutaraldehyde water solution of 0.05 mol sodium bicarbonate, requires the pH value to be 7.9-8.3.Crosslinking reaction is stirred gently at 20-30 ℃, in 2 hours reaction times, promptly obtains porous support, polyethylene polyamine and glutaraldehyde upholder.This biological catalyst upholder is contacted with specific enzyme solution, be suitable under the pH of this enzyme, stirred gently 4 hours under the temperature 20-30 ℃ of condition, promptly obtain the conjugation immobilized enzyme, preserve in the suitable damping fluids in 4 ℃, standby.
Implementing the present invention need be embedded in above-mentioned conjugation immobilized enzyme that obtains and the microorganism cells that produces this enzyme (or enzyme) in one or more embedding mediums jointly, constitutes complex solidifying enzyme.
Implement example 1 of the present invention and at length narrated the method for preparing compound fixed glucose isomerase.With acidophilic streptomycete (Streptomyces acidophilus) fermentative production glucose isomerase, be regular according to its intracellular enzymes of variation of actual conditionses such as fermentation period length and extracellular enzyme ratio and change.Generally along with fermentation period prolongs, the ratio of intracellular enzyme and extracellular enzyme progressively successively decreases.For intramycelial glucose isomerase and the glucose isomerase that is discharged in the fermented liquid all can fully be reclaimed, design then among the present invention about compound process for fixation with the acidophilic streptomycete cell and the glucose isomerase that produces.By embodiment 1, this notion of complex solidifying enzyme becomes more clear and clearer and more definite.What need spell out is: the microorganism cells that complex solidifying enzyme is not limited to only to fix a kind of enzyme and produces this enzyme, more than one enzyme and more than one the compound immobilization of microorganism cells that produces this enzyme respectively also belong within this scope.
The process for fixation of various enzymes and the existing in the literature a large amount of records of process for fixation that produce the various microorganism cellss of enzyme, by as listed above go out about glucose isomerase research and all kinds of bacterial classifications, the zymotechnique that are adopted in producing and the various process for fixation that are associated with it, can clearly find out the development track at this specific field such as bacterial classification, fermentation, immobilization technology.
Process for fixation about generation enzyme microorganism cells is a lot, wherein commonly used with entrapping method.The embedding medium that is adopted has: carrageenin, xanthan gum, gelatin, agar, diacetate fiber, triacetate fiber.
Implementing the first step of the present invention is resolvase in the fermentative production to be fixed in porous support be prepared into the conjugation immobilized enzyme.Second step be will be loaded with the porous support of conjugation immobilized enzyme be embedded in jointly in the various embedding mediums with the microorganism cells that produces this enzyme (or enzyme), this embedding medium can be single also can be that several embedding mediums mix and use.
Example 1,
Need do following processing before being used for fixing of porous particle diatomite: 40 grams, 120～180 order porous particle diatomite are handled with 1N HCl, 1N NaOH respectively, all use the deionized water thorough washing after each the processing, treat that the diatomite post precipitation inclines washing water to go out gently, then with diatomite vacuum-drying.
Prepare compound fixed glucose isomerase by following processing method by the acidophilic streptomycete fermented liquid:
1, preparation conjugation fixed glucose isomerase:
(1) will wash and 250 milliliters of thorough mixing of the 0.2% polyethylene polyamine aqueous solution of vacuum drying granular diatomaceous earth 40 grams and pH9.8, put in 500 ml shake flasks in 28～30 ℃ gently vibration stirred 4 hours, natural subsidence then, inclining gently supernatant liquor.
(2) with the deionized water thorough washing to get rid of the polyamine-polyethylene polyamine be not attached on the diatomite.Use the reactive material glutaraldehyde of amine to combine then: 250 milliliters of 0.5% glutaraldehyde solutions that will contain 0.05 mol sodium bicarbonate with pendant amine groups attached to the polyamine on the diatomite, pH8.2, put in 500 ml shake flasks with the diatomite that adheres to polyamine, vibrated gently 2 hours, incline gently then and glutaraldehyde solution, use the deionized water thorough washing, remove unreacted glutaraldehyde
(3) glucose isomerase enzyme work is reached 20,000 IGIU(International Glucose Isomerase Unit)/200 milliliters of the acidophilic streptomycete fermented liquids of L, through centrifugation, the 4 ℃ of preservations of wet mycelium that obtain after centrifugal are standby.Get the filtrate and the above-mentioned upholder that contains diatomite, polyethylene polyamine and glutaraldehyde that contain resolvase, pH6.8 puts in 500 ml shake flasks, vibrated gently 4 hours in 28 ℃, inclining liquid, promptly makes the conjugation fixed glucose isomerase, standby with deionized water washing back.
2, with conjugation immobilized enzyme and the common embedding of acidophilic streptomycete cell, make compound fixed glucose isomerase.
(1) wet mycelium that above-mentioned centrifugation obtained 25 grams disperse to be suspended in 8%100 milliliters of magnesium citrate solution, vibrate gently 2 hours in 40 ℃, and centrifugation makes the mycelium suspension with 25 ml physiological salines in 45 ℃ after the washing again.
(2) with carrageenin 3.0 grams and agar 3.0 grams, under 70 ℃ of conditions, be dissolved in 50 ml physiological salines, waiting to be chilled to 40 ℃ pre-fixes above-mentioned conjugation immobilized enzyme and magnesium citrate in cell, mycelium suspended liquid is in the lump in the colloid mixture of impouring carrageenin and agar, stir, be cooled to 10 ℃, static at least 2 hours, this gel is dipped in the 0.3M KCl solution, 10 ℃ after 4 hours, granulating and forming.
(3) molded particle is handled each more than 8 hours with 0.2% polyethylene polyamine and 0.5% glutaraldehyde respectively, remove unnecessary polyethylene polyamine and glutaraldehyde is promptly made compound fixed glucose isomerase with deionized water washing, total yield can reach 92%, enzyme throughput can reach 4,500 gram high fructose syrup (butt)/grams (immobilized enzyme).
Example 2:
With peracid, alkaline purification, deionized water washing and vacuum drying cenosphere is carrier, is prepared the complex solidifying enzyme of glucose isomerase as follows by the acidophilic streptomycete fermented liquid:
1, with 40 milliliters of cenospheres and pH9.8,250 milliliters of mixing of the 0.2% polyethylene polyamine aqueous solution are put in 500 ml shake flasks in 30 ℃ of vibrations and were stirred 4 hours, then sedimentation and incline and supernatant liquor.
2, with the deionized water thorough washing to get rid of the polyethylene polyamine be not attached on the cenosphere.Then with 250 milliliters of 0.5% glutaraldehyde solutions that contains 0.05 mol sodium bicarbonate, pH8.2, mix with cenosphere and to insert in 500 ml shake flasks through above-mentioned processing, vibrated gently 2 hours, after static, isolate glutaraldehyde solution gently, use the deionized water thorough washing, obtain containing the upholder of cenosphere, polyethylene polyamine and glutaraldehyde.
3, with acidophilic streptomycete fermented liquid 200 milliliters of the filtrate of containing resolvase and above-mentioned upholders that contain cenosphere, polyethylene polyamine and glutaraldehyde through centrifugation, put in 500 ml shake flasks and vibrated 4 hours down in 28 ℃, separating gently inclines liquid, promptly makes the conjugation fixed glucose isomerase with the deionized water washing again.
4, with above-mentioned conjugation fixed glucose isomerase and the wet mycelium that obtains through centrifugation, this mycelium needs through 8% magnesium citrate solution-treated, processing method according to example 1 is embedded in the mixed gel of carrageenin and gelatin composition jointly then, make compound fixed glucose isomerase, the total yield 94% of enzyme, complex solidifying enzyme throughput after drying reach 4800 gram high fructose syrup (butt)/grams. immobilized enzyme.
Example 3:
The treatment step of cenosphere is identical with example 2.The first step prepare contain cenosphere, polyethylene polyamine and glutaraldehyde its method of upholder also with example 2.
100 milliliters of the fermentation of Aspergillus niger liquid of glucoamylase will be produced, centrifugation, obtain filtrate, transferring to pH4.8 with the 0.2M acetate buffer mixes this filtrate of containing glucoamylase and places 500 ml shake flasks to vibrate gently under room temperature 4 hours with the above-mentioned upholder that contains cenosphere, polyethylene polyamine and glutaraldehyde, isolate cenosphere gently, with the deionized water washing, promptly make the conjugation fixation glucose amylase.
The wet thallus of gained after the centrifugation according to the method for example 1, is embedded in the mixed gel of carrageenin and agar composition jointly with above-mentioned conjugation fixation glucose amylase, makes compound fixation glucose amylase.
Example 4
Organic porous support chitin (chitin) prepares as follows and handles: 20 gram crab shells add 1N HCl100 milliliter, at room temperature handled 2 hours earlier, boiled again 8 hours, remove residual protein, room temperature is placed and is spent the night, with deionized water washing, be dipped in then in 0.5% potassium permanganate solution 1 hour, filter, washing, drying at room temperature, grinding is sieved, and 120～160 order particles are standby.
The upholder that the porous organic carrier chitin of above-mentioned preparation is prepared chitin, polyethylene polyamine and glutaraldehyde by example 1 processing method.100 milliliters of glucose oxidase fermented liquid centrifugations are obtained mycelium and filtrate two portions, filtrate and the above-mentioned upholder that contains chitin, polyethylene polyamine and glutaraldehyde, put in 500 ml shake flasks in 25 ℃ and vibrated gently 4 hours, elimination liquid is promptly made the conjugation immobilized glucose oxidase.
Wet thallus is suspended in the deionized water, under room temperature, makes 15 milliliters of suspension, in 50 milliliter of 0.5% sodium alginate of impouring and the 0.5% gelatin mixed gel, stir evenly be cooled to 10 ℃ at least 2 hours, clamp-on 2.5MCaCl with shaped device
2In the solution, put refrigerator overnight, handle, with the deionized water washing, make compound immobilized glucose oxidase again with 3% citric acid solution.
Claims (10)
1, a kind of method for preparing complex solidifying enzyme, it comprises following content:
(1) preparation conjugation immobilized enzyme, its step is as follows:
(a) porous support contacts with having pendant amine groups polyamino compound solution, and polyamine is attached on the porous support;
(b) wash with deionized water, the polyamine that excludes solvent and disperse wherein not adhere to, contact with the reactive material of porous support of having handled and amine then, this solution is a kind of multifunctional aldehyde, a kind of multi-functional Organohalogen compounds, a kind of polyfunctional acid anhydride or a kind of multi-functional azo-compound, one of them amine reactive group and pendant amine groups are reacted, and staying another amine reactive group can further react;
(c), exclude solvent and any being dissolved in wherein and the reactive material of unreacted amine with the deionized water washing.The porous support of having handled contacts with at least a enzyme solution, with amido and the unreacted amine reaction-ity group reaction that causes enzyme, by the form immobilized enzyme of covalent linkage.
(2) conjugation immobilized enzyme and the microorganism cells that produces this enzyme are embedded in one or more embedding mediums jointly, make complex solidifying enzyme.
2, method according to claim 1, wherein porous support comprises:
(1) granular diatomaceous earth.
(2) cenosphere.
(3) chitin.
3, method according to claim 1, wherein polyamine is polyethylene diamines, polymine, polymethylene diamines, polyhexamethylene diamines, poly-tetren, polyethylene polyamine.
4, method according to claim 1, wherein the reactive material of amine is a glutaraldehyde.
5, method according to claim 1, wherein enzyme is glucose isomerase, glucoamylase, α-Dian Fenmei, beta-amylase, Starch debranching enzyme, transglucosidase, glucose oxidase.
6, the conjugation immobilized enzyme comprises the following reaction product that is attached to porous support: have the amine reactive group phase reaction in the reactive material of pendant amine groups polyamino compound and amine-a kind of dual-function compound glutaraldehyde, amine reactive group that it another do not reacted and the reaction of the free amino of enzyme or enzyme, and the formation conjugation immobilized enzyme that links with it.
7, conjugation immobilized enzyme according to claim 6, wherein porous support comprises:
(1) granular diatomaceous earth.
(2) cenosphere.
(3) chitin.
8, conjugation immobilized enzyme according to claim 6, wherein polyamine is polyethylene diamines, polymine, polymethylene diamines, polyhexamethylene diamines, poly-tetren, polyethylene polyamine.
9, method according to claim 1, wherein embedding medium is: carrageenin, xanthan gum, gelatin, agar, Lalgine, diacetate fiber, triacetate fiber, polyacrylamide.
10, a kind of method for preparing compound fixed glucose isomerase and isomerization glucose, this method comprises the steps:
(1) porous support is contacted with having the pendant amine groups polyamino compound, polyamine is attached on the porous support;
(2) wash with deionized water, but the polyamine that excludes solvent and disperse wherein not adhere to, contact with the reactive material solution of amine, this solution is a kind of dual-function compound, as glutaraldehyde, the pendant amine groups of one of them amine reactive group and polyamine reacts, and staying another amine reactive group can further react;
(3), exclude solvent and any being dissolved in wherein and the reactive material of unreacted amine with the deionized water washing.The porous support of having done above-mentioned processing contacts with the glucose isomerase enzyme solution, makes the amido of enzyme and another amine reaction-ity group reaction of the reactive material of amine, by the form fixing glucose isomerase of covalent linkage, constitutes the conjugation fixed glucose isomerase.
(4) above-mentioned conjugation fixed glucose isomerase and the acidophilic streptomycete cell that produces this enzyme are embedded in the embedding medium jointly, and react with the polyamino compound that has pendant amine groups, glutaraldehyde respectively again, make compound fixed glucose isomerase.
(5) compound fixed glucose isomerase is packed in the reaction column, contact, make glucose isomerase to turn to fructose with Liquid Glucose.
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| CN 90108304 CN1059759A (en) | 1990-10-20 | 1990-10-20 | The preparation method of complex solidifying enzyme |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1068503C (en) * | 1994-04-07 | 2001-07-18 | 雀巢制品公司 | Immobilised beta-mannanase |
| WO2003100052A1 (en) * | 2002-05-23 | 2003-12-04 | Beijing University Of Chemical Technology | A anionic laminar material immobilized enzyme and preparation method thereof |
| CN100383242C (en) * | 2005-10-08 | 2008-04-23 | 北京化工大学 | Miniature enzyme reactor with controllable size and property and its prepn |
| CN101278047B (en) * | 2005-09-30 | 2012-12-12 | 诺维信公司 | Immobilization of enzymes |
| CN103224926A (en) * | 2013-04-03 | 2013-07-31 | 大连医诺生物有限公司 | Method of preparing immobilized lipase |
| CN104911225A (en) * | 2015-06-05 | 2015-09-16 | 浙江工业大学 | Method for preparing gabapentin with chemo-enzymatic method |
| CN105349520A (en) * | 2015-11-26 | 2016-02-24 | 青岛大学 | Hollow microsphere immobilized laccase and preparation method thereof |
| CN106399290A (en) * | 2016-10-08 | 2017-02-15 | 甘琦 | Method of utilizing polysaccharide plant gum to prepare embedded microorganisms |
| CN108421366A (en) * | 2018-04-04 | 2018-08-21 | 北京理工大学 | Gelatin hydrolysate removes aldehyde composite material and preparation method with diatomite covalent bonding |
-
1990
- 1990-10-20 CN CN 90108304 patent/CN1059759A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1068503C (en) * | 1994-04-07 | 2001-07-18 | 雀巢制品公司 | Immobilised beta-mannanase |
| WO2003100052A1 (en) * | 2002-05-23 | 2003-12-04 | Beijing University Of Chemical Technology | A anionic laminar material immobilized enzyme and preparation method thereof |
| CN101278047B (en) * | 2005-09-30 | 2012-12-12 | 诺维信公司 | Immobilization of enzymes |
| CN100383242C (en) * | 2005-10-08 | 2008-04-23 | 北京化工大学 | Miniature enzyme reactor with controllable size and property and its prepn |
| CN103224926A (en) * | 2013-04-03 | 2013-07-31 | 大连医诺生物有限公司 | Method of preparing immobilized lipase |
| CN104911225A (en) * | 2015-06-05 | 2015-09-16 | 浙江工业大学 | Method for preparing gabapentin with chemo-enzymatic method |
| CN105349520A (en) * | 2015-11-26 | 2016-02-24 | 青岛大学 | Hollow microsphere immobilized laccase and preparation method thereof |
| 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 |
| CN108421366A (en) * | 2018-04-04 | 2018-08-21 | 北京理工大学 | Gelatin hydrolysate removes aldehyde composite material and preparation method with diatomite covalent bonding |
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