CN1772915A - Chitosn producing process with immobilized enzyme - Google Patents
Chitosn producing process with immobilized enzyme Download PDFInfo
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- CN1772915A CN1772915A CN 200410087507 CN200410087507A CN1772915A CN 1772915 A CN1772915 A CN 1772915A CN 200410087507 CN200410087507 CN 200410087507 CN 200410087507 A CN200410087507 A CN 200410087507A CN 1772915 A CN1772915 A CN 1772915A
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Abstract
The present invention relates to the preparation of chitin oligose, and is especially chitin oligose producing process with immobilized enzyme. The chitin oligose producing process includes the following steps: 1. activating immobilized medium through reaction of immobilized medium with coupler in 3-30 vol% at 20-110 deg.c for 5-24 hr; 2. preparing immobilized enzyme through reaction of enzyme and the activated immobilized medium in buffering phosphate solution to fix the enzyme onto the surface of the medium; and 3. preparing chitin oligose through the enzymolysis reaction between chitosan and immobilized enzyme dissolved in reaction medium, with the excessive immobilized enzyme being recovered through filtering after reaction. The present invention has the advantages of low cost and repeated use of immobilized enzyme, and is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation of oligochitosan, specifically a kind of method of producing oligochitosan with immobilized enzyme.
Background technology
Oligochitosan is meant what β-1,4 connected, and by the oligose that 2-10 2-glucosamine residue constitutes, it has enhancing body immunizing power, suppresses tumor growth; Promote bifidus bacillus propagation, suppress the harmful intestinal tract bacteria growing; Obstruction growth of pathogenic bacteria breeding strengthens plant to the defence capability of disease and pest or the like multiple function, thereby is widely used in a plurality of fields such as medicine, protective foods and agriculture production.Oligochitosan is obtained by degradation of chitosan usually, and degradation method has acid degradation, oxidative degradation and enzyme liberating, and enzyme liberating is a degradation method commonly used now.
Resolvase degrade chitosan method is adopted in the production of oligochitosan now more, because resolvase is dissolved in the reaction system, does not still have simple method that it is separated from reaction system, and enzyme only uses once just have been lost; Enzyme can't reclaim after DeR is finished, and causes the production cost height of oligochitosan, the product price height; Therefore, in the production cost of oligochitosan, enzyme occupies quite great proportion, and reducing this departmental cost is the key that reduces total cost.Owing to the water-soluble feature of resolvase, the resolvase degrade chitosan adopts intermittently stirring reaction mode more again, and efficient is low, and the cycle is long, is unfavorable for suitability for industrialized production.
Summary of the invention
The method that the object of the present invention is to provide a kind of enzyme to reclaim and to reuse with immobilized enzyme degrade chitosan production oligochitosan.
For achieving the above object, the technical solution used in the present invention is:
With the method that immobilized enzyme is produced oligochitosan, operate as follows:
1) activation of entrapment media
Entrapment media and coupling molecule are reacted in liquid phase medium, and temperature of reaction is 20-110 ℃, and reaction times 5-24 hour, the consumption of coupling molecule was the 3%-30% (v/v) of reaction system volume;
2) preparation of immobilized enzyme
Enzyme and step 1) activatory entrapment media are reacted in 0-0.2M phosphoric acid salt or acetate buffer solution, make enzyme be fixed on the surface of medium, the pH of this reaction system is 3.0-8.0, temperature of reaction is 0-25 ℃, reaction times is 24-48 hour, and the amount of enzyme is 2-10mg enzyme/g weight in wet base entrapment media;
3) preparation of oligochitosan
Under agitation condition, to be dissolved in the chitosan in reaction medium water or the weakly acid soln, under 30-50 ℃, with step 2) immobilized enzyme of preparation carries out enzyme liberating reaction, the weightmeasurement ratio of chitosan and reaction system is 0.5-3.0% (w/v), the weightmeasurement ratio of immobilized enzyme and reaction system is 2-10% (w/v), reaction times 10-50 hour, promptly makes oligochitosan; After reaction finishes, the filtered and recycled immobilized enzyme.
Entrapment media can be inorganic or organic porous or poromerics, as molecular sieve, aluminum oxide, granulated glass sphere, polymeric adsorbent or ion exchange resin etc.; Coupling molecule is aminopropyl triethoxysilane, epoxy chloropropane or glutaraldehyde etc.; Described enzyme is generally a kind of enzyme of chitinase, chitoanase, cellulase, bromeline, neutral protease, N,O-Diacetylmuramidase, lipase, helicase etc. or the mixture of several enzymes.Intermittence or the continuously stirring reaction of enzyme liberating reaction for can in stirring tank, carrying out; Or in the reactors such as packed bed, fluidized-bed or membrane bioreactor that immobilized enzyme packed into, chitosan solution is pumped into intermittence or the successive reaction that reactor carries out; Liquid phase medium can be organic phase or water, and wherein organic phase is toluene or dimethylbenzene; Described weakly acid soln is rare mineral acid or micromolecular organic acid soln, as dilute hydrochloric acid, formic acid, acetate, pyruvic acid, lactic acid solution etc.
The present invention produces the oligochitosan method with the resolvase degrade chitosan and compares, and has following advantage:
1. cost is low, and immobilized enzyme can be reused.The present invention adopts immobilized enzyme to produce oligochitosan because with enzyme immobilization, realized DeR after enzyme can reclaim and reuse, thereby reduce the production cost of oligochitosan greatly; In addition, the used entrapment media of the present invention is cheap, and the immobilized enzyme method is simply effective, also makes the cost of this method reduce.
2. be adapted to large-scale production.The immobilized enzyme reaction process is than resolvase reaction process flexible operation, optional majority kind reactive mode and reactor, this method adopts immobilized enzyme, multiple reactive mode such as be fit to intermittently or continuous, pack in packed bed or the reactors such as fluidized-bed or membrane bioreactor as intermittence of in stirring tank, carrying out or continuously stirring reaction or with immobilized enzyme, chitosan solution is pumped into intermittence or the successive reaction that reactor carries out, and the realization suitability for industrialized production is highly advantageous to.
Embodiment
Below by embodiment technology of the present invention is further specified.
Embodiment 1: the microsphere immobilized chitoanase of porous alumina
With 18 order porous alumina microballoons of the dehydration degassing in advance Glass Containers of packing into, the toluene solution that adds 30% (v/v) aminopropyl triethoxysilane is to submergence porous alumina microballoon, and heating makes refluxing toluene 12h; Get and react the porous alumina microballoon that finishes, use toluene, Virahol, water washing successively, then this porous alumina microballoon is joined in 25% (v/v) glutaraldehyde water solution, 30 ℃, stirring reaction 1h, washing is activatory porous alumina microballoon; Activatory porous alumina microballoon is added (0.2M, pH6.0 in the phosphate buffered saline buffer of chitoanase; Enzyme concn 2mg/ml, total enzyme amount 6mg enzyme/gram weight in wet base activatory porous alumina microballoon), 4 ℃ of reaction 24h intermittently stir.Enzyme yield 40% alive.
Embodiment 2: molecular sieve immobilized chitoanase
Get 3 , 4 , 5 molecular sieves,, activatory molecular sieve is added (0.2M, pH6.0 in the phosphate buffered saline buffer of chitoanase with embodiment 1 method activation; Enzyme concn 2mg/ml, total enzyme amount 4mg enzyme/g weight in wet base activatory molecular sieve), 4 ℃ of reaction 24h intermittently stir.Enzyme yield 20-30% alive.
Embodiment 3: Bio-Glas immobilization chitoanase
Get Bio-Glas with embodiment 2 method immobilization chitoanases, enzyme yield 30% alive.
Embodiment 4: polymeric adsorbent immobilization chitoanase
Get in water, 0.02M pH6.0,0.1M pH6.0 and the 0.02M pH8.0 phosphate buffer soln that each 5g of polymeric adsorbent (weight in wet base) joins 5ml respectively, chitoanase added in above-mentioned four kinds of solution, with four kinds of solution at the optical density value (OD of 280nm place
280) detect the amount of polymeric adsorbent D3520, D4006, X-5, HZ801, HZ802, HZ803 absorption chitoanase, the adsorptive capacity difference of chitoanase in different polymeric adsorbents and different solutions, from 2mg enzyme/g weight in wet base polymeric adsorbent to the 8mg enzyme/g weight in wet base polymeric adsorbent, wherein HZ801 adsorptive capacity maximum in 0.1M pH6.0 phosphate buffer soln is 8mg enzyme/g weight in wet base polymeric adsorbent.The HZ801 polymeric adsorbent of absorption chitoanase is suspended in water again, and 20 ℃, whipped state drips 8% (v/v) glutaraldehyde water solution down, and reaction 2h is fixed on the polymeric adsorbent chitoanase, enzyme yield 60% alive.
Embodiment 5: polymeric adsorbent immobilization chitinase
Get chitinase and be fixed on the polymeric adsorbent with embodiment 4 methods, HZ801 polymeric adsorbent adsorptive capacity is 8mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 60% alive.
Embodiment 6: the polymeric adsorbent immobilized cellulase
The extracting cellulose enzyme is fixed on the polymeric adsorbent with embodiment 4 methods, and HZ801 polymeric adsorbent adsorptive capacity is 7mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 40% alive.
Embodiment 7: polymeric adsorbent immobilization bromeline
Get bromeline and be fixed on the polymeric adsorbent with embodiment 4 methods, HZ801 polymeric adsorbent adsorptive capacity is 8mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 60% alive.
Embodiment 8: polymeric adsorbent immobilization neutral protease
Get neutral protease and be fixed on the polymeric adsorbent with embodiment 4 methods, HZ801 polymeric adsorbent adsorptive capacity is 8mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 70% alive.
Embodiment 9: the polymeric adsorbent immobilized lysozyme
Get N,O-Diacetylmuramidase and be fixed on the polymeric adsorbent with embodiment 4 methods, HZ801 polymeric adsorbent adsorptive capacity is 8mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 40% alive.
Embodiment 10: the polymeric adsorbent immobilized lipase
Get N,O-Diacetylmuramidase and be fixed on the polymeric adsorbent with embodiment 4 methods, HZ801 polymeric adsorbent adsorptive capacity is 8mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 20% alive.
Embodiment 11: polymeric adsorbent immobilization helicase
Get helicase and be fixed on the polymeric adsorbent with embodiment 4 methods, HZ801 polymeric adsorbent adsorptive capacity is 8mg enzyme/g weight in wet base polymeric adsorbent, glutaraldehyde immobilized enzyme yield 80% alive.
Embodiment 12: the fixed on ion exchange resin chitoanase
Get weak anion exchange resin D301R, D392, use ethanol successively, water, 1N sodium hydroxide, 5% sodium-chlor, 1N hydrochloric acid, 1N sodium hydroxide, water cleans; The degassing; Every milliliter of resin drips 20 milliliters of 7% glutaraldehyde water solutions, stirring at room reaction 24h; Water is suspended in pH5.0 with resin after cleaning, and in the 0.2M acetate buffer, adds 4 ℃ of reactions of chitoanase 24h, intermittently stirs, and enzyme dosage is the 5mg/ml resin.
Embodiment 13: the fixed on ion exchange resin helicase
Get helicase and be fixed on the ion exchange resin D392 with embodiment 12 methods, enzyme dosage is the 8mg/ml resin.
Embodiment 14: the fixed on ion exchange resin neutral protease
Get the snail neutral protease and be fixed on the ion exchange resin D392 with embodiment 12 methods, enzyme dosage is the 8mg/ml resin.
Embodiment 15: immobilized enzyme degrade chitosan reaction 1
In glass reactor, the 1g chitosan is dissolved in 1% acetum of 100ml, add 2ml embodiment 12 described immobilization chitoanases, 38 ℃ of stirring reactions; Stopped reaction behind the 2h filters, and water cleans immobilized enzyme, again this immobilized enzyme is dropped into reactor and carries out above-mentioned reaction; Repeat 15 secondary responses altogether, activity of the immobilized enzyme does not have considerable change.
Embodiment 16: immobilized enzyme degrade chitosan reaction 2
In stainless steel reactor, the 20g chitosan is dissolved in 1% acetum of 2000ml; 80ml embodiment 13 described immobilization helicases are packed in the glass column, make immobilized enzyme column; Chitosan solution is pumped into immobilized enzyme column react, the reaction stream fluid is got back in the stainless steel reactor, circulating reaction like this, 50 ℃ of temperature of reaction, reaction times 20h; After reaction is finished, bleed off reaction solution, water cleans immobilized enzyme column, restarts above-mentioned reaction; Carry out 28 batch reactions altogether, activity of the immobilized enzyme still keeps more than 80%.
Claims (7)
1. method of producing oligochitosan with immobilized enzyme is characterized in that operating as follows:
1) activation of entrapment media
Entrapment media and coupling molecule are reacted in liquid phase medium, and temperature of reaction is 20-110 ℃, and reaction times 5-24 hour, the consumption of coupling molecule was the 3%-30% of reaction system volume;
2) preparation of immobilized enzyme
Enzyme and step 1) activatory entrapment media are reacted in 0-0.2M phosphoric acid salt or acetate buffer solution, make enzyme be fixed on the surface of medium, the pH of this reaction system is 3.0-8.0, temperature of reaction is 0-25 ℃, reaction times is 24-48 hour, and the amount of enzyme is 2-10mg enzyme/g weight in wet base entrapment media;
3) preparation of oligochitosan
Under agitation condition, to be dissolved in the chitosan in reaction medium water or the weakly acid soln, under 30-50 ℃, with step 2) immobilized enzyme of preparation carries out enzyme liberating reaction, the weightmeasurement ratio of chitosan and reaction system is 0.5-3.0%, the weightmeasurement ratio of immobilized enzyme and reaction system is 2-10%, reaction times 10-50 hour, promptly makes oligochitosan; After reaction finishes, the filtered and recycled immobilized enzyme.
2. according to the described method of producing oligochitosan with immobilized enzyme of claim 1, it is characterized in that: described entrapment media is molecular sieve, aluminum oxide, granulated glass sphere, polymeric adsorbent or ion exchange resin.
3. according to the described method of producing oligochitosan with immobilized enzyme of claim 1, it is characterized in that: described coupling molecule is aminopropyl triethoxysilane, epoxy chloropropane or glutaraldehyde.
4. according to the described method of producing oligochitosan with immobilized enzyme of claim 1, it is characterized in that: described enzyme is a kind of enzyme of chitinase, chitoanase, cellulase, bromeline, neutral protease, N,O-Diacetylmuramidase, lipase, helicase or the mixture of several enzymes.
5. according to the described method of producing oligochitosan of claim 1, it is characterized in that: intermittence or the continuously stirring reaction of described enzyme liberating reaction in stirring tank, carrying out with immobilized enzyme; Or immobilized enzyme packed in packed bed, fluidized-bed or the membrane bioreactor reactor, chitosan solution is pumped into intermittence or the successive reaction that reactor carries out.
6. according to the described method of producing oligochitosan with immobilized enzyme of claim 1, it is characterized in that: described liquid phase medium is organic phase or water.
7. according to the described method of producing oligochitosan with immobilized enzyme of claim 1, it is characterized in that: described weakly acid soln is dilute hydrochloric acid, formic acid, acetate, pyruvic acid or lactic acid solution.
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| CNB2004100875071A CN1321191C (en) | 2004-11-10 | 2004-11-10 | Chitosn producing process with immobilized enzyme |
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| CNB2004100875071A CN1321191C (en) | 2004-11-10 | 2004-11-10 | Chitosn producing process with immobilized enzyme |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102978263A (en) * | 2012-12-12 | 2013-03-20 | 石狮市华宝海洋生物化工有限公司 | Method for producing high-purity N-acetylglucosamine |
| CN106011127A (en) * | 2016-06-25 | 2016-10-12 | 陈毅忠 | Method for preparing chitosan oligosaccharide by using immobilized snailase |
| CN106755207A (en) * | 2016-12-23 | 2017-05-31 | 浙江海洋大学 | A kind of method of shitosan high solids content enzymolysis generation narrow molecular-weight activity chitosan oligosaccharide |
| CN109468353A (en) * | 2018-11-27 | 2019-03-15 | 浙江海洋大学 | A kind of method that supercritical carbon dioxide fluid enzymatic hydrolysis prepares chitosan oligosaccharide |
| CN109517869A (en) * | 2018-12-22 | 2019-03-26 | 大连海洋大学 | A method of oyster ace inhibitory peptide is produced with immobilised enzymes |
| CN109609571A (en) * | 2018-12-21 | 2019-04-12 | 青岛颂田生物技术有限公司 | A kind of preparation method of anacidity chitosan oligosaccharide |
| CN111979206A (en) * | 2019-05-24 | 2020-11-24 | 深圳瑞德林生物技术有限公司 | Immobilized fusion enzyme and method for preparing glutathione by using same |
| CN112011530A (en) * | 2020-08-31 | 2020-12-01 | 羚鲨贸易(东莞)有限公司 | Adsorption resin material for adsorbing bromelain and preparation method thereof |
| CN112980905A (en) * | 2021-03-11 | 2021-06-18 | 烟台泓源生物肥料有限公司 | Preparation method of chitosan oligosaccharide |
| CN113045683A (en) * | 2021-04-16 | 2021-06-29 | 中国科学院海洋研究所 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
| CN113832203A (en) * | 2021-10-28 | 2021-12-24 | 湛江市博泰生物化工科技实业有限公司 | Chitosan oligosaccharide and preparation method thereof |
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| CN1126756C (en) * | 2000-01-05 | 2003-11-05 | 中国科学院大连化学物理研究所 | Process for preparing chitoligose by coupling enzyme degradation of chitosan with membrane separation |
| CN1177856C (en) * | 2001-08-14 | 2004-12-01 | 华东理工大学 | Process for industrial production of oligochitose and chitooligose |
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2004
- 2004-11-10 CN CNB2004100875071A patent/CN1321191C/en not_active Expired - Fee Related
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978263A (en) * | 2012-12-12 | 2013-03-20 | 石狮市华宝海洋生物化工有限公司 | Method for producing high-purity N-acetylglucosamine |
| CN106011127A (en) * | 2016-06-25 | 2016-10-12 | 陈毅忠 | Method for preparing chitosan oligosaccharide by using immobilized snailase |
| CN106755207A (en) * | 2016-12-23 | 2017-05-31 | 浙江海洋大学 | A kind of method of shitosan high solids content enzymolysis generation narrow molecular-weight activity chitosan oligosaccharide |
| CN109468353A (en) * | 2018-11-27 | 2019-03-15 | 浙江海洋大学 | A kind of method that supercritical carbon dioxide fluid enzymatic hydrolysis prepares chitosan oligosaccharide |
| CN109609571A (en) * | 2018-12-21 | 2019-04-12 | 青岛颂田生物技术有限公司 | A kind of preparation method of anacidity chitosan oligosaccharide |
| CN109517869A (en) * | 2018-12-22 | 2019-03-26 | 大连海洋大学 | A method of oyster ace inhibitory peptide is produced with immobilised enzymes |
| CN111979206A (en) * | 2019-05-24 | 2020-11-24 | 深圳瑞德林生物技术有限公司 | Immobilized fusion enzyme and method for preparing glutathione by using same |
| CN112011530A (en) * | 2020-08-31 | 2020-12-01 | 羚鲨贸易(东莞)有限公司 | Adsorption resin material for adsorbing bromelain and preparation method thereof |
| CN112980905A (en) * | 2021-03-11 | 2021-06-18 | 烟台泓源生物肥料有限公司 | Preparation method of chitosan oligosaccharide |
| CN113045683A (en) * | 2021-04-16 | 2021-06-29 | 中国科学院海洋研究所 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
| CN113045683B (en) * | 2021-04-16 | 2022-10-11 | 中国科学院海洋研究所 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
| CN113832203A (en) * | 2021-10-28 | 2021-12-24 | 湛江市博泰生物化工科技实业有限公司 | Chitosan oligosaccharide and preparation method thereof |
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