CN1772912B - Biologically catalytic hydroxyacetic acid production - Google Patents
Biologically catalytic hydroxyacetic acid production Download PDFInfo
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- CN1772912B CN1772912B CN2004100681409A CN200410068140A CN1772912B CN 1772912 B CN1772912 B CN 1772912B CN 2004100681409 A CN2004100681409 A CN 2004100681409A CN 200410068140 A CN200410068140 A CN 200410068140A CN 1772912 B CN1772912 B CN 1772912B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The present invention discloses biologically catalytic method of producing hydroxyacetic acid. The present invention produces hydroxyacetic acid with hydroxyacetonitrile as initial material and microbe enzyme obtained through fermenting culture of Brevibacterium casei CGMCC No. 0887 and possessing nitrile hydrolyzing activity as the catalyst and through hydrolysis under certain conditions. The method of the present invention has the features of mild reaction condition, simple technological path, no pollution and capacity of continuous industrial production.
Description
Technical field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of method of biologically catalytic hydroxyacetic acid production.
Background technology
Oxyacetic acid has another name called oxyacetic acid or glycolic acid, is the meticulous Organic Chemicals that a kind of large-tonnage is produced, and has purposes widely.Can be applicable to sticker, produce degradation polymer, metal detergent, electroplating additives, the agent of milk preparation vessel cleaning, dyeing auxiliary, well sanitising agent, building, weaving, washing composition, pharmacy, agricultural chemicals, tanning etc.; Its verivate; Like pure acid anhydride (ether), salt, ester is the raw material of many meticulous organic chemical industry's products; Picture oxyacetic acid zinc, the oxyacetic acid butyl ester has been used to the production of vehicle paint base.Oxyacetic acid is used by a large amount of in the production of boilercompound, and wherein most representative is the removing that the mixture of oxyacetic acid and formic acid is used for the super critical boiler incrustation scale, and effect is fine, has become the standard method of passing through in the world.It is the simplest linear aliphatic adoption ester of structure that PGTA or be called gathers NSC 403079, is a commercial the earliest polymeric articles of degradable in vivo, is applied to many fields such as engineering in medicine material and polymer degradable material.
Industry is at present gone up the production oxyacetic acid and is mainly chemosynthesis; Method has: the Mono Chloro Acetic Acid hydrolysis method; Mono Chloro Acetic Acid hydrolysis under strong alkaline condition generates oxyacetic acid, and this method exists shortcomings such as raw material consumption height, product yield are low, product purification complicacy, and corrosion and seriously polluted; The formaldehyde carbonyl process, the industrial production of ethyl alcohol acid method of du pont company initiative, this method needs high pressure, liquid acid catalysis,, seriously corroded very high to equipment requirements, the separation of the finished product, refining complicated, catalyzer can not reuse, and pollutes also serious; The electrolytic reduction of oxalic acid is realized industriallization the nineties in 20th century, and this method energy consumption is high, product is complicated, the cost of production oxyacetic acid is high; Hydrolysis when hydroxyacetonitrile acid-hydrolysis method, hydroxyl acetonitrile aqueous solution exist in sulfuric acid, phosphoric acid, nitric acid or mixing acid acid obtains product through processes such as extraction, desalination, back extraction, dehydrations.
There is document to propose applying biological catalysis process synthesis of hydroxy acetate, advantages such as selectivity is good, product purity is high because biological catalysis has, reaction conditions gentleness.Patent U.S.3,940,316 disclose the mikrobe Bacillus with nitrile hydrolytic activity, Bacteridium, the method for the synthetic acid of Micrococcus and the hydrolysis of Brevibacterium catalysis nitrile compound, hydroxyacetonitrile as one of them substrate; Patent JP 09028390 report utilizes Rhodococcus or Gordona nitrilase catalysis hydroxyl acetonitrile hydrolysis synthesis of hydroxy acetate; Patent U.S.6,037,155 utilizes mikrobe Variovorax spp. and the synthetic corresponding alcohol acid of Arthrobacter NSSC104 catalysis hydroxyl nitrile hydrolysis, classifies hydroxyacetonitrile as one of them substrate; Patent U.S.6,416,980 utilize the lytic enzyme synthesis of hydroxy acetate of Acidovorax facilis 72W and variant thereof, and production concentration can be accumulated and reach 1M.The meliority that these enzymatic methods of conversion demonstrated, for the instead of chemical method has been showed good prospect, but these reaction cumulative production concentrations are also not high, the report of no suitability for industrialized production aspect, state of the art has satisfied not industrial production requirement.
Summary of the invention
The objective of the invention is to overcome the above-mentioned deficiency of prior art; Thereby a kind of method of biologically catalytic hydroxyacetic acid production is provided; Be specially the nitrilase that obtains through fermentation culture brevibacterium casei (Brevibacterium casei) CGMCC No.0887 and come the hydrolysis reaction of catalysis hydroxyacetonitrile to obtain the product oxyacetic acid, reaction formula is following:
For achieving the above object, practical implementation process of the present invention is following:
One, preparation biological catalyst nitrilase
(1) we are geographic from the Shanghai gold mountain contains that seed selection obtains a bacterial strain the nitrile waste water; And in submission China Microbial Culture Preservation Commission common micro-organisms center (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica) preservation on January 20th, 2003, classification called after brevibacterium casei; The Latin name is called Brevibacterium casei, and preserving number is CGMCC No.0887.This bacterial classification is carried out shake-flask culture for we or the top fermentation jar is cultivated, and obtain fermented liquid;
Shake-flask culture: an amount of substratum (10-70%) of in triangular flask, packing into, a certain amount of bacterial classification (1-10%) is inserted in the sterilization back, under 20-40 ℃, cultivates 30-120 hour in the rotary shaking table (100-300rpm), obtains fermented liquid, and it is subsequent use to remove agar.
Wherein, the shake-flask culture base is formed (%): glucose: 1-2.0; Yeast extract paste: 0.2-1; NaCl:0.05-0.15; K
2HPO
4: 0.05-0.3; MgSO
47H
2O:0.01-0.3; Agar: 2.0, pH is 7.0-7.5.
Last jar of fermentation culture: an amount of seed culture medium (10-70%) of in seeding tank, packing into; A certain amount of bacterial classification (1-10%) is inserted in the sterilization back, at temperature 20-40 ℃, cultivates 30-120 hour under the condition of mixing speed 100-300rpm; Obtain seed liquor, at 50L-20m
3Fermentor tank in an amount of fermention medium (40-70%) of packing into; The seed liquor of 2-30% is inserted in real jar of sterilization back, at air flow 1: 0.2-1: 1, mixing speed 50-400rpm; Tank pressure 0.03-0.06MPa; Under temperature 20-40 ℃ the condition, fermentation culture 30-200 hour, acquisition contained the fermented liquid of enzyme.
Wherein, last jar of fermentation culture formed:
Seed culture medium is formed (%): glucose: 0.5-2.0; Yeast extract paste: 0.2-1; KH
2PO
4: 0.05-0.3; K
2HPO
4: 0.05-0.3; MgSO
47H
2O:0.01-0.3; PH6.5-7.5.
Fermention medium is formed: glucose: 1.0-2.5; Yeast extract paste: 0.2-1; K2HPO4:0.03-0.1; KH2PO4:0.03-0.1; MgSO47H2O: inductor I:0.01-0.5; Inductor II:0.01-0.5; Growth hormone: 0.1-2; PH6.5-7.5.Inductor I can be hexanolactam, cyanobenzene, benzyl cyanide, beta-lactam, vinyl cyanide, n-Butyronitrile, trimethylene cyanide, cigarette nitrile, butyrolactam, alpha-amino group pentanedioic acid lactan, laurolactam, adiponitrile, acetonitrile or isopropyl cyanide etc.; Inductor II can be urea, peptone, Carnis Bovis seu Bubali cream, Fructus Hordei Germinatus extract or steeping water etc., and growth hormone can be CoCl
2, FeCl
3, FeCl
2, halfcystine, MnSO4, CuCl
2, l-asparagine, glycocoll, ZnSO4, thiophenol, sulfur alcohol, thioglycolic acid, α, β-dithioglycerol, MBAMT or methionine(Met) etc.
(2) fermented liquid carries out the enzyme cell harvesting through centrifugal or membrane filtration system again, prepares biological catalyst then;
The enzyme cell harvesting: fermented liquid is centrifugal under the condition of separation factor >=5000, abandoning supernatant, and cell is centrifugal again after with deionized water wash, collects spissated wet cell liquid; Perhaps fermented liquid carries out the membrane sepn filtration with membrane filtration system; And the deionized water wash of 2-3 times of fermentating liquid volume of adding; Collect spissated wet cell liquid, membrane filtration system wherein can be hollow-fibre membrane or rolled film or ceramic membrane or ultra-filtration membrane or Ultra-flo.
Preparation biological catalyst: the containing the enzyme cell and can directly be used as catalyzer of collection; Or through being used as catalyzer after the immobilizations such as ordinary method such as sodium alginate to embed method; Or the cytoclasis liquid that cell is obtained after with fragmentations such as ordinary method such as UW is as catalyzer; Or the centrifugal enzyme clear liquid of removing behind the cell debris is directly as catalyzer; The zymoprotein that thick enzyme that maybe will passing through purifies obtains or purity are higher is used as catalyzer.
Two, biocatalysis hydrolysis reaction:
Hydroxyacetonitrile biocatalysis hydrolysis reaction comprises two steps of substrate pre-treatment and hydrolysis reaction.
(1) pre-treatment of substrate hydroxyacetonitrile: with highly basic or basic carbonate such as NaOH, KOH, Ba (OH), Ca (OH)
2, Ca (HCO
3)
2Or CaCO
3) neutralization, or with highly basic or weak base anion resins such as D201 type, D202 type or D301 type, to pH6-8.
(2) the biocatalysis hydrolysis reaction of hydroxyl acetonitrile aqueous solution: in water or phosphoric acid buffer or water-organic medium or little water organic reaction medium, add biological catalyst; React after adding substrate; PH is controlled between the 2.0-10.0; Temperature of reaction is controlled between 0-70 ℃, and stirring velocity is controlled between the 50-500rpm.Reactive mode can be batch formula reaction or successive reaction, and substrate can disposablely add in batch formula reaction, perhaps intermittently adds several times, and perhaps stream adds, and concentration of substrate is controlled at 0-50% (wt%).Reaction times decides according to the particular case of reaction, and reaction should guarantee that concentration of substrate is low as far as possible when finishing, and this can prolong the reaction times through stopping reinforced back, or reaches this requirement through adding a small amount of biological catalyst reaction for some time.In successive reaction, substrate can disposablely add, and perhaps intermittently adds several times, and perhaps stream adds; The add-on that all must guarantee substrate at any time can not make concentration of substrate >=50% (wt%) in the reaction solution, when the production concentration accumulation reaches certain value >=10% (wt% is in oxyacetic acid), and can be disposable through membrane filtration system; Perhaps several times off and on, perhaps distribute the reaction clear liquid continuously, be preferably the continuous partial flow method; Simultaneously can be disposable, perhaps intermittently several times, perhaps add reaction medium and substrate continuously; Be preferably continuous replenishment method, and according to the situation of reacting, can be disposable; Perhaps intermittently several times, perhaps quantitatively add new biological catalyst continuously, can carry out continuously to guarantee reaction.
Embodiment
The present invention further specify below through embodiment in order to explain better.
Percentage concentration listed in following examples all is weight percentage.
Enzyme is lived and defined: the hydroxyacetonitrile with a microgram in 1 hour is converted into the needed enzyme amount of oxyacetic acid, the unit microgram/hour.
Embodiment 1
Prepare shake flask fermentation culture medium A, B, C respectively, wherein substratum is formed as follows:
A: glucose 1.0%, yeast extract paste 0.2%, NaCl0.05%, K
2HPO
40.05%, MgSO
47H
2O0.3%, agar 2.0%, pH7.0;
B: glucose 1.5%, yeast extract paste 0.5%, NaCl0.10%, K
2HPO
40.15%, MgSO
47H
2O0.15%, agar 2.0%, pH7.2;
C: glucose 2.0%, yeast extract paste 1.0%, NaCl0.15%, K
2HPO
40.30%, MgSO
47H
2O0.01%, agar 2.0%, pH7.5;
Respectively above prepared culture medium is poured into the 30mL portion in the triangular flask of 250mL, in autoclave, sterilized 20 minutes for 120 ℃.Inoculate brevibacterium casei (Brevibacterium casei) preservation bacterial classification after the cooling respectively in each substratum, in 28 ℃, 250rpm rotating speed concussion was down cultivated 96 hours.After cultivating end, collect fermented liquid respectively, get 10ml, add the 1ml hydroxyacetonitrile, reacted 10 minutes, adding 5M hydrochloric acid soln, termination reaction, HPLC (Tianjin, island LC-10A of company) measures the amount of oxyacetic acid, calculates enzyme (down together) alive, and the result lists in table 1.
Table 1
Embodiment 2
The preparation seed culture medium, it consists of: glucose 0.6%, yeast extract paste 0.2%, K
2HPO
40.05%, KH
2PO
40.05%, MgSO
47H
2O0.05%, pH7.2;
Prepare fermention medium A, B, C, D more respectively, substratum consists of:
A: glucose 1.0%, yeast extract paste 0.2%, K
2HPO
40.03%, KH
2PO
40.05%, MgSO
47H
2O0.05%, beta-lactam 0.05%, urea 0.5%, CoCl
20.1%, pH7.0;
B: glucose 1.5%, yeast extract paste 0.5%, K
2HPO
40.05%, KH
2PO
40.10%, MgSO
47H
2O0.03%, adiponitrile 0.2%, peptone 0.35%, halfcystine 0.5%, pH7.5;
C: glucose 2.0%, yeast extract paste 0.8%, K
2HPO
40.08%, KH
2PO
40.03%, MgSO
47H
2O0.10%, alpha-amino group pentanedioic acid lactan 0.4%, Carnis Bovis seu Bubali cream 0.15%, l-asparagine 1.0%, pH6.5;
D: glucose 2.5%, yeast extract paste 1.0%, K
2HPO
40.10%, KH
2PO
40.08%, MgSO
47H
2O0.08%, cigarette nitrile 0.5%, steeping water 0.05%, thioglycolic acid 2.0%, pH7.2;
The seed culture medium 70% of in the 5L seeding tank, packing into, the bacterial classification of an eggplant bottle is inserted in the sterilization back, 28 ℃ of temperature, cultivates 48 hours under the condition of mixing speed 400rpm, obtains seed liquor, at 50L
3Fermentor tank in pack into fermention medium A, B, C, D each 70%, 10% seed liquor is inserted in real jar of sterilization back, air flow 1: 0.5, mixing speed 300rpm, tank pressure 0.05Mpa, under the condition that temperature is 28 ℃, fermentation culture 48 hours.Fermentation culture is collected fermented liquid respectively after finishing, and measures enzyme and lives, and the result lists in table 2.
Table 2
Embodiment 3
By the fermented liquid 3.5L that embodiment 2 methods obtain, use low-temperature and high-speed centrifugal respectively, 6 ℃ with the 15000rpm rotating speed under frozen centrifugation 15 minutes or tubular fibre membrane filtration, flow 180mLmin
-1, pressure 0.8Kg/cm
2Or rolled film filters flow 180mLmin
-1, pressure 16Kg/cm
2Or ceramic membrane filter, flow 180mLmin
-1, pressure 4Kg/cm
2Or Ultra-flo filters flow 180mLmin
-1, pressure 3Kg/cm
2And use 4L deionized water wash cell respectively, and collect concentrating cells liquid, get a certain amount ofly, return to the original fermented solution volume with the deionized water dilution, measure enzyme and live, the result lists in table 4
Table 2
Embodiment 4
By the fermented liquid that embodiment 2 methods obtain, press the tubular fibre membrane filtering method of embodiment 3,4 times of washing concentrating, the concentrating cells that obtains is further handled, and obtains the multi-form biological catalyst with nitrilase activity.1., after the sodium-alginate of getting concentrating cells and 3.2% mixes, be injected into the CaCL of 0.2M
2Carry out the gelation embedding in the solution, make immobilized cell.2., get concentrating cells with the ultrasonic cell-break device with cytoclasis, condition 800W, 8 ℃ of temperature, each fragmentation volume 100mL, accumulative total ultrasonic time 1 hour obtains cytoclasis liquid.3., the cytoclasis liquid got 2. is centrifugal down in 15000g, 6 ℃, collects supernatant, obtains acellular enzyme liquid.4., the supernatant of getting 3. passes through the different concns ammonium sulfate precipitation; Collect the deposition component under the 20-50% saturation ratio, place the 10mM potassium phosphate buffer of pH7.0 to dialyse 24 hours, and then pure water dialysis 24 hours; Obtain crude enzyme liquid, promptly obtain thick enzyme powder through after the vacuum freezedrying.5., the thick enzyme powder of getting is 4. handled with the DEAE-Sephadex post of crossing through the 10mM potassium phosphate buffer pre-equilibration of pH7.0; 10mM potassium phosphate buffer gradient elution with the pH7.0 that contains 0-0.6M Repone K; Collection has the component of nitrilase activity; Abundant dialysis desalting promptly gets the higher relatively enzyme solution of purity in pure water, promptly obtains the enzyme powder through after the vacuum freezedrying.With deionized water various multi-form biological catalysts are returned to initial fermentating liquid volume, measure enzyme and live, the result lists in table 3.
Table 3
| Catalyst mode | Fermented liquid | Enchylema | The fixation cell cytosol | Cytoclasis liquid | Acellular enzyme liquid | Crude enzyme liquid | Enzyme liquid |
| Enzyme lives (* 10 4 UmL -1·hr -1) | 7.5428 | 6.788 | 4.526 | 6.1092 | 4.88736 | 2.930 | 1.759 |
Embodiment 5
Get hydroxyl acetonitrile aqueous solution (pH≤2.5, concentration 50%) 100mL, drip the concentrated sodium hydroxide neutralization, make pH ≈ 7.0, place under the low temperature subsequent use; Get identical hydroxyl acetonitrile aqueous solution 100mL, drip dense yellow soda ash neutralization, make pH ≈ 7.0, place under the low temperature subsequent use; Get identical hydroxyl acetonitrile aqueous solution 100mL, last resin column depickling is handled, and the glass column of φ 3 * 50 is equipped with the D202 strong base anion resins of 250mL, discards preliminary examination stage effluent, collects the bottoms stream fluid, concentration 45%, and pH＞6.0 place under the low temperature subsequent use; Handle substrate by same method with the D301 weak base anion resins, collect the bottoms stream fluid, concentration 46%, pH＞6.0 place under the low temperature subsequent use.Handle the concentrating cells liquid that obtains by the tubular fibre membrane filtering method of embodiment 3 and be used as catalyzer, concentrating cells returns to original volume with deionized water, gets 100mL respectively; Each adds 1% (wt%; By volume 100mL calculates) the hydroxyacetonitrile handled of aforementioned different modes, oscillatory reaction is 30 minutes under 30 ℃ and 200rpm condition, HPLC stratographic analysis (Tianjin, island LC-10A of company; Down together), result such as table 4.
Table 4
Embodiment 6
Cytoclasis liquid by the method for embodiment 4 obtains returns to the preliminary examination fermentating liquid volume with deionized water with cytoclasis liquid, gets 50mL respectively; Add 2% substrate (wt% through the sodium hydroxide neutralizing treatment; By volume 50mL calculates), in differing temps, oscillatory reaction under the rotating speed of 200rpm; React after 30 minutes HPLC stratographic analysis result such as table 5.
Table 5
Embodiment 7
Enchylema by the method for embodiment 4 obtains returns to initial fermentating liquid volume with deionized water with enchylema, gets 1.5L in three mouthfuls of reaction flasks of 2L, adds the 0.5% hydroxyacetonitrile solution (wt% through the sodium hydroxide neutralizing treatment; By volume 1.5L calculates), under 28 ℃ of bath temperatures and 150rpm mixing speed condition, react, intermittently add substrate, when concentration of substrate is lower than 0.1%; By 0.5% adding substrate, to react 68 hours, substrate accumulative total adds indegree 55 times; Add 412.5g altogether, about 825mL, reaction back substrate residual concentration＜0.05%; Transformation efficiency＞99.0%, end product concentration 3.13M, oxyacetic acid productive rate＞98.0%.
Embodiment 8
Enchylema by the method for embodiment 4 obtains returns to initial fermentating liquid volume with deionized water with enchylema, gets 2.0L in three mouthfuls of reaction flasks of 3L, adds the hydroxyacetonitrile solution through the sodium hydroxide neutralizing treatment with peristaltic pump, the about 8-15mLmin of stream rate of acceleration
-1, under 30 ℃ of bath temperatures and 200rpm mixing speed condition, reacting, trace analysis is regulated the stream rate of acceleration according to the height of concentration of substrate; Guarantee concentration of substrate≤0.6%, when the product cumulative concentration reaches the 3.0M left and right sides, stop to feed in raw material; Continue reaction and making base consumption intact, termination reaction when substrate residual concentration＜0.05%, 56 hours entire reaction course time; Transformation efficiency＞99.0%, end product concentration 3.47M, oxyacetic acid productive rate＞98.0%.
Embodiment 9
The preparation seed culture medium, it consists of: glucose 0.6%, yeast extract paste 0.4%, K
2HPO
40.05%, KH
2PO
40.05%, MgSO
47H
2O0.05%, pH7.2.Prepare fermention medium again, it consists of: glucose 2%, yeast extract paste 0.4%, K
2HPO
40.05%, KH
2PO
40.05%, MgSO
47H
2O0.05%, inductor I 0.1%, inductor II 0.5%, growth hormone 0.5%, tank pressure 0.05Mpa, pH7.2.The seed culture medium 70% of in the 5L seeding tank, packing into, the bacterial classification of an eggplant bottle is inserted in the sterilization back, 28 ℃ of temperature, cultivates 48 hours under the condition of mixing speed 400rpm, obtains seed liquor.The fermention medium 70% of in the fermentor tank of 50L, packing into, 10% seed liquor is inserted in real jar of sterilization back, air flow 1: 0.5, mixing speed 300rpm, tank pressure 0.05Mpa, under the condition that temperature is 28 ℃, fermentation culture 48 hours.Fermentation culture is collected fermented liquid respectively after finishing, and filters with flat plate ultrafiltration membrane; The twice deionized water wash is collected concentrating cells, and deionized water is diluted to original volume; In the reactor drum of the 10L that has membrane separation apparatus, add enchylema 6L, 30 ℃ of jacket water (J.W.) bath temperatures, mixing speed 300rpm; Pump into hydroxyacetonitrile solution with peristaltic pump, the about 8-15mLmin of stream rate of acceleration through the sodium hydroxide neutralizing treatment
-1, the HPLC chromatogram tracking is analyzed, and regulates the stream rate of acceleration according to the height of concentration of substrate; Guarantee that concentration of substrate about 0.5%, when the product cumulative concentration reaches 15% (2.0M) left and right sides, stops to feed in raw material; Continuing reaction for some time makes base consumption intact, when concentration of substrate＜0.05%, leaches the reaction clear liquid through membrane filtration system; Add deionized water to reactor drum then, current adding substrate continues reaction again; And add concentrating cells at any time according to response situation, so successive reaction constantly obtains containing the reaction clear liquid of product, the reaction clear liquid that contains the product oxyacetic acid that obtains.
The present invention adopts the method for biocatalysis to produce oxyacetic acid, utilize nitrilase that hydroxyacetonitrile is converted into corresponding acid, but its operating process is simple, reaction conditions is gentle, the little industriallization continuous production of environmental pollution.
The present invention compares with existing method; Have tangible characteristics and progress: the new bacterial strain of bacterium producing multi enzyme preparation for obtaining from the screening of natural source; Be accredited as brevibacterium casei (Brevibacterium casei) at present; Its preserving number is CGMCC No.0887, and through secular breeding work, enzyme activity has reached 100,000 units per ml fermented liquids; In Catalyst Production, introduce membrane separation technique, production can be carried out in serialization; Good reaction selectivity, raw material availability are high, environmental pollution is little, production cost is low; The product cumulative concentration is high, but industrializing implementation production.
Claims (10)
1. the method for a biologically catalytic hydroxyacetic acid production; Comprise through nitrilase and come the hydrolysis reaction of catalysis hydroxyacetonitrile to obtain oxyacetic acid, it is characterized in that described nitrilase obtains through fermentation culture brevibacterium casei (Brevibacterium casei) CGMCC No.0887.
2. the method for claim 1 is characterized in that described nitrilase is the fermented liquid that fermentation culture obtains to be carried out the enzyme cell harvesting through centrifugal or membrane filtration system obtain.
3. method as claimed in claim 2 is characterized in that described membrane filtration system is hollow-fibre membrane or rolled film or ceramic membrane or ultrafiltration membrance filter system.
4. method as claimed in claim 3, said ultrafiltration membrance filter system is the Ultra-flo filtering system.
5. the method for claim 1 is characterized in that described hydroxyacetonitrile carries out pre-treatment earlier before hydrolysis reaction, handles promptly with highly basic or basic carbonate neutralization, or with highly basic or weak base anion resins, to pH6-8.
6. method as claimed in claim 5 is characterized in that described highly basic is selected from: NaOH, Ca (OH)
2, Ba (OH)
2Or KOH.
7. method as claimed in claim 5 is characterized in that described basic carbonate is selected from: CaCO
3, Na
2CO
3, NaHCO
3Or Ca (HCO
3)
2
8. method as claimed in claim 5 is characterized in that described highly basic or weak base anion resins are selected from: D201 type, D202 type or D301 type.
9. the method for claim 1, it is characterized in that the condition of described hydrolysis reaction is following: temperature is 0-70 ℃, and pH is 2-10, and the concentration of substrate hydroxyacetonitrile is controlled at 0～50% (wt%), and wherein the concentration of substrate hydroxyacetonitrile is not 0% (wt%).
10. a brevibacterium casei (Brevibacterium casei) that is used for biologically catalytic hydroxyacetic acid production is characterized in that preserving number is CGMCC No.0887.
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| CN101186933B (en) * | 2007-11-09 | 2011-03-30 | 华东理工大学 | Culture of bacillus alcaligenes and method for preparing glycolic acid by using the same to hydrolyzing nitrile |
| CN101875907B (en) * | 2009-04-28 | 2013-07-10 | 上海市农药研究所 | Microbial strain capable of hydrolyzing 3-hydroxybutyronitrile |
| CN110923223B (en) * | 2019-11-25 | 2021-10-08 | 中山大学 | Novel nitrilase and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3940316A (en) * | 1973-09-19 | 1976-02-24 | Agence Nationale De Valorisation De La Recherche (Anvar) | Process for the production of organic acids by biological hydrolysis |
| US6416980B1 (en) * | 2001-02-23 | 2002-07-09 | E. I. Du Pont De Nemours & Company | Method for producing glycolic acid from glycolonitrile using nitrilase |
-
2004
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3940316A (en) * | 1973-09-19 | 1976-02-24 | Agence Nationale De Valorisation De La Recherche (Anvar) | Process for the production of organic acids by biological hydrolysis |
| US6416980B1 (en) * | 2001-02-23 | 2002-07-09 | E. I. Du Pont De Nemours & Company | Method for producing glycolic acid from glycolonitrile using nitrilase |
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