CN102277322B - Arthrobacter nitroguajacolicus and method for preparing iminodiacetic acid by utilizing same to catalyze iminodiacetonitrile - Google Patents
Arthrobacter nitroguajacolicus and method for preparing iminodiacetic acid by utilizing same to catalyze iminodiacetonitrile Download PDFInfo
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- CN102277322B CN102277322B CN 201110224295 CN201110224295A CN102277322B CN 102277322 B CN102277322 B CN 102277322B CN 201110224295 CN201110224295 CN 201110224295 CN 201110224295 A CN201110224295 A CN 201110224295A CN 102277322 B CN102277322 B CN 102277322B
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- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 33
- BSRDNMMLQYNQQD-UHFFFAOYSA-N iminodiacetonitrile Chemical compound N#CCNCC#N BSRDNMMLQYNQQD-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 241001540716 Paenarthrobacter nitroguajacolicus Species 0.000 title claims description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 241000186063 Arthrobacter Species 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
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- 210000001822 immobilized cell Anatomy 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
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- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- KVNRLNFWIYMESJ-UHFFFAOYSA-N butyronitrile Chemical compound CCCC#N KVNRLNFWIYMESJ-UHFFFAOYSA-N 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 5
- 238000006555 catalytic reaction Methods 0.000 claims description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- JBKVHLHDHHXQEQ-UHFFFAOYSA-N epsilon-caprolactam Chemical compound O=C1CCCCCN1 JBKVHLHDHHXQEQ-UHFFFAOYSA-N 0.000 claims description 3
- LRDFRRGEGBBSRN-UHFFFAOYSA-N isobutyronitrile Chemical compound CC(C)C#N LRDFRRGEGBBSRN-UHFFFAOYSA-N 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
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- 230000009466 transformation Effects 0.000 description 8
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- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 3
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- 102000018120 Recombinases Human genes 0.000 description 3
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- 235000010413 sodium alginate Nutrition 0.000 description 3
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- CSIFGMFVGDBOQC-UHFFFAOYSA-N 3-iminobutanenitrile Chemical compound CC(=N)CC#N CSIFGMFVGDBOQC-UHFFFAOYSA-N 0.000 description 2
- 241000588813 Alcaligenes faecalis Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- JZVZLMSUWQYJRJ-UHFFFAOYSA-N C1(=CC=CC=C1)O.[K].[Br] Chemical compound C1(=CC=CC=C1)O.[K].[Br] JZVZLMSUWQYJRJ-UHFFFAOYSA-N 0.000 description 2
- 239000005562 Glyphosate Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for preparing iminodiacetic acid by utilizing a microbe to catalyze iminodiacetonitrile and a bacterial strain. The method is characterized by carrying out enzyme production culture on the bacterial strain producing nitrilase to obtain nitrilase and taking the nitrilase as a biocatalyst to biologically catalyze the iminodiacetonitrile to prepare the iminodiacetic acid. The method has the following beneficial effects: the method provides a basis for producing the iminodiacetic acid by biologically catalyzing the iminodiacetonitrile and has important application prospect.
Description
(1) technical field
The present invention relates to a kind of method of producing the preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes of nitrilase, and the bacterial strain with nitrilase activity of screening acquisition.
(2) background technology
Iminodiethanoic acid (IDA) is a kind of important industrial chemicals, have very strong complex ability energy and many kinds of metal ions complexing and form inner complex, can form blue huge legendary turtle compound with cupric ion, be commonly used for complexing agent and tensio-active agent, being usually used in organic synthesis, also is the important intermediate that glyphosate (Glyphosate) is produced.Because contain imino-and carboxyl in its molecule, chemical property is very active, is widely used in the fields such as plating, dyestuff, medicine, electronics.It is a kind of important chemical intermediate.
At present the preparation iminodiethanoic acid generally adopts chemical process, mainly contains chloroactic acid method, nitrilotriacetic acid method, ammonia for chloroactic acid method, prussic acid method, monoethanolamine process, diethanolamine method etc. according to the difference of raw material.Traditional chemical process exists the generation cost high, seriously polluted, high to equipment requirements, perhaps uses the weakness such as prussic acid of severe toxicity.Utilize the biocatalysis iminodiacetonitrile to produce iminodiethanoic acid and compare with chemical hydrolysis, it is advantageous that: (1) reaction conditions is gentle.Chemical hydrolysis need to carry out about 100 ℃, must be equipped with heating and temperature control unit, severe reaction conditions, and energy consumption is high and high to equipment requirements.And biological catalysis generally all is to carry out at normal temperatures, and reaction conditions is gentle, and equipment cost is relatively low.(2) raw material consumption significantly reduces, and does not substantially produce soluble salt.Utilize nitrilase one one-step hydrolysis to generate iminodiethanoic acid, saved alkaline hydrolysis and acidification technique, avoided a large amount of uses of NaOH and concentrated hydrochloric acid.Utilize NH in the biological process
4OH regulates the pH value of transformation system, adopts regeneration techniques in product reclaims, and can effectively reduce the discharging of soluble salt.(3) discharge of wastewater is few.According to present chemical hydrolysis process, produce iminodiethanoic acid per ton and will produce at least 8 tons of waste water.After adopting biological process, calculate (reuse 5 times) with nitrilase enzyme activity 100,000 units, the wastewater flow rate of producing iminodiethanoic acid per ton can be controlled in below 2 tons, and does not have the difficult impurity such as salt in the waste water, and biodegradability is strong.
Utilize nitrile invertase biocatalysis iminodiacetonitrile to prepare iminodiethanoic acid, can overcome the defective that chemical method is produced iminodiethanoic acid.Biocatalysis is the catalyst system optionally of the most efficient, the tool known to the mankind up to now, can provide many traditional chemical methods can not or to be difficult for the synthetic method of the synthetic chipal compounds that obtains, demonstrate good chemo-selective, regioselectivity and stereoselectivity.And bioconversion reaction mild condition, reaction product purity be high, separate easily and purifying, in the production of pesticide intermediate, demonstrates huge development potentiality.
(3) summary of the invention
The present invention seeks to the cultivation of production by biological enzyme and obtain the preparation method that nitrilase catalysis iminodiacetonitrile prepares iminodiethanoic acid.The present invention another purpose provide the microorganism of the product nitrilase that obtains of screening.
The technical solution used in the present invention is:
Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, 430072, deposit number CCTCCNo:M 208252, preservation date on December 11st, 2008.
Above-mentioned bacterial strains separates acquisition by bio-engineering research institute of Zhejiang Polytechnical University from soil.
The invention still further relates to the method for utilizing aforementioned strain microorganism preparing iminodiacetic acid by catalyzing iminodiacetonitrile, described method is to utilize the bacterial strain that produces nitrilase, the multiparity enzyme is cultivated and is obtained nitrilase, prepares iminodiethanoic acid take this enzyme as biological catalyst iminodiacetonitrile.The process that the biocatalysis iminodiacetonitrile is produced iminodiethanoic acid is as follows:
The method for preparing iminodiethanoic acid of the present invention is specific as follows:
(A) described Arthrobacter CCTCC No:M 208252 is seeded to culture medium, adds inductor a and under 100~200r/min, 20~35 ℃ of conditions, carry out fermentation culture 2~5d; Described inductor a is n-Butyronitrile, isopropyl cyanide or hexanolactam; Described culture medium final concentration consists of: glycerine 8g/L, yeast extract paste 6g/L, NaCl 1g/L, K
2HPO
45g/L, MgSO
40.2g/L solvent is water, pH value 7.0; The final concentration of described inductor a is 5g/L;
(B) get the aqueous solution that iminodiacetonitrile is mixed with mass concentration 0.5%~6%, transferring initial pH is 7.0~7.5, as the enzymic catalytic reaction substrate solution; The wet thallus that obtains with step (A) fermentation, the crude enzyme liquid that cytoclasis obtains or the immobilized cell that obtains through immobilization are as the enzyme source, and enzyme source add-on is counted 0.1~10g wet thallus/10mL substrate solution with wet thallus; The control temperature of reaction is that 30 ℃, reaction pH are 6.0~9.0, carries out conversion reaction 8~16h, and after reaction finished, reaction solution obtained iminodiethanoic acid through separation and purification.
The described fermentation culture of above-mentioned steps (A) is preferably carried out fermentation culture 3d under 30 ℃ of conditions; The described control temperature of reaction of step (B) is preferably 30 ℃, carries out conversion reaction 10h.
The microorganism of product nitrilase of the present invention is screened acquisition by the following method, and described method comprises primary dcreening operation and multiple sieve:
(1) primary dcreening operation: inoculation to be screened to the primary dcreening operation substratum, is cultivated 1~4d for 25~37 ℃, produce the bacterial strain of yellow variable color circle in the picking colony, be inoculated into the LB slant medium, get bacterial strain behind 25~30 ℃ of cultivation 2~5d and carry out multiple sieve; Described primary dcreening operation substratum final concentration is composed as follows: imido grpup diacetonitrile 5g/L, yeast extract paste 6g/L, NaCl1g/L, K
2HPO
41g/L, MgSO
41g/L, bromine potassium phenol violet 1g/L, agar powder 20g/L, solvent are water, pH 7.0; Be that the every 1L of described primary dcreening operation substratum prepares by following composition: imido grpup diacetonitrile 5g, yeast extract paste 6g, NaCl 1g, K
2HPO
41g, MgSO
41g, bromine potassium phenol violet 0.1g, agar powder 20g, solvent are water, pH 7.0;
28~30 ℃ of the described culture temperature of preferred steps (1) primary dcreening operation are cultivated 3~4d.
(2) multiple sieve: the inoculation that primary dcreening operation is obtained is to sieving again substratum, and 100~200r/min, 25~35 ℃ of lower 2~7d that cultivate collect the growing amount that thalline is measured iminodiethanoic acid, collects to generate the iminodiacetic acid (salt) acid content greater than 10% microorganism; Described to sieve again the substratum final concentration composed as follows: glycerine 8g/L, yeast extract paste 6g/L, NaCl 1g/L, K
2HPO
45g/L, MgSO
40.2g/L n-Butyronitrile 5g, solvent are water, pH 7.0, and namely the described every 1L of substratum that sieves again prepares by following composition: glycerine 8g, yeast extract paste 6g, NaCl 1g, K
2HPO
45g, MgSO
40.2g inductor b 5g, solvent are water, pH 7.0.
It is 30~32 ℃ that preferred steps (2) is sieved described culture temperature again.
Inductor a of the present invention and inductor b are represented all is inductor in the microbial cultivation process, and a and b just appear at different step in order to distinguish inductor here.
Beneficial effect of the present invention is mainly reflected in: provide a kind of bacterial strain multiparity enzyme that produces nitrilase that utilizes to cultivate, obtain nitrilase, prepare the method for iminodiethanoic acid as biological catalyst iminodiacetonitrile take this enzyme, but High-efficient Production iminodiethanoic acid, the present invention also provides the screening method that can produce the microorganism of nitrilase, and obtained the bacterial strain of multiple product nitrilase, provide the foundation for adopting the biocatalysis iminodiacetonitrile to produce iminodiethanoic acid, have the important application prospect.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the primary dcreening operation that produces the nitrilase bacterial classification
The every 1L of primary dcreening operation substratum prepares by following composition: yeast extract paste 6g, NaCl 1g, K
2HPO
41g, MgSO
41g, agar powder 20g, pH 7.0 water complement to 1000mL, are cooled to 50 ℃ after the sterilization and add 5g iminodiacetonitrile and 1g purpurum bromocresolis, and mixing is to dull and stereotyped.
Collect bacterium and screen, coat on the flat board, 28~30 ℃ of temperature, cultivate 3~4d, produce the bacterial strain of yellow variable color circle in the picking colony, be inoculated into LB slant culture (25~28 ℃);
The microorganism strains of yellow variable color circle has: edge pseudomonas ZJB09121 (Pseudomonas marginalis ZJB09121), pseudomonas putida ZJB09134 (Pseudomonas putia ZJB09134), pseudomonas putida ZJB09129 (Pseudomonas putia ZJB09129), pseudomonas putida ZJB09135 (Pseudomonas putia ZJB09135), Pseudomonas fluorescens ZJB09137 (Pseudomonas fluorescens ZJB09137), acid-producing Klebsiella bacterium ZJB09123 (Kelbsiella ocytoca ZJB09123), Fu Shi rhodococcus ZJB09124 (Rhodococcus wratislaviensis ZJB09124), have a liking for pyridine rhodococcus ZJB09126 (Rhodococcus pyridinivorans ZJB09126), prunosus red coccus ZJB09125 (Rhodococcus rhodochrous ZJB09125), Rhodococcus ruber ZJB09127 (Rhodococcus rubber ZJB09127), micrococcus luteus ZJB09131 (Micrococcus luteus ZJB09131), brevibacterium halotolerans ZJB09132 (Brevibacterium halotoerans ZJB09132), quick acidovorax facilis ZJB09122 (Acidovorax facilis ZJB09122), Bacillus foecalis alkaligenes ZJB09138 (Alcaligenes faecalis ZJB09138), Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 (being CCTCC No:M 208252).
Embodiment 2: the multiple sieve that produces the nitrilase bacterial classification
With produced metachromasia and the large bacterial strain of yellow variable color circle that screens arranged among the embodiment 1, be inoculated in the multiple sieve substratum 30~32 ℃, 100~200r/min, 500mL triangular flask liquid amount 150mL, fermentation culture 3d, centrifugal crude enzyme liquid or the wet thallus of obtaining of fermented liquid.Respectively get the 1g wet thallus, place the 500ml triangular flask, add the reaction substrate iminodiacetonitrile aqueous solution of 50ml 2% (w/w), react 60min under 30 ℃ of shaking table 150r/min conditions, centrifugal removal thalline, the growing amount of mensuration iminodiethanoic acid, calculate transformation efficiency, the results are shown in Table 1.
Sieving again the every 1L of substratum prepares by following composition: glycerine 8g, yeast extract paste 6g, NaCl 1g, K
2HPO
45g, MgSO
40.2g, n-Butyronitrile 5g, pH value 7.0, water complements to 1000mL; The inductor n-Butyronitrile also can isopropyl cyanide or hexanolactam replacement.
Table 1: the multiple sieve result who produces the nitrilase bacterial classification
Sequence number | Strain name | Transformation efficiency (%) |
1 | Edge pseudomonas (Pseudomonas marginalis) ZJB09121 | 26.5 |
2 | Quick acidovorax facilis (Acidovorax facilis) ZJB09122 | 21.9 |
3 | Acid-producing Klebsiella bacterium (Kelbsiella ocytoca) ZJB09123 | 22.3 |
4 | Fu Shi rhodococcus (Rhodococcus wratislaviensis) ZJB09124 | 23.6 |
5 | Prunosus red coccus (Rhodococcus rhodochrous) ZJB09125 | 24.7 |
6 | Have a liking for pyridine rhodococcus (Rhodococcus pyridinivorans) ZJB09126 | 30.0 |
7 | Rhodococcus ruber (Rhodococcus rubber) ZJB09127 | 23.3 |
8 | Pseudomonas putida (Pseudomonas putia) ZJB09129 | 24.4 |
9 | Pseudomonas putida (Pseudomonas putia) ZJB09135 | 23,9 |
10 | Micrococcus luteus (Micrococcus luteus) ZJB09131 | 24.7 |
11 | Brevibacterium halotolerans (Brevibacterium halotoerans) ZJB09132 | 25.8 |
12 | Pseudomonas putida (Pseudomonas putia) ZJB09134 | 13.1 |
13 | Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09137 | 14.3 |
14 | Bacillus foecalis alkaligenes (Pseudomonas aeruginosa) ZJB09138 | 54.8 |
15 | Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 | 26.4 |
[0032] Embodiment 3: cultivation and the biocatalysis of producing the nitrilase microorganism prepare iminodiethanoic acid
The every 1L of culture medium prepares by following composition: glycerine 8g, yeast extract paste 6g, NaCl 1g, K
2HPO
45g, MgSO
40.2g water complements to 1000mL; Other adds inductor n-Butyronitrile 5g.
Culture medium is sub-packed in the 500mL triangular flask, liquid amount 100mL/ bottle, and with Autoclave heat sterilization 20min, temperature is 121 ℃.
With the bacterial strain that obtains among the embodiment 1, inoculate respectively bacterial classification that 1 ring cultivates through slant activation in the culture medium that has added inductor, be 200r/min at the shaking flask rotating speed, temperature is under 30 ℃, cultivates 3d, collects wet thallus as the thick enzyme of nitrilase.In the iminodiacetonitrile aqueous solution of 100mL 3% (w/w), add the reaction that is hydrolyzed of 5g wet thallus, 30 ℃ of temperature of control, mixing speed 200r/min, the ammoniacal liquor auto-feeding is regulated pH 7.0, measure the productive rate of product iminodiethanoic acid and calculate transformation efficiency behind the hydrolysis 10h, the results are shown in Table 2.
Table 2: the productive rate and the transformation efficiency that produce nitrilase bacterial classification iminodiethanoic acid
Sequence number | Strain name | Transformation efficiency (%) |
1 | Edge pseudomonas (Pseudomonas marginalis) ZJB09121 | 23.7 |
2 | Quick acidovorax facilis (Acidovorax facilis) ZJB09122 | 25.1 |
3 | Acid-producing Klebsiella bacterium (Kelbsiella ocytoca) ZJB09123 | 20.8 |
4 | Fu Shi rhodococcus (Rhodococcus wratislaviensis) ZJB09124 | 24.5 |
5 | Prunosus red coccus (Rhodococcus rhodochrous) ZJB09125 | 26.2 |
6 | Have a liking for pyridine rhodococcus (Rhodococcus pyridinivorans) ZJB09126 | 30.7 |
7 | Rhodococcus ruber (Rhodococcus rubber) ZJB09127 | 23.9 |
8 | Pseudomonas putida (Pseudomonas putia) ZJB09129 | 23.1 |
9 | Pseudomonas putida (Pseudomonas putia) ZJB09135 | 24.2 |
10 | Micrococcus luteus (Micrococcus luteus) ZJB09131 | 24.8 |
11 | Brevibacterium halotolerans (Brevibacterium halotoerans) ZJB09132 | 25.7 |
12 | Pseudomonas putida (Pseudomonas putia) ZJB09134 | 13.0 |
13 | Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09137 | 14.7 |
14 | Bacillus foecalis alkaligenes (Pseudomonas aeruginosa) ZJB09138 | 54.9 |
15 | Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 | 26.5 |
[0038] Embodiment 4: the free cell biocatalysis is produced iminodiethanoic acid
In order to investigate the stability of the nitrilase that bacterial isolates produces that obtains among the embodiment 1, press the method for embodiment 3, the bacterial isolates that obtains among the embodiment 1 is carried out repeatedly in batches enzymic hydrolysis test of free cell, measure the content of iminodiethanoic acid behind the hydrolysis 10h, and centrifugal collection thalline, the thalline of centrifugal collection is used further to hydrolysis reaction, 3 times repeatedly.Test-results such as table 3.
Table 3: the free cell of product nitrilase bacterial classification is in batches enzymic hydrolysis test-results repeatedly
Embodiment 5: the immobilized cell biocatalysis is produced iminodiethanoic acid
In order further to investigate the stability of the nitrilase that bacterial isolates produces that obtains among the embodiment 1, the part bacterial strain is carried out cell fixation, and carry out repeatedly in batches enzymic hydrolysis test.
Take by weighing the edge pseudomonas ZJB09121 (Pseudomonas marginalis ZJB09121) that cultivates by embodiment 3, pseudomonas putida ZJB09134 (Pseudomonas putia ZJB09134), pseudomonas putida ZJB09129 (Pseudomonas putia ZJB09129), pseudomonas putida ZJB09135 (Pseudomonas putia ZJB09135), Pseudomonas fluorescens ZJB09137 (Pseudomonas fluorescens ZJB09137), acid-producing Klebsiella bacterium ZJB09123 (Kelbsiella ocytoca ZJB09123), Fu Shi rhodococcus ZJB09124 (Rhodococcus wratislaviensis ZJB09124), Fu Shi rhodococcus ZJB09126 (Rhodococcus pyridinivorans ZJB09126), prunosus red coccus ZJB09125 (Rhodococcus rhodochrous ZJB09125), Rhodococcus ruber ZJB09127 (Rhodococcus rubber ZJB09127), micrococcus luteus ZJB09131 (Micrococcus luteus ZJB09131), brevibacterium halotolerans ZJB09132 (Brevibacterium halotoerans ZJB09132), quick acidovorax facilis ZJB09122 (Acidovorax facilis ZJB09122, Bacillus foecalis alkaligenes ZJB09138 (Alcaligenes faecalis ZJB09138), the somatic cells of Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99, be mixed with bacteria suspension with deionized water, be 2% (w/w) sodium alginate heating for dissolving with concentration, to be made into the even mixed solution of 6% (g wet thallus/ml sodium alginate soln) in somatic cells and the sodium alginate soln of wet thallus after the cooling, and mixed solution be splashed into the CaCl of 2% (w/w)
2In the solution, immobilization 12h; Leach particle, clean with physiological saline, above-mentioned immobilized cell uses the glutaraldehyde of 0.2% (w/v)-HCl solution (to contain 30mM CaCl again
2) stir process 24h under the room temperature, with distillation washing 3 times, obtain crosslinked immobilized cell afterwards.With the crosslinked immobilized cell of above-mentioned different strains, be used for repeatedly batch hydrolysis and split, 3 times repeatedly, result such as table 4.
Table 4: the free cell of product nitrilase bacterial classification is in batches enzymic hydrolysis test-results repeatedly
Embodiment 6: the resolvase biocatalysis is produced iminodiethanoic acid
With the bacterium thalline that obtains through fermentation among the embodiment 1, utilize 0.9% physiological saline washing 3 times, collecting thalline is resuspended in Tris-HCl pH 7.0 damping fluids, regulate cell concentration and count 10% (g/m1) with wet thallus, utilize ultrasonic wave, high pressure cracker, high pressure homogenizer that cell is carried out fragmentation, obtain containing the crude enzyme liquid of free nitrilase, utilize resolvase to carry out biocatalytic reaction.In the iminodiacetonitrile aqueous solution of 9m13% (w/w), add the reaction that is hydrolyzed of 1ml crude enzyme liquid, 30 ℃ of temperature of control, mixing speed 200r/min, the ammoniacal liquor auto-feeding is regulated pH 7.0, behind the hydrolysis 10h, behind the centrifugal 20min of 10000rpm, measure the productive rate of product iminodiethanoic acid in the supernatant liquor and calculate transformation efficiency, the results are shown in Table 5.
Table 5: free nitrilase biocatalysis is produced the iminodiethanoic acid result
Sequence number | Strain name | Transformation efficiency (%) |
1 | Edge pseudomonas (Pseudomonas marginalis) ZJB09121 | 22.9 |
2 | Quick acidovorax facilis (Acidovorax facilis) ZJB09122 | 26.7 |
3 | Acid-producing Klebsiella bacterium (Kelbsiella ocytoca) ZJB09123 | 22.3 |
[0052]
4 | Fu Shi rhodococcus (Rhodococcus wratislaviensis) ZJB09124 | 24.1 |
5 | Prunosus red coccus (Rhodococcus rhodochrous) ZJB09125 | 27.2 |
6 | Have a liking for pyridine rhodococcus (Rhodococcus pyridinivorans) ZJB09126 | 29.1 |
7 | Rhodococcus ruber (Rhodococcus rubber) ZJB09127 | 24.7 |
8 | Pseudomonas putida (Pseudomonas putia) ZJB09129 | 26.5 |
9 | Pseudomonas putida (Pseudomonas putia) ZJB09135 | 28.3 |
10 | Micrococcus luteus (Micrococcus luteus) ZJB09131 | 31.2 |
11 | Brevibacterium halotolerans (Brevibacterium halotoerans) ZJB09132 | 25.6 |
12 | Pseudomonas putida (Pseudomonas putia) ZJB09134 | 20.1 |
13 | Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09137 | 15.3 |
14 | Bacillus foecalis alkaligenes (Pseudomonas aeruginosa) ZJB09138 | 55.9 |
15 | Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 | 28.7 |
Reach a conclusion by above embodiment: the pseudomonas (Pseudomonas) of acquisition that the present invention screens, klebsiella (Kelbsiella), rhodococcus (Rhodococcus), micrococci (Micrococcus), bacillus (Alcaligenes), tyrothricin (Brevibacterium), Arthrobacter (Arthrobacter), bacterial strain and mutant strain thereof that acidovorax facilis (Acidovorax) etc. belong to, all effective to producing iminodiethanoic acid, free cell no matter, resolvase after the cytoclasis, immobilized cell has good conversion iminodiacetonitrile to produce the effect of iminodiethanoic acid.The stability of the nitrilase that the free cell of these bacterial strains and immobilized cell produce better, so these bacterial strains can be used for biocatalysis hydrolysis iminodiacetonitrile, and then produce iminodiethanoic acid.
Claims (5)
1. Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, 430072, deposit number CCTCC No:M 208252, preservation date on December 11st, 2008.
2. method of utilizing the described Arthrobacter CCTCC of claim 1 No:M 208252 preparing iminodiacetic acid by catalyzing iminodiacetonitrile, described method is as follows:
(A) described Arthrobacter CCTCC No:M 208252 is seeded to culture medium, adds inductor a and under 100 ~ 200 r/min, 20 ~ 35 ℃ of conditions, carry out fermentation culture 2 ~ 5d; Described inductor a is n-Butyronitrile, isopropyl cyanide or hexanolactam; Described culture medium final concentration consists of: glycerine 8 g/L, yeast extract paste 6 g/L, NaCl 1 g/L, K
2HPO
45 g/L, MgSO
40.2 g/L, solvent are water, pH value 6.0 ~ 8.0; The final concentration of described inductor a is 5 g/L;
(B) get the aqueous solution that iminodiacetonitrile is mixed with mass concentration 0.5% ~ 6%, transferring initial pH is 7. 0 ~ 7. 5, as the enzymic catalytic reaction substrate solution; The wet thallus that obtains with step (A) fermentation, the crude enzyme liquid that cytoclasis obtains or the immobilized cell that obtains through immobilization are as the enzyme source, and enzyme source add-on is counted 0.1 ~ 10 g wet thallus/10 mL substrate solutions with wet thallus; The control temperature of reaction is that 30 ℃, reaction pH are 6.0 ~ 9.0, carries out conversion reaction 8 ~ 16 h, and after reaction finished, reaction solution obtained iminodiethanoic acid through separation and purification.
3. method as claimed in claim 2 is characterized in that described step (B) gets the aqueous solution that iminodiacetonitrile is mixed with mass concentration 0.5% ~ 6%, transfers pH as 7. 0 ~ 7. 5 take ammoniacal liquor, NaOH or the HCl aqueous solution.
4. method as claimed in claim 2 is characterized in that the described fermentation culture of described step (A) carries out fermentation culture 3d under 30 ℃ of conditions.
5. method as claimed in claim 2 is characterized in that the described control temperature of reaction of described step (B) is 30 ℃, carries out conversion reaction 10 h.
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